NEP INHIBITORS FOR THE TREATMENT OF FEMALE SEXUAL DISFUNCTION

COMPOUNDS FOR THE TREATMENT OF FEMALE SEXUAL DYSFUNCTION

The present invention relates to pharmaceutical preparation, which is useful for treatment of female sexual dysfunction ( ZhSD ), in particular disorder sexual excitation of in women ( RPVZh ).

The present invention also relates to method of treating ZhSD, in particular RPVZh.

The present invention also relates to analysis for screening compounds, mineral in treatment of ZhSD, in particular RPVZh.

For convenience of list of rate, which are used in below the following text, driven before division formula of the invention.

Phase of excitation of sexual response in women difficult discrimination between from attraction of, until begin physiological of changing in vagina and klitore, and also in other genital organs. Floor excitation and pleasure accompanied combination of vascular and nervous-muscular phenomena, which lead to overflow blood of the clitoris, lips and wall of the vagina, enhancement of lubrication of vagina and expansion of vaginal lumen ( Levin, 1980; Ottesen, 1983 ; Levin, 1991; Levin, 1992 ; Sjoberg, 1992;

Wagner, 1992; Schiavietal., 1995; Mastersetal., 1996; Bermanetal., 1999).

Overflow blood vagina creates conditions for transudation, and this process responsible for amplification of lubrication vagina. Transsudatsiya makes possible current plasma through epithelium on the surface of the vagina, driving force which is amplified circulation in vaginal capillary bed during excited state. Besides, overflow blood leads to increase of the length of the vagina and diameter of lumen, especially in distal 2/3 vaginal channel. Expansion of lumen vagina is outcome combination of relaxation of smooth muscle of its wall and relaxation of skeletal muscles bottom of pelvic cavity. Is considered, that some sexual pain disorder, such as vaginizm, at least partially are result of inadequate relaxation, preventing expansion of vagina; remains to be still establish, whether this in first turn problem smooth or skeletal muscles ( Levin, 1980;

Ottesen, 1983; Levin, 1991 ; Levin, 1992; Sjoberg, 1992 ; Wagner, 1992; Schiaviet al., 1995; Masterseta ! ., 1996; Bermanetal., 1999).

Vascular and mikrososudistaya network vagina innerviruetsyanervami, containing neiropeptidy, and other neuromediators-candidates. They include sibling gene calcitonin peptide (CGRP), neuropeptide Υ (NPY), synthase nitrogen oxide (NOS), substance p and vazoaktivnyiintestinalnyi peptide (VIP) ( Hoyleet al., 1996). Peptides, which are present in klitore, discusses below. Synthase nitrogen oxide, which frequently is localized together with vip, immunologically elicits more a strong expression in deep arteries and veins, than in blood vessels proprio ( Hoyleet al., 1996).

Is known, that some individuals can suffer female sexual dysfunction ( ZhSD ).

ZhSD best to determine as embarrasment or inability of a woman to produce satisfaction in sexual expression. ZhSD is a term for several different sexual disorders in women ( Leiblum, 1998, Bermanet al., 1999). In women may be absent attraction, may be difficulties with excitation or orgazmom, pain at floor snoshenii or a combination of these problems. Some types of diseases, drugs, damage or psychological problem can cause ZhSD.

Of investigating, Readers sexual dysfunction in pairs, show, that up to 76% women have complaints on sexual dysfunction, and that 30-50% women in SShA is tested ZhSD.

Subtypes ZhSD include disorder gipoaktivnogo sexual attraction, disorder sexual excitation of in women, orgazmicheskoe disorder, and disorder sexual attraction.

Devised therapy directed to treatment of specific subtypes ZhSD, mainly disorders attraction and exciting.

Category ZhSD best determine, opposing thus their phases of normal sexual reaction in women: attraction, excitation and orgasm ( Leiblum, 1998). Floor attraction, or libido, is engine sexual expression, and its manifestations often include sexual ideas or in public life of interest partner, either under action of other erotic stimuli. In contrast to this, excitation represents vascular reaction on the genital stimulation, an important component of which is lubrication of vagina and stretching vagina. So, floor excitation, in contrast to sex alcoholic, represents reaction, related to genitalnomu (for example vaginalnomu and kpitornomu ) blood flow and optionally to sensitivity. Orgasm represents release of sexual voltage, which reaches culmination for in excitation time. Consequently, ZhSD usually arises, when female experts shows inadequate or unsatisfactory response in any of these phases, usually in phase of attraction, of excitation or orgasm. Category ZhSD include disorder gipoaktivnogo sexual attraction, disorder sexual of excitation, orgazmicheskoe disorder, and sexual pain disorder.

Disorder gipoaktivnogo sexual attraction has place, if female experts has no or has weak desire be sexual, or has no or has low sexual thoughts or fantazii. Cause of this type ZhSD may be low level of testosterone due to or natural menopause, or surgical menopause. Other ratio are disease, drugs, fatigue, depression and anxiety.

Disorder sexual excitation of in women ( RPVZh ) is characterised by inadequate genital by reaction on the genital stimulation. Genitals (for example vagina and/or clitoris) are not subjected to overflow blood, which characterizes normal floor excitation. Vaginal wall is insufficient are lubricated by, due to which floor intercourse becomes painful. This can be used as obstacle offensive orgasm. Disorder of excitation may be caused Your savings estrogen at menopause or after delivery and in lactation period, and also diseases with vascular components, such as diabetes and atherosclerosis. Other causes derive from treatment diuretics, antigistaminami, antidepressants, for example SSRIs, or antigipertenzivnymi agents. RPVZh more detail discusses below.

Sexual pain disorder (which include dispareuniyu and vaginizm ) are characterized by pain in a result of penetration and can be caused by drugs, which loosened lubrication, endometriosis, inflammation organs in the pelvis, inflammatory intestinal disease or problems urinary path.

Prevalence of ZhSD difficult to estimate, since this term embraces several types of problem, some of which difficult-to-determine, and also in communication with the, that of interest in treatment of ZhSD is relatively recent. Numerous sexual problem in women are connected or directly with process of ageing of a woman, or with chronic diseases, such as diabetes and hypertension.

In messages about of occurrence and the scope of ZhSD there are large divergence, which partially are accounted use of various criteria evaluating, however the majority of researchers is imparted about the fact, that significant share of the healthy in other respect women has symptoms of one or more than one subgroup ZhSD. For example, comparative of investigating sexual dysfunction in pairs, shown, that 63% women have dysfunction of excitation or orgazmicheskuyu dysfunction compared with 40% men with erectile or eyakulyatornoi dysfunction ( Flanketa ! ., 1978).

However prevalence of disorder sexual excitation of in women considerably varies from examination to survey. By data of recent a report on examination national health and social life 19% women reported about difficulties with ozonides, and in outpatient gynecological clinic about similar difficulties with ozonides reported 14% women ( Roseneta ! ., 1993).

Some of investigating revealed also dysfunction of sexual excitation of in women diabetics (up to 47%), and this included attenuation of lubrication vagina ( Winczeet and! ., 1993). Connection between neuropathy and sexual dysfunction obstructed.

Much numerical of investigating shown also, from 11 to 48% of all women may have age Your lu sexual attraction. Also from 11 to 50% women is about problem with excitation and ozonides and is tested pain at floor act. Vaginizm much less than is spread, infecting approximately 1% of women.

Of investigating sexually experimental women shown, that 5-10% have primary anorgazmiyu. Other 10% have a small orgazmy, and more 10% is tested their disjointed fashion ( Spectoret a/., 1990).

Since ZhSD consists of several subtypes, at which symptoms are manifested in separate phases cycle of sexual reaction, single therapy, not exists. Modern therapy ZhSD is focused mainly on psychological plan and plan relations. Therapy ZhSD gradually generates, since all exceeds clinical and fundamental research is dedicated investigation of this medical problem. Complaints women on sexual disorder are not completely psychological in pathophysiology, especially for those individuals, which may have component dysfunction of vascular genesis (for example RPVZh ), affects common complaint of a woman on sexual disorder. At the present time there is no drugs, permitted for treatment of ZhSD. Empirical medicinal therapy comprises introducing estrogen (locally or in the form gormonozamestitelnoi therapy), androgen or drugs, influencing on mood, such as buspirone or trazodone. These possible versions of treatment of frequently are unsatisfactory from-because of low efficiency or unacceptable adverse effects.

Since interest to pharmacological treatment of ZhSD is relatively recent, therapy consists of the following: psychological cases, sex lubricating means, sold without prescribing the recipe, and candidates for research, including drugs, endorsed for other states. These drugs include hormonal agents, or testosterone, or combination of estrogen and testosterone, and with recent time vascular of drug, efficiency which had at erectile dysfunction in men. Nor for one their these agents is not demonstrated high efficiency of treatment ZhSD.

Reaction of sexual of excitation includes boss of blood in the pelvic region, lubrication of vagina and tension and swelling external sexual organs. Disturbance causes notable distress and/or interrelations difficulties. Of investigating, Readers sexual dysfunction in pairs, show, that exists large number of women, breast dysfunction sexual excitation of, otherwise known as disorder sexual excitation of in women ( RPVZh ).

Instruction on diagnostics and statistics (DSM) iv american association psychiatrists determines disorder sexual excitation of in women ( RPVZh ) as follows:

" Stable or A repeating inability of reach or maintain adequate reaction lubrication-swelling of at floor excitation till completion of sexual activity. This disturbance can cause notable distress or interrelations difficulties ".

RPVZh is widely occurring sexual disorder, projectile women during pre-, periipostmenopauzy (± HRT ). It is coupled with attendant disorders, such as depression, cardio-vascular disease, diabetes and urogenitalnye ( UG ) disorder.

Primary consequences RPVZh are failure overfilling blood/swelling of, insufficiency of lubrication and absence of sensation of pleasure in genitalia. Secondary consequences RPVZh are Your lu sexual attraction, pain during sexual intercourse, embarrasment in achieving orgasm.

Recently was is pushed hypothesis, that symptoms RPVZh in at least part of patients have vascular base ( Goldsteinet al., 1998), and data on animals is fixed with this point of vision (park et al., 1997).

Drugs-candidates for treatment of RPVZh, that are in the stage examinations on efficiency, are, first of all, therapy of erectile dysfunction, which promote krovoobrashcheniyu in male genitalia. They include two types of preparations-oral or sublingual drugs (apomorphine, phentolamine, sildenfil) and prostaglandin ( PGE1 - Alprostadil ), which injected or introduced transuretralno men and locally in genitals due to.

The present invention is directed to creation of effective agents of treatment of ZhSD, and in particular RPVZh.

Fundamental by opening of the present invention is the possibility of treat, the woman, stradayushchuyuZhSD (preferably RPVZh ), with use of and: nep.

In accordance with the present invention, on and: nep according to the present invention present as on "agent according to the present invention".

Agent according to the present invention can be used also in combination with one or more than one additional pharmaceutically active agent. On the additional pharmaceutically active agent, if it is present or is used in combination with agent according to the present invention, may be reference as on "additional agent". One or more than one of these additional agents may be one or more than one of the following: and: pde, other and: nep, Η: ΝΡΥ. Combination of agents more detail discusses below.

If additional agent according to the present invention represents and: pde, for some ways to make your said pde represents pde, hydro lizuyushchuyu camp (and possibly hydro lizuyushchuyu cgmp). Term "hydro lizuyushchaya camp" includes also metabolism and/or destruction of camp. Term "hydro lizuyushchaya camp (and possibly cgmp)" means, that this additional agent can be is able hydro mobilization cgmp in addition to camp. Here term "hydro lizuyushchaya cgmp" also includes metabolism and/or destruction of cgmp. However relative to some ways to make your of the present invention should have in form, that additional agent according to the present invention not necessarily should be is able hydro mobilization cgmp.

Here common references to agents can be used to additional agents, also as and to agents according to the present invention.

In accordance with the present invention the agent according to the present invention acts on target, preferably specifically to certain target. On this target sometimes is reference as on "target according to the present invention". However agent according to the present invention may operate on one or more than one other target. On these other target may be references as on "additional target". Also, if use additional agent, then action of this additional agent may be directed on the same target according to the present invention and/or on additional target (which is not necessarily should represent same additional target, on which acts this agent according to the present invention). Target are described in this invention description. Should have in form, that in this invention description common references on target can be used to additional targets, so same as and to target according to the present invention.

The following fundamental by opening of the present invention is the possibility of amplifying genital (for example vaginal or klitornyi ) blood flow in women with use of agent along the present invention.

In their experiments authors-find, that RPVZh is coupled with weakening genital blood flow, in particular with attenuation circulation in vagina and/or klitore. Consequently, treatment of women with RPVZh may be achieved by enhancing genital blood flow vazoaktivnymi agents. In their research authors invention revealed, that camp mediate vaginal and klitornuyuvazorelaksatsiyu, and that genital (for example vaginal and klitornyi ) blood flow can be enhanced/potentiate by means of diffusion condensate levels of camp. This one more fundamental opening.

In this with respect to nobody earlier not anticipated, that RPVZh can be treat so, i.e. right or indirect diffusion savings levels of camp. More addition, in this engineering there is no theories, which allow call, that RPVZh is coupled with negative modulation of activity and/or levels of camp or camp responsible behind mediation vaginal and klitornoivazorelaksatsii. Therefore the present invention is even more unexpected.

Besides, authors-find, that, applying agents according to the present invention, can be enhanced overflow blood genitalia and treat RPVZh -for example, to enhance lubrication into vagina and to increase sensitivity in the vagina and klitore.

So, in wide aspect, the present invention relates to the use of Using camp, for treatment of ZhSD, in particular RPVZh.

The present invention has advantage of, which creates agent for for restoring normal reaction sexual of excitation, in other words: amplification of genital blood flow, leading to overflow blood vagina, the clitoris and lips. This leads to increased vaginal lubrication by means of transudation of plasma, diffusion Exploration vaginal compliance and diffusion Exploration genital (for example vaginal and klitornoi ) of sensitivity. Consequently, according to the present invention proposes means for restoration or potentiation of normal reaction sexual of excitation.

Detailed description of aspects of the present invention in one aspect, the present invention relates to a pharmaceutical composition for use (or when it is used) in treatment of ZhSD, in particular RPVZh, containing agent, capable of potentiate camp in the sexual genitalia of a woman, affected ZhSD, in particular RPVZh, which possibly mixed with pharmaceutically acceptable carrier, diluent or excipient, and which represents agent according to the present invention, as it is defined here. Here this composition (like any other composition, mentioned here) can be is packed for subsequent application in treatment ZhSD, in particular RPVZh.

In another aspect, the present invention relates to the use of agent in drug production (such as pharmaceutical composition) for treatment of ZhSD, in particular RPVZh ; where this agent is able potentiate camp in the sexual genitalia of a woman, affected ZhSD, in particular RPVZh ; and where said agent represents agent according to the present invention, as it is defined here.

In the following aspect, the present invention relates to a method of treating of a woman, affected ZhSD, in particular RPVZh, at which this woman is introduced agent, which can potentiate camp in sexual genitalia, in such amount, to cause taking antilogarithms camp in sexual genitalia this woman; at that this agent possibly mixed with pharmaceutically acceptable carrier, diluent or excipient; and said agent represents agent according to the present invention, as it is defined here.

In the following aspect, the invention relates to a method of analysis for for identifying agent, which can be used for treatment of ZhSD, in particular RPVZh, at which, whether this agent directly or indirectly potentiate camp; at that taking antilogarithms camp in the presence of this agent is an index addition, that this agent can be useful in treatment of ZhSD, in particular RPVZh ; and said agent represents and: nep.

One of, the invention relates to a method of analysis for for identifying agent, which can directly or indirectly potentiate camp, to treat ZhSD, in particular RPVZh, at which agent is brought in contact with component, capable of act on activity and/or levels of camp, and measured activity and/or levels of camp; at that taking antilogarithms camp in the presence of this agent is an index addition, that this agent can be useful in treatment of ZhSD, in particular RPVZh ; and said agent represents and: nep.

As one more example, the invention relates to a method of analysis for for identifying agent, which can directly or indirectly potentiate camp for addition, to treat ZhSD, in particular RPVZh, at which agent is brought in contact with camp and measured activity of camp; at that taking antilogarithms camp in the presence of this agent is an index addition, that this agent can be useful in treatment of ZhSD, in particular RPVZh ; and said agent represents and. NEP.

In the following aspect, the invention relates to a method, including stage, on which : (and) analysis according to the present invention ; (b) is identified one or more than one agent, which can directly or indirectly potentiate activity of camp; and (in) is obtained some amount of one or more than one identified agent; and where said agent represents and: nep.

According to this aspect, agent, identified at stage (b), can be modified with so, to, for example, maximize activity, and then stage (and) is to be repeated. These steps possible to repeat the course of, until desirable activity or pharmacokinetic profile will not be achieved.

So, in the following aspect, the invention relates to a method, including stage, on which : (a1) analysis according to the present invention ; (61) is identified one or more than one agent, which can directly or indirectly potentiate activity of camp ; (62) is modified one or more than one said identified by agent ; (a2) possibly is repeated stage (a1); and (in) is obtained some amount of one or more than one identified agent (i.e. the, which were are modified); and where said agent represents and: nep.

In the following aspect, the present invention relates to a method of treating ZhSD, in particular RPVZh, by potentiation of camp using agent in vivo; where the agent is capable of directly or indirectly potentiate camp at a method for analysis in vitro \ where this method of analysis of in vitro represents method of analysis according to the present invention; and where said agent represents and: nep.

In the following aspect, the present invention relates to the use of agent in preparing a pharmaceutical composition for treatment of ZhSD, in particular RPVZh ; where this agent is able potentiate camp directly or indirectly analysis of in vitro method of analysis according to the present invention; and where said agent represents and: nep.

In the following aspect, the present invention relates to animal model, used for identification of agents, capable of treat ZhSD (in particular RPVZh ), including anestezirovannuyu females animal together with means for measuring changes of vaginal and/or klitornogo blood flow in of said animal after stimulation its pelvic nerve; where said agent represents and: nep.

In the following aspect, the invention relates to a method of analysis for for identifying agent, which can directly or indirectly potentiate camp for addition, to treat RPVZh, at which agent is added animal model according to the present invention and measured any taking antilogarithms camp and/or amplification of circulation in vagina and/or klitore of said animal; and where said agent represents and: nep.

In the following aspect, the present invention relates to a method of diagnosing, at which in women are taken specimen, is determined, has whether this sample object, present in such amount, to cause ZhSD, preferably RPVZh, or whether it in such amount, to cause ZhSD, preferably RPVZh ; where this object produces direct or indirect effect on level or activity of camp in sexual genitalia this woman; and where said object can be modulated for achieving favorable effect by using agent; and where said agent represents and: nep.

In the following aspect, the present invention relates to composition or set of for diagnosis of, containing (it) agent for object detection in taken in women sample, which can be used for determining, has whether this sample this object and in such amount, to cause ZhSD, preferably RPVZh, or whether it in such amount, to cause ZhSD, preferably RPVZh ; where this object produces direct or indirect effect on level or activity of camp in sexual genitalia this woman; and where said object can be modulated for achieving favorable effect by using agent; and where said agent represents and: nep.

For convenience of these and subsequent aspects of the present invention now discusses under corresponding headers sections. However then, that indicated in each partition not necessarily is limited content of each specific interface.

Agent according to the present invention preferably represents agent for treatment of RPVZh.

Agent according to the present invention preferably represents mediator genital (for example vaginal or klitornoi ) vazorelaksatsii in women.

In one embodiments agent according to the present invention preferably represents agent for oral administration.

In other embodiments agent according to the present invention can be agent for local administration.

Agent according to the present invention represents and: nep (sometimes write them and: nep]).

For some applications agent preferably represents selective and: nep.

For some applications agent preferably represents and: nep, where said nep represents EC 3.3.24.11.

For some applications agent preferably represents selective and: nep, where said nep represents EC 3.3.24.11.

Preferably agent according to the present invention represents inhibitor-i.e. it is able exerting inhibitory function.

Preferably agent according to the present invention produces indirect potentiating effect on camp. In alternative expression for some applications agent preferably does not direct potentiating effect on camp. Obvious, that agent produces indirect potentiating effect on camp, while acting on natural and naturally localised directly acting agents, such as natural and localized vip.

For some applications agent according to the present invention may be introduced in combination with another pharmaceutically active agent. Here this combined introduction there is no necessity make simultaneously, not speaking already on the same track. Are together entered composition may be composition, which contains agent according to the present invention and additional agent, this additional agent can exhibit direct potentiating effect on camp. Examples of combinations of discusses below.

For some applications additional agent preferably has indirect potentiating effect on camp. Examples of such additional agents include and: nep and/or Η: ΝΡΥ. In alternative expression for some applications this additional agent preferably does not direct potentiating effect on camp. Obvious, that this agent can exhibit indirect potentiating effect on camp, while acting on natural and naturally localised directly acting agents-such as natural and naturally localized vip.

For some applications additional agent preferably has direct potentiating effect on camp. Examples of such additional agents include and: pde.

Additional agent preferably represents inhibitor, i.e. it is able exerting inhibitory function.

For some applications additional agent represents antagonist.

For some applications additional agent preferably represents and: pde (sometimes write them as pde]).

For some applications additional agent preferably represents selective and: pde.

For some applications additional agent preferably represents and: FDE1 or and: FDE2 (sometimes write them and: FDEP, or FDEP, , or FDE20, or and: FDEZ, or and: pde4, or and: FDE7, or and: FDE8, more preferably this agent represents and: FDE2.

For some applications additional agent preferably represents selective and: FDEI (sometimes write them kakFDE2 ).

For some applications additional agent preferably represents and: nep (sometimes write them as nep ^.

For some applications additional agent preferably represents selective and: nep.

For some applications additional agent preferably represents and: nep, where said nep represents EC 3.4.24.11.

For some applications additional agent preferably represents selective and: nep, where said nep represents EC 3.4.24.11.

For some applications additional agent preferably represents Η: ΝΡΥ (sometimes write them as NPYj ).

For some applications additional agent preferably represents Η: ΝΡΥ Y1, or ΐ / ΐ : ΝΡΥ Y2, or Η: ΝΡΥ Y5, more preferably this agent represents Η: ΝΡΥ Y1.

For some applications additional agent preferably represents selective Η: ΝΡΥ.

For some applications additional agent preferably represents Η: ΝΡΥ Y1.

For some applications additional agent preferably represents selective Μ: ΝΡΥ Y1.

For some applications agent does not cause-either introduced so, that it does not cause-long fall of blood pressure (for example during period of approximately 5 minutes or more). In this embodiments, if agent should be introduced locally, then this agent can be capable of to cause drop of blood pressure (as, for example, if its either intravenously), provided that at topical administration minimal levels agent pass in blood stream. For oral agent preferably, to this agent had reigned long fall of blood pressure.

In preferable aspect, agent according to the present invention causes no-or its is introduced so, that it does not cause-large of change of frequency of heart contractions.

Should have in form, that in this invention description all references to treatment include one or more than one from the following: curative, palliativnuyu and prophylactic therapy. Preferably term "treatment" includes at least curative therapy and/or palliative care services.

Term "Ferragamo genitals" is used in accordance with determination, which given in gray's Anatomy, C.D. Ciemente, 13rh american edition:

" Genitalia include inner and outer group. Internal organs are located within pelvis and include ovaries, uterine of pipe, uterus and vagina. External members are surface and are located against of urogenital diaphragm and at the bottom of the pelvic vault. They include lobok, large lip and small lip, clitoris, vestibule, onion bulb vestibule and large gland vestibule ".

Synthesis of endogenous camp

In especially preferred embodiments agent according to the present invention-potentiated synthesis of endogenous camp, for example-potentiated levels of endogenous camp.

Here term "synthesis of endogenous camp" means camp, which is result of sexual stimulation (sexual excitation of). Consequently, this term not embraces levels of camp, which increased independently from sexual attraction.

So, according to the present invention, treatment of RPVZh is achieved by direct or indirect potentiation of endogenous camp of alarm, that, in its turn, enhances vaginal blood flow/lubrication and/or klitornyi blood flow, thus increasing natural reaction sexual of excitation. So, method of treatment according to the present invention restores or-potentiated normal reaction of excitation.

At method for treatment according to the present invention-achieve, using inhibitor of nep (EC 3.4.24.11).

Here is animal model testing. This animal model testing can be used for determining amplification of genital blood flow in a result of potentiation of camp. In this animal model of stimulate pelvic nerve, which causes effect, which imitates physiology of sexual of excitation/reaction. In these experiments agents according to the present invention cause amplification of blood above control of amplification after, as this nerve was stimulirovan. In the absence of stimulation of these agents have no effect (or possess negligibly small effect) to cause amplification of blood flow. Usually in these experiments nerve stimulate, to obtain basic amplification of blood flow. Then agentkandidat (or real agent) give animal systematically or locally, for example intravenous, local or oral by. Amplification of blood above control of amplification, consequently, is an index agent along the present invention.

The present invention also embraces introduction agent along the present invention before and/or during sexual stimulation. Here term "floor stimulation of" may be synonym term "floor excitation". This aspect of the present invention creates advantage, since it provides systemic selectivity. Natural cascade takes place only in genitalia, and not in other lokalizatsiyakh, for example in the heart and T. d. Consequently, achievement of selective effect on genitals will be possible.

So, in some aspects of the present invention extremely preferably, to have place stage sexual stimulation. Authors-find, that this stage can provide systemic selectivity. Here "floor stimulation of" can represent one or more than one from the following: Visual stimulation of, physical stimulation of, sound stimulation of or myslennaya stimulation of.

So, agents according to the present invention preferably is introduced before or during sexual stimulation, especially when these agents are intended for oral administration.

Consequently, in this preferable aspect, according to the present invention the use of agent along the present invention in manufacturing of medicine for treatment RPVZh ; where this agent is able potentiate camp in the sexual genitalia of a woman, affected RPVZh ;

and where the said female experts is subjected to sexual stimulation before or during introduction of a drug said.

Preferably, according to the present invention the use of agent along the present invention in manufacturing of medicine for treatment RPVZh ; where this agent is able potentiate camp in the sexual genitalia of a woman, affected RPVZh ; where said female experts is subjected to sexual stimulation before or during introduction of a drug said; and where specified drug is introduced said woman perorally.

Besides, in this preferable aspect, according to the present invention proposes method of treatment of of a woman, affected RPVZh, at which this woman is introduced agent according to the present invention, which can potentiate camp in sexual genitalia, in such amount, to cause taking antilogarithms camp in sexual genitalia this woman; at that agent possibly mixed with pharmaceutically acceptable carrier, diluent or excipient; and at said female experts is subjected to sexual stimulation before or during introduction of the indicated agent.

Preferably, according to the present invention proposes method of treatment of of a woman, affected RPVZh, at which this woman is introduced agent according to the present invention, which can potentiate camp in sexual genitalia, in such amount, to cause taking antilogarithms camp in sexual genitalia this woman; at that agent possibly mixed with pharmaceutically acceptable carrier, diluent or excipient; said female experts is subjected to sexual stimulation before or during introduction of the indicated agent; and said agent is added said woman perorally.

Taking antilogarithms camp

At usage here referring to a camp term "taking antilogarithms" includes one or more than one from the following: diffusion lu effectiveness of camp, diffusion lu levels of camp, diffusion lu activity of camp, Your lu level of splitting of camp, Your lu level of inhibition of camp.

Potentiating effect can be right effect. Example of direct effect can be positive regulate the levels of camp agent, which increases its expression.

Alternatively, potentiating effect can be indirect effect. Example of such effect may be action on substance, which in otherwise inhibited least and/or limited levels of and/or activity of camp. Other example of such effect may be amplification effect of substance, which increases efficiency of camp, increases the levels of camp, enhances activity of camp, reduces level of splitting of camp or reduces level of inhibition of camp.

Example Ptsdmf may be and: pde, such as and: FDEP.

Mimetic of camp

In some aspects, the present invention additional agent can act as mimetic of camp.

Used here term "mimetic of camp" means agent, which may operate like a camp (for example have like biological profile and effect) in sexual genitalia of a woman and produces one or more than one of the following actions: increases efficiency of camp-like components, increases levels of camp-like components, enhances activity of camp-like components, reduces level of splitting of camp-like components, reduces level of inhibition of camp-like components.

Example of mimetic of camp can be forscoline. Now authors-find, that forscoline-enhanced vaginal and klitornyi circulation and can act also as vaginal relaksanta.

In preferable aspect, mimetic of camp is administered orally.

Activator of camp

Used here term "activator of camp" means substance, which controls or releases the camp in the sexual genitalia of a woman. This control can be straight (for example directed on proper camp) or indirect (for instance through activation of camp). For convenience of authors-present on these substance as on Atsamf -

Term "target", used here with a reference to the present invention, means any substance, which represents a camp, Atsamf, Itsdmf or NMtsamf. Otherwise consisting, on target according to the present invention may be reference as on target Ptsamf.

Target according to the present invention and/or additional target can represent amino acid sequence and/or nucleotide sequence, encoding it, and/or unit of expression, responsible for its expression, and/or its modulator.

Target may be also combination of such targets.

Agent

Agent according to the present invention may be any suitable agent, which may act as Ptsamf.

Agent (i.e. agent according to the present invention and/or additional agent) can represent amino acid sequence or its chemical derivative. This substance may also represent organic compound or other chemical substance. Agent may also be a nucleotide sequence, which may be semantic sequence of or describes anti-sense sequence of. Agent may also represent antibody.

So, term "agent" includes, but not is limited them, compound, which may be obtained from any suitable of source or synthesized by any suitable source, as natural, and non-natural.

Agent can be is constructed or is obtained from library of compounds, which may include peptides, and also other compounds, such as small organic molecules, such as compounds lead.

One, agent can represent natural substance, biological macromolecule or extract, made from biological materials, such as bacteria, fungi, or cells or tissues of animals (in particular mammals), organic or inorganic molecule, synthetic agent, semisynthetic agent, structural or functional mimetic, peptide, peptidomimetic, derived agent, peptide, otshcheplennyi from whole protein, or peptides, synthesized by synthetic (such as, for example, or using synthesizer peptides, or recombinant methods, or their combinations), recombinant agent, antibody, natural or non-natural agent, fused protein or its equivalent, and also mutants, their derivatives or combination.

Used here term "agent" may represent a separate unit, or it may represent combination of agents.

If additional agent represents organic compound, then for some applications, such as if the agent represents and: nep, this organic compound typical may contain amide group (i.e.-Ν(Η)-0(0)-or also -0(0)-Ν(Η) -) and one or more than one hydrocarbon group. Here term "hydrocarbon group" means group, containing at least with and n, and optionally containing one or more than one other suitable substituent. Examples of such substituents may include halogeno-, alkoxy-, nitro-, al ' kiliuyu group, cyclic group and T. d. In addition to the fact, that provide substituents, representing cyclic group, combination of substituents may form cyclic group. If hydrocarbon group has more than one with, then these carbon atoms not necessarily must be are connected to each other. For example, at least two carbon atoms may be are connected through suitable element or group. So, hydrocarbon group may contain heteroatoms. Suitable heteroatoms obvious for specialists in this area and include, for example, sulfur, nitrogen and oxygen. For some applications agent preferably has at least one cyclic group. For some applications agent preferably has at least one cyclic group of, connected with the other hydrocarbon group by means of amide bond. Examples of such compounds are presented here in partition examples.

If additional agent represents organic compound, then for some applications, such as if the agent represents and: pde, this organic compound typical may contain two or more than two connected hydrocarbon groups. For some applications agent preferably contains at least two cyclic group, besides possibly one of these cyclic groups may be condensed cyclic circular structure. For some applications at least one of these cyclic groups preferably represents heterocyclic group. For some applications this heterocyclic group preferably has at least one n in ring. Examples of such compounds are presented in partition examples.

If agent represents organic compound, then for some applications, such as if the agent represents Η: ΝΡΥ, this organic compound typical may contain two or more than two connected hydrocarbon groups. For some applications agent preferably contains at least two cyclic group, one of them may be condensed cyclic circular structure. For some applications preferably at least one of these cyclic groups represents a heterocyclic group. For some applications this heterocyclic group preferably has at least one n in ring. Examples of such compounds are presented here in partition examples.

Agent can contain galogenogruppy. Here "halogen" means fluoro, chloro, bromo or under.

Agent can contain one or more than one alkyl, alkoxy, alkenyl, alkylene or alkenylene group, which may be non-branched or branched.

Agent may be in the form of pharmaceutically acceptable salt, such as acid addition salt or salt of base, or its solvate, including hydrate. Suitable salt are processed in Bergeet al. .J.pharm.sci., 1977, 66, 1-19.

Suitable acid addition salts are formed from acids, which form nontoxic salts, examples of which are salt hydro chloride, hydro bromide of, hydro iodide, sulfate, bisulfate, nitrate, phosphate, hydro phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, saccharate, benzoate, methanesulfonate, etansulfonat, benzene, ptoluolsulfonat and pamoat.

Suitable salts of bases are formed of bases, which form nontoxic salts, examples of which are sodium salt, potassium, aluminum, calcium, magnesium, zinc and diethanolamine.

Pharmaceutically acceptable salt agent along the present invention easily can be to produce by mixing together solutions agent and desired acid or base, where as is suitable. This salt can be deposited from solution and assemble by filtration, or its one can discern evaporation of solvent.

Agent can existing in polymorphous.

Agent can contain one or more than one asymmetric carbon atom and, consequently, of existing in two or more stereoisomeric forms. In case of, when agent contains alkenyl or alkenylene group, may have place cis ( e) and trans (Z) isomerism. Present invention includes individual stereoisomers agent and, if this are typical of, its individual tautomeric forms together with their mixtures.

Separation of diastereoisomers or cis and transisomers of can be successfully made traditional methods, for example fractional crystallization, by chromatography or VEZhKh stereoisomeric mixture of agent or its suitable salt or derivative. Individual enantiomer of agent may be obtained also from corresponding optically pure intermediate compound or separation of, for example VEZhKh, corresponding racemate using a suitable chiral stationary phase, or fractional crystallization 3 salts, formed by reacting corresponding racemate with a suitable optically active acid or base, where as is suitable.

The present invention also includes all suitable isotope variety agent or its pharmaceutically acceptable salt. Izotopnuyu subkind agent along the present invention or its pharmaceutically acceptable salt is determined as subkind, in which at least one atom replaced atom, having the same atomic number, but having nuclear mass, different from nuclear mass, usually detected by the in nature. Examples of isotopes, which can be inserted into agent and its pharmaceutically acceptable salts, include isotopes

hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as^{2 n},^{3 n},^{13 with},^{14 with},^{15 n},^{17 0},^{18 0},^{31 p},^{32 p},^{35 s},^{18 f and36 ci} respectively.

Some isotope variety agent and its pharmaceutically acceptable salts, for example those, in which is introduced radioactive isotope, such as^{3 n or14 with}, useful in studies of tissue distribution of medical agent and/or substrate. Tritievye, i.e.^{3 n}, and carbon -14, i.e.^{14 with}, isotopes especially are preferable in connection with the simplicity of their production and detection of. Besides, replacement of isotopes, such as deuterium, i.e.^{2 n}, to produce certain therapeutic advantages due to more high metabolic stability, for example, diffusion nnomu period of-life in vivo or lower nnoi demand dosage, and therefore at some circumstances it may be preferable. Isotope variety agent and its pharmaceutically acceptable salts, as a rule, can be obtained traditional methods using corresponding isotopic varieties of suitable reagents.

For specialists in this area obvious, that agent can be form from the prodrug. Examples prodrugs include objects, which have definite protective group (group of) and which can not possess pharmacological activity alone, but in certain cases of their may be introduced (for example orally or parenterally), after which they undergo transformation metabolism in organism with formation of agent, which is pharmacologically active.

Besides, obvious, that some groups, known as " progruppirovki", for example as described in "design care Prodrugs", n. Bundgaard, Elsevier, 1985 (opens here by references), may be placed on corresponding functional groups of agents. Such prodrugs also are connected in volume-.

Ptsamf can one or more than one from the following:

directly or indirectly increase efficiency of camp, directly or indirectly to increase levels of camp, directly or indirectly to enhance activity camp, directly or indirectly to decrease the level of splitting of camp, directly or indirectly decrease of level of inhibition of camp.

Preferably agent according to the present invention directly or indirectly-enhanced levels of camp in the sexual genitalia of a woman, affected RPVZh.

More preferably agent according to the present invention directly or indirectly selectively increases levels of camp in the sexual genitalia of a woman, affected RPVZh.

More preferably agent according to the present invention directly or indirectly selectively increases levels of camp, where said camp represents camp, induced sexual excitation of.

In especially preferable aspect, agent according to the present invention increases the relative quantity of camp, induced sexual excitation of.

For some applications agent according to the present invention selectively treat RPVZh.

In one aspect, agent can act on suitable target as inhibitor or antagonist of and thereby to increase levels of camp in the sexual genitalia of a woman. In this text authors invention is term "inhibitor of" for designation of inhibitor and/or antagonist.

In another aspect, the agent can act on suitable target activator or agonist and thereby to increase levels of camp in the sexual genitalia of a woman. In this text authors invention is used terms "activator" and "positive regulator" for designation of activator and/or positive regulator and/or agonist.

So, agent can act on suitable target as agonist, antagonist, positive regulator or inhibitor.

Agent according to the present invention can represent one unit, which is capable of exerting two or more than two of these properties. Alternatively or additionally agent according to the present invention can be a combination of agents, which are capable of exerting one or more than one of these properties.

Preferably agent can act on suitable target as selective agonist, selective antagonist, selective positive regulator or selective inhibitor.

Preferably agent can act on selective suitable target as selective agonist, selective antagonist, selective positive regulator or selective inhibitor.

Agent may also be capable of exerting one or more than one other favorable functional property. For example, agent according to the present invention can potentiate camp, and also potentiate cgmp.

For some applications (for example local application) agent may also exerting inhibitory action in relation to apf (angiotensin-converts enzyme). Analysis of apf is presented here in experimental partition. For some applications (for instance in specific types of patients) such agents (i.e. agents, which also show inhibitory action in relation to apf) may be unsuitable for oral administration.

For some applications agent can exerting also inhibitory action in relation to EPF (endothelium-converts enzyme). Analysis EPF problems in this engineering.

Agent according to the present invention can be used in combination with one or more than one another pharmaceutically active agent, such as Ptsgmf (such inhibitor of phosphodiesterase type 5, for example Sildenafil, or donor of nitrogen oxide, or precursor nitrogen oxide, for example l-arginine, or inhibitors of arginase) and/or acting on the central nervous system pharmaceutical agent (for example receptor agonists dopamine, such as apomorphine, or selective receptor agonists D2 dopamine, such as PNU-95666, or agonist melanocortin receptors, such as melanotan ii). Indicating by use of apomorphine as pharmaceutical preparation can be find in us-a- 5945117. In this specific document apomorphine is administered sublingually. Additionally or alternatively agent can be used in combination with one or more than one inhibitor of FDE5 (for example sildenafilom, vardenafilom ( Bayer wa 38-9456) and IC351 ( Cialis, IcosLilly )), one or more than one donor nitrogen oxide (for example NMI-921), one or more than one agonist dopamine receptors (for example apomorphine, uprima, iksenom ), one or more than one heterocyclic amine, such as heterocyclic amines, described in general outlined and particularly WO 00/40226, in particular examples 7,8 and 9, one or more than one agonist melanocortin receptors (for example melanotanom ii or RT14 ), one or more than one otkryvatelem potassium channel (for example otkryvatelem potassium channel Kdtf (for example minoksidilom, nikorandilom ), and/or activated calcium otkryvatelem potassium channel (for example BMS-204352), one or more than one antagonist a1-adrenoceptor antogonists (for example fentolaminom, vazofemom, vazomaksom ), one or more than one agonist receptors vip or analogue vip (for example ro -125-1553) or fragments of vip, one or more than one antagonist a- adrenotseptorov in combination with vip (for example invikorpom, aviptadilom ), one or more than one antagonist a2-adrenoceptor antogonists (for example iokhimbinom ), one or more than one estrogen, estrogen and medroksiprogesteronom or medroxyprogesterone acetate (mpa), or agent gormonozamestitelnoi therapy estrogens and metiltestosterona (for example HRT (hormone-substitution therapy), in particular premarin, senestin, estrofeminal, ekvin, estras, estrofem, elleste solos, estring, estraderm, estradermTTS, estraderm matrix, dermestril, premfaz, prempro, prempak, premik, estratest, estratest hs, tibolon ), one or more than one zamestitelnym agent testosterone (inc DHEA (dehydro androstedion ), testosterone ( Tostrell ) or testosteronovyi implant ( Organon )), one or more than one of testosterone/estradiol agent, one or more than one agonist of estrogens, for example lazofoksifenom, one or more than one agonist or antagonist of serotonin receptors (for example agonist and antagonist receptors 5ΗΤ1Α, 5NT2S, 5NT2A and 5NTZ, as they are described in WO 2000/28993), one or more than one agonist prostanoidnykh receptors (for example Muse, alprostadil, misoprostol), one or more than one agonist purinergicheskikh receptors (in particular P2Y2 and P2Y4 ), one or more than one agent-antidepressant (for example bupropionom ( Velbutrin ), mirrtazapinom, nefazodonom ).

Structure of IC351 represents:

IC351 ( IcosLilly )

If is introduced combination of active agents, then they may be introduced simultaneously, separately or in series.

Combination with VIR

According to the present invention agent is vip (or preferably not represents its analogue or its fragment). However in some embodiments agent according to the present invention may be introduced together with vip, or with its analog or fragment.

In very preferable aspect, vip, or its analogue or its fragment, is not introduced. Its is not introduced in connection with the fact, that was the message about the fact, that infusion vip lead to considerable undesirable serdechnososudistym effects, such as increased heart beat rate and Your lu diastolic blood pressure ( Ottesen 1983, 1987, 1995).

Besides, and even despite, that Ottesen and its sponsors demonstrated, that vip induces amplification of vaginal blood flow and lubrication in healthy Volunteers, mechanism, by means of which vip shows their effects, unclear. In literature there is a row of examples of VIR of alarm through different secondary system mediator, for example cgmp/ guanilattsiklazu ( Ashur - Fabian, 1999), carbon monoxide (with)/ gemoksigenazu (fan et aj., 1998) and camp/adenylatcyclase ( Foda, 1995;

Schoeffer, 1985; Gu, 1992). Examples this are given in recent message, which discloses, as vazorelaksiruyushchie effects vip in uterine artery can be explain release of nitrogen oxide ( Jovanovic, 1998). Again same, has a confirmation vip modulation of nitrogen oxide (g \ program)/ cgmp in male urogenital function ( Kim, 1994).

Besides, in literature was message about the fact, that vip not acts on levels of camp in cultures smooth muscle cells vagina (cm. Traish, and., Moreland, roption. b., Huang, Υ., et al (1999). Development of human and rabbitvaginalsmoothmuscle cell cultures : Effects care vasoactive agents on intracellular levels of cyclic nucleotides. Mol. cell. Biol. res. comm., 2, 131-137).

More addition, in subsequent investigations Ottesen and its sponsors (cm. Palle, Bredkjaer, Ottesen and Fahrenkrug, 1990. Clinical and experimental Pharmacology and Physiology, vol. 17, 61-68) is imparted to, impact vip on vaginal blood flow independently from path of introducing is rather part of systemic sosudorasshiryayushchego effect, and not local response. Besides, they is about some vascular adverse effects, associated with vip, in other words: about pokrasnenii, hypotension and tachycardia.

For some applications agent according to the present invention (and possibly possible additional agent) preferably has value Kj less than approximately 100 nm, preferably less than approximately 75 nm, preferably less than approximately 50 nm, preferably less than approximately 25 nm, preferably less than approximately 20 nm, preferably less than approximately 15 nm, preferably less than approximately 10 nm, preferably less than approximately 5 nm.

Value K

For some applications agent according to the present invention (and possibly possible additional agent) preferably has value K less than approximately 100 nm, preferably less than approximately 75 nm, preferably less than approximately 50 nm, preferably less than approximately 25 nm, preferably less than approximately 20 nm, preferably less than approximately 15 nm, preferably less than approximately 10 nm, preferably less than approximately 5 nm.

Value Κα

For some applications agent according to the present invention (and possibly possible additional agent) preferably has value_{to a less than approximately} 100 nm, preferably less than approximately 75 nm, preferably less than approximately 50 nm, preferably less than approximately 25 nm, preferably less than approximately 20 nm, preferably less than approximately 15 nm, preferably less than approximately 10 nm, preferably less than approximately 5 nm.

In some embodiments of the present invention agents according to the present invention (and possibly possible additional agent, for example and: nep) preferably have log d from -2 to +4, more preferably from -1 to +2. log d may be determined by standard methods, known in this area, such, which are described in j. Pharm. Pharmacol. 1990.42: 144.

In addition or alternatively, in some embodiments agents according to the present invention (and possibly possible additional agent, for example and: nep) preferably are characterized by be saso -2 more than 2x10 '^{6 cm with}'^1, more preferably more than 5x10 '^{6 cm-} with'^1.

Value of saso can be determined by standard methods, known in this area, such as described in j. Pharm. Sci. 79, 7, ρ.595-600, and Pharm. res. vol. 14, no. 6 (1997).

For some applications agent according to the present invention (and possibly possible additional agent) preferably has at least approximately 100-fold selectivity to the desired target, preferably at least approximately 150-fold selectivity to the desired target, preferably at least approximately 200-fold selectivity to the desired target, preferably at least approximately 250-fold selectivity to the desired target, preferably at least approximately 300-fold selectivity to the desired target, preferably at least approximately 350-fold selectivity to the desired target.

For some applications agent according to the present invention (and possibly possible additional agent) preferably has at least about 400-fold selectivity to the desired target, preferably at least approximately 500-fold selectivity to the desired target, preferably at least approximately 600-fold selectivity to the desired target, preferably at least about 700-fold selectivity to the desired target, preferably at least approximately 800-fold selectivity to the desired target, preferably at least approximately 900-fold selectivity to the desired target, preferably at least about 1000-fold selectivity to the desired target.

In a typical event agent can be obtained by methods of chemical synthesis.

Agent or its target or variants, homologs, derivatives, fragments or mimetics can be obtained by the chemical methods for synthesis of agent is fully or partially. For example, peptides can be synthesized solid-phase methods, cleaved from resin and cleaned by means of preparative highly effective liquid stnoi chromatography (for example Creighton (1983) ProteinStructures and Molecular principles, WHFreeman and co, new york NY ). Composition of these synthetic peptides can be confirm amino acid analysis or sequencing (for example by technique of splitting Edmana ; Creighton, cm. above).

Direct synthesis of agent, or its variants, homologues, derivatives, fragments or mimetics may be implemented using various solid-phase methods ( Roberge jy et and! (1995) science 269 : 202-204), and automatic synthesis can be successfully made for instance using synthesizer peptides ABI 43 1 a ( PerkinElmer ) in accordance with instructions, vested in by manufacturer. Additionally amino acid sequences of, components of agent or any its part, may be varied in process of direct synthesis of and/or combined, using chemical methods, with the sequence from other subunits or any their part, to produce versions agent or target, such as, for example, version pde.

In alternative embodiments of the invention coding sequence agent, its target or variants, homologues, derivatives, fragments or mimetics can be to synthesize, is fully or partially, using chemical methods, known in this engineering (cm. Caruthers min et and! (1980) NucAcids res SympSer 215-23, Horn Τ et al (1980)NucAcids res SympSer 225-232).

Mimetic

Used here term "mimetic" relates to any chemical compound, which includes, but not is limited by them, peptide, polypeptide, antibody or other organic chemical compound, which has the same quantitative activity or same effect, as and mentioned agent, relative to a target.

Term "derivative" or "derivative", as it is used here, involves chemical modification of agent. Illustration of such chemical modifications may be replacement of hydrogen galogenogruppoi, alkyl group, acyl group or amino group.

In one embodiments of the present invention agent may be a chemically modified agent.

Chemical modification agent may or amplifying, or to interaction with formation of hydrogen communication, interaction of charges, hydro fobnoe interaction, van-der vaalsovo interaction or dipole interaction between agent and target.

In one aspect, identified by agent can act as a model (for example of matrix) for development of other compounds.

In a typical event agent for use in analysis according to the present invention can be obtained according to techniques of recombinant dna.

Taking antilogarithms iGMF

Used here referring to a cgmp term "taking antilogarithms" includes any from the following: diffusion lu efficiency cgmp, diffusion lu levels of cgmp, diffusion lu activity cgmp, Your lu level of splitting of cgmp, Your lu level of inhibition of cgmp.

Potentiating effect can be a direct effect. Alternatively it may be a secondary effect and/or downward effect.

At that agent, which-potentiated cgmp, preferably acts on Itsgmf and/or on nm_{centner gmp}, ; modulator cgmp possesses negative action on cgmp, so that this agent reduces and/or eliminates and/or masks and/or directs in other side undesirable effect and_{centner gmp} and/or NMtsgmf, directed on cgmp.

Therefore the present invention embraces the combination of one or more than one and: Itsamf and one or more than one and: and_{centner gmp}. In one aspect, and: and_{centner gmp} represents and: pde_{centner gsh} >.

IpASh and/or NMiAMD )

Authors of the invention shown, that camp mediate genital (for example vaginal or klitornyi ) circulation, and by amplification of camp signaling them It enhance genital (for example vaginal or klitornyi ) circulation in animal model. So, agent, which is positively controls/enhances mediated camp vazorelaksatsiyu, should be effective in treatment of RPVZh. For convenience of authors-present on these substance as on Itsamf and/or NMtsamfZdesItsamf and NMtsamf have negative effect on levels of or activity of camp.

So, this agent may represent any one or more than one from: and: Itsd_{MF and/or} and: NMtsamf -

This agent can represent one unit, which is capable of exerting two or more than two of these properties. Alternatively or additionally this agent can be a combination of agents, which are capable of exerting one or more than one of these properties.

Examples Itsdmf and NMtsamf include nep and one or more than one pde or any component, connected with them. This connected component may represent, for example, receptor and/or cofactor.

So, agent according to the present invention can be used in combination with one or more than one from the following: and: FDEtsamf, Π : ΝΡΥ (sometimes write them as NPYj ), Η: ΝΡΥ y_{n (} sometimes write them as ΝΡΥ y_{n i}).

Similarly, this agent can represent one unit, which is capable of exerting two or more than two of these properties. Alternatively or additionally this agent can be a combination of agents, which are capable of exerting one or more than one of these properties.

And: Ipam <ь И/ИЛИ И: НМцдма>

In accordance with the present invention, authors-find, that RPVZh possibly treat and/or prevention of, using agent, which reduces and/or eliminates and/or masks and/or directs in other side and/or prevents undesirable effect Itsdmf and/or NMtsamf, directed on camp. This agent can even to reduce levels of camp, which were lowered Itsamf and/or NMtsamfDlya convenience of authors-present on these substance as on and: Itsamf and/or and: NMtsamf -here and: Itsamf and.' NMtsamf prevent or is reduced negative impact on levels of or activity of camp.

So, in one preferable aspect, the agent represents and: Itsamf and/or and: NMtsamf, ; NMtsamf possesses undesirable effect on camp.

In accordance with the present invention, authors-find, that one of important cases RPVZh is outcome of low levels or low activity of camp in female genitalia.

So, agent can represent etc: Atsa_{m 2}.

So, agent according to the present invention may preferably also be capable of act as and: ATs, a: vip_r, a: VlP_n, and: and : \ / 1p_{g or} and: and : \ / 1p_U, and/or its can be used in combination with one or more than one from a: ATs, a: vip_r, a: vip_n, m:l/l: vip_{r or} and: and : \ LR_U.

This agent can represent one unit, which is capable of exerting two or more than two of these properties. Alternatively or additionally this agent can be a combination of agents, which are capable of exerting one or more than one of these properties.

ΠΡ : Αμαμφ

In other with respect to additional target can represent component, enhances level of camp. Consequently, agent may also act as etc: ATs.

Consequently, for example agent according to the present invention may also be capable of act as etc: Atsamf, and : Ats, a: vip_r, a: vip_n, h:h: vip_{r or} h:l/i: VIPn, and/or its can be used in combination with one or more than one agent, which can be any of: etc.' Atsamf, and: Ats, a: vip_r, a: VIPn, and: dwg/1p_{g or} and: dwg/1p_U.

One target can represent proper alone camp, or ATs or vip (or their combinations).

Combination and: and "amf and/or and: m" dmf and/or ΠΡ : α,. αμφ

In another aspect, the agent according to the present invention can be used with combination of potentsiatorov camp. For example, agent according to the present invention can be used in combination with one or more than one from:

And: FDEtsdmf

And: FDEPtsamf

Η: ΝΡΥ

Η: ΝΡΥ y_n

AND: NEP

Etc: Atsamf

AND: ATs

A: vip_r

A: vip_n

H:h: vip_r

Η: Η : νΐΡη

mimetic of camp

Term "inhibitor", as it is used here with respect to agent along the present invention, means agent, which may reduce and/or eliminate and/or a and/or to prevent undesirable action Itsamf and/or undesirable action Mtsamf, directed to camp.

Term "activator", as it is used here with respect to agent along the present invention, means agent, which can increase and/or to produce and/or demaskirovat and/or develop and/or provide action camp and/or Atsdmf. This activator may act as agonist.

In another aspect, the agent according to the present invention may also be in combination with one or more than one other active component, such as one or more than one from agents, capable of potentiate cgmp.

Used here term "amino acid sequence" is synonym term "polypeptide" and/or term "protein". In some cases term "amino acid sequence" is synonym term "peptide". In some cases term "amino acid sequence" is synonym term "protein".

Amino acid sequence can be to produce, selecting it from suitable source of, or its can be to produce by synthetic, or its can be to produce by using techniques of recombinant dna.

In one aspect, according to the present invention invention proposes amino acid sequence, which can act as target in analysis for identification of one or more than one agent and/or its derivative, capable of act on this amino acid sequence, to potentiate camp for treatment of RPVZh.

Used here term "nucleotide sequence" is synonym term "polynucleotide".

Nucleotide sequence may represent dna or rna or genome, or synthetic, or recombinant origin. Nucleotide sequence may be double-stranded or of a single-stranded, representing either sense, or antisense thread, or their combinations.

For some applications nucleotide sequence preferably represents dna.

For some applications nucleotide sequence is preferably produced, using procedures of recombinant dna (for example recombinant dna).

For some applications nucleotide sequence preferably represents cdna.

For some applications nucleotide sequence may preferably be of the same, as occurring in nature mould for this aspect.

In one aspect, according to the present invention invention proposes a nucleotide sequence, encoding substance, capable of act as target in analysis of (such as analysis of two hybrids yeast) for identification of one or more than one agent and/or its derivative, able to act on this amino acid sequence, to potentiate camp for treatment of RPVZh.

For specialists obvious, that much numerical different nucleotide sequences can to encode target in a result of vyrozhdennosti genetic code. Besides, obvious, that specialists may, using routine techniques, make nucleotide replacement of, which substantially have no influence on activity, encoded this nucleotide sequence according to the present invention, to reflect use of codons any specific organism host, in which is expressed target. So, terms "variant", "homologue" or "derivative" with respect to nucleotide sequence, given in applied lists sequences, include replacement of, variation of, modification of, replacement of, deletion or addition of one (or more than one) nucleic acid from this sequence or to this sequence, set to nucleotide sequence, encoding functional target according to the present invention (or even agent according to the present invention, if _AOE280A2AO> agent contains nucleotide sequence or amino acid sequence).

As indicated above, with respect to homology of sequence has place preferably at least 75%, more preferably at least 85%, more preferably at least 90% homology with sequences, given in list of sequences. More preferably is at least 95%, more preferably at least 98% homology. Of comparison of nucleotide homology can be carried out, as described above. Preferable program comparing sequences is program GCGWisconsinBestfit, circumscribed above. By default ratable matrix has matched value of 10 for each identical nucleotides long and -9 for each non-conforming. Zanizhe lu for formation of gepa by default is

-50, and zanizhe lu for elongating gepa by default is -3 for each nucleotide.

The present invention also covers nucleotide sequences, which are capable of selectively hybridize with represented here sequences of, or with any of their variant, fragment or derivative, or with complement of any higher than the said. Length of nucleotide sequences is preferably at least 15 nucleotides, more preferably at least 20, 30, 40 or 50 nucleotides. These sequences can be used as probes, such as in a diagnostic set.

Except mentioned here specific amino acid sequences and nucleotide sequences, the present invention also covers their application variants, homologues and derivatives. Here term "homology" can to equate to the term "identity".

In a context of the present invention is accepted, that homologous sequence includes amino acid sequence, which may be at least 75,80 or 90% identical, preferably at least 95 or 98% identical. In particular, usually should be consider homology with respect to the regions, which are known as essential for activity. At homology may also be viewed from the point of view of similarity (i.e. amino acid residues, having like chemical properties/function), in a context of the present invention preferably express homology in terms of identity of sequence.

Comparison of homology can be carried out eyes or, more usually, with the aid of public programs of comparing sequences. These commercially available computer program may calculate % homology between two or more than two sequences.

% homology can be calculate between adjoining sequences, i.e. one sequence is leveled with the other sequence of, and each amino acid in one sequence of directly compared with the amino acid in other succession one residue. This is called leveling "without gepov". Usually such equalizing without gepov only in the range of relative to small number of residues.

At this method is very simple and reliable, it can not take into account the then, that for example in identical in other with respect to pair of sequences one insertion or deletsiya will lead to the fact, that the following amino acid residues will not be are aligned, that, possibly, will lead to large Your Exploration % homology during global of levelling. Consequently, the majority of methods of comparing sequences is developed so, to produce optimal equalizing with consideration of possible insertsii and deletsii without undue zanizhe condensate of the general evaluation homology. This is achieved by insertion of " gepov" in leveling sequences so, to try to maximize local homology.

However, at these more complex methods is determined "punitive glasses gepov" for each gepa, which is seen at the flattening, so, for one and the same number of identical amino acids at equalisation sequence at as possible smaller including gepov -that reflects higher osculatory between two sravnivaemymi sequences of-will be achieved more high evaluation, than at a large number gepov. Used is usually "price kinship by gepam" (" affine gap costs"), which is relatively high "price" behind existence gepa and smaller "punitive glasses" for each of the following residue in gepe. This system for estimating gepov widely used. High "punitive glasses" for gepov, end, will lead to optimized equalization of at gepakh size smaller. The majority of programs of levelling provide possibility to modify "punitive glasses" for gepov. However using such software for comparing sequences preferably value by default. For example, at packet use programs GCGWisconsinBestfit (cm. below) "punitive glasses" for gepa by default make -12 for gepa and -4 for each elongation.

Calculation of maximum % homology, consequently, first of all require obtain optimal equalizing with account of "penalty glasses" for gepov. Suitable a computer program for equalizing is package of programs GCGWisconsinBestfit ( University care Wisconsin, U.S.A. ; Devereuxet al, 1984, NucleicAcids research 12:387). Examples of other software, which can to comparison of sequences, include, but are not limited to, packet programs BLAST (cm. Ausubelet and! ., 1999ibid- chapter 18), FASTA ( Atschulet al., 1990, j. Mol. Biol., 403-410) and set of programs of comparison GENEWORKS. As BLAST, and FASTA are accessible as for the autonomous, and for operative for searching (cm. Ausubelet al., 1999ibid, pages 7-58 to 7-60). However preferably program GCGWisconsinBestfit. Also is suitable a new program, said BLAST 2 Sequences, for comparing protein and nucleotide sequence (cm. FemsMicrobiolLett 1999,174 (2): 247-50;

FemsMicrobiolLett 1999,177 (1): 187-8 and tatiana@ncbi.nlm.nih.gov).

At final % homology can be measured from the point of view of identity of, proper alone process of equalizing not usually is based on comparison of steam by principle_{: "} all or nothing". Instead, as a rule, it is used matrix for evaluating with scale of similarity, which assigns glasses for each pairwise comparison, based on chemical modeled or progressive distance. Example of such widely used matrix is matrix BLOSUM62 -matrix by default for the set programs BLAST. In programs GCGWisconsin, as a rule, is used or public value by default, or made to order comparative table of symbols, if it is furnished (additional details cm. in guide for user). Preferably public value by default for package of programs GCG or by default BLOSUM62 in case of other software.

As only software revealed optimal leveling, possibly calculation of % homology, preferably % of identity sequence. Usually software performs this calculation as part of comparing sequences and gives quantitative result.

In sequences of may also be deletions, insertion or replacement of amino acid residues, which lead to molchashchei replacement of and is given as a result of functionally equivalent substance. Deliberate amino acid replacement of can be to produce based on similarity of polarity, charge, solubility of, hydro fobnosti, hydro filnosti and/or amfipaticheskoi nature of these residues, remains secondary binding activity of this substance. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar main groups, having similar value of hydro filnoe™, include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

Conservative replacement of can be make, for example, according to given below table. Amino acids in one and the same unit in the second graph and preferably in one and the same line in the third graph can be replace the on each other._;

The present invention also embraces possibility of realization homologous replacement of (terms "replacement" and "replacement of" both are used here for designation of exchange of existing amino acid residue on alternative residue), i.e. replacement of like on like, for example main on main, acid on acid, polar on polar and T. d. May also occur negomologichnaya replacement of, i.e. from one class of residues to other, or such, in which alternatively involved switching of unnatural amino acids, such as ornithine (here on it present both on z), diaminomaslyanaya acid ornithine (here on it present both on in), norleucine ornithine (here on it present both on about), piriilalanin, tienilalanin, naphthylalanine and phenylglycine.

Replacement may also be made on unnatural amino acids, alpha * and alpha-disubstituted * amino acid, n- alkilaminokisloty *, lactic acid *, halide derivatives of natural amino acids, such as triftortirozin *, U-c1-phenylalanine *, U- Vg -phenylalanine *, U-1-phenylalanine *, l-allyl-glycine *, p-alanine *, l-a-aminobutyric acid *, 1_ -in--aminobutyric acid *, l-a-aminoisobutyric acid *, 1_ -8-aminocaproic acid *, 7- aminogeptanovuyu acid *, l-methionine sulphon # *, l-norleucine *, l-norvaline *, U-nitro- 1_ -phenylalanine *, 1_ -hydro ksiprolin #, l- tioprolin *, methyl derivatives of phenylalanine (phe), such as 4-methyl- Re *, pentamethyl-phe *, l-phe (4-amino) #, l-tyr (methyl) *, l-phe (4-isopropyl) *, l- Tic (1.2, 3, 4-tetrahydro isoquinoline-3-carboxylic acid) *, l- diaminopropionovuyu acid * and l-phe (4-benzyl) *. Designation * used in purposes above given discussion (relative to homologous and negomologichnoi

replacement of), to indicate on hydro fobnuyu nature of this derivative, whereas the designation # used, to indicate on hydro filnuyu nature of this derivative, # * indicates amfipaticheskie characteristics.

Variant amino acid sequences may include suitable spacer group, which can be inserted in between any two amino acid residues sequence of, including alkyl groups, such as methyl, ethyl or propyl group, in addition to amino acids- speiseram, such as residues of glycin or βalanina. Next mould variation, in which involved the presence of one or more than one of the amino acid residues in peptoidnoi form, should be well known professionals in this area. To avoid doubt " peptoidnaya mould" is used for references to variant amino acid residues, where group-substituent and-carbon is rather on nitrogen atom of this residue, and not on a-carbon,. Methods for preparing peptides in peptoidnoi the form of are known in this area (for example, SimonRJ et al., PNAS (1992) 89 (20), 9367-9371, and Horwell dc, TrendsBiotechnol. (1995) 13 (4), 132-134).

The present invention also embraces use of sequences, which can hybridize with represented here sequences of targets, so, as if the agent represents antisense sequence.

Term "hybridisation", as it is used here, must include "process, as a result of which the thread nucleic acid connected with complementary thread by means of coupling bases", and also the process of amplification, which occurs at technologies polymerase chain reaction (PCR).

Nucleotide sequences according to the invention, capable of selectively hybridize with represented here nucleotide sequences or with their complement, should be, as a rule, at least 75%, preferably at least 85 or 90% and more preferably at least 95% or 98% homologous corresponding represented by the here complementary nucleotide sequences of on the region of at least 20, preferably at least 25 or 30, for example at least 40, 60, 100 or more adjacent nucleotides. Preferred nucleotide sequences by invention will contain zones, homologous nucleotide sequence, presented in SEQ id NO 2 lists of sequences according to the present invention, preferably at least 80 or 90%, and more preferably at least 95% homologous to nucleotide sequence, presented in SEQ id NO 2 lists of sequences according to the present invention.

Term "selectively hybridised" means, that the nucleotide sequence, when its is used as probe, is used in conditions of, at which is detected, that the nucleotide sequence of-the target is hybridised with this probe at the level of, considerably exceeding background. Background hybridisation can take place in connection with the presence of other nucleotide sequences, for example in cdna or genomic dna library, which is subjected to screening. In this case under background imply level of signal, generated by interaction between probe and nonspecific dna-member library, which is less than 10 times, preferably less than 100 times intensive, than specific interaction, observed with dna-target. Intensity of interaction can be measured, for example, by means of radioactive labeling of probe, for example^{32 p}.

Conditions for hybridization are based on melting temperature (T_Product) complex binding nucleic acids ( Berger and Kimmel, 1987, guide to MolecularCloningtechniques, Methods in Enzymology, vol 152, academic press, san Diego ca), and confer a certain "stringency", as explained below.

Maximum stringency usually is created at approximately T_{pp -5}^{0 with} (on 5° with below T_{Product of probe}); high stringency-at temperature of approximately 5° with -10 ° with below T_Product; intermediate stringency-at temperature approximately 10° to -20 ° with below T_Product; and low stringency-at temperature of approximately 20° with -25 ° with below T_Product. As is known specialists in this area, hybridization maximum of rigor can be used for identification or detection identical nucleotide sequences, whereas the intermediate (or low) stringency hybridization can be used for identification or detection similar or related polynucleotide sequences.

In preferable aspect, the present invention embraces nucleotide sequences, which can hybridize with nucleotide sequence according to the present invention in stringent conditions (for example 65 and 0,1xSSC {1xSSC = 0, 15ΜNaCI, 0, 015ΜYaztsitrat, ph 7.0). If nucleotide sequence is double-stranded, both threads of this duplex or individually, or in combination, enter volume of the present invention. If nucleotide sequence is of a single-stranded, should have in form, that complementary sequence of this nucleotide sequence also is connected in volume of the present invention.

Nucleotide sequences, which are not on 100% homologous sequences of according to the present invention, but enter volume of invention, can be obtained near methods. Other modifications described here sequences can be obtained, for example, by probing of dna libraries, created from row of sources. Besides, can be obtained other viral/bacterial or cellular homologs, in particular cellular homologs, detected in cells of mammals (for example in cells rat, mouse, bovine and of pimates), and such homologs and their fragments, as a rule, will are capable of selectively hybridize with sequences, represented here in list of sequences. Such sequence can be obtained by probing of cdna libraries, created from other types of animals, or genome dna libraries from other types of animals, and probing such libraries probes, completely or partially containing presented by here nucleotide sequence, in conditions of rigor from middle to high. Similar considerations is used to production of travel homologs and allelic variants of amino acid and/or nucleotide sequences according to the present invention.

Versions and shtammovye /species homologs can also be produced with the use of degenerate PCR, in which will be used primers, constructed to sequences of-targets within versions and homologs, encoding conservative amino acid sequences within sequences according to the present invention. Conservative sequence can be estimating, for example, by balancing the amino acid sequences of several variants/homologues. Equalizing sequences may be performed, using computer software, known in this area. For example, employed program GCGWisconsinPileUp. Primers, used in degenerate PCR, will contain one or more than one degraded position, and their will be used at conditions of more low rigor, than conditions, which are used for kloniruyushchikh sequences with primers single sequences against known sequences.

Alternatively, such nucleotide sequences can be obtained by site-directional mutagenesis described sequences, such as nucleotide sequence, presented on SEQ id NO 2 lists of sequences according to the present invention. This can be useful, if, for example, silent of replacement of codons required for sequences, to optimize preferableness codons for specific-cell host, in which is expressed this nucleotide sequence. Other change of sequence can be large, to introduce sites recognition enzymes restriction, or to change activity of protein, being encoded these nucleotide sequences.

Nucleotide sequences according to the present invention can be used for producing primer, for example PCR primer, primer for alternative amplification reaction, of probe, for example marked vyyavlyayushchei mark traditional methods with the use of radioactive or nonradioactive marks, or these nucleotide sequences can be clone in vectors. Length of such primers, probes and other fragments should be at least 15, preferably at least 20, for example at least 25,30 or 40 nucleotides, and they also are embraced by term nucleotide sequence by invention, as it is used here.

Nucleotide sequences, such as dna polynucleotides and probes by invention, can be to produce recombinant, by synthetic or any methods, available specialists in this area. Their may also be clone using standard methods.

As a rule, primers is obtained synthetic methods, including step-by-step production of desired nucleic acid sequence one nucleotide at a time. Techniques for realization of this with the use of automated technologies, readily available in the given engineering.

Longer nucleotide sequences, as a rule, is obtained, using recombinant methods, for example using PCR (, polymerase chain reaction) techniques cloning. Such methods include creation of pairs of primers (for example approximately from 15 to 30 nucleotides), flanking region sequence-target, which preferably clone, bringing these primers in contact with mrna or cdna, obtained from cells animal or human, performing polymerase chain reaction (PCR) in conditions of, which lead to amplification of desired region, isolation of amplified fragment (for example by cleaning reaction mixture on agarose gel) and isolation of amplified dna. Primers can be are constructed so, that they contained suitable sites recognition enzymes restriction, so that amplified dna can be clone in suitable cloning vector.

Due to which is intrinsic genetic aspect code degraded ™ for cloning and expression of sequence-targets can be used other dna sequence, which are encoded substantially same or functionally equivalent amino acid sequence. As is known specialists in this area, for some systems expression may be favorable to obtain sequence-target with codons, not triggering in nature. Codons, preferable for specific prokaryotic or eucaryotic host ( Murray e et al (1989)NucAcids res 17 : 477-508), can be chosen, for example, to to increase speed of expression of target or to obtain recombinant rna transcripts, having desirable properties, such as more prolonged period of time of half-decay, than in transcripts, produced by with sequence, found in nature.

Nucleotide sequence for use as target or for expression of target can be included in recombinant vector displacement. This vector can be used for replication of nucleotide sequence in compatible cell-host and/or its expression of compatible kpetki -host. Expression can be adjusted, using regulatory sequence, which include promoters/ enkhansery and other signals regulation of expression. Can be used prokaryotic promoters and promoters, functional in eukaryotic cells. Can be used tkanespetsifichnye or stimulspetsifichnye promoters. May also be use of chimeric promoters, containing elements sequence of from two or more than two different promoters, described above.

Protein, generated recombinant cell-warlock by means of expression of nucleotide sequence, can be sekretiruemym or can contain vnutrikletochno depending on sequence and/or from used vector. Encoding sequence can be are constructed with signal sequences, which are directed secretion of substance, being encoded these sequences, through membrane specific procaryotic or eucaryotic cells.

Amino acid sequence of-the target may be obtained in the form of fused protein, for example to enable its separation and purification. Examples proteins-partners fusion of include glutathione-e-transferase (GST), 6xHis, GAL4 (dna-binding domain and/or domain transcription activation of) and p--galactosidase. Can also be convenient include site of proteolytic cleavage between protein-partner fusion and of interesting protein sequence of, to give possibility to remove sequence of fusion protein. Preferably fusion protein not conceals activity of target.

Fused protein can contain antigen or antigenic determinants, fused with substance according to the present invention. In this embodiments this fused protein may represent not seldom frequency in nature fused protein, containing substance, which can act as adjuvant in sense provide general immune system stimulation. Antigen or antigenic determinants can be connect or to amino, or to carboxy end of substance.

In other embodiments of the invention amino acid sequence can be ligirovat with of heteroduplex sequence of, in order to code fused protein. For example, for screening of peptide libraries on agents, capable of effect on activity substance, can be useful to encode chimeric substance, ekspressiruyushchee heterologous epitope, which is identified by a commercially available antibody.

In one embodiments of the present invention agent can represent antibody. Additionally or alternatively target can represent antibody. Additionally or alternatively means for detection of target can represent antibody.

Antibodies may be obtained standard methods, such as immunization substance or use of phage display library.

For the purposes of-term "antibody", if not indicated the other, includes, but not is limited by them, polyclonal, monoclonal, chimeric, single-chain, fab fragments, fragments, produced library of fab expression, and also their mimetics. Such fragments include fragments of holistic antibodies, which are stored their binding activity with respect to substance target, fv, f (ab ') and f (ab')_{2 fragments}, and also one-chained antibodies (scfv), fusion proteins and other synthetic proteins, which contain antigen-binding site antibody. Besides, antibodies and their fragments can be humanised antibodies. Nuetralising antibodies, i.e. those, which inhibit biological activity of substances polypeptides, are particularly preferable for diagnosis and treatment of.

If desirable are polyclonal antibodies, selected animal (for example a mouse, rabbit, on goats, hoarse and T. d.) is subjected to immunization of immunogenic polypeptide, carrying epitope (epitopes), obtained from identified agent and/or substances according to claimed invention.

Depending on types-host for reinforcing immunological response can be used different adjuvants. Such adjuvants include, but are not limited to, adjuvant Freinda, mineral gels, such as hydro ksid aluminium, and surface-active substances, such as lizoletsitin, plyuronovye polyols, polyanions, peptides, oil emulsions, Keyhole from blyudechka and dinitrophenol. Bcg( BacilliCalmette - Gueriri ) andcorynebacterium parvum are potentially useful adjuvants person, which may be used, if purified substance polypeptide is introduced individuals with immunological risk of, in stimulation of systemic protection.

Serum from immunized animal is collected and are treated by known techniques. If serum, containing polyclonal antibodies to epitope, obtained from identified agent and/or substance according to the present invention, contains antibodies to other agents, these polyclonal antibodies can be to purify with the help of immunoaffinity chromatography. Procedures of obtaining and processing of polyclonal antisera are known in this engineering. To such antibodies can be made, according to the invention also polypeptides or their fragments, gaptenizirovannye with other polypeptide, for use as immunogens to the in animals or people.

Technician in this area can also easily to produce monoclonal antibodies, directed against epitopes, received from identified agent and/or substances according to claimed invention. Total technique for peer making monoclonal antibodies by hybridomas is well known. Timeless antibody-producing cell lines can be to create with the aid of cellular fusion of, and also with the help of other techniques, such as direct transformation of in lymphocytes oncogenic dna or transfection with epstein- Barra. Group of monoclonal antibodies, produced by against spherical epitopes, may be subjected to screening on different properties; i.e. on isotype and affinity to epitope.

Monoclonal antibodies to substance and/or identified agent can be to produce, using any methods, which is developed for producing molecules of antibodies continuous cellular lines in culture. They include, but are not limited to, gibridomnuyu methods, newly described Koehler and Milstein (1975 Nature 256 : 495-497), technique of Βkletochnykh human hybridomas ( Kosboret al (1983)Immunol. Today 4:72; Coteet al (1983) proc. Natl. Acad. Sci. USA 80 : 2026-2030) and technique of EBV -hybridomas (cole et al (1985)MonoclonalAntibodies and cancer therapy, Alan r Liss inc., ρρ. 77-96). Besides, can be used techniques, developed for producing "chimeric antibodies", splicing genes murine antibodies with genes of human antibodies to produce molecules with suitable antigenic specificity and biological activity ( Morrisonet al (1984) proc. Natl. Acad. Sci. USA 81 : 6851-6855 ; Neubergeret al (1984)Nature 312 : 604-608 ; Takedaet al (1985)Nature 314 : 452-454). Alternatively, techniques, described for producing single-stranded antibodies (patent SShA№ 4946779) may be adapt for producing specific to substance single-stranded antibodies.

Antibodies, as monoclonal, so and polyclonal, which are directed against epitopes, available from identified agent and/or substances, particularly useful in diagnostics, and those, which are neutralizing, are useful in passive immunotherapy. Monoclonal antibodies, in particular, can be used, to cause protivoidiotipicheskie antibody. Protivoidiotipicheskie antibodies represent immunoglobulins, which carry "inner image of" substance and/or agent, against which is protection. Procedures of creating protivoidiotipicheskikh antibodies are known in the given engineering. These protivoidiotipicheskie antibodies may also be useful in therapy.

Antibody can be also to produce, inducing their in vivo production in the lymphocyte population, or with the help of screening libraries of recombinant immunoglobulins or groups of highly specific binding reagents, as described in Orlandiet and / (1989, Rgos. Natl. Acad. Sci. 86 : 3833-3837) and winter g. And Milstein s. (1991 ; Nature 319 : 293-299).

May also be to create fragments of antibodies, which contain specific sites of binding for substance. For example, such fragments include, but are not limited to, f (ab ') 2 fragments, which can be to produce by enzymatic hydro lipolysis molecule with pepsin, and fab fragments, which can be to create with the help of removing di-sulphide bridges f (ab')_{2 fragments}. Alternatively can be construct library fab expression, that allows quickly and easily identify monoclonal fab fragments with the desired specificity of ( HuseWDet al (1989) science 256:1275-1281).

Analytical methods (and also skriningakh ) according to the present invention can be used a wide variety of repoters, ; preferred reportery provide convenient detected signals (for example spectroscopy). One reporter gene can to encode enzyme, which catalyzes, modifying the properties of light absorption.

Examples reporter molecules include, but are not limited to, p--galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, p- glyukuronidazu, exo-glucanase and glucoamylase. Alternatively, labeled radioactive mark or labeled fluorescent label nucleotides can be include in newly formed transcripts, which then is identified at binding with oligonucleotide probes.

In one preferred embodiments production of reporter molecules is measured by enzymatic activity of reporter gene product, such as p-galactosidase.

In this engineering is known multiple protocols for detecting and measuring expression of target, for instance using or polyclonal, or monoclonal antibodies, specific to this protein. Examples include immunofermentative solid-phase analysis (ELISA), radioimmunologicheskii analysis (RIA) and activated fluorescence analysis of cells (FACS). Preferable is dvukhsaitovyi immunological analysis on monoklonalnoi base, using monoclonal antibodies, reactive to two not interferiruyushchim epitopes on polypeptides, but may also be used competitive binding analysis. These and other analysis are described, among other things, in Hampton r et al ( 1990, SerologicalMethods, a Laboratory manual, aps press, st paul ΜΝ ) and Maddox de et al ( 1983, j. exp. med. 15,8:121 1).

Wide variety of marks and techniques of conjugation is known specialists in this area, and their can be used in various analysis of nucleic acids and amino acids. Methods for preparing labeled probes hybridization or PCR for detection of polynucleotide sequence-targets include oligomechenie, niktranslyatsiyu, end tagging or PCR amplification using labeled nucleotide. Alternatively, coding sequence or any its part can be clone in vector for producing mrna of probe. Such vector are known in this engineering, commercially are accessible, and their can be used for rna synthesis of probes in vitro addition of suitable rna polymerase, such as t7, TZ and SP6, and labeled nucleotides.

A number of companies, such as PharmaciaBiotech ( Piscataway, NJ ), Promega ( Madison, Wl ) and us BiochemicalCorp ( Cleveland, it) delivered grade sets and protocols for these techniques. Suitable reporternye molecules or labels include radionuclides, enzymes, fluorescent, chemiluminescent or khromogennye agents, and also substrates, cofactors, inhibitors, magnetic particles and the like. Patents, in which there are indicate by use of such marks, include us-a- 3817837 ; us-a- 3850752 ; us-a- 3939350 ; us-a- 3996345 ; us-a- 4277437 ; us-a- 4275149 and us-a- 4366241. Recombinant immunoglobulins can be also to produce, as shown in us-a- 4816567.

Additional methods of quantitative determination of expression of specific molecules include radioactively-labeled ( Melby pc et al 1993 j. Immunol. Methods 159 : 235-44) or biotinylated ( Duplaa c et al 1993Anal. Biochem. 229-36) nucleotides, koamplifikatsiyu control nucleic acid and standard curves, on which are interpolated, experimental results. Quantitative determination of multiple samples may be increased with the help of analysis in format ELISA, where interest oligomer is presented in various dilutions, and spectrophotometric or colorimetric response gives rapid quantitative determination.

At presence/absence of expression of marker gene allows call, that gene of interest also is present, its presence and expression of necessary to prove. For example, if the nucleotide sequence is built inside the sequence of marker gene, recombinant cells, containing this sequence, may be identified by absence of function of marker gene. Alternatively, marker gene can be arranged in tandem with encoding sequence-target under control of one promoter. Expression of marker gene in response to induction or selection usually indicates also on expression of target.

Alternatively, cells-hosts, which contain coding sequence for target and express encoding zones of this target, may be identified with the help of various techniques, known professionals in this area. These techniques include, but are not limited to, dna-dna or dna-rna hybridization, and also procedures of biological or immunological dough on protein, which include technology based on membranes, based on solutions of or based on granules for detection and/or quantitative determination of nucleic acid or protein.

Ability of tested agent potentiate camp can be determined by measuring corresponding diffusion condensate or Your condensate level of target. Additionally or alternatively, ability of the tested agent potentiate camp can be determined by measuring corresponding diffusion condensate levels of camp. For example, may be adapt the technique of Smithet al 1993 ( Appl. Biochem. Biotechnol. 41 : 189-218). There are also commercially available sets of immunological analysis for measuring camp (for example Amersham international, ArlingtonHeights, il and DuPont, Boston, ΜΑ ). Details by suitable analysis of camp are given in experimental partition.

Any one or more than one suitable target, such as amino acid sequence or nucleotide sequence, can be used for identification of Ptsamf in any of various techniques of screening of drugs. Target, used in such dough, can freely be in solution, may be fixed on solid carrier, its may carry cell surface, or it may be localized inside cells. This target may even be inside of animal model, where said target may be exogenous target or introduced target. This animal model usually is not human animal model. Undoing activity of target or formation of complexes binding between target and testing agent can be measured.

Methods of screening drugs can be are based on method, described Geysen in the european patent claim 84/03564, published 13 September 1984. In transcribed, big amount of various small peptide both compounds are synthesized on solid substrate, such as plastic rods or what-either another surface. These peptide both compound is subjected to interaction with suitable target or its fragment and washed. Are then determined connected substance, for example appropriate way adapted methods, process by conventional in this engineering. Purified target can also be applied directly on boards for use in techniques screening of drugs. Alternatively, for trapping peptide and immobilization of its on solid carrier may be used not nuetralising antibodies.

According to the invention is also to use competitive skriningovye analysis medicinal agents, at which nuetralising antibodies, able to bind with target, specifically competed with testing compound behind binding with target.

Other technique screening is developed for screening with high carrying capacity (HTS) agents, with suitable binding affinity to substances, and is based on method, are set out in WO 84/03564.

It is expected, that these methods of analysis according to the present invention are suitable both for small, and for widely scale screening of tested compounds, and also in quantitative analysis.

So, the present invention also relates to a method for identification of agents, which potentiate camp, at which suitable target is brought in contact with agent, and then is measured activity and/or levels of camp.

The present invention also relates to a method for identification of agents, which selectively potentiate camp in the sexual genitalia of a woman, at which is brought in contact suitable target sexual genitalia of a woman, and then is measured activity and/or levels of camp.

The present invention also relates to a method for identification of agents, which potentiate camp, at which suitable target is brought in contact with agent, and then is measured activity and/or levels of this target.

The present invention also relates to a method for identification of agents, which selectively potentiate camp in the sexual genitalia of a woman, at which is brought in contact suitable target sexual genitalia of a woman, and then is measured activity and/or levels of this target.

In preferable aspect, screening of according to the present invention includes at least the following stages (not necessarily must be in the same order of sequence), on which : (and) is performed in vitro screening, to determine, whether agent-candidate corresponding activity (such as modulation of nep, such as nep from kidney dog) ; (b) is one or more than one screening of selectivity, to determine selectivity said agentakandidata (for example see, whether said agent also inhibitor of apf, for example using protocol analysis, represented here); and (in) is performed in vivo screening of with said agent-a candidate (for example using functional animal model). Usually, if agentkandidat passes screening of (and) and screening of (b), then is performed for screening (in).

According to the present invention is also diagnostic composition or set for detection of predisposition to RPVZh. In this with respect to this composition or kit contains object, which can indicate on the presence of one or more than one-or absence of one or more than one-from targets in test sample. Preferably this tested specimen is produced from sexual genitalia of a woman or their secretion.

For example, diagnostic composition may contain any nucleotide sequence, said here, or its variant, homologue, fragment or derivative, or sequence, capable of hybridize with any full-or partial sequence of.

To provide base for diagnosis of disease, should be to establish normal or standard value for target. This can be realized by combining liquid posts organism or cell extracts, taken of normal subjects, or in animal, or in human, with antibody to a target in conditions of, suitable for complex formation, which problems in this area. Number of standard complex formation can be calculate by comparison with its series of dilutions of positive testing runs, where known amount of antibody with known concentrations of purified target. Then standard value, obtained with normal samples, can be compare with values, obtained with samples, taken in subjects, , potentially affected RPVZh. By deviation between standard value and value in subject presence of diseased state.

Proper target or any part of it can provide base for diagnostics and/or therapeutic compound. In the diagnosis can be used polynucleotide sequences target, to detecting and quantitatively to determine gene expression in states, disorders or diseases, in which may be involved RPVZh.

A polynucleotide sequence, encoding target, can be used for diagnosis of RPVZh as a result of expression of this target. For example, for detection of anomaly in expression of target polynucleotide sequences, encoding target, can be used in hybridization or PCR analysis of tissues from biopsies or autopsii, or biological liquid posts. Types of such qualitative or quantitative methods may include SauzerniliNozern -analysisaocommab.dashaoblot or other technology on membrane base; PCR technology; technology submersion of rod, pin or granules; and ELISA or other technology format multiple specimens. All these technology well known in this area engineering and actually make base many commercially available diagnostic sets of.

Such analysis can be adapted for evaluation of efficiency of a specific mode of therapeutic treatment and can be used in research animals, in clinical tests or in monitoring treatment of individual patient. To provide base for diagnosis of disease, should be to establish normal or standard profile for expression of target. This is performed by combining liquid posts organism or cell extracts, taken of normal subjects, or in animal, or in human, with target or its part, in conditions of, suitable for hybridization or amplification. Standard hybridization can be determined quantitatively by comparison of the values, received for normal subjects, with a series of dilutions of positive kontrolen, made in the same experiment, where there is used the known amount of purified target. Standard value, obtained with normal samples, can be compare with values, obtained with samples from subjects, potentially affected disorder or disease, coupled with expression of encoding sequence target. By deviation between standard value and value of subject presence of diseased state. If disease is installed, is existing therapeutic agent, and can be vision statement presented ve therapeutic profiles or value. Finally, this analysis of possible to repeat the course on regular basis, to evaluate, progressive these value or returned to normal or standard patternu. Successful therapeutic profiles can be used for detecting of treatment efficiency period of several days or several months.

So, in one aspect, the present invention relates to the use of polypeptide target or its versions, homolog, fragment or derivative, for producing antibodies against target, which can be used, for example in diagnostic purposes, for detection and quantitative determination of levels of target at states RPVZh.

Furthermore, according to the present invention are diagnostic analysis and sets for detection of target in cells and tissues, containing purified target, which may be used as positive control, and antibodies against target. Such antibodies can be used in based on solutions, based on membranes or based on tissues technologies for detection of any diseased state, either state of, relating to expression of protein-target or expression of its deletsii, or its variant, homolog, fragment or derivative.

Diagnostic compositions and/or methods and/or sets of can be used in the following techniques, which include, but are not limited to, competitive and not competitive analysis, radioimmunologicheskii analysis, biolyuminestsentnye and chemiluminescent analysis, flyuorimetricheskie analysis, sandwich-analysis, immunoradiometricheskie analysisaocommab.dashaoblot analysis, enzyme analysis, including ELISA, mikrotitrovalnye boards, covered with antibodies strip or rods for submersion for fast monitoring of urine or blood, immunogistokhimiyu and immunotsitokhimiyu.

For example, set for immunohistochemistry may also be used for localization of activity nep in tissue genitalia. This set for immunohistochemistry makes it possible to determine the localization of nep in slices of tissue and cultured cells using as light, and electronic microscopy, which can be used as in research, and in clinical purposes. Such information can be useful in diagnostic and, possibly, in therapeutic purposes for detecting and/or preventing and/or treating ZhSD, such as RPVZh. For each set of range, sensitivity, accuracy, reliability, specificity and reproducibility of analysis. Variation inside analysis and between analyses is installed in points 20%, 50% and 80% on standard curves size or activity.

Probes

The other aspect of the present invention is creation of probes hybridization of nucleic acids or PCR, which are capable to detect (in particular probes, which are capable of selectively detect) polynucleotide sequences, including genomic sequences, encoding encoding area of target, or close related molecules, such as alleles. Specificity of probe, i.e. its occurrence from highly conservative, conservative or nekonservativnogo region or domain, and stringency hybridization or amplification (high, intermediate or low), should determine, identifies whether this probe only occurring in nature coding sequence target or related sequence. Probes for detecting related nucleic acid sequences is selected from conservative or highly conservative nucleotide regions of members of family of target, and such probes can be used in bullet degenerated probes. For detection of identical nucleic acid sequences or there, where is maximum specificity, probes of nucleic acids is selected from nekonservativnykh nucleotide regions or unique regions polynucleotides target. Used here term " nekonservativnyi nucleotide region" relates to nukleotidnomu district, which is unique for encoding sequence target, described here, and not meets in related members of this family of.

PCR, as described in us-a- 4683195, us-a- 4800195 and us-a- 4965188, involves additional application for oligonucleotides, based on sequences of target. Such oligomers, as a rule, are synthesized chemically, but their can be construct enzymatic by or to produce from recombinant source. Oligomers, as a rule, contain two nucleotide sequences, one with semantic orientation (5 '-> 3') and one with describes anti-sense orientation (3 '< -5'), used in optimised conditions for identification of particular gene or state. The same two oligomer, built sets of oligomers or even degenerate pool of oligomers can be used in less stringent conditions for detection and/or quantitative determination of close related dna or rna sequences.

Nucleic acid sequence for target may also be used, to construct probes hybridization, as described earlier, for mapping of endogenous genomic sequence. This sequence can be mapping on particular chromosome or in specific area of chromosome, using known techniques. They include in situ hybridization with Pap chromosomes ( Vermaet al (1988) human Chromosomes : a manual care basic Techniques, Pergamon press, new york city), chromosomes, mechanically selected with the help of flow cytometry, or artificial khromosomnymi structures, such as YAC, bacterial artificial chromosome (bac), bacterial pi structure or library " dna separate chromosomes.

in situ hybridisation of chromosomal preparations and procedures of physical mapping, such as analysis of adhesion with use of installed of chromosomal markers, invaluable in development of genetic maps. Examples of genetic maps can be find in science 1995,270:41 care and 1994 ; 265 : 1981Τ frequently placing gene on chromosome another kind of mammals can detect conjugated markers, even if the number or arm of a specific human chromosome unknown. New sequence of can be bind to the arms of the chromosomes or their sections with means of physical mapping. It provides security information for researchers, dealing with looking for genes of disease, using positional cloning or other methods of investigating gene. After coarse localization of disease or syndrome by means of genetic of clutch in a specific area of genome, any sequence of, kartirovannye in this region of, can represent conjugated or regulatory genes for further study. Nucleotide sequence of object of the invention may also be used, to to detect differences in the chromosomal localization due to translocation, inversion of and T. d. between normal an individual, carrier and patients.

Term " kpetka -host" relative to the present invention includes any cell, which may contain target for agent.

So, in the following embodiments of the present invention proposes-cell hosts, transformed or transfected polynucleotide, which represents or expresses target. Preferably, said polynucleotide is transferred in vector for replication and expression polynucleotides, which should be the target or should to express this target. These cells should be select so, that they are with said vector, and they may represent, for example, prokaryotic (for example bacterial), mushroom, yeast or plant cells.

Gram bacteria e. coli widely used as host for of heterologous gene expression. However considerable amount of heterologous protein have tendency to accumulation of inside cells. Subsequent purification of the desired protein from the whole mass of intracellular proteins phagolysis coli sometimes happens zatrudnitelna.

In contrast to e. coli, bacteriaof bacillus genus are for suitable for use as heterologous host in connection with their ability secrete proteins in culture medium. Other bacteria, suitable for use as host, are bacteria from delivery streptomyces and pseudomonas.

Depending on the nature of the polynucleotide, encoding polypeptide according to the present invention, and/or from desirability of further processing of expressed protein, preferable may be eucaryotic hosts, such as yeast or other fungi. In general, yeast cells prefer other mushroom cells in connection with the fact, that with them lighter-than-operate. However, some proteins or slightly secreted from yeast cells, or in some cases not undergo transformation regular Fast (for example giperglikozilirovanie in yeast). In these cases of organism-host should be select from other fungi.

Examples suitable host expression in the framework of the present invention are fungi, such as mushrooms of species aspergillus ( such as those, which are described in er-a- 0184438 and ep-a- 0284603 ) and type of trichoderma;

bacteria, such as species bacteria bacillus ( such as those, which are described in er-a- 0134048 and ep-a- 0253455 ), type of streptomyces and type ofpseudomonas; and yeast, such as yeast type of Kluyveromyces ( such as those, which are described in er-a- 0096430 and ep-a- 0301670 ) and type of saccharomyces. For example, typical hosts expression may be selected from aspergillus niger, aspergillus niger var. tubigenis, aspergillus niger var. awamori, aspergillus aculeatis, aspergillus nidulans, aspergillus oryzae, trichoderma reesei, Bacillussubtilis, bacillus licheniformis, bacillus amyloliquefaciens, Kluyveromyces lactis andsaccharomyces cerevisiae.

Use of suitable kpetok -host, such as yeast, mushroom and vegetable-cell hosts, can provide posttranslational modification (for example miristoilirovanie, glycosylation, shortening, lapidatsiyu and phosphorylation at tyrosine, an or threonine), which may require for imparting optimal biological activity of recombinant products expression according to the present invention.

Term "organism" relative to the present invention includes any organism, which may contain target and/or products, produced from it. Examples of organisms may include a fungus, yeast or plant.

Term "transgenic organism" relative to the present invention includes any organism, which contains target and/or products.

Transformation of cells-host/organisms-host

As indicated above, organism-host may be prokaryotic or eukaryotic organism. Examples suitable eucaryotic organisms include phagolysis coli andbacillus subtilis. Indicating by transformation of prokaryotic host well documented in this engineering (cm. for example Sambrooket al (MolecularCloning : a Laboratory manual, 2^{nd edition}, 1989, ColdSpringHarborLaboratory press) and Ausubelet al., current protocols in Molecular biology (1995), john Wiley & Sons, inc).

If use prokaryotic host, nucleotide sequence may require suitable modification before transformation, such as removal of introns.

In other embodiments transgenic organism can be a yeast. In this with respect to yeast also widely used as carrier for of heterologous gene expression. Type saccharomyces cerevisiae has a long history of industrial use, including its use for of heterologous gene expression. Observation expression of heterologous genes in saccharomyces cerevisiae is madeGoodey et al (1987, Yeast biotechnology, d r Berry et al, concentrate, ρρ 401-429, allen and Unwin, London ) and king et al (1989, Molecular and cell biology care Yeasts, ε f Walton and g Τ Yarronton, concentrate, ρρ 107-133, Blackie, Glasgow ).

Along several reasons saccharomyces cerevisiae are good of heterologous gene expression. In-first, they do not patogenny for humans and are not capable to produce certain endotoxin. In-second, they have long history of safe use on the centuries of commercial application for various purposes. This to wide public recognition. -Third, intensive commercial application and of investigating, devoted to this organism, led as a result of to wealth of knowledge genetics and physiology of, and also characteristics of large scale fermentation saccharomyces cerevisiae.

Observation principles of heterologous gene expression in saccharomyces cerevisiae and secretion of gene products is made e. Hinchcliffe, E. Kenny (1993, " Yeast as a vehicle for the expression care heterologousgenes", Yeasts, vol. 5, Anthony Η rose and j StuartHarrison, concentrate, 2^{nd edition}, academic press ltd.).

Contains there is several types of yeast vectors, including integrativnye vectors, which is required recombination with an alphavirus host for their maintaining, and independently replicating plasmid vectors.

To obtain transgenic saccharomyces, structure expression is made by insert nucleotide sequence according to the present invention in the construction, intended for expression in yeast. Proposed several types of structures, used for of heterologous expression. These constructs contain promoter, active in yeast, fused with nucleotide sequence according to the present invention. Used is usually promoter of yeast origin, such as promoter GAL1. Used is usually signal sequence of yeast origin, such as sequence, encoding signal peptide SUC2. Terminator, active in yeast, completes expression system.

For transformation of yeast is developed several protocols transformation. For example, transgenic saccharomyces according to the present invention can be to produce, following instructions Hinnen et al {1978, Proceedings care the national Academy care sciences care the USA, 75,1929); Beggs, J.D. (1978, Nature, London, 275,104); and ito, Η. et al (1983, j Bacteriology 153, 163-168).

Transformed yeast cells are selected, using different selective markers. Among markers, used for transformation of, is a row of auxotrophic markers, such as LEU2, HIS4 and TRP1, and dominant markers of resistance to antibiotics, such as markers of aminoglycoside antibiotics, for example G418.

Other organism-host represents plant. Main principle for design genetically modified plants is the genetic information into the plant genome so, to obtain stable maintenance of this built-in genetic material. For insertion of this genetic information exists several techniques, at that, two main principles is direct introduction of genetic information and introduction of genetic information by using vector system. View of common techniques can be used in articles Potrykus ( Annu rev PlantPhysiolPlantMolBiol 42:

205-225) and Christou ( Agro -food-industry hi-tech march/april 1994 17-27). Additional indicating by plant transformation can be find in er-a- 0449375.

So, in this invention also relates to a method of transformation of cells-host nucleotide sequence, which should be the target or to express target. -cell hosts, transformed this nucleotide sequence, can be-possibility of in conditions of, suitable for expression and isolation of being encoded protein from cell culture. Protein, generated recombinant cell, can be sekretiruemym, or can contain vnutrikletochno depending on used sequence and/or vector of. For specialists in this area obvious, that expression vectors, containing encoding sequence, can be construct with signal sequences, which are directed secretion of encoding sequences through specific prokarioticheskuyu or eukaryotic membrane. Other recombinant structure can connected coding sequence to nucleotide sequence, encoding domain polypeptide, which may enable purification of soluble proteins ( Kroll dj et al (1993) dna cell Biol 12:441-53).

According to the present invention is also pharmaceutical composition, containing therapeutically effective amount of agent along the present invention and pharmaceutically acceptable carrier, diluent or excipients (including combinations thereof).

Pharmaceutical compositions can be earmarked for use in medicine and veterinary science and typical contain one or more than one pharmaceutically acceptable carrier, diluent or excipient. Acceptable carriers or diluents for therapeutic application problems in pharmaceuticals and described are, for example, in Remington's Pharmaceutical sciences, Mack publishing co. (a. roption. Gennaro edit. 1985). Selection of pharmaceutical carrier, excipient or diluent can be made with consideration of assigned path of introducing and standard pharmaceutical practice. Pharmaceutical compositions may contain as carrier, excipient or diluent or in addition to it any suitable binding agent (s), lubricating agent (s), suspending agent (s), coating agent (s), a solubilizing agent (s).

Preserving agents, stabilizers, dyes, and also correcting agents can be included in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters pgidroksibenzoinoi acid. Can be used also antioxidants and suspending agents.

Depending on various systems of delivery to composition/preparation may be different requirements. For example, pharmaceutical composition according to the present invention can be prepared in the form of preparation for delivery with use of mini-pump or mukozalnym by, for example in the form of a nasal spray or aerosol for inhalation, or swallowed solution of, or for delivery of the by, for which purpose composition are manufactured in form of preparation in injectable form for delivery of for example intravenous, intramuscular or subcutaneous by. Alternatively, may be created preparation for introduction with means of and and other by.

If agent required deliver through mucosa, through gastro-intestinal mucosa, it should possess capable of keeping stability during transport through zheludochnokishechnyi tract; for example, it must be stable to proteolytic cleavage of, stable at acid ph and stable to impact detergents bile.

By circumstances, pharmaceutical compositions may be introduced by inhalation; in the form of a suppository or pessary; locally in the form of a lotion, solution, cream, ointment or sprinkling, use of skin plaster;

perorally in form of tablets, containing excipients, such as starch or lactose, or in capsules or ovulyakh, or some, or in mixture with excipients, or in the form of elixirs, solutions or suspensions, containing corrective or staining agents; or their can be be grouted parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral composition introduction best used in the form of sterile aqueous solution, which may contain other substances, for example sufficient amount of salts or monosaccharides, to make this solution isotonic with blood. For transbuccal or sublingual composition introduction may be introduced in the form of tablets or flat cakes, preparations which can be prepared in the usual way.

In some embodiments agents may also be used in combination with cyclodextrin. Is known, that cyclodextrins form inclusion complexes and complexes not with molecules of drug. Formation of complex drug-cyclodextrin can modify the solubility, dissolution rate, biological availability and/or stability of molecules of a medicinal agent. Complexes medicinal agent-cyclodextrin, as a rule, are useful for the majority of medicinal forms and ways of introduction. As alternative to direct complexing with medicinal agent cyclodextrin can be used as additional additive, for example as carrier, diluent or solubiliser. Alpha-, betaigammatsiklodekstriny is used happens, and suitable examples are described in WO -a- 91/11172, WO -a- 94/02518 and WO -a- 98/55148.

In preferable embodiments agents according to the present invention is delivered systemic (for example orally, transbukkalno, sublingually, more preferably orally.

Therefore agent preferably is in the form, which is suitable for oral delivery of.

For some ways to make your agent, when its is used, preferably not acts on the central nervous system.

For some ways to make your agent, when its is used, preferably acts enhanced efficiency by peripheral route.

Term "introduction" includes delivery with the help of viral and nevirusnykh techniques. Mechanisms of viral delivery include, but are not limited to, adenovirusnye vectors, adenoassotsiirovannye viral (AAV) vectors, gerpesvirusnye vectors, -transducing retroviral vectors, lentivirusnye vectors and proposes vectors. Mechanisms nevirusnoi delivery include transfection, mediated lipids, liposomes, immunoliposomy, lipofektin, cationic fascial amphiphiles (CFA) and their combinations.

Agents according to the present invention may be introduced alone, but, as a rule, their is introduced in the form of pharmaceutical composition-for example when agent is in mixture with suitable pharmaceutical excipient, diluent or carrier, selected with consideration of assigned path of introducing and standard pharmaceutical practice.

For example, agent may be introduced (for example orally or locally). in form of tablets, capsules, ovulei, elixirs, solutions or suspensions, which may contain corrective or staining agents, for applications immediate, delayed, modifitsiruemogo, long, pulse or controlled release.

Tablets can contain excipients, such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dikaltsiiortofosfat and glycine, leavens, such as starch (preferably corn, potato or manioca starch are taken as starch source), starch sodium glycolate, sodium-croscarmelose and some complex silicates, and binding for granulation of, such as polyvinylpyrrolidone, hydro ksipropilmetiltsellyuloza ( GPMTs ), hydro ksipropiltsellyuloza ( GPTs ), sucrose, gelatin and gum arabic. Besides, can be included lubricating agents, such as magnesium stearate, stearic acid, glitserilbegenat and talc.

Solid compositions like can be used as fillers in gelatin capsules. Preferable in this ratio of excipients include lactose, starch, cellulose, milk sugar or high molecular polyethylene glycols. For aqueous suspensions of and/or elixirs agent can be combined with different podslashchivayushchimi or flavor agents, coloring substance or dyes, with emulsifying and/or suspending agents and with diluents, such as water, ethanol, propylene glycol and glycerol, and their combinations.

Path of introducing (delivery) include, but are not limited to, one or more than one of the following: peroral (for example in the form of tablet, capsule or in the form swallowed solution of), local, through mucosa (for example in the form of a nasal spray or aerosol for inhalation), nasal, parenteral (for instance by means of injectable shape), gastro-intestinal, vnutripozvonochnyi, intraperitoneal, intramuscular, intravenous, vnutrimatochnyi, intraocular, intracutaneous, intracranial, vnutritrakhealnyi, intravaginalnyi, intratserebroventrikulyarnyi, intratserebralnyi, subcutaneous, oftalmicheskii (including vnutristeklovidnyi and vnutrikamernyi ), skin-through, rectal, transbukkalnyi, vaginal, an epidural, sublingual path.

Should have in form, that not entire agent necessary to introduce one and the same by. Also, if the composition contains more than one active component, then these components may be introduced different routes.

If agent according to the present invention is parenterally administered, then examples of such administration include one or more than one of the following: intravenous, intraarterial, intraperitoneal, vnutriobolochkovoe, intraventrikulyarnoe, intrauretralnoe, intrasternalnoe, intracranial, intramuscular or on agent; and/or using technique infusion.

For parenteral administration agent preferably used in the form of sterile aqueous solution, which may contain other substances, for example sufficient amount of salts or glucose, to make this solution isotonic with blood. Aqueous solutions should be properly zabuferit (preferably to ph from 3 to 9), if necessary. Preparation of suitable parenteral preparations in sterile conditions easily is standard pharmaceutical techniques, well known professionals in this area.

As indicated, agent according to the present invention can be administrated by intranasal route or inhaling, and conveniently deliver its in the form of dry powder inhaler or aerosol sprayed preparation from container under pressure, pump, a spray device or sprayer with the use of proper propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane synthesis, hydro ftoralkana, such as 1.1, 1, 2-tetrafluoroethane ( HFA 134a ™ ) or 1.1, 1, 2, 3, 3, 3-heptafluoropropane ( HFA227EA™ ), carbon dioxide or other suitable gas. In case of of aerosol under pressure unit dosage can be determined by means of valve for delivery of metered amount of. Container under pressure, pump, a sprayer or sprayer may contain solution or suspension of active compound, for instance using mixture of ethanol and propellant as solvent, which may additionally contain lubricating agent, for example trioleate sorbitol. In preparations capsules and cartridges (made, for example, gelatin) for use in inhaler or insufflator may be include powdered mixture agent and suitable powder base, such as lactose or starch.

Alternatively, agent can be introduced in the form of a suppository or pessary, or its can be used locally in the form of gel, hydro gel, lotion, solution, cream, ointment or sprinkling. Agent may be introduced also skin or percutaneously, for example by using skin patch. Can be to pulmonary or rectal by. Their may be introduced eye by. For oftalmicheskogo application preparations compounds can be prepared the form of micronised suspensions in isotonic sterile physiological solution with installed ph or, preferably, in the form of solutions in isotonic sterile physiological solution with installed ph, possibly in combination with preservative, such as benzilalkoniya chloride. Alternatively, they may be introduced in composition of an ointment, such as vaseline.

For applying locally on skin agent can be prepared in the form of suitable ointment, containing active compound, suspended or dissolved, for example, in mixture with one or more than one from the following:

mineral oil, liquid vaseline, white Vaseline, propylene glycol, polyoxyethylene- polioksipropilenovoe connection, high wax and water. Alternatively, it may be prepared in the form of suitable lotion or cream, suspendiruya or to dissolve, for example, in the mixture of one or more than one of the following: mineral oil, monostearate sorbitol, polyethylene glycol, liquid paraffin, polysorbate 60, wax from tsetilovykh esters, cetearyl alcohol, 2- oktildodekanol, benzyl alcohol and water.

Compositions according to this invention may be introduced with the help of direct injection.

For some applications agent is added preferably perorally.

For some applications agent is added preferably locally.

Usually doctor will determine actual dosing, most suitable for individual subject. Specific level of doses and frequency of dosage for any specific patient can variation, and will depend on range of factors, including the activity of specific used compound, metabolic stability and duration of action of compound, from age, body weight, total health state, floor, diet, type of and time of administration, rate of injection, combination of medicinal agents, gravity specific state and individual therapy. Agent and/or pharmaceutical composition according to the present invention may be introduced in accordance with the mode of from 1 to 10 times daily, for example once or twice a day.

For oral and parenteral administration patients-people daily level of dosage agent may be in a single dose or separate doses.

Depending on requirements agent may be introduced in a dose from 0.01 to 30 mg/kg of body weight, for example from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg of body weight. Naturally, mentioned here dosage are example of average case. Undoubtedly, may be individual cases, when will are preferable higher or more low ranges of dosage.

Agent can be prepared in the form of a pharmaceutical composition, for example by mixing with one or more than one suitable carrier, diluent or excipient on techniques, which are known in this engineering.

Below is presented several not limiting examples of preparations. Preparation 1: tablet is manufactured, using the following ingredients:

Mass/Mg

Agent 250

Microcrystalline cellulose, 400

Silicon dioxide, white soot 10

Stearic acid 5

The whole 665

These components are mixed and pressed for formation of tablets, each of which weighs 665 mg.

Preparation 2: intravenous preparation can be prepared as follows:

Agent 100 mg isotonic physiological solution 1000 ml

Agent can be introduced in the form of pharmaceutically acceptable salt. Usually pharmaceutically active salt of easily can be to produce, using desired acid or base, by circumstances. This salt can be deposited from solution and assemble by filtration, or one can discern its by evaporation of solvent.

In vivo model can be used for investigating and/or mining of therapy or therapeutic agents for treatment of RPVZh. These model can be used for investigating effect of various agents/driving compounds on a number of parameters, which indicate on reaction sexual of excitation. These animal control model may be used as analysis or analysis of according to the present invention. This animal control model will be not homine animal control model.

Contains there is a row of animals models of vasculogenic female sexual dysfunction, which can be used.

For example, can be reference to invasive animal model (for example cm. Park et al., 1997). Here vaginal and klitornuyu hemodynamic of reaction can be register directly after stimulation pelvic nerve in normal and atherosclerotic females rabbit. In vivo effects of agents, potentsiruyushchikh camp can be it possible to study or of normal animals, or in animals with RPVZh.

As one more example can be reference to non-invasive animal model (for example cm. observation Goldsteinet al., 1998; Laanet al., 1998):

Here pulse wave ultrasonic Doppler echography provides detection means blood circulation changes in vaginal and klitornoi arteries. This model can be used for investigating vaskulogennykh effects during pharmacological introduction vasodilatators.

Other non-invasive techniques, which can be used, include vaginal fotopletizmografiyu, which provides quantitative measurement of overfilling blood of vaginal mucosa, and procedures of vaginal thermal clearance, which are based on the principle, that change of vaginal blood flow can be register by measuring transfer from vnutrivlagalishchnogo of probe, supported at constant temperature.

In their research authors-developed healthy reproduced model physiology of sexual of excitation. In this model is used anestezirovannogo rabbit and is used laser Doppler technology for monitoring genital blood flow, simultaneously routinely recording cardio-vascular parameters. Authors of the invention are able to measure small changes in vaginal (and also klitornom ) blood stream, caused by stimulation of pelvic nerve or infusion vip in absence and in the presence of tested agents.

Authors invention is accepted as, that their animal model directly reflects the clinical data. Consequently, this model can be used for studying agents-candidates for treatment of RPVZh, for example measuring amplification of vaginal or klitornogo blood flow.

According to the present invention for measuring klitornogo and vaginal blood flow can be used different techniques. For example, can be used vaginal fotopletizmografiyu, technique of vaginal heat transfer, MRV (magnetic-resonance visualisation) with diffusion nnoi contrast clitoris and vaginal, pulse laser Doppler visualization of the clitoris/vulva and kpitornuyu ultrasonic ekhografiyu.

Quantitatively vaginal lubrication can also be measured according to techniques, known in this area, such as (and) weighing vaginal tampons before and after stimulation and (b) measurement of ph vaginal liquid sti. With respect to the latter aspect normal acid medium into vagina in rest becomes more alkaline, since it reaches ph blood, when during sexual stimulation occurs transsudatsiya liquid sti.

According to one aspect of the present invention additional target represents target Ptsdmf, while this target Ptsdmf is soboik nep.

Nucleotide sequences and amino acid sequences of nep are described in literature. Some sequences are presented in applied here lists sequences.

In one aspect, nep represents nep (EC 3.4.24.11) (known also as enkefalinaza or endopeptidaza -2). Now authors-find mrna nep EC 3.4.24.11 and expressed protein into vagina human and rabbit.

Here authors invention is accepted as, in women, including the, breast RPVZh, vip cleaved by nep EC 3.4.24.11. Consequently, inhibitors of nep should potentiate synthesis of endogenous vazorelaksiruyushchii effect vip, is in excitation time. This should lead to treatment of RPVZh, for example by means of amplification overfilling blood vagina. Authors of the invention shown, that selective inhibitors of nep EC 3.4.24.11 promote to VIPindutsirovannomu enhancement of genital (for example vaginal or klitornogo ) blood flow at stimulation of pelvic nerve. Besides, these selective inhibitors of nep amplified mediated vip and nerve relaxation isolated vaginal wall.

Preconditions by nep are presented Victor a. McKusik et al on htpp :/ / www3.ncbi.nlm.nih.gov / Omim / searchomim.htm. From this source extracted next information, considering nep:

" Antigen total acute lympholeukosis ( AOOLL ) is an important marker cell surface in diagnostics acute lympholeukosis in human (all). It is present on leukaemia cells of pre-phenotype, which represent 85% of cases of all. AOOLL, however, not is limited leikoznymi cells and is detected in some normal tissues. AOOLL represents glycoprotein, which especially is presented in kidneys, where it is present on brush kaemke proximal kidney tubules and on glomerulyarnom epithelium. Letarte et al. (1988) cloned cdna, encoding AOOLL, and shown, that amino acid sequence, brought on base cdna sequence of, is identical sequence of membrane-connected neutral endopeptidase human (nep: EC 3.4.24.11), known also as enkefalinaza. Nep splits peptides amino-side hydro fobnykh residues and inactivates some peptide hormones, including glucagon, enkefaliny, substance p, neirotenzin, oxytocin and bradykinin. With the help of transfektsionnogo analysis cdna Shipp et al. (1989) have confirmed, that AOOLL represents functional neutral endopeptidazu type, which previously was name enkefalinazoi. Barker et al. (1989) demonstrated, that gene AOOLL, which encodes 100 kd transmembrane glycoprotein type ii, exists in single copy more than 45 T. u. about., which is not subject to reconstruction in malignant tumours, expressing AOOLL cell surface. This gene localised on chromosome 3 human study of hybrids of somatic cells and in situ hybridizationraionirovan in position of 3q21-q27. Tran database option- Paterson et al. (1989) also strapped this gene to chromosome 3 at Southern-blot-analysis of dna from hybrids of somatic cells human and rodents. D' Adamio et al. (1989) demonstrated, that gene AOOLL propagates more than 80 T. u. about. and consists of 24 exons. "

And: nep

As indicated above, additional agent may be any suitable agent, which may act as and: nep. Additionally or alternatively agent according to the present invention may also act as and: nep.

Detail along suitable system for analyzing for identification and/or investigation and: nep are presented in the following partition.

AND. Nep discusses in the following windows articles:

Pathol. Biol., 46 (3), 1998,191.

Current Pharm. Design, 2 (5), 1996,443.

Biochem. Soc. trans., 21 (3), 1993,678.

Handbook Exp. Pharmacol., 104/1, 1993, 547.

Tips, 11, 1990, 245.

Pharmacol. Rev., 45 (1), 1993,87.

Curr. Opin. Inves. drugs, 2 (11), 1993,1175.

Antihypertens. Drugs, (1997), 113.

Chemtracts, (1997), 10 (11), 804.

ZincMetalloproteases health dis. (1996), 105.

Cardiovasc. Drug rev., (1996), 14 (2), 166.

Gen. Pharmacol., (1996), 27 (4), 581.

Cardiovasc. Drug rev., (1994), 12 (4), 271.

Clin. exp. Pharmacol. Physiol., (1995), 2291), 63.

Cardiovasc. Drug rev., (1991), 9 (3), 285.

Exp. Opin. Ther. Patents (1996), 6 (11), 1147.

And: nep are described in the following documents:

EP- 509442A

US-192435

US-4929641

ΕΡ - 599444Β

US-884664

ΕΡ - 544620Α

US-798684

J. med. Chem. 1993,3821. Circulation 1993. 88 (4). 1.

EP-136883

JP-85136554

US-4722810

Curr. Pharm. design, 1996, 2, 443. EP-640594

J. med. Chem. 1993,36 (1), 87.

EP- 738711 -A

JP-270957

CAS # 115406-23-0

DE-19510566

DE-19638020

EP-830863

JP-98101565

EP-733642

W09614293

JP-08245609

JP-96245609

WO9415908

JP05092948

WO-9309101

WO-9109840

EP-519738

EP-690070

Bioorg. med. Chem. Letts., 1996,6 (1), 65.

Preferable and: nep are described in the following documents:

EP-A- 0274234

JP-88165353

Biochem. Biophys. res. comm., 1989, 164. 58.

ΕΡ - 629627 -Α

US-77978

Perspect. med. Chem. 91993), 45.

ΕΡ - 358398 -Β

Preferred examples and: nep are selected from following structures:

More preferable and: nep are selected from following structures:

These compounds were produced on techniques, in experimental partition (cm. below). These compounds tested as agents and find, that they are suitable for potentiation of camp and, consequently, are useful for treatment of RPVZh. Some from experimental data, concerning these compounds, are presented in experimental partition (cm. below).

Soluble nep is produced from of cortical substance kidney and analyzed activity by measuring speed of splitting substrate nep Abz -d-arg-arg-leu- EDDnp with creation of its fluorescent product Abz -d-arg-arg.

1. MATERIALS

Entire water twice deionizirovana.

1.1. Tissue

Kidney human monument ( Pennsylvania. U.S.A. )

Kidney rat

Kidney rabbit

Kidney dog

1.2. Medium for homogenization

100 mm mannitol and 20 mm tris n ph 7.1

2.42 g tris ( Fisher t/ R630 /60) is diluted in 1 liter of water and is brought ph to 7.1, using 6m HCI at room temperature. To this is added 18.22 g mannitol ( Sigma m -9546).

1.3. Tris buffer (nep buffer)

50 ml of 50 mm tris ph 7.4 ( SigmaT2663 ) is diluted in 950 ml of water.

1.4. Substrate ( Abz -D-Arg-Arg-Leu- EDDnp )

Is obtained by order from SNPE and stored in the form of powder at -20 ° with. 2 mm initial solution is prepared soft resuspension substrate in tris buffer. Its should not be agitated on vortex or subjected to treatment.

600 mcl aliquots of this 2 mm initial solution stored at -20 ° with up to one month ( Medeiros, M.A. S., franca, M.S.F. et al., (1997) BrazilianJournal care medical and Biological research, 30, 1157-1162).

1.5. Total product

Samples, corresponding to 100% conversion of substrate in product, include plotting board, determination % conversion of substrate. Total product is formed at incubation 1 ml of 2 mm substrate with 20 mcl initial solution enzyme in for 24 hours at 37° with.

1.6. Feet-Solution

Is prepared 300 mcm initial solution fosforamidona ( Sigma R7385) in nep buffer and stored aliquots at 50 mcl at -20 ° with.

1.7. Dimethylsulfoxide (Dmso)

1.8. Magnesium chloride- MgCl2.6H20 ( FisherM0600 /53)

1.9. Black 96 well flat bottom analytical boards ( Costar 3915)

1.10. Topseal a ( Packard 6005185).

1.11. Centrifuge test tubes

2.1. Centrifuge Sorvall RC-5B (rotor SS34 GSA, preliminarily cooled to 4° with).

2.2. Mixer mini homogenizer Braun.

2.3. Centrifuge Beckman CS-6R.

2.4. Fluostar Galaxy.

2.5. Jointing incubator Wesbart 1589.

3. METHODS

3.1. PREPARING THE PREPARATION OF TISSUE

3.2. Nep dog, rat, rabbit and human is produced from of cortical substance kidney, using adapted method Booth, A.G. & Kenny, A.J. (1974) Biochem. j. 142, 575-581.

3.3. Frozen pochkam is allowed to thaw at room temperature, and korkovoe substance cut off from cerebral layer.

3.4. Korkovoe substance finely cut and homogenized approximately in 10 volumes of buffer for homogenization of (1.2), using mini homogenizer Braun (2.2).

3.5. To gomogenatu is added magnesium chloride (1.8) (20.3 mg/g of tissue) and stirred in ice bath for 15 minutes.

3.6. Homogenate is centrifuged at 1500 X g (3820 rev/min) for 12 minutes in centrifuge Beckman (2.3), after which the supernatant are transferred in pure centrifuge test tube, and precipitate discarded.

3.7. Supernatant centrifuged at 15000 x g (12100 rev/min) for 12 minutes in centrifuge Sorvall (2.1), and supernatant discarded.

3.8. Pale-pink layer on the surface of the remaining precipitate is removed and is resuspended in buffer for homogenization, containing magnesium chloride (9 mg MdS in 5 ml buffer on 1 g of tissue).

3.9. This suspension is centrifuged at 2200 x d (4630 rev/min) for 12 minutes in centrifuge Beckman (2.3), and the precipitate discarded.

3.10. Supernatant centrifuged at 15000 x g (12100 rev/min) for 12 minutes, using centrifuge Sorvall (2.1), and supernatant discarded.

3.11. Final residue is resuspended in buffer for homogenization, containing magnesium chloride (0.9 mg MgCl_{2 in} 0.5 ml buffer on 1 g of tissue). Homogeneous suspension is obtained, using mini homogenizer Braun (2.2). Then it is frozen in 100 mcl aliquots, to to analyze on activity nep.

Activity nep, preliminarily divided into aliquots, is measured by its ability to cleave specificity to nep peptide substrate.

4.1. Is prepared 4% solution dmso/nep buffer (4 ml dmso in 96 ml nep buffer).

4.2. Concentrated solutions substrate, total product, enzyme and fosforamidona is left on ice to thawing.

4.3. In each is added 50 mcl 4% solution of dmso/nep buffer.

4.4 . 2 mm concentrated solution of substrate is diluted 1:40 to obtain 50 mcm of solution. In each is added 100 mcl 50 mcm substrate (final concentration 25 mcm).

4.5. For initiation of reaction is added 50 mcl dilution series of enzyme (used is usually 1:100,1:200,1:400,1:800,1:1600 and 1:3200). In pure holes is added 50 mcl nep buffer.

4.6 . 2 mm total product is diluted 1:80 to produce 25 mcm of solution. In the first four holes new board is added 200 mcl 25 mcm product.

4.7. Boards are incubated at 37° with in shaking incubator for 60 minutes.

4.8 . 300 mcm initial solution fosforamidona diluted 1:1000 to 300 nm. Reaction is stopped addition of 100 mcl 300 nm fosforamidona and incubated at 37° with in shaking incubator for 20 minutes until red color on Fluorstar ( ℮χ320 / ℮γπ420 ).

5.1. Initial solutions substrate, total product, enzyme and fosforamidona is left on ice to thawing.

5.2. Is prepared initial solutions of compound in 100% dmso and diluted with 1:25 in nep buffer with produce 4% dmso of solution. All further dilution is performed in 4% dmso solution (4 ml dmso in 96 ml nep buffer).

5.3. In 96-alveolar plotting board is added 50 mcl compound in two repetitions and 50 mcl 4% dmso/nep buffer in check and pure holes.

5.4 . 2 mm initial solution substrate diluted 1:40 in nep buffer with produce 50 mcm solution of (275 mcl 2 mm substrate on 10.73 ml buffer is sufficient for 1 plate).

5.5. Initial solution of enzyme is diluted in nep buffer (is determined from checks on activity).

5.6 . 2 mm total product is diluted 1:80 in nep buffer with produce 25 mcm of solution. In the first four holes separate board is added 200 mcl.

5.7 . 300 mcm initial solution fosforamidona diluted 1:1000 to obtain 300 nm solution of (11 mcl fosforamidona on 10.99 ml nep buffer).

5.8. In each well of 96 - ; board is added the following:

5.9. Reaction is initiated addition of nep of enzyme, and then are incubated at 37° with for 1 hour in shaking incubator.

5.10. Reaction is stopped 100 mcl 300 nm fosforamidona and incubated at 37° with in shaking incubator for 20 minutes until red color on Fluorstar ( ℮χ320 / ℮γπ420 ).

6. CALCULATION OF

Activity nep of enzyme is determined in the presence and absence compounds and is expressed in percents.

Average fu kontrolen -average fu pure testing runs khYuO average fu summary-average fu pure testing runs

Average fu compound-average fu pure testing runs khYuO average fu summary-average fu pure testing runs

Control activity in %

Sigmoid curve dose-response corresponds to % activities (%) against concentration of compound, and value of IC50 is calculated, using LabStats curves of correspondence in Excel.

Pde ( FosFodiesteraza1

According to the present invention target represents Ptsamf target, while this Ptsamf target represents pde (phosphodiesterase), which represents the pde, hydro lizuyushchuyu camp (and possibly hydro lizuyushchuyu cgmp).

Is known, that cyclic nucleotides, such as camp and cgmp, are important intracellular secondary middlemen. Phosphodiesterase cyclic nucleotides-otherwise known as pde represent family of enzymes, which catalyze cleavage of cyclic nucleotides and are one of cellular components, which are adjusted concentration of cyclic nucleotides.

In the last years at least seven pde enzymes (such as FDE1 -pde \ / 11), and also multiple subtypes of these enzymes, were determined based on substrate affinity and kofaktorakh ( Beavo ja and ReifsnyderDH, TrendsPharmacol. Sci. 11:150; Beavo j in:

cyclic NucleotidePhosphodiesterases : structure, regulation and Drug action, Beavo j and Housley md (concentrate. ) Wiley : Chichester, ρρ. 3-15).

Examples pde include: FDE1, which represents ca^{2 + /}kalmodulin -dependent pde; FDEP, which represents the camp and cgmp stimulated by pde; FDESh, which represents cgmp ingibiruemuyu pde; FDE1 \ /, which is highly affine camp-specific pde; and pde \ /, which represents tsGMFspetsifichnuyu pde. FDE1 and T. d. sometimes called pde type i and T. d. or pde type 1 and T. d.

Each family of pde can contain two or more than two isoforms (i.e. can existing two or more than two pde isozyme). For example, is known, that pde iv mammals, homologue gene DunceDrosophila ( chen cn et ai, proc. Natl. Acad. Sci. (USA) 83:9313) has four isoforms in rat ( SwinnenJVet a/., proc. Natl. Acad. Sci. (USA) 86:5325). Is known also, that human pde match in form of isoforms and have versions splicing. For example, about cloning one human isoforms pde iv of monocytes may impart to in 1990 ( Livi gp et ai, Mol. cell. BIO ., 10:2678). As one more example, other researchers have independently cloned three versions of splicing pde iv, which at the present time are designated by hPDEIV -b 1, hPDEIV -b 2 and hPDEIV -b 3.

Information about fosfodiesterazakh cyclic nucleotides may also be find in us-a- 5932423 and us-a- 5932465.

Information on one more phosphodiesterase-cyclic nucleotides, in other words cn PCDE8, can be find in WO -a- 97/35989. Cn PCDE8 has two isozyme, which were marked as cn PCDE8A and cn PCDE8. On term "isozyme" in this engineering sometimes present as on "isoform".

According to WO -a- 97/35989 identified numerous inhibitors of various pde, and some proposed clinical evaluation. For example, inhibitors of FDE1I is developed as protivotromboticheskikh agents, as antihypertensive agents and as kardiotonicheskikh agents, in treating congestive heart failure. Rolipram, inhibitor of FDE1P, has been used in treatment of depression, and other inhibitors of FDE1I proposed clinical estimation of as anti-inflammatory agents. Was shown also, that rolipram inhibits induced by lipopolysaccharides (lps) tnf-alpha, which, as was shown, promotes replication hiv -1 in vitro. Consequently, rolipram can to inhibit replication hiv -1 (angel et al. 1985AIDS 9:1137-44). Besides, on the basis of its ability to suppress production of FIO alpha and beta, and also interferon gamma, is indicated efficiency roliprama in treatment of encephalomyelitis, experimental animal model of multiple sclerosis ( Sommeret al., 1995 nat med 1:244-248) and probable efficiency in treatment of late dyskinesia ( Sasakietal., 1995 Eur j Pharmacol 282:71-76).

According to WO -a- 97/35989 exists also multiple nonspecific inhibitors of pde, such as theophylline, used in treatment of bronchial asthma and other respiratory diseases, and Pentoxifillin, used in treatment of intermittent lameness and induced diabetes mellitus of peripheral vascular disease. Is considered, that theophylline acts on function of smooth musculature respiratory tracts, and also in anti-inflammatory and immunomodulyatornoi functional activity in treatment of respiratory diseases (banner et al., 1995Respir j 8:996-1000), where is considered, that it acts by means of inhibition of as hydro lipolysis camp cn pde, and hydro lipolysis cgmp (banner et al., 1995Monaldi arch Chest dis 50:286-292). Pentoxifillin, on which also is known, that it blocks the production of tnf-alpha, may to inhibit replication hiv -1 (angel et al, cm. above). List of inhibitors of cn pde driven into Beavo 1995, cm. above.

Instead, that selective inhibitors of pde in addition to their izozimam and subrange will lead to more effective therapy with less adverse effects. For example, cm. reviews Wieshaar re et al ( j.

Med. Chem., 28:537), GiembyczΜΑ ( Biochem. Pharm., 43:2041) and Lowe ja and ChengJB (drugs of the future, 17:799-807).

So, for some applications preferably have selective inhibition of individual type pde.

Preconditions by pde are presented Victor a. McKusik et al on htpp :/ / www3.ncbi.nlm.nih.gov / Omim / searchomim.htm. From this source extracted next information, considering FDEI or cgmp-stimulating pde:

" Cyclic nucleotides are used as secondary mediator, which mediate row of cellular responses to extracellular signals, such as hormones, light and neuromediators. Phosphodiesterase cyclic nucleotides (pde) play role in signal transduction by means of regulation of cellular concentration of cyclic nucleotides. Mammalian cells contain multiple pde, which are divided by at least on 7 of families on the basis of their substrate affinity and their selective sensitivity to kofaktoram and an inhibitor medicinal agents. These of family represent : (I) ca^{2 + /}kalmodulinzavisimye pde ; (II) cgmp- stimuliruemye pde ; (III) cgmp- ingibiruemye pde;

(IV) camp-specific pde ; (V) cgmp-specific pde ; (VI) fotoretseptornye pde; and (VII) highly affine camp-specific. From amino acid sequences of is seen, that all these of family of pde contain sibling domain, which is considered to be the catalytic domain, approximately with 30% Identity sequence of between groups. Lets one and the same of family have more close cousinage; they is separated from 60 to 80% identity of sequence on the whole coding region.

Michael et al. (1993) developed highly sensitive functional screening for isolating " dna, encoding camp phosphodiesterase, by means of those parishes defects in strain saccharomyces cerevisiae, in which there are both endogenous gene camp pde, FDE1 and FDE2. Three groups of cdna, corresponding 3 separate human diagnosed, encoding camp-specific pde, were separated from cdna library glioblastoma human with use of this functional screening. Two of these genes have close cousinage with tsAMFspetsifichnoi pde " dunce" Drosophila. Third gene, on which Michael et al. (1993) alluded as on NSR1, kodiroval new camp-specific pde. NSR1 its amino acid sequence, -sequences of catalytic domains of all pde cyclic nucleotides. This pde represents highly affine camp-specific pde, which, however, not divides other properties tsAMFspetsifichnogo of family of pde. Pde activity NSR1 has not been is sensitive to cgmp or other inhibitors cgmp- ingibiruemykh pde. Biochemical and pharmacological properties of NSR1, which is member not earlier open of family of pde cyclic nucleotides, which is marked as family of vii. suggested Michael et al. (1993). Nozern -blot-analysis indicated on the presence of high levels of NSR1RNK in skeletal muskulature human.

With the help of Southern-blot-analysis of somatic hybrid cell lines Milatovich et al. (1994) kartirovali locus NSR1 in chromosome 8 ; by studying hybrid somatic cell lines, which contained different zones chromosome 8, they understand referencing gene, 8ς13 - ς22. Han et al. (1998) kartirovali gene PDE7A in 8q13 with the aid of fluorescent in situ hybridization. With the help of mezhvidovogo analysis of return crossbreeding they kartirovalimyshinyi gene Pde7A in proximal the region of chromosome 3. "

Preconditions by FDE2 are presented Jennifer p. Macke et al on

htpp :/ / www3.ncbi.nlm.nih.gov / Omim / searchomim.htm. From this source

extracted next information, considering cgmp-stimulating FDE2 :

" Rosman et al. (1997) cloned kDIK, corresponding to human FDE2A. This gene FDE2A encodes polypeptide from 941 amino acids with prediction molecular mass 106 kd. Protein sequence on 90% is identical to sequences of bovine and rat FDE2A. Nozern - blotanaliz proved, that FDE2A expressed in the form of 4.2 T. u. about. mrna at doorstep ranging levels in all Tested human tissue with the high expression in brain. Of investigating expression revealed, that FDE2A hydro lizuet camp and cgmp and inhibited FDE2A -specific inhibitor of EHNA. "

Nucleotide sequences and amino acid sequence for pde are described in literature. Some sequences are presented in given here lists sequences.

In one aspect, pde target is selected from one or more than one of the following pde enzymes: FDEtsamf1, FDEtsamf2, FDEtsamfZ, FDEtsamf4, FDEtsdmf7 and FDEtsamf8.

In more preferable aspect, pde target is selected from one or more than one of the following pde enzymes: FDEtsamf1, FDEtsdmf2, FDEtsamfZ and FDEtsamf4.

Preferably for the present invention pde for target is at least FDE2.

And: pde

As indicated above, additional agent may be any suitable agent, which may act as and: pde. In addition or as an, agent according to the present invention may also act as and: pde.

Examples and: pde are disclosed in ep-a- 091133 and ep-a- 07711799.

Preferably and: pde represents and: FDE2. Consequently, preferable examples of compounds are those, which are presented in er-a- 0771799.

For convenience of authors of the invention in this moment is repeated station 1 ep-a- 0771799 :

Derivative of purine-6-one with common formula (I):

where:

R¹

R²

R^{3 and r4}

A

R^{6 and r7}

R^{3 and/or} r⁴

R^{3 and/or} r^{4 in}

Τ represents hydrogen, or normal or branched alkyl, containing up to 8 carbon atoms;

represents a normal or branched acyl, containing up to 4 carbon atoms, or normal or branched alkyl, containing up to 8 carbon atoms, possibly substituted hydro ksilom, azido or group of formula-nr^{3 r4 or-}0S0_{2 r}^5, where

are identical or different and represent, cycloalkyl, containing from 3 to b carbon atoms, hydrogen, formyl, or normal or branched alkyl, containing up to 6 carbon atoms, possibly substituted normal or branched alkoxy or alkoxycarbonyl, respectively containing up to 6 carbon atoms, or a group of formula-(CO)_a- nr^{6 r7,} where represents a number of 0 or 1;

are identical or different and represent hydrogen, formyl, hydro ksil, phenyl, or normal or branched alkyl, containing up to 6 carbon atoms, possibly substituted hydro ksilom, or normal or branched alkoxy, containing up to 5 carbon atoms;

or

represent normal or branched alkoxycarbonyl, containing up to 6 carbon atoms, carboxyl, or normal or branched acyl, containing up to 6 carbon atoms, possibly substituted hydro ksilom, or normal or branched alkoxy, containing up to 4 carbon atoms; or

represent residue of formula-(CO)_b-t-nr^{8 r9,}-co-r^10,

-S0_{2 r}^{11 or-} s0_{2 nr}^{12 r13,} where

has, this higher for and, and is identical to it or different from it;

can represent normal or branched r^{8 and r9 r10}

R^{11 r12 and r13}

R^{3 and r4}

R^{14 r5}

A

alkyl, containing up to 5 carbon atoms, or, when b about, may also represent communication;

have the values, given higher for r^{6 and r7,} and are identical them or differing from them;

represents saturated, partially unsaturated or unsaturated 5-7-members heterocycle, containing up to 3 heteroatoms, selected from s, n and/or about, which may be possibly substituted n function normal or branched alkyl, alkoxy or alkoxycarbonyl, containing up to 4 carbon atoms, carboxyl, benziloksikarbonilom or hydro ksilom ;

represents a normal or branched alkyl, containing up to 5 carbon atoms, benzyl or phenyl;

are identical or different and represent hydrogen, phenyl, or normal or branched alkyl, containing up to 6 carbon atoms; or

together with nitrogen atom form 5ili 6-membered saturated, partially unsaturated or unsaturated heterocycle, which may contain up to 3 heteroatoms, selected from s, n and/or about or residue-nr^14, and which possibly substituted carbonyl, normal or branched alkoxycarbonyl, containing up to 5 carbon atoms, or normal or branched alkyl, containing up to 5 carbon atoms, which in its turn may be substituted hydro ksilom, carboxy or normal or branched acyl, alkoxy or alkoxycarbonyl, respectively containing up to 6 carbon atoms; where

represents hydrogen, carbonyl, or normal or branched alkyl or alkoxycarbonyl, respectively containing up to 5 carbon atoms; and

represents phenyl, or normal or branched alkyl, containing up to 5 carbon atoms;

represents a normal or branched alkylene or alkenylene chain, respectively containing up to 6 carbon atoms;

are identical or different and represent aryl, containing from 6 to 10 carbon atoms, or 5-7-membered aromatic, possibly benzokondensirovannyi heterocycle, containing up to 3 heteroatoms, selected from s, n and/or about, possibly substituted up to 3 times, identically or-differently, halogen, hydro ksilom, nitro, trifluoromethyl, carboxy, normal or branched alkyl, alkoxy or alkoxycarbonyl, respectively containing up to 6 carbon atoms, or group of formula-(V)_c- nr^{15 r16 and/or}-or^{17 ;}

where

represents a number of 0 or 1;

represents a residue of formula-with or-so2;

are identical or different and have value, given for r^{3 and r4 above};

represents hydrogen, normal or branched alkenyl, containing up to 8 carbon atoms, or normal or branched alkyl, containing up to 8 carbon atoms, possibly substituted up to 3 times, identically or-differently, hydro ksilom, carbonyl, or normal or branched alkoxycarbonyl, containing up to 5 carbon atoms; and/or cycles possibly are substituted by aryl, containing from 6 to 10 carbon atoms, or 5-7- chlennym aromatic, possibly benzokondensirovannym heterocycle, containing up to 3 heteroatoms, selected from s, n and/or about, which in its turn possibly substituted up to two times, identically or-differently, halogen, hydro ksilom, nitro, carboxyl, trifluoromethyl, or normal or branched alkyl, alkoxy or alkoxycarbonyl, respectively containing up to 5 carbon atoms, or group of formula-(v ') crNR^{1s r19 ;} where

has, this higher for and, and is identical r^{18 and r19}

V'

R^{20 and r21}

Ε

Υ

ζ

D

to it or different from it;

have the values, given higher for r^{3 and r4,} and are identical them or differing from them;

has, this higher for v, and is identical to it or different from it; and/or

represents a ring system, this below for d, possibly substituted normal or branched acyl, containing up to 5 carbon atoms, possibly substituted hydro ksilom, normal or branched alkoxy, containing up to 5 carbon atoms, or group of formula-nr^{20 r21 ;} where

are identical or different and have the values, data higher for r^{3 and r4 ;} or

represents a residue of formula-ch_2-y- z-; where

represents a bond or oxygen or sulfur atom, or group-nh-;

represents a normal or branched alkyl chain, containing up to 5 carbon atoms;

represents a residue of the formula

ABOUT

\ \ / G "⁰

and its tautomers and salt.

Preferable and: pde are selected from following structures:

According to one aspect of the present invention additional target represents Ptsamf target, while this Ptsamf target represents npy or one of binding with it receptors.

Nucleotide sequences and amino acid sequence for npy and its receptors are described in literature. Some sequences are presented in given here lists sequences.

Now authors-find, that neuropeptide Υ (NPY) shows inhibitory control effect on mediated vasoactive intestinalnym peptide (VIP) vazorelaksatsiyu. Consequently, inhibition of npy receptor will lead as a result of to diffusion nnomu mediated pelvic nerve and vip-mediated enhancement of genital (for example of vaginal and klitornogo ) blood flow. Clinically this should lead to enhancement of overfilling blood of vagina and/or the clitoris, which results in the final analysis enhancement of lubrication by means of transudation plasma and diffusion Exploration vaginal compliance. Consequently, suitable target for treatment of RPVZh is npy or one of binding with it receptors.

So, in preferable aspect, additional target represents npy Υΐ Y2 or y_{5 antagonist}, preferably peroral npy y_{1 Y2} or y_{5 antagonist}. This agent will treat RPVZh by enhancement of genital (for example vaginal or klitornogo ) blood flow and amplification lubrication.

Npy-mediated antagonism vip-induced amplification of blood flow, consequently, represents a potential therapeutic target, by means of which can be to effect on circulation in female genital tracts. Mechanism, along which arises this antagonism, most probable through npy Υΐ receptor- indutsirovannuyuGj /_{0 activation}. Shown, in other physiological systems receptors npy Y-i involves in mediation vasoconstriction and in inhibition of release of sympathetic of mediator ( Lundberget and! ., 1996; effect npy Y2 because avoiding). Authors of the invention is considered, in female genital tracts npy inhibits vazorelaksatsiyu through direct inhibition of directly ingibiruemogoadenilattsiklazoi release of VIR or sympathetic neurotransmission.

As indicated, additional Ptsdmf target represents one of the npy receptor.

Neironalnoe release of npy regulates vip- indutsirovannuyuvazorelaksatsiyu vaginal vascular bed. Most probable it occurs by means of presinapticheskogo mechanism, in which receptors npy Y-i, at because avoiding postsinapticheskii type of action. Npy antagonist will potentiate vip- indutsirovannuyuvazorelaksatsiyu vaginal vascular bed. Clinically this will lead to diffusion Exploration for lubrication and pliability of vagina by overfilling blood vaginal wall.

In taken investigations of receptor expression npy authors-identified npy Υΐ Y2 and y_{5 subtypes} receptor within human vagina.

Consequently, in one aspect, additional Ptsdmf target represents one or more than one from npy Y1 Y2 and Y5 receptor subtypes.

Preconditions by npy and binding with it receptors are presented Victor a. McKuzik et al. on htpp :/ / www3.ncbi.nlm.nih.gov / Omim / searchomim.htm. From this source extracted the following text, touching npy.

" Neuropeptide Υ (NPY) represents peptide, which is in abundance and widely is spread in nervous system of mammals. It has homology of sequence with peptide yy and more than 50% homology amino acid and nucleotide sequence with pancreatic polypeptide ( PNP ; 167780). Npy represents a 36-amino acid peptide. Minth et al. (1984) cloned gene npy proceeding from mrna pheochromocytoma. Takeuchi et al. (1985,1986) earmarked cdna clones of genes npy and PNP from pheochromocytoma and endocrine tumor pancreas, respectively. Using these cdna probes for analysis of genomic dna of panels of chromosomes of somatic cell hybrids chelovekmysh, they then investigated question, whether these genes sintenichnymi. These of investigating shown nesintenichnost, at that npy is on 7pter - 7q22, a PNP on 17p11.1-17qter. With the help of research return crossing with mus spretusBahary et al. (1991) kartirovali a gene npy on fusing murine chromosome 6. Since mouse chromosome 6 has homology with human 7q, probable, that gene npy in human localised in the area of 7cen-q22. Meisler et al. (1987) excluded close adhesion between loci for mucoviscidosis (219700) and neuropeptide Υ. Terenghi et al. (1987) identified distribution of mrna, encoding npy, in neurons cerebral cortex in preparations surgical biopsies and posthumous brain with the help of techniques of in situ hybridization. They can show loaded localization of transcription and gene expression npy in normal mature cortical neurons. Baker et al. (1995) with the aid of fluorescent in situ hybridization of shown, that gene npy localised on 7ρ 15.1 and exists in single copy. They expressed opinion, that npy is one of the most highly conservative known peptides, for instance with only 3 amino acid differences between by one shark. Neuropeptide y represents neiromodulyator, involves to regulation of energy balance, and its sverkhprodutsirovanie occurs in hypothalamus in mice ob/ob. To determine as npy in answering deficiency of leptin (164160), Erickson et al. (1996) constructed mice ob/ob with insufficiency npy. In the absence of npy of mouse ob/ob less than suffered obesity in connection with smaller assimilation food and diffusion nnym consumption of energy, and were less subjected mellitus, sterility and somatotropnym defects. These results interpreted as indication on then, that npy is central effektorom insufficiency of leptin. Analysis of genetic of clutch in rats, which selection demonstrated relative to alcoholic preference, identified chromosomal region, which included a gene npy ( Carr et al., 1998). Rat, preferring alcohol, have low levels of npy in several sections of brain compared with rats, not preferring alcohol. Therefore Thiele et al. (1998) investigated consumption of alcohol mice, in which completely absent npy as a result of directed interruption of gene ( Erickson et al., 1996). They find, that npy-scarce of mouse show diffusion nnoe compared with mice wild type consumption of solutions, containing 6%, 10% and 20% (vol./ rev. ) ethanol. Npy-scarce of mouse were also less than are to sedative/hypnotic effects of ethanol, that maintains more rapid waking up from induced ethanol sleep, even despite, that ethanol concentration in plasma fields do not differ considerably from those in controls. In contrast to this transgenic mouse with by overexpression of labeled gene npy in neurons, in which it usually is expressed, possessed low preference ethanol and were more are to sedative/hypnotic effects of ethanol, than examinations. These data give direct the, that consumption of alcohol and resistance are in reverse depending on levels of npy in brain. As part of ongoing genetic base of obesity Karvonen et al. (1998) identified 1128T -with polymorphysm, which led to substitution of leucine for proline in the remnant the 7 signal peptide section πρ℮ - προ - ΝΡΥ. This polymorphism was not is connected with obesity or energy exchange, but was considerably and connected in series with high whey total and LNP (lipoproteins low density) levels of cholesterol as in Finns with normal weight and with obesity, and in subjects, -Danes with obesity. Uusitupa et al. (1998) find polymorphysm rgo7 in 14% Finns, but only in 6% Danes. Subjects with rgo7 in npy have in average 0.6-1.4 mmole/l more high levels of total serum cholesterol content, than subjects without this gene variant. Since action pro7 npy for levels of serum cholesterol content can be detected via in Danes with normal weight, can be the, that people with obesity can be more are to effect this gene variant. Was calculated, that probability of possession rgo7 in npy can be 50% to 60% in subjects, with obesity at total syvorotochnomkholesterine, equal or above 8 mmol/l. At least among Finns rgo7 mould npy is one of strongest genetic factors, influencing on levels of serum cholesterol content. Cm. also allen and bloom (1986); Dockray (1986); Maccarrone and Jarrott (1986); Minth et al. (1986). "

As indicated, preconditions no npy and binding with it receptors are presented Victor a. McKuzik et al (there). From this source extracted the following text, touching NPYR1.

" Neuropeptide Υ (npy; 162640) is one of the abundant neiropeptidov in nervous system of mammals and demonstrates varied range of important physiological activities of, including action on psikhomotornuyu activity, assimilation of food, regulation central endocrine secretion and strong vasoactive action on cardio-vascular system. Two main subtype npy (Y1 and Y2) are determined by with pharmacological criteria. Receptors npy Y1 identified in a number of tissues, including brain, spleen, small intestine, kidney, testicles, placenta and smooth muscles aorta. Y2 receptor is detected mainly in central nervous system. Herzog et al. (1992) reported about cloning cdna, encoding human npy receptor, which, as they proved, is member subfamily receptors, associated with g protein. At expression in cells of chinese hamster ovary (cho) or embryonic kidneys human this receptor demonstrated typical ligandnuyu specificity. In cell line kidneys this receptor contacted with sensitive to pertussis toxin g protein, which is as a proxy inhibition of accumulation of cyclic amf. In cell line cho, with the other side, this receptor was is connected not with inhibition of adenylate cyclase, and rather with diffusion savings intracellular calcium. So, secondary the intermediary of, binding to npy receptor, was specific to cell type depending on particular composition g of proteins and effector systems, present in the cellular type of. Larhammar et al. (1992) independently cloned and described receptor of neuropeptide Υ. Herzog et al. (1993) identified molecular organization and regulation of human gene receptor npy Y1. They find, that, in contrast to continuous structure of the majority of genes receptors, binding with g protein, gene receptor npy Y1 has 3 an exon of the. They also identified usual Pstl polymorphysm in the first introne of this gene. By fluorescent in situ hybridization of high resolution they lokalizovali this gene in 4q31.3- q32. Herzog et al. (1997) find, that genes NPY1R and NPY5R (602001) together are localised on chromosome 4q31-q32. These 2 gene transkribiruyutsya in opposite directions with total of the promoter region. One of the exons of alternative splicing 5-prim gene Y1 receptor is section encoding sequence Y5 receptor. This unusual organization enabled Herzog et al. (1997) call, that these 2 gene formed in a result of event duplication gene, and that their expression may be coordinated. With the help of mezhvidovogo analysis of return crossing Lutz et al. (1997) kartirovali genes Npylr and Npy2r in conservative groups of adhesion at chromosomes of mouse 8 and 3, respectively, which corresponds to distal district human chromosome 4q. "

As indicated, preconditions by npy and binding with it receptors are presented Victor a. McKuzik et al (there). From this source extracted the following text, touching NPYR2.

" Neuropeptide Υ (NPY) performs signal through family of receptors, binding with g protein, present in encephalon and sympathetic neurons. At least 3 type receptors neuropeptide y determined based on pharmacological criteria, tissue distribution and structure from the encoding gene (cm. 162641 and 162643). Rose et al. (1995) reported about expression cloning in cos cells cdna of human receptor type 2, NPY2R. Transfected cells show high affinity to npy (16240), peptide ΥΥ (pyy; 600781) and fragment npy, comprising amino acids with 13 at 36. Predicted 381-amino acid peptide has 7 transmembrane domains, characteristic for receptors, binding with g protein, and only on 31% identical to human Y1 receptor (NPY1R; 162641). On Nozern - blotakh tissue samples of several sections nervous system find 4 T. u. about. mrna. Gerald et al. (1995) cloned cdna, corresponding to human Y2 receptor from cdna expressing library hippocampus human, using binding analysis radioactively marked of pyy. They expressed gene Y2 in COS-7 cells and exercised binding analysis hormone, which proved, that Y2 receptor binds (from the highest to the lowest affinity) hormones of pyy, npy and pancreatic polypeptide (PP; 167780). Ammar et al. (1996) cloned and described human gene, encoding receptor npy type 2. Transcript occupies 9 T. u. about. genome sequence and is encoded in 2 ekzonakh. Since in gene npy receptor type 1 5-prim netransliruyushchii area of NPY2R interrupted 4.5 T. u. about. intermediate sequence. Ammar et al. (1996) with the help of Southern-analysis of cell hybrids rodent-man with subsequent fluorescent in situ hybridization (FISH) demonstrated, that gene NPY2R mapped in 4q31, the same area, which contains gene NPY1R, assuming, that these subtypes could form by as a result of duplication gene, despite their structural difference. With the help of mezhvidovogo analysis of return crossing Lutz et al. (1997) kartirovali genes Npylr and Npy2r in conservative groups of adhesion at chromosomes of mouse 8 and 3, respectively, which corresponds to distal district human chromosome 4q. "

Analysis for determining, whether supposed or actual agent to bind with npy, is presented in WO -a- 98/52890 (cm. . 96, line 2-28 description-).

Η : ΝΡΥ

As indicated above, additional agent may be any suitable agent, which may act as Η: ΝΡΥ (sometimes present as on antagonist npy). Additionally or alternatively, agent according to the present invention can also act as Η: ΝΡΥ.

Η: ΝΡΥ (in particular, antagonists npy) discusses in the following windows articles:

Dunlop j, Rosenzweig - Lipson s: Therapeuticapproaches to obesity exp OpinTherPat 1999 8 12 1683-1694;

Wang s, Ferguson ks, Burris topt, Dhurandhar nv:8^{th annual} international conference on obesity and non- insulindependentdiabetesmellitus : noveldrugdevelopments. Exp OpinInvest drugs 1999 8 7 1117-1125;

Ling al: NeuropeptideΥreceptorantagonists exp OpinTherPat 1999 9 4,375-384;

Adham Ν, Tamm j, du Ρ, Hou c et al: identification care residuesinvolved in the binding care the antagonist snap 6608 to the Υ5receptorSocNeurosciAbstr 1998 24 part 2 626.9;

ShuΥΖ, CutroneJQ, Klohr se, Huang s: BMS-192548, a tetracyclicbindinginhibitor care neuropeptideΥreceptors, from subclause aspergillus niger WB2346. II. Physicochemical properties and structural characterization j Antibiot 1995 48 10 1060-1065;

Rigollier Ρ, Rueger Η, Whitebread s, YamaguchiΥ, Chiesi Μ, Schilling w, Criscione l: synthesis and sar care CGP71683Α, a potent and selectiveantagonist care neuropeptideΥΥ5receptor. Int Symp med Chem 1998 15^{th Edinburgh} 239;

Criscione l, Rigollier Ρ, Batzi - Hartmann c, Rueger Η, Stricker - Krongard a et al: food uptake in free- feeding and energy- deprivedleanrats is mediated by the neuropeptideΥ5receptor. J ClinInvest 1998 102 12 2136-2145;

NeurogenCorp : NGD 95-1 ClinTrials monitor 1996 5 10 ab 19244;

Buttle la: anti-obesity drugs: on target for huge market sales. Exp OpinInvest drugs 1996 5 12 1583-1587;

Gehlert dr, Hipskind Pa: NeuropeptideΥreceptorantagonists in obesity. Exp OpinInvest drugs 1996 7 9 1827-1838;

Goldstein dj, Trautmann me: treatments for obesity. Emerging drugs 1997 2-1-27;

Hipskind Pa, Lobb kl, Nixon ja, Britton tc, Bruns rf, Catlow j, DieckmanMcGinty dk, Gackenheimer sl, Gitter bd, Iyengar s, Schober da et al.: Potent and selective 1.2,3- trisubstitutedindoleΝΡΥ Y-1 antagonists. J med Chem 1997 40 3712-3714;

Zimmerman Dm, Cantral Be, Smith Ecr, Nixon Ja, Bruns Rf, Gitter Bd, Hipskind Pa, Ornstein Pl, Zarrinmayeh Η, Britton Tc, Schober Da, Gehlert Dr:

Structure-activity relationships, a series of 1-substituted-4- methylbenzimidazoleneuropeptide Y-1 receptorantagonistsBioorganic med ChemLett 1998 8 5,473-476;

Zarrinmayeh Η, Nunes a, Ornstein pl, Zimmerman d, Arnold MB et al.:

Synthesis and evaluation care a series care novel 2 - [(4-chlorophenoxy) methyl ] benzimidazoles as selectiveneuropeptideΥΥ1receptorantagonists. J med Chem 1998 41 2709-2719;

Britton tc, Spinazze pg, Hipskind Pa, Zimmerman dm, Zarrinmayeh Η, Schober da, Gehlert dr, Bruns rf: structure-activity relationships, a series of benzothiophene -derived ΝΡΥ - Υ 1 antagonists : optimization of the C2 side chain Bioorganic med ChemLett 1999 9 3,475-480;

Zarrinmayeh Η, Zimmerman dm, Cantrell be, Schober da, Bruns rf, Gackenheimer sl, Ornstein pl, Hipskind Pa, Britton tc, Gehlert dr: structure-activity relationship care a series care diaminoalkyl substituted benzimidazole as neuropeptideΥΥ1receptorantagonistsBioorganic med ChemLett 1999 9 5,647-652;

MurakamiΥ, Naga Η, Okada Τ, Hashizume Η, Kii Μ, IshiharaΥ, Ishikawa Μ, Mihara S-l, Kato g, Hanasaki Κ, Hagishita s, Fujimoto Μ: 1.3- disubstitutedbenzazepines as novel, potent, selectiveneuropeptideΥΥ1receptorantagonists j med Chem 1999 42 14 2621-2632;

Rudolf Κ, Eberlein w, Engel w, Wieland ha, Willim kd, Entzerorh Μ, Wienen w, BeckSickinger ag, DoodsΗΝ : the first highlypotent and selective non- peptideneuropeptideΥΥ1receptorantagonist : BIBP3226 eur j Pharmacol 1994 271 2-3 R11-R13;

Wieland ha, Willim kd, Entzerorh Μ, Wienen w, Rudolf Κ, Eberlein w, Engel w, DoodsΗΝ : subtype page selectivity and antagonist profilescript care the non- peptideneuropeptideΥΥ1receptorantagonist : BIBP3226 j Pharmacol exp Ther 1995,275 1,143-149;

Wright j, Bolton g, Creswell Μ, Downing d, Georgic l, Hefner Τ, Hodges j, McKenzie r, Wise l:8- amino -6 - ( arylsulphonil ) - 5- nitroquinolines : novel non- peptideneuropeptideΥΥ1receptorantagonistsBioorganic med ChemLett 1996 6 15 1809-1814;

Capurro d, Huidobro - ToroJP : the involvement care neuropeptideΥΥ1receptors in the Blood pressure baroreflex : studies with BIBP 3226 and BIB 3304. Eur j Pharmacol 1999,376 3,251-255;

DumontΥ, Cadieux a, Doods Η, Quiron r: new tools to investigateneuropeptideΥreceptors in the central and peripheral nervous systems: BIBO -3304 ( Υ1 ), BIIE -246 ( Υ2 ) and-gr- 231118 ( Υ1 / Υ4 ). SocNeurosciAbstr 1999 25 part 1 abs 74,11;

Hegde ss, BonhausDW, Stanley w, Eglen rm, MoyΤΜ, Loeb Μ et al:

Pharmacologicalevaluation care 12291) 91, a high affinity and selectiveneuropeptideΥ ( ΝΡΥ )- Υ1receptorantagonistPharmacol res 1995 31,190;

Matthews je, Chance wt, GrizzleΜΚ, Heyer d, Daniels aj: food uptakeinhibition and body weight loss in ratstreated with gl 264879Α, an ΝΡΥ - Υ 1 receptor. SocNeurosciAbstr 1997 23 pt 2 1346;

DoodsΗΝ, Willim K-D, SmithSJ : BIBP 3226 : a selective and highlypotentΝΡΥ - Υ 1 antagonist proc br PharmacolSoc 1994 13-16 dec. C47;

Rudolf Κ, Eberlein w, Engel w, Wieland ha, Willim kd, Entzerorh Μ, Wienen w, BeckSickinger ag, DoodsΗΝ : the first highlypotent and selective non- peptideneuropeptideΥΥ1receptorantagonist : BIBP3226 eur j Pharmacol 1994 271 2-3 R11-R13;

Serradelil -le- Gal c, Valette g, Rouby pe, Pellet a, Villanova g, Fouion l, Lespy l, Neliat g, ChambonJP, MaffrandJP, le- Fur g:sr 12010Α and SR120819A: two potent and selective, orally - effectiveantagonists for ΝΡΥΥ1receptorsSocneurosciAbstr 1994 20 pt 1,907-abs 376,14;

Hong Υ, Gregor v, Ling al, Tompkins ev, porter j, Chou ts, Paderes g, Peng Ζ, Hagaman c, Anderes Κ, Luthin d, May j: synthesis and biologicalevaluation care novelguanylureacompounds as potentΝΡΥΥ1receptorantagonist acs 1999,217 AnaheimMEDI 108.

Η: ΝΡΥ (in particular ΝΡΥ antagonists) are described in the following documents:

WO-98/07420

WO-94/00486

WO-96/22305

WO-97/20821

WO-97/20822

WO-96/14307

JP-07267988

WO-96/12489

US-5552422

WO-98/35957

WO-96/14307

WO-94/17035

EP- 0614911

WO-98/40356

EP- 0448765

EP- 0747356

WO-98/35941

WO-97/46250

EP- 0747357

Preferable examples h. npy are selected from following structures. These compounds were tested, and was recognized, that they useful in potentsirovanii camp and therefore are useful for treatment of RPVZh. Some experimental data, concerning these compounds, are presented in experimental partition (cm. below).

According to one aspect of the present invention additional target represents Ptsdmf target, while this Ptsamf target represents vip or one of binding with it receptors. In modern classification of/bill on them present both on VPAC1, vpac2 and pacap.

Nucleotide sequences and amino acid sequence for vip and its receptors are described in literature. Some sequences are presented in given here lists sequences.

Authors of the invention shown, that VPAC1 and vpac2 present in vagina human and rabbit. Pacap absent in the vagina as rabbit, so and human.

Vip is main endogenous neiromediatorom, liberated during sexual of excitation, which responsible behind indutsirovannuyu nerve vaginal vasodilation vascular bed, raspolozhennennogo in the wall of the vagina. These vasorelaxants effects mediated activation of adenylate cyclase and produtsirovaniem camp. If not be limited to theory, this effect can be mediated through receptor subtypes VPACi, VPAC_{2 or} pacap (peptide, activated hypophysis adenilattsiklazoi ) receptors vip. Receptors VPAC_{2 and} pacap widely Invention provides expression of in cns, and these receptors, despite the fact, that they Invention provides expression of in pituitary gland, at visible, have no widespread biological function.

Agent can potentiate vip and/or act as mimetic vip or its analogue. This agent will, so, potentiate and/or repeat vazorelaksiruyushchie effects of endogenous VIR, is during sexual of excitation. This agent can also have additive effect on vip- indutsirovannuyu relaxation of smooth musculature vagina. Clinically this will lead to treatment RPVZh, at least amplification lubrication vagina by overfilling blood and pliability of vaginal wall. In this embodiments mimetic or analog will not possess, however, harmful properties vip, which discussed above.

Preconditions by vip and binding with it receptors are presented Victor a. McKuzik et al on htpp :/ / www3.ncbi.nlm.nih.gov / Omim / searchomim.htm. From this source extracted the following text, touching vip.

" Vazoaktivnyiintestinalnyi peptide (VIP), 28-amino acid peptide, are initially isolated from duodenum pig, is present not only in gastro-intestinal tissues, but also in nerve tissues, possibly in as neuromediator, and has different biological action. Since VIR has similarities with glucagon, sekretinom and gastric an inhibitor peptide (GIP), its is considered member of family of glucagon-are injected subcutaneously. Primary translation product of mrna, encoding VIR ( prepro -vip), has molecular mass 20 Dalton. As a result of cloning dna sequence of, complementary mrna, encoding human vip, Itoh et al. (1983) find, that precursor vip has not only vip, but also new peptide from 27 amino acids, denoted RNM27, which has aminoterminalnyi histidine and karboksiterminalnyi methionine. It differs from PHI17, isolated from intestine pig, on 2 amino acids; PHI27 as indicated in its notation, has karboksiterminalnyi isoleucine. Linder et al. (1987) earmarked human gene vip and RNM27 and investigated its expression in different tissues rat. Heinz - Erian et al. (1985) expressed assumed, that insufficient renewal of sweat glands in patients with mucovisceidosis by VIR neuropeptide can be main mechanism lower nnogo water content and relative impermeability epithelium for chlorine ions and of other ions, that characterizes cystic fibrosis. To check this hypothesis, Gozes et al. (1987) taken approach " kandidatnogo gene". Bodner et al. (1985) shown, that vip and RNM -27 are encoded adjacent ekzonami. Gozes et al. (1987) is encoding RNM -27 genomic fragment, to to detect the presence of gene vip on 6q21- qter. So, they excluded defective gene vip as candidate for primary causes mucoviscidosis (which is encoded by chromosome 7). With the help of techniques of in situ hybridizationGozes et al. (1987) strapped gene vip to 6q24. So vip placed in area of MYB (189990), which mapped on 6q22. Gozes et al. (1987) investigated functional relationships between 2 genes in of nervous tissue. They watched acute peak mrna MYB in hippocampus 3-day rats, preceding peak mrna vip, which appears in this area in the age of 8 days. Omary and Kagnoff (1987) find nuclear receptors vip in cell line adenocarcinoma of human large intestine. Gotoh et al. (1988) strapped vip to chromosome 6 by means of spot-blot hybridization of molecular-cloned fragment of this gene with sorted chromosomes. Localization of clarified as 6q26-q27 with the help of in situ hybridization. "

As indicated, preconditions by vip and binding with it receptors are presented Victor a. McKuzik et al on htpp :/ / www3.ncbi.nlm.nih.gov / Omim / searchomim.htm. From this source extracted the following text, touching VIR.

" Vazoaktivnyiintestinalnyi peptide (vip; 192320) represents oktakozamernyi neuroendocrine mediator, detected mainly in cholinergic presinapticheskikh neurons central nervous system and in peripheral peptidergic neurons, viscera innervating different tissue. Among many neuroendocrine peptides with immunological functions VIR is its capacity directly act as on-cells, and on T-cells. Separate subgroup nerve, respiratory, gastro-intestinal and immune cells carry specific highly affine receptors for vip, which are connected with guanidinnukleotid -binding (G) protein, capable of activating adenylatcyclase. Libert et al. (1991) proposed 4 new receptor from family receptors, binding with g protein, with the aid of selective amplification and cloning from " dna of thyroid gland. One of them, said RDC1, Streedharan et al. (1991) identified as receptor vip. Libert et al. (1991) kartirovali gene VIPR in 2q37 with the help of in situ hybridization. More late information shipped under questioned, as gene, mapping in 2q37, in validity is receptor genome vip ( Vassart, 1992). Wenger (1993) element has detected, that sequence, which was is indicated as GPRN1Sreedharan et al. (1991) and kartirovana in 2q37, does not bind vip. Sreedharan et al. (1995) earmarked gene receptor vip authentic type i and by means of in situ hybridizationlokalizovali its in disk Zr22 in the area of, coupled with small cell lung cancer. With the help of mezhvidovogo analysis of return crossing Hashimoto et al. (1999) kartirovalimyshinyi gene VIPR1 in distal region of chromosome 9, the area of, which shows homology sintenii with human chromosome 3ρ. Sreedharan et al. (1993) cloned human gene intestinal receptor vip; brought amino acid sequence has 84% identity with rat pulmonary receptor vip. Couvineau et al. (1994) earmarked 2 cdna clone VIPR from cdna library of human toshchekishechnykh of epithelial cells. One of them encodes receptor vip, consisting of 460 amino acids and having 7 supposed transmembrane domains, as and other receptors, binding with g protein. Other encodes 495-amino acid vip receptor-sibling protein, showing 100% homology with functional receptor vip more than 428 amino acids in with-end the area of, but containing completely different 67-amino acid n-terminal domain. At expression in COS-7 cells second protein does not bind with labelled with radioactive with 10d vip, at it normally adresovalsya to plasma membrane, as was given in immunofluorestsentnykh research. Sreedharan et al. (1995) find, that receptor vip type i, also said receptor pacap type ii (cm. the other type of receptor pacap in 102981), occupies approximately 22 U. about. and is contained in 13 ekzonakh (are in the range from 42 to 1400 U. about. ) and 12 intronakh (are in the range from 0.3 to 6.1 τ. π. ο.). Sreedharan et al. (1995) also described promoter and 5-prim flanking region of this gene. "

As indicated, preconditions by VIR and binding with it receptors are presented Victor a. McKuzik et al. (there). From this source extracted the following text, touching VIPR2 and vpac2.

" Vazoaktivnyiintestinalnyi peptide (vip; 192320) and polypeptide, activating pituitary adenilattsikpazu (pacap; 102980) represent homologous peptides, which function as neuromediator and neuroendocrine hormones. At receptors for VIR and pacap have homology, they differ in their substrate specificity and patternam expression. Cm. Vipr1 (192321) and ADCYAP1R1 (102981). Svoboda et al. (1994) conducted pcr low rigidity, using primers, based on sequences of, conservative among receptors vip. They cloned human gene receptor VIP2 from cdna library limfoblastov. This gene kodiroval 438-amino acid polypeptide, which has 86% identity with rat receptor VIP2. They expressed human receptor VIP2 in cells chinese hamster ovary and find, that it binds with pacap -38, pacap -27, vip and kheloderminom, and that binding of this receptor with any of these peptides activates adenilattsikpazu. Was recognized, that binding of peptides inhibited GTF. Adamou et al. (1995) cloned gene receptor VIP2 from cdna library of human placenta. Nozern - blotting showed, that VIPR2 is expressed in the form of 2 transcripts 4.6 T. u. about. and 2.3 T. u. about. at high levels in skeletal muskulature and at more low levels of in heart, brain, placenta and pancreas. Mackay et al. (1996) used is a fluorescent in situ hybridization for mapping of gene VIPR2 in human chromosome 7q36.3. Further mapping with the use of cell lines, isolated in patients with goloprozentsefaliei type 3 ( NREZ ; 142945), revealed, that gene VIPR2 lies within minimum critical region NREZ. Mackay et al. (1996) installed, that, at VIPR2 can make a contribution into NREZ phenotype, it is not the only responsible for it factor. "

Au ( adenilatiikpaza )

According to one aspect of the present invention additional target represents Ptsdmf target, ; Ptsamf target represents ATs.

Nucleotide sequences and amino acid sequence for ATs can be find in literature.

For confirm, that vip induces vazorelaksatsiyu by means of diffusion condensate intracellular levels camp and subsequent activation of adenylate cyclase, authors-measured vaginal concentration of camp during vip stimulation and is forscoline, activator of adenylate cyclase, that to imitate effects of activation of metabolic track camp/adenylate cyclase.

In these research authors-find, that treatment of vip and treatment of forskolinom is increased intercellular concentration of camp in isolated tissue vagina.

Authors invention also find, that forscoline enhances vaginal blood flow in animal model of sexual of excitation.

Besides, authors-find, that forscoline induces relaxation in isolated vagina.

Preconditions by ATs are presented Victor a. McKuzik et al on htpp :/ / www3.ncbi.nlm.nih.gov / Omim / searchomim.htm. From this source extracted the following text, touching ATs.

" Adenililtsiklaza (EC 4.6.1.1) catalyzes conversion of ATP in cyclic amf. Enzymic activity is under control of several hormones, and different polypeptides participate in transmission of signal from receptor to catalytic component. Stimulyatornye and inhibitory receptors (rs and ri) interact with g proteins (gs and gi), which inhibit GTFaznuyu activity, and they are modulated catalytic activity subunit adenililtsiklazy. Parma et al. (1991) cloned cdna, corresponding adenililtsiklaze brain, designated them as NVAS1. With the help of in situ hybridization with Pap of metaphase chromosomes, using cdna probe human brain, Stengel et al. (1992) shown, that this gene localised on 8ς 24.2. Highly a gene ADCY2 (103071) strapped to 5ρ 15.3 in the same way. "

At in common mentioned here procedures of problems in this engineering, can be reference, in particular, on Sambrooket al., MolecularCloning, a Laboratory manual (1989) and Ausubelet al., short protocols in Molecular biology (1999) 4^{th ed}, john Wiley & Sons, inc. PCR described in us-a- 4683195, us-a- 4800195 and us-a- 4965188.

In combination, the present invention relates to the use of 14 : ΝΡΥ for treatment of ZhSD, in particular, RPVZh.

Now the present invention will be described only as is an example of, in which there is a link on the following graphic materials:

Figure 1, which is a graph;

Figure 2, which is a graph;

Figure 3, which is a graph;

Figure 4, which is a graph;

Figure 5, which represents the diagram;

Figure 6, which represents the diagram;

Figure 7, which is a graph;

Figure 8, which is a graph;

Figure 9, which is a graph;

Figure 10, which represents the diagram;

Figure 11, which represents the diagram; and

Figure 12, which represents the diagram.

Should be understandable, that agent according to the present invention represents and: NPE. Are presented also scientific data on and: pde and Η: ΝΡΥ, which being clearly indicate technician in this field on then, that agent according to the present invention can be used in combination with one or both agents of these types of for achieving favorable effect, mentioned here. These additional scientific data also are connected as additional confirmation basic open of authors of the invention.

Now graphic materials discusses more detail.

Figure 1: electric stimulation of pelvic nerve induces chastotozavisimoe amplification of vaginal blood flow in model of sexual excitation of anestezirovannogo rabbit. Increased stimulation frequency induces larger amplification of blood flow. Monitoring changes to, using laser Doppler technology.

Figure 2 : Vazoaktivnyiintestinalnyi peptide (VIP) induces amplification of vaginal blood flow in model of sexual excitation of anestezirovannogo rabbit. Figure 2 and illustrates, as amplified by vaginal blood flow depending on the concentration of a result of infusions vip (intravenous bolus). Figure 26 demonstrates, that 2 repeated infusion vip is Resembling amplification of vaginal blood flow. Overviews, that duration of the reaction are also similar. Monitoring all changes to, using laser Doppler technology.

Figure 3 : Vazoaktivnyiintestinalnyi peptide (VIP) reduces average arterial blood pressure in model of sexual excitation of anestezirovannogo rabbit. This graph illustrates typical action of combined vasoactive agents and of stimulation parameter, used for investigation of vaginal blood flow, on average arterial pressure in anestezirovannogo rabbit. These observed influence are typical trends, observed in all tested animals. Vip induced considerable drop of mean arterial pressure, while stimulation of pelvic nerve, control infusion gepsalina or inhibitors of FDEtsamf or nep not provided effect on blood pressure. Overviews, that Your lu blood pressure, coupled with infuziyami vip, also with great magnification frequency of heart contractions.

Figure 4: activation of metabolic track camp/adenylate cyclase imitates mediated vip vazorelaksatsiyu and relaxation of smooth musculature in tissue vagina. Figure 4 and illustrates, that infusion forscoline (40 nmol/kg in. in. bolus, mimetic of camp) induces significant amplification of vaginal blood flow. Overviews, that amplitude and duration of the response characteristic of induced vip (20.0 mcg/kg, in. in. bolus). Interesting, that effect on blood flow has greater action duration on external vaginal wall. Monitoring all changes to, using laser Doppler technology. Figure 46 demonstrates, that both vip (0.1 mcm), and forscoline (10 mcm) considerably enhances intercellular concentration of camp compared with basal levels of into vagina rabbit. Figure 4 in shows, that forscoline induces a strong relaxation preliminarily reduced (1 mcm phenylephrine) strips of vagina rabbit at IC50 -300 nm. All change was quantitatively, using in vivo laser Doppler technology, biochemical enzymatic immunoassay of camp, or relaxation of tissue in vitro.

Figure 5: infusion vip enhances klitornyi circulation, and activation of metabolic track camp/adenylate cyclase imitates vip-mediated klitornuyuvazorelaksatsiyu in model of sexual excitation of anestezirovannogo rabbit. Infusion vip (60-200 mcg/kg) induces dependent from concentration of amplification of kpitornogo circulation. 115% amplification klitornogo blood watched after in. in. infusion 200 mcg/kg vip. Effects vip on klitornyi blood flow can be to imitate by infusion mimetic of camp forscoline (SCF, 40 nmol/kg, in. in. bolus). 156% amplification klitornogo blood watched after in. in. infusion of 40 nmol/kg forscoline. All amplification of considerably differ in positive side from control infusions ( gepsalin ). Overviews, that amplitude response of same amplitude, induced vip (200 mcg/kg, in. in. bolus) and is comparable with amplitude, observed in vaginal blood Figure 2 and Figure 4. All change was quantitatively, using in vivo laser Doppler technology, and they were in significant degree are compared with infuziyami carrier ( gepsalina ).

Figure 6: selective inhibitor of nep EC 3.4.24.11-enhanced amplification of vaginal blood flow in a result of stimulation of pelvic nerve ( STN ) in model of sexual excitation of anestezirovannogo rabbit. Repeated STN through 15-minute-long intervals induce reproducible amplification of vaginal blood flow (white strip). Administering the inhibitor nep (Polonina strip) enhances peak amplification of vaginal blood flow, induced submaksimalnymi frequencies stimulation (for example 4 hz), compared with amplification, observed during corresponding control stimulyatsii controls-carriers (crosshatched strip). Watched the following dozozavisimye amplification of-dose of 0.3 mg/kg. in. induced 40% amplification, and the dose of 1.0 mg/kg in. in. induced 91% amplification (average U = 3). Inhibitor of nep not provided effect on basal (without stimulation) vaginal blood flow (data not shown). Monitoring all changes to, using laser Doppler technology.

Figure 7: selective inhibitors of nep EC 3.4.24.11 is increased vip-induced amplification of vaginal blood flow in model of sexual excitation of anestezirovannogo rabbit. Repeated STN through 30-minute-long intervals induce reproducible amplification of vaginal blood flow (cm. figure 26). Inhibitor of nep and-potentiated amplitude and increased duration of the amplification of blood flow, when this amplification is induced submaksimalnymi doses vip, for example 60 mcg/kg. At dose vip, which induce maximum amplification of vaginal blood flow, for example 60 mcg/kg, inhibitors of nep only potentiate duration of the amplification of vaginal blood flow. Vip-induced amplification in the presence of inhibitor nep shown in the form of solid triangles, while control vip responses shown in the form of empty triangles. Control infusion gepsalina not would strike action on amplitude of these answers. Monitoring all changes to, using laser Doppler technology.

Figure 8: selective inhibitor of FDEtsdmf type 2-enhanced amplification of vaginal blood flow at stimulation of pelvic nerve ( STN ) in model of sexual excitation of anestezirovannogo rabbit. Repeated STN through 15-minute-long intervals induce reproducible amplification of vaginal blood flow (white squares). Administering the inhibitor FDEtsdmf type 2 enhances peak amplification of vaginal blood flow, induced submaksimalnymi frequencies stimulation (black squares; 4 hz) compared with amplification, observed during corresponding control stimulyatsii controls-carriers (empty squares). Infusion of inhibitor FDE2 (500 mcg/kg) induced 86.8+21.9% amplification of vaginal blood flow (mean ± standard deviation average, U = 2). Monitoring all changes to, using laser Doppler technology.

Figure 9: selective inhibitors of FDEtsamf type 2 is increased vip-induced amplification of vaginal blood flow in model of sexual excitation of anestezirovannogo rabbit. Repeated infusion vip through 30-minute-long intervals induce reproducible amplification of vaginal blood flow (cm. figure 26). Selective inhibitor of FDEtsamf type 2 (25 mcg/kg, in. in. bolus)-potentiated duration of blood current, induced vip (60 mcg/kg, in. in. bolus). Vip-induced amplification in the presence of inhibitors of FDEtsamf shown in the form of solid triangles, and control vip responses shown in the form of empty triangles. Control infusion gepsalina not would strike action on amplitude of these answers. Monitoring all changes to, using laser Doppler technology.

Figure 10: selective antagonist receptors ΝΡΥ Y1-enhanced amplification of vaginal blood flow at stimulation of pelvic nerve ( STN ) in model of sexual excitation of anestezirovannogo rabbit. Repeated STN through 15-minute-long intervals induce reproducible amplification of vaginal blood flow (data not shown). Administration of ΝΡΥ Y1 (grey column) enhances peak amplification of vaginal blood flow, induced submaksimalnymi frequencies stimulation (for example 4 hz), compared with amplification, observed during corresponding control stimulyatsii controls-carriers (hatched column). Watched the following dozozavisimye amplification of-dose of 0.01 mg/kg. in. induced 15.8±19.6% amplification ; dose of 0.03 mg/kg. in. induced 35.1±17.17% amplification ; dose of 0.10 mg/kg. in. induced 60.1±16.9% amplification, and the dose of 0.3 mg/kg. in. induced 91.9±27.4% amplification (mean ± standard deviation average, U = 3). Antagonist ΝΡΥ Y1 not provided effect on basal (without stimulation) vaginal blood flow (data not shown). Monitoring all changes to, using laser Doppler technology.

Figure 11 represents total diagram for some given here of data, which indicates, that agents absolutely are useful for reinforcing vaginal blood flow by means of potentiation of endogenous levels of camp.

Figure 12: selective inhibitor of nep EC 3.4.24.11-enhanced amplification of kpitornogo blood flow at stimulation of pelvic nerve ( STN ) in model of sexual excitation of anestezirovannogo rabbit. Administering the inhibitor nep (grey column) enhances peak amplification klitornogo blood flow, induced submaksimalnymi frequencies stimulation (for example 4 hz) compared with amplification, observed during corresponding control stimulyatsii controls-carriers (hatched column). Watched the following dozozavisimye amplification of-dose of 1.0 mg/kg in. in. induced 131% amplification (average, U = 3). Inhibitor of nep not provided effect on basal (without stimulation) vaginal blood flow (data not shown). Monitoring all changes to, using laser Doppler technology.

Measurement of camp from tissue samples vagina using set of for immunoferment analysis (iea) Biotrack camp ( Amersham life sciences RPN 225).

Levels of camp is measured in samples of tissue vagina with the help of iea. Iea is based on competition between an unmarked camp and fixed amount of labeled peroxidase camp behind limited amount of tsAMFspetsifichnogo antibody.

1. MATERIALS

If not indicated otherwise, all materials is provided set of for iea camp Amersham life sciences ( RPN 225).

1.1. Mikrotitrovalnyi plotting board -96-alveolar plotting board, coated with igg donkey against rabbit.

1.2. Analytical buffer -0, 05Μ sodium-acetate buffer, ph 5.8, containing 0.02% bovine serum albumin and 0.5% preservative after pererazbavleniya. The content of the bottle is transferred in graded cylinder, using 3x15 ml flushing distilled water. Then final volume is brought to 500 ml.

1.3. Standard camp (for method acetylation). Camp in concentration of 10.24 pmole/ml in 0, 05Μ acetate buffer, ph 5.8, containing 0.02% bovine serum albumin and 0.5% preservative after pererazbavleniya. For use standard is dissolved in 2.5 ml of analytical buffer.

1.4. Antiserum. Antibody anti-camp in 0, 05Μ acetate buffer, ph 5.8, containing 0.02% bovine serum albumin and 0.5% preservative after pererazbavleniya. Before using antibody is diluted with 11 ml of analytical buffer and mixed calming overturning before dissolution content.

1.5. Conjugate of camp. camp horse radish peroxidase in 0, 05Μ acetate buffer, ph 5.8, containing 0.02% bovine serum albumin and 0.5% preservative after pererazbavleniya. Before using antibody is diluted with 11 ml of analytical buffer and mixed calming overturning before dissolution content.

1.6. Flushing buffer. 0.01 m phosphate buffer ph 7.5, containing 0.05% (vol./ rev. ) Tween ™ 20 after pererazbavleniya. The content of the bottle is transferred in graded cylinder, using 3x15 ml flushing distilled water. Then final volume is brought to 500 ml.

1.7. TMB substrate. 3.3 ', 5.5' -tetramethylbenzidine ( TMB )/ hydrogen peroxide in 20% (vol./ rev. ) dimethylformamide.

Is always ready to use.

1.8. Atsetiliruyushchii reagent. 2 ml of acetic anhydride, 4 ml of triethylamine, prepared as required.

1.9. Sulfuric acid (1 μ a). 1 m solution of sulfuric acid is prepared from 18M initial solution (BDH). 1.11 ml of acid is added in 18.8 ml of distilled water.

2.1. Disposable 5 ml glass test tubes for testing.

2.2. Spectrophotometric readout device for boards ( Spectra max 190).

2.3. -for microtiter trays ( Luckham R100).

3. PROCEDURES

-Preparation of cloth sample. Tissue is corresponding predobrabotke in samples by 5 ml for example agonists, camp mimetics and T. d in physiological saline solution. After treatment samples immediately freeze in liquid nitrogen, and then smashing of hammer. Powder soskrebali in centrifuge test tube and a glucose 0,5m cooled in ice perchloric acid ( PKhA ). Specimen stirring on vortex and left on ice on 1 hours.

-Extraction of camp from tissue samples. Samples is centrifuged at 10000 g for 5 min at 4° with. Supernatant benefit and hostages in other centrifuge test tubes. Residue kept for analysis for protein at -80 ° with. Samples of supernatant then neutralized remnant to ph ~6, using K3RO4. Is centrifuged at 10000 g for 5 min at 4° with. Separated supernatant and are washed 4 times 5 volumes of (by 5 ml) diethyl ether, water-saturated. Upper ether layer should be discard after each washing. Water layer have weathered in short of finely stennuyu glass test tube and was dried in flow of nitrogen at 60° with. Dried extract was dissolved in 1 ml of analytical buffer and kept in a refrigerator of, until it be required (or can be freeze).

-Parent reagents balanced in advance to room temperature, and then is prepared working solutions.

-camp protocols is prepared in glass test tubes, marked 2, 4, 8, 16, 32, 64, 128, 256 and 512 fmol. This is achieved by addition of 1 ml of analytical buffer in each test tube except standard 512 fmol. Then to two highest standards (256 and 512 fmol) is added 1 ml of standard acetylation (10.24 pmole/ml). Standard 256 fmol stirred on vortex, and 1 ml is transferred in standard 128 fmol. This procedure is continued up to standard 2 fmol, from which is 1 ml of solution. Is installed a test tube zero standard, containing 1 ml of analytical buffer.

-Samples tissue extracts of thawed on ice (if necessary) and diluted with 1 in 100 (10 mcl sample on 990 mcl analytical buffer) in marked glass test tubes.

-camp in all standards and samples acetylated in exhaust cabinet addition of 100 mcl atsetiliruyushchego of reagent, which is added down along wall of the test tubes, and then immediately stirred on vortex,

- 50 mcl all standards and specimens is added in corresponding wells of 96 - ; board and added 150 mcl analytical buffer in holes for nonspecific binding ( NSS ).

-100 mcl antiserum is added in all holes except pure kontrolen

(In) and NSS, and then incubated for 2 hours at 3-5° with.

-After incubation 100 mcl conjugate camp-peroxidase is added in all holes except in, and then incubated more 1 hour at 3-5° with.

-Boards dried, inverting them upward bottom and promokaya on by blotting paper, and then washed with each well four times 400 mcl washing buffer. After each washing boards again promokayut, is, that the whole remaining otmyvochnyi buffer removed. Then 200 mcl TMB immediately is distributed in all lunkam.

-Boards is placed on-for boards on 30 minutes at room temperature, and then in all holes is 100 mcl 1m of sulfuric acid. Optical density is read on Spectra max 190 at 450 nm within 30 minutes.

4. PROTOCOLS

At each analysis is installed the following standard test tubes:

Peak for known quantity of camp is obtained in analytical buffer, to determine efficiency analysis. 70 pmole/ml camp is added in analytical buffer, which is equivalent to 35 fmol/hole in analysis, and it is average value on curve dose-response.

For making up 1 ml of standard: 68.4 mcl 521 fmol/hole standard

931.6 mcl analytical buffer

4.2. Effect of compounds on plotting board

Is installed protocols for determining, possesses whether compound, used in functional studies, some-either action on 96-alveolar plotting board or effect whether it on binding camp. They include:

-Production of peak for connection only in analytical buffer for influence of this compound directly on plotting board.

-Production of peak compound in plasma, containing basal levels of camp, for evaluating action of this compound on binding camp with the table.

Produced peaks 5 nm concentrations of compound in each standard. 5 nm concentration of selected therefore, that summary levels of drug in the end of infusion earlier prepared approximately 150-300 nm. Samples of diluted 1:100 before analysis, therefore 5 nm gives possibility for any concentrations of medicinal agent, which exceeds, than expected in the end of infusion.

5. CALCULATION OF

Readout device for boards Spectra max reads optical density (OD) at 450 nm.

Standard curve is obtained by vision statement presented Details graph of dependence 5-b/in_{0 (} axis in) from camp fmol/hole (its X axis) on Spectra max.

% In/in_{0 (} % binding) for each sample and standard is calculated as follows:

In_{0 =} zero standard (cm. methods of 3.2)

5Β/Β U = (standard or specimen od- NSS od) khYuO

(In_{0 od}- NSS od)

Then value of fmol/hole can be read directly with standard curve for each sample. Then value is transferred in pmole/ml, and then is taken mean for each pair of samples.

fmol in pmole = for 1000

Volume in pit = 50 Micn... So (X1000}

50

Specimen diluted 1/100, therefore the whole = 1 X 1000/1000 x 100/50 = 2

So, all values fmol/hole multiplied by 2 to obtain pmole/ml.

Taking antilogarithms effects of cyclic adenosine -3 ', 5' monophosphate (camp) leads to increased vaginal blood flow in model of sexual excitation of ANESTEZIROVANNOGO rabbit

1.0 target

1. To develop and substantiate animal model of female sexual of excitation.

2. To identify mechanism (s), responsible for regulation of genital blood flow in anestezirovannogo rabbit.

3. To identify potential approaches to increased vaginal and klitornogo blood flow.

4. Study of mechanism (s), which lie in base relaxation of smooth muscles vagina, and identify potential approaches to increased vaginal relaxation.

2.0 introduction

Normal reaction sexual excitation of consists of a number of physiological reactions, which are watched during sexual of excitation. These changing, such as overflow blood vagina, lips and of the clitoris, are result amplification of genital blood flow. Overflow blood leads to increased lubrication vagina by means of transudation of plasma, to diffusion Exploration pliability of vagina (relaxation of vaginal smooth muscle) and to diffusion Exploration sensitivity of vagina and of the clitoris.

Disorder sexual excitation of in women ( RPVZh ) is widely occurring sexual disorder, projectile to 40% women in pre-, perii postmenopausal (+ HRT ). Primary consequence RPVZh attenuation overfilling blood or swelling of genitalia, which is seen in insufficiency of lubrication of vagina and in absence of sensation of enjoying in genitalia. Secondary consequences include lower nnoe floor attraction, pain during sexual intercourse and embarrasment in achieving orgasm. The most common cause RPVZh attenuation genital blood flow, which leads to lower nnomu overflow blood vagina, lips and of the clitoris (park, 1997;

Goldstein, 1998; Berman, 1999a ; Werbin, 1999).

As explained here, according to the present invention proposes means for restoration or potentiation of normal reaction sexual excitation of in women, suffering from RPVZh, by enhancement of genital blood flow.

In their research authors-identified camp (cyclic adenosine -3', 5-monophosphate) as mediator vaginal vazorelaksatsii, using laser Doppler technology for measuring small changes in genital blood flow. Using inhibitor metabolism vip (inhibitor of nep EC 3.4.24.11), authors invention demonstrated also, that amplification of genital blood flow, observed during stimulation of pelvic nerve (i.e. sexual excitation of) indirectly vip. In connection with this was is developed animal model of sexual of excitation, and was demonstrated, that these data reflect the physiological change of, observed by during sexual excitation of a woman. This model then is used for identification of and substantiation of mechanisms, which amplified genital blood flow, for example direct or indirect taking antilogarithms camp-mediated vazorelaksatsii.

3.0 techniques

3. 1. Protocol anesthesia

Female sables NZ rabbits (~2.5 kg) to premedication combination of medetomidina ( Domitor ®) 0.5 ml/kg. m. and ketamine ( Vetalar ®) 0.25 ml/kg. m., keeping supply of oxygen through the face mask. Rabbits trakheotomirovali, using endotracheal tube without cuff 3 id Protex™, connected with fan and supported at rate of ventilation 30-40 breathings minute with air volume, obmenivaemogo at one breathing, approximately 18-20 ml and at maximum pressure in tube 10 cm H_{2 0}. Then anesthesia pereklyuchali on isoflurane and continued ventilation 0_{2 at} 2 l/min. Right marginal ear vein at a dose of kanyulirovali, using catheter 23G and 24G, and execute perfusion Ringer with lactate at 0.5 ml/min. Rabbit kept on 3% izoflurane for invasive operation, decreasing to 2% for maintenance of anesthesia.

3.2. Kanyulirovanie vessels

Left inguinal area rabbit vybrivali and did vertical incision approximately 5 cm in length along thigh. Femoral vein and artery exposed, insulated, and then kanyulirovali PVC (polyvinyl chloride) catheter (17G) for infusion medicinal agents and compounds. Kanyulirovanie repeated for femoral artery, to catheter on depth of 10 cm, is, that the catheter reaches the abdominal aorta. This arterial catheter soedinyali with system Goulda for recording blood pressure. Sample for analysis of blood gases also took through arterial catheter. Measured systolic and diastolic pressure and evaluate a formula when you evaluate mean arterial blood pressure, using the formula (diastolic X 2 + systolic)* 3. Heart rate measured by means of device pulse and system of software data collection po-ne-mah (PonemahPhysiology platform, GouldInstrument systems inc).

Did ventralnyi incision along the middle line in abdominal cavity. This incision was approximately 5 cm in length directly above lobkom. To tupoe separation of fatty tissue and muscle, to detect podzheludochnyi nerve, which passes downwards body cavity. Important was ad closed side curve of wall pubis, to avoid damage of femoral vein and artery, which lie above lobkom. Sciatic and pelvic nerves, which lie deeper, lokalizovali after further dissection rabbit from back. Immediately after identification sciatic nerve easily lokalizovali pelvic nerve. Term "pelvic nerve" is used non-precisely; in anatomical books according to this object is absent sufficiently detailed determination of nerves. However stimulation of this nerve causes amplification of vaginal and kpitornogo circulation and innervation pelvic area. Pelvic nerve absolve from the surrounding tissue, and around nerve have bipolar stimulating electrode Harvard. Nerve slightly raised, to create some voltage, then electrode fixed in this position. Around nerve and electrode hostages approximately 1 ml of lung paraffin oil. It acts as protective lubricant nerve and prevents contamination of electrode blood. Electrode connected to stimulyatoruGrass S88. Pelvic nerve stimulated, using the following parameters : - 5-b, width pulse of 0.5 ms, duration of stimulus 10 seconds and range of frequencies from 2 to 16 hz. Reproducible responses obtained, when nerve stimulated every 15-20 minutes.

Curve of frequency response was in beginning of each experiment, to determine optimal frequency for use as submaksimalnogo response, usually 4 hz. Infusion of compound (th), which tested, was implemented through femoral vein, using pump for infusion Harvard 22, creating possibility of continuous 15-minute stimulation cycle.

Did ventralnyi incision along the middle line in tail end pubis, to unsheathe the lobkovuyu area. Connecting tissue had been expelled, to unsheathe the envelope of the clitoris, providing release of wall from small blood vessels. External vaginal wall also exposed removal of the whole of connective tissue. One movable laser doppler sensor either inside sheath on 3 cm, so that half of rod transducer was yet visible. Second sensor placed so, that it calve-directly above the outer wall of the clitoris. Position of these sensors then govern of, until obtained signal. Second sensor hostages directly above surface of blood vessel on the external vaginal wall. Both sensors clamped in this position.

Vaginal and klitornyi circulation registered or in the form of numbers directly with speed meter blood flow, using the software data collection po-ne-mah (PonemahPhysiology platform, GouldInstrument systems inc), or indirect with recorded curve graphic recording device Gould. Calibration of-in the beginning of experiment (0-125 ml/min/100 g of tissue).

3.5. Infusion vasoactive intestinal polypeptide (VIP)

Dose of vip ( Bachem, n- 3775 ) carrying 2.0, 6.0, 20.0, 60.0 mcg/kg. in., and its infusion execute in volume of 0.5 ml of physiological solution. Infusion vip execute, using pump Harvard 22, with the rate of 500 mcl/min through 3-running cock in femoral vein. After infusion VIR catheter is washed with heparinized physiological solution ( gepsalinom ), so that vip not remained in catheter.

For experiments using infusions vip was required initial sensitizing curve dose-response (2-60 mcg/kg), to can be to obtain reproducible responses. Initial infusion gepsalina (50 ul/ml) to, that it would as negative control.

Inhibitors of nep (neutral endopeptidaza EC 3.4.24.11), inhibitors of phosphodiesterase type 5 ( FDE5 ) and antagonists ΝΡΥ Y1 was dissolved in physiological solution or in 5% glucose solution (200 mcl 50% glucose in 1.8 ml of water per injection). Inhibitors of FDEtsamf was dissolved in 40% ethanol solution (200 mcl 50% glucose in 1.8 ml of water/ethanol per injection). Infusion of inhibitors and testing runs-carriers of to with the same rate, that and vip. Inhibitors of nep left on 30 min before curve dose-response VIR ; Besides, inhibitors of nep, receptor antagonists ΝΡΥ Y1 and inhibitors of FDEtsamf left on 15 min to stimulation of pelvic nerve.

3.7(a). In vitro preparation of vaginal rabbit :-females NZ white rabbits (2.0-3.0 kg) were beaten by cervical dislocation of. Vskryvali abdominal cavity and carved vagina. Tissue strip enhance longitudinally in 5 ml siliconized chambers organs Wesley with. wicker silk threads for wounds (size of 6/0) at initial residual tension 1.5 g in bicarbonate buffer Krebsa, was kept at 37 and is passed gas 95% 0g /5% S0_2. Upper ligature each tissue strip of saddles to sensor movement, caused by force load of 10 g, and changes in isometric force measured and registered, using in vitro system of data collection DART. Tissues gave uravnovesitsya for 1.5 hours and regularly is washed with buffer Krebsa.

3.7(6). Induced vasoactive intestinalnym peptide relaxation vagina rabbit :-each fabric reduce, using fenilerfin in concentration of 1 mcm on tub. When sokratitelnyi response attains stable plateau (~15 minutes), vip cumulative a glucose to chamber for member in logarithmic units for producing concentration from 0.1 to 100 nm. Relaxation responses measured through 5 minutes after adding vip each concentration; during this time the maximum relaxation if when walking usually. Then tissue obtained or tested agent (for example inhibitor of nep or pde), or dmso-carrier (synchronized control).

3.7(b). Analysis of the data experiments on vip-relaxation :-for each curve concentration of vip-relaxation response, relaxation responses, induced vip, expressed in the form of percent from maximum induced fenilerfinom reduction of. Then these values is on graph against log concentration of vip and points built sigmoidalnye curves. In drawing curve by points at least relaxation response conditionally bent behind 0%, and the maximum relaxation to which not kept. Was concentration of vip, required for producing 50% relaxation fenilerfinovogo reduction of (EC_{50 pe}).

3.7 (d). Stimuliruemaya electric field relaxation vagina rabbit :-vaginal strip of rabbit obtained, as described in section 3.7 (a). These tissue strip enhance between two platinum electrodes, placed on top and bottom of chamber for organs on distance approximately 4 cm from each other. Each fabric reduce, using fenilerfin in concentration of 1 mcm on tub. When sokratitelnyi response attains stable plateau (~15 minutes), tissue is predobrabotke stimulation electric field ( EPS ), inducing relaxation curve. This was implemented between 40-60 voltami at successive frequencies of 2, 4, 8 and 16 hz, supplied in the form of 10-millisecond of series of pulses duration 0.5 milliseconds. Tissues gave to return to base line predsokratitelnogo tension in gaps between frequencies (5 minutes), and registered value of relaxation response.

After completion of vision statement presented Details curve EPS response-defined pre-all tissues are washed for 15 minutes, allowing tissues to return to base line of tension. Then tissue obtained or tested agent (for example inhibitor of nep or pde), or carrier-dmso (synchronized control). Tissue repeatedly reduce fenilerfinom (1 mcm) through 15 min after addition of compounds or carrier and was curve EPS -induced relaxation response, as described above.

For EPS experiments into buffer Krebsa a glucose atropine (10 mcm) and guanetidin (150 mcm), to cancel which-either cholinergic or adrenergic neural innervation vagina.

Measurement of concentrations of camp to in extracts of vaginal tissues, using set of for immunoferment analysis (iea) BiotracksAMR ( Amersham life sciences RPN 225). Samples of isolated vaginal tissues treated testing agents (for example forskolinom or vip). Through 5 minutes these samples of instantly freeze, using liquid nitrogen, homogenized and allocate camp. Levels of camp measured with the help of iea. Iea is based on competition between an unmarked camp and fixed amount of labeled peroxidase camp behind limited amount of tsAMFspetsifichnogo antibody.

Tsitozolnye extracts vaginal wall person obtained from abs inc., Delaware (age donors 41 and 60 years). Izofermenty pde shared using anion-exchange chromatography mono-q and characterized on the basis of their substrate selectivity, sensitivity to allostericheskim modulators and selective inhibitors. To also western-analysis, using specific antibodies to izofermentam pde, for detection of expression pde into vagina human.

All data are given as mean ± standard deviation average. Using t-criterion Styudenta, identified considerable change of.

4.0. P ezultaty and discussion

In their research authors invention developed on rodents reproduced model physiology of sexual of excitation. Using this model anestezirovannogo rabbit authors-It measure small changes in genital blood flow, using laser Doppler technology. Stimulation of pelvic nerve is used, to to stimulate neural effects sexual of excitation.

Authors-find, that stimulation of pelvic nerve induces chastotozavisimoe amplification of vaginal and klitornogo blood flow (cm. figure 1). This amplification of vaginal blood flow is essential at recording on the inner and outer walls vagina. Stimulation of pelvic nerve in 2 hz induced average maximum diffusion condensate vaginal blood flow 10.3±1.8; at 4 hz -20.0±4.6, at 8 hz -36.3±4.8 and at 16 hz -46.6±4.7 ml/min/100 g of tissue (η = 4 ; 15- 20V, 0.5 ms, 10 with) and amplification klitornogo blood 14.7+3.6 at 2 hz, 29.4±1.4 at 4 hz and 69.7±2.1 at 8 hz. These values have amplitude, similar amplitude, observed earlier examination of human and animals models of excitation ( Berman, 1999a; park, 1997).

Authors-find, that submaksimalnaya stimulation of pelvic nerve leads to reproducible enhancement of genital blood flow (for example stimulation of 4 hz every 15 minutes yielded average amplification of vaginal blood flow 8.50±0.10 ml/min/100 g of tissue, U = 8, and mean amplification klitornogo blood 13.65±0.86 ml/min/100 g of tissue, η=11). Reproducibility is maintained up to 5 hours. Authors invention can use reproducibility of these answers for investigating and) identity of endogenous combined vasoactive agents/mechanisms, which indirectly overflow blood genitalia, and b) effect of medicinal agents, which may be effective in amplification vaginal and/or klitornogo blood flow.

Authors-find, in anestezirovannogo rabbit no undesirable cardio-vascular effects, connected with stimulation pelvic nerve (cm. figure 3).

Genital blood flow is enhanced during sexual excitation of ( Berman, 1999) by means of diffusion nnogo arterial blood supply vaginal artery, vaginal branch uterine artery, inner lobkovaya artery of and middle branches of middle rectal artery all involves in blood supply of vagina and of the clitoris. Pelvic nerve, delivered from S2/S4 spinal areas, innerviruet Ferragamo genitals and has branches, terminating in lower part of vagina, klitore and corresponding blood vessels. By means of stimulation of pelvic nerve authors invention may stimulate effect on blood flow, observed during sexual of excitation, i.e. amplification of arterial genital blood flow. Interesting, that amplification of arterial blood flow causes no venous drainage, allowing capillary grids be flooded blood. Overflow blood vagina results to smazyvaniyu vagina by means of diffusion condensate transudation of plasma, and this is one of first pelvic answers, observed during sexual stimulation. Neuromediators, which are released at stimulation of pelvic nerve or during sexual excitation of, at the present time not identified. Nerves, containing neiropeptidy and other neuromediators-candidates, which innerviruyut vascular network and mikrososudistuyu network of vagina and the clitoris, identified immunohistochemical. These of investigating indicate then, that sibling gene calcitonin peptide (CGRP), neuropeptide Υ (NPY), synthase nitrogen oxide (NOS), substance p and vazoaktivnyiintestinalnyi peptide (VIP) all present in nerves, which innerviruyut vagina and clitoris human ( Hoyle, 1996; Hauser - Kronberger, 1999).

To change data on blood flow, obtained using this model, in data, observed on human model of sexual of excitation, authors-conducted direct comparison of their data with data via vaginalnomu blood flow and cardio-vascular data, obtained in predklinicheskikh research.

Authors-find, that infusion VIR shows the following action in model of sexual excitation of rabbit:

-Exogenous vip (in. in. bolus) induces considerable dependent from concentration of amplification of vaginal blood flow (cm. figure 2 and). Value of this amplification of considerably exceed basal value of blood flow, when their registered on the inner and outer wall of vagina. Vaginal blood flow in significant degree noise on 24.7±3.6 ml/min/100 g tissue at intravenous administration vip (60 mcg/kg). Blood flow is diffusion nnym compared with basal for approximately 11 minutes after infusion. More low dose of induced less than amplification of, for example dose of 6.0 mcg/kg raised circulation at 7.5+1.3 ml/min/100 g of tissue, and blood flow was diffusion nnym for 7 minutes after infusion.

-Repeated infusion similar doses vip (in. in. through 30 minute-long intervals) induce significant reproducible amplification of vaginal blood flow (cm. figure 26). ...

-Vip (in. in. ) in significant degree increases heart rate and reduces mean arterial blood pressure (cm. figure 3). In dose of 6.0 mcg/kg (in. in. ) Vip had significant Your lu mean arterial blood pressure 13.2±0.7 mm Hg. ct. and significant diffusion lu frequency of heart contractions 16 ± 4 impacts in min.

This animal model directly displays clinical data, observed at infusion vip healthy volunteers, i.e. amplification of vaginal blood flow, Your lu blood pressure and diffusion lu frequency of heart contractions. Consequently, this model can be used for investigating mechanism (s), which lays in the basis of physiological changes, which are during sexual excitation of, and, besides, for substantiation of new approaches to increased vaginal blood flow and, consequently, treatment of RPVZh.

Ottesen and its sponsors demonstrated, that vip induces amplification of vaginal blood flow and lubrication in healthy volunteers. However mechanism, by means of which vip shows these action, unclear. In literature there are several examples of vip-alarm through different system of secondary mediator, including cgmp/ guanilattsikpazu ( Ashur - Fabian, 1999), carbon monoxide/ gemoksigenazu (fan, 1998) and camp/adenylatcyclase ( Schoeffer, 1985; Gu, 1992 ; Foda, 1995). Examples of this are given in recent message, in which described, as relaxation effects vip in uterine artery can be explain release of nitrogen oxide ( Jovanovic, 1998). Interesting, that a there is a also certificate vip modulation of no/cgmp in male urogenital function ( Kim, 1994), and a direct the, that treatment of crops smooth muscle cells vagina human vip (0.5 mcm) not is increased levels of camp ( Traish, 1999 ibid).

In this examination of authors of the invention shown, that vip induces vazorelaksatsiyu by means of diffusion condensate intracellular levels of camp. During a series of functional experiments authors-measured blood flow and relaxation of smooth musculature in addition to biochemical measurements of intracellular concentrations of camp. Authors invention is forscoline, activator of adenylate cyclase or mimetic of camp, to reproduce effects of activation of metabolic track camp/adenylate cyclase. Vip and forscoline show identical action on physiological excitation, on vaginal blood flow and relaxation.

Vir (20 mcg/kg) and forscoline (40 nmol/kg) induce significant amplification of vaginal blood flow 13.2 and 12.7 ml/min/100 mg tissue respectively (cm. figure 2 a and 4a). These amplitude change, induced vip and forskolinom, differed insignificantly. These values amplification in largely above basal values of blood flow, when their registered or on inner, or on external vaginal wall.

Vip (0.1 mcm) and forscoline (10 mcm) both considerably Establish intercellular concentration of camp above basal levels of in isolated tissue vagina (cm. figure 46).

Vip (0.1 mcm) and forscoline (10 mcm) Establish basal concentration with 276 nm on 156% and 238%, respectively. Difference in these amounts reflect the difference in used concentrations VIR and forscoline, for example vip in concentration of 0.1 mcm relax preliminarily and reduced isolated vagina approximately 80%, while sufficient for complete relaxation of isolated vagina amount of forscoline is 10 mcm.

Besides, authors of the invention shown, that vip and forscoline induce relaxation of isolated tissue vagina at values ES50 18.8+0.6 nm and 320 ± 20 nm, respectively (cm. figure 4 in).

These data prove, that VIR induces vaginal vazorelaksatsiyu through metabolic path camp/adenylate cyclase. Consequently, this model can be used for producing data, results of whether stimulation of pelvic nerve, i.e. floor excitation, to release vip/activation of metabolic track camp/adenylate cyclase. Besides, may also be examined approaches to increased vaginal blood flow during sexual of excitation, for example by direct or indirect amplification of camp-alarm.

4.4. iAMF is wherein vaginal vazooelaksaiii

Candidates neuromediator and secondary proxy, responsible for amplification of vaginal blood flow during sexual excitation of, at the present time not identified. To present time scientists consolidated his attention on metabolic track nitrogen oxide ( YOUtsGMF. In accordance with the present invention, authors invention demonstrated, that: 1) metabolic path camp/adenylate cyclase mediate vip-induced amplification of vaginal blood flow; 2) vip is endogenous neiromediatorom, which is released during sexual excitation of; and 3) endogenously released vip induces their effects vazoleksatsii by means of diffusion condensate camp.

Neiromediator, responsible for relaxation wall of the vagina, at the present time not identified. Authors of the invention shown, that vip is neiromediatorom, which is released at stimulation of pelvic nerve, and that camp mediate vip- oposreduemuyuvazorelaksatsiyu. Agents, which prevent metabolism vip or directly amplified tsAMFsignalizatsiyu, is increased spurred by pelvic nerve amplification of vaginal blood flow, for example inhibitors of nep or inhibitors of FDEtsamf respectively (cm. the following sections).

In their research authors-find, which can be to exclude as N0 in vip-induced vaginal relaxation. Strongly acting and selective inhibitor of pde type 5 possesses minimum action on vip-induced relaxation of isolated smooth muscle vagina (30% amplification of vip-induced relaxation; cm. table 1).

Table 1: amplification of vip-mediated relaxation of isolated vagina rabbit. This table illustrates percentage of diffusion condensate EC_{50 for} vip-induced relaxation of preliminarily reduced smooth muscle vagina (1 mcm fenilerfin ). Selective inhibitors of FDEtsamf types of 1, 2, 3 and 4 all in significant degree potentsirovali vip-indirect relaxation, and selective inhibitor of FDEtsgmf type 5 or control-carrier does not provided any action on vip-indirect relaxation.

Authors of the invention shown, that vip is also endogenous not adrenergic, not kholinergicheskim ( NANKh ) neiromediatorom, partially responsible for EPS -induced relaxation of isolated smooth muscle vagina. High dose of inhibitor of nitrogen oxide synthase (l- NOARG, 300 mcm) inhibits only 50% EPS -induced relaxations. Inhibitor of nep (1 mcm), which will prevent nep--induced metabolism vip and, consequently, of strengthening vip-signalling, enhances not oksidazotnuyuNANKh relaxation, indutsirovannuyuEPS. Authors of the invention shown, that and N0, and vip is adjusted tone of smooth muscle in the wall of the vagina. Therapeutically will possibly amplifying relaxation of vaginal smooth muscle agents, which amplified mediated no/cgmp and/or vip/camp signalling.

4. 5 vip induiiouetklitoonuyuvazooelaksaiiyu by means of metabolic track iAMF

Candidates neuromediator and secondary proxy, responsible for amplification of kpitornogo blood flow during sexual of excitation, in

time not identified. In parallel with the current examination vaginal blood flow operation was focuses on metabolic track nitrogen oxide ( 1MO )/ cgmp. No messages about the fact, that VIR plays a part in mediating klitornogo blood flow/overfilling blood, at in tissue of the clitoris visualised neurons, containing vip ( Hauser - Kronbergeret al., 1999).

In this examination of authors invention demonstrated, that:

1. Infusion vip enhances klitornyi blood flow.

2. Vip-induced amplification of klitornogo blood is mediated metabolic by camp/ adenilattsikpaza.

3. Vip is endogenous klitornymneiromediatorom, which is released during sexual excitation of:

1. Infusion vip (60-200 mcg/kg, in. in. bolus) induces dependent from concentration of amplification of klitornogo blood flow (Figure 5). 115% amplification klitornogo blood watched after in. in. infusion 200 mcg/kg vip. This considerably exceed control infusion ( gepsalin ).

2. Action vip on klitornyi blood flow can be to imitate by infusion mimetic of camp forscoline (40 nmol/kg in. in. bolus, Figure 5). 156% amplification klitornogo blood watched after in. in. infusion of 40 nmol/kg forscoline. This considerably exceed control infusion ( gepsalin ). Overviews, that amplitude response of same amplitude, induced vip (200 mcg/kg, in. in. bolus) and is comparable with the observed on vaginal blood Figure 2 and 4.

3. Selective inhibitors of nep EC 3.4.24.11 in of clinically pertinent doses considerably enhances induced pelvic nerve amplification klitornogo blood flow (cm. figure 12). Inhibitor of nep raising peak amplification klitornogo blood flow on value up to 131% compared with values of amplification for monitoring carrier.

These data prove, that vip shows ability of strengthening klitornyi blood flow/ vazorelaksatsiyu, and that this effect can be to imitate by activation of metabolic track camp/adenylate cyclase. Opening addition, that inhibitor of nep EC 3.4.24.11 (responsible for metabolism vip)-induced pelvic nerve amplification klitornogo blood flow, demonstrates, that vip is neiromediatorom, which is released during stimulation of pelvic nerve/sexual of excitation.

RPVZh is coupled and, possibly, is a result of lower nnogo genital blood flow. Potential approaches to treatment of this disorder rotate about amplification of genital blood flow. Selecting, that camp is as a proxy genital vazorelaksatsii, and that diffusion lu camp is a result of is from neurons vip, authors of the invention is considered, that if amplified by camp signaling, then, as a result, will be increased genital blood flow, to restoration of genital blood flow up to normal levels and treatment of RPVZh.

In especially preferable aspect, authors invention is selected three target for direct or indirect amplification of camp-mediated vazorelaksatsii -inhibitors FDEtsamf, for example inhibitors of FDEtsamf type 2, inhibitors of nep (EC 3.4.24.11) and receptor antagonists neuropeptide Υ Y1 ( ΝΡΥ Y1).

4.6.1. Inhibitors of neutral endopeptidase (nep EC 3.4.24.11)

Nep EC 3.4.24.11 metaboliziruet vip and, consequently, stops vip-mediated biological activity. Inhibitors of nep potentiate synthesis of endogenous relaxing effect vip, is by excitation time. Clinical effect of this-enhanced overfilling blood genitalia.

Previously in literature is not messages about localization nep EC 3.4.24.11 or its functional role in tissue vagina or role of penis excitation.

Selective inhibitors of nep EC 3.4.24.11 in of clinically pertinent doses considerably enhances induced pelvic nerve amplification of vaginal blood flow (cm. figure 6).

Inhibitor of nep raising peak amplification of vaginal blood flow on value of up to 53% compared with synchronized control amplification. This amplification of sub-maximum frequencies stimulation (for example 4 hz) was dose-dependent, for example dose of 0.1 mg/kg in. in. induced 35.0±7.6% amplification ; dose of 0.3 mg/kg. in. induced 42.60+27.7% amplification; and dose of 1.0 mg/kg in. in. induced 52.8±32.5% amplification. Inhibitors of nep not provided any effect on basal (without stimulation) vaginal blood flow. Consequently, agents according to the present invention rather amplified excitation by means of potentiation of camp signaling, induce excitation in absence of sexual attraction, i.e. by direct diffusion condensate camp of alarm.

Selective inhibitors of nep EC 3.4.24.11 in of clinically pertinent doses considerably enhances induced pelvic nerve amplification klitornogo blood flow (cm. figure 12). Inhibitor of nep raising peak amplification klitornogo blood flow on value up to 131% compared to amplification of checking carrier. Inhibitors of nep not provided any effect on basal (without stimulation) vaginal blood flow. This additionally confirms opinion of authors invention, that agents according to the present invention rather amplified excitation by means of potentiation of camp signaling, induce excitation in absence of sexual attraction, i.e. by direct potentiation of camp of alarm.

Selective inhibitors of nep EC 3.4.24.11 in of clinically pertinent doses is increased vip-induced amplification of vaginal blood flow compared with synchronized controls. In sub-maximum doses vip (for example 6.0 mcg/kg) is considerable taking antilogarithms as in peak amplification (95 ± 6%), and extended duration of this amplification (approximately 140% -from 7 to more than 17 minutes; cm. figure 7). Inhibitors of nep considerably continued duration vip-induced amplification of vaginal blood flow, when their give in combination with a dose vip, which produces maximum values of blood current (approximately 80% increasing-from 11 to 20 minutes).

Selective inhibitors of nep EC 3.4.24.11 in of clinically pertinent doses considerably enhances vip-induced and indirect nerve relaxation in isolated tissue. ES50 for VIR is considerably reduced with 18.8±0.6 nm to 2.9±0.3 nm in the presence of selective inhibitor of nep (1 mcm). Effect of inhibitor nep depends on concentration of.

mrna the intermediary of and protein nep EC 3.4.24.11 is expressed, and it was identified in the vagina human and rabbit with means of Nozern and western-analysis.

4.6.2. Phosphodiesterase inhibitors (pde)

camp cleaved camp-hydro lizuyushchimi pde, i.e. FDEtsamf, inhibitors of FDEtsamf will potentiate synthesis of endogenous vazorelaksiruyushchii effect of camp, is in excitation time. This should give a clinical effect amplification of overfilling blood vagina.

In literature no messages about localization FDEtsamf or functional role of these isoenzymes of in tissue vagina or role of penis excitation. Pde profiling vagina human and rabbit authors of the invention shown, that there are the following izofermentyFDEtsamf 1, 2, 3, 4, 7 and 8. Inhibitors of these FDEtsamf represent potential agents for amplification of vaginal blood flow and/or relaxation of smooth muscles vagina.

Selective inhibitor of FDEtsamf, inhibitor of type 2, in of clinically pertinent doses considerably increases induced pelvic nerve amplification of vaginal blood flow (cm. figure 8). Inhibitor of FDEtsamf type 2 raising peak amplification of vaginal blood flow on 86.8±21.9% compared with values of amplification, observed during synchronized of checking (@ 4 hz).

Selective inhibitor of FDEtsamf type 2 considerably increased duration vip-induced (60 mcg/kg) amplification of peak vaginal blood flow on value above 100% (measured at 50% amplitude; cm. figure 9). Selective inhibitor of FDEtsamf type 2 considerably improves peak amplification of blood flow, induced vip-stimulation (approximately 15 ± 3% [200 mcg/kg]).

Selective inhibitor of FDEtsamf type 2 considerably increased duration vip-induced amplification of peak vaginal blood flow on value above 100% (measured at 50% amplitude; cm. figure 8). Selective inhibitor of FDEtsamf type 2 considerably improves peak amplification in circulation, induced stimulation pelvic nerve (approximately 15 ± 3% [200 mcg/kg] at 4 hz).

Selective inhibitors of FDEtsamf amplified vip- indutsirovannuyu relaxation preliminarily brief isolated smooth muscle vagina (1 mcm fenilerfin ; cm. table 1). Selective inhibitors of FDEtsamf types of 1, 2, 3 and 4 all considerably potentsirovali vip-indirect relaxation (210% @ 76 nm, 130% @ 8 nm, 220% n 3.4 mcm and 160% @ 686 nm taking antilogarithms values of EC_{5 about} vip). These inhibitors either in dose, which, as is known, is selective with respect to specific interest FDEtsamf. Selective inhibitor of pde_tsGmf type 5 or control-carrier does not provided visible action on vip-indirect relaxation.

4.6.3. Receptor antagonists ΝΡΥ Y1

Npy shows inhibitory effect on vip-mediated vazorelaksatsiyu, and antagonists npy Y1 will enable vazorelaksiruyushchemu effect endogenous vip, is by excitation time. Clinical effect of this will amplification of overfilling blood vagina.

In literature no messages about localization npy receptor or functional role of these receptors in tissue vagina or role of penis excitation.

Of investigating receptors expression npy with the help of Nozerni western-analysis of identified, that subtypes receptor npy Υΐ y_{2 and y3 present in} vagina human and rabbit.

Selective inhibitors of npy Y1 in of clinically pertinent doses considerably enhances induced pelvic nerve amplification of vaginal blood flow (cm. figure 10). Antagonist npy Y1 raising peak amplification of vaginal blood flow on value of up to 92% compared to values of synchronized control of amplification. This amplification of sub-maximum frequencies stimulation (for example 4 hz) was dose-dependent, for example dose of 0.01 mg/kg. in. induced 15.8±19.6% amplification ; dose of 0.03 mg/kg. in. induced 35.1 ±17.17% amplification ; dose of 0.10 mg/kg. in. induced 60.1±16.9% amplification ; and dose of 0.3 mg/kg induced 91.9±27.4% amplification (mean ± standard deviation average, U = 3). Antagonists npy Y1 not provided any effect on basal (. without stimulation) vaginal blood flow. This reinforces opinion of authors of the invention, that they rather amplified excitation by means of potentiation of camp signaling, induce excitation in absence of sexual attraction, i.e. by direct diffusion condensate camp of alarm.

Search for oral therapy RPVZh preferably, that is not associated with it undesirable cardio-vascular effects, for example of action on blood pressure or heart rate. In their research authors-find, that infusion vip considerably is reduced average arterial blood pressure (cm. figure 3) and considerably is increased heart rate. Consequently, in especially preferable aspect, agent is vip. Stimulation of pelvic nerve and inhibitors of FDEtsdmf and nep, however, no any effect on blood pressure. In dose of 6.0 mcg/kg vip (in. in. ) had considerable Your lu mean arterial blood pressure 13.2±0.7 mm Hg. ct. and considerable diffusion lu frequency of heart contractions 16 ± 4 impacts in minute. In more high doses, such as 60.0 mcg/kg, vip (in. in. ) had considerable Your lu average arterianogo blood pressure 14.7±1.37 mm Hg. ct., and this was is coupled with considerable diffusion savings frequency of heart contractions 111 ± 30 impacts in minute, that then enhances mean arterial blood pressure of blood 8.5±1.4 mm Hg. ct.

A series of above-mentioned compounds tested in accordance with the present invention, and was recognized, that they are effective in accordance with the present invention, i.e. they can act as Ptsamf, to treat ZhSD, in particular RPVZh.

These compounds included includes:

Compound of the formula la (" Fla")-i.e. 5 - [4 - (diethylamino) benzyl] - 1-methyl-3-propyl -6,7-dihydro -1h-pyrazolo [4,3-c1] pirimidin-7-on. Compound Fla can be obtained as ukaazano in er-a- 0911333 (is an example of 50 this application).

Compound of the formula ii (" FII")-i.e. 9 - (1-acetyl-4-phenylbutyl) - 2 - [(3.4-dimethoxyphenyl) methyl] - 1_{1 9-dihydro} -6h- purin-6-on. Compound FII can be obtained as specified in er-a- 0771799 (is an example of 100 this application).

Compound of the formula iii ("fill")-i.e. milrinon. Compound fill is commercially available product.

Compound of the formula iv (" FIV")-i.e. rolipram. Compound FIV is commercially available product.

Compound of the formula v ("fv")-i.e. 3 - [[ [1 - (2-carboxy-4- pentenil ) cyclopentyl] carbonyl] amino]-cyclohexane carboxylic acid 1 ethyl ester. Compound fv can be obtained as specified in er-a- 0274234 (is an example of 300 of this application).

Compound of the formula vi (" FVI")-i.e. 3 - [[ [1 - (2-carboxy-4- pentenil ) cyclopentyl] carbonyl] amino]- tsiklogeksankarbonovaya acid. Compound FVI can be obtained as specified in er-a- 0274234 (is an example of 379 this application)

In particular, Fla, FII, fill and FIV are inhibitors of FDEtsdmf. Fla and: FDE1, FII and: FDEI, fill and '. FDESh and FIV and: FDE1U.

Data for these compounds are presented above in the previous partition examples-for example, cm. table 1.

Being clearly, that these inhibitors of FDEtsdmf amplified vip-induced relaxation of isolated tissue.

Compound FII, which is selective and: FDEI, -enhanced vip-induced amplification of vaginal blood flow in of clinically pertinent doses.

Compound FII also increases induced pelvic nerve amplification of vaginal blood flow in of clinically pertinent doses.

Compound fv and FVI are by selective inhibitors of nep EC 3.4.24.11.

Data, presented above in the previous partition examples, are data for compound FVI. However similar results were produced for connection fv.

Being clearly, that compound fv and FVI is increased vip-induced amplification of vaginal blood flow in of clinically pertinent doses.

Compound fv and FVI also is increased by pelvic nerve amplification of vaginal blood flow in of clinically pertinent doses.

Compound fv and FVI also amplified vip- indutsirovannuyu and mediated nerve relaxation of isolated tissue in of clinically pertinent doses.

Additional compound, which tested and proved, that they are effective, include:

2 - [(1 - {[ (^{1-benzyl -6}-oxo -1,6-dihydro-3-pyridinyl) amino] carbonyl} cyclopentyl) methyl] - 4- metoksibutanovuyu acid (F57),

2- {n-( {[^3- (2-oxo-1-pyrrolidinyl) propyl] amino} karboniltsiklopentil ] methyl} - 4- fenilbutanovuyu acid (F58),

(+) 2 - {[ 1 - ({[ 2 - (hydro ksimetil ) - 2,3-dihydro -1h- inden-2-il ] amino} carbonyl) cyclopentyl] methyl} - 4- fenilbutanovuyu acid (F59),

2 - [(1 - {[ (5-methyl -1, 3, 4- tiadiazol-2-il ) amino] carbonyl} cyclopentyl) methyl] - 4- fenilbutanovuyu acid (F60),

cis-3 - (2-methoxyethoxy) - 2 - [(1 - {[ (4 - {[ (phenylsulfonyl) amino] carbonyl} cyclohexyl) amino] carbonyl} cyclopentyl) methyl] propane acid (F61),

(+) - 2 - {[ 1 - ({[ 2 - (hydro ksimetil ) - 2,3-dihydro -1h- inden-2-il ] amino} carbonyl) tsikpopentil ] methyl} pentanoic acid (F62),

(+) - 2 - [(1 - {[ (5-ethyl -1, 3, 4- tiadiazol-2-il ) amino] carbonyl} cyclopentyl) methyl] pentanoic acid (F63),

2- ({1 - [3- benzilanilino ) carbonyl] cyclopentyl} methyl) pentanoic acid (F64),

2 - [(1 - {[ (1-benzyl-6-oxo -1,6-dihydro-3-pyridinyl) amino] carbonyl} cyclopentyl) methyl] pentanoic acid (F65),

2- {[ '^|-( {[ (^{1^} > 33,4k) - 4 - (aminocarbonyl) - 3-butylcyclohexyl] amino} carbonyl) cyclopentyl] methyl} pentanoic acid (F66).

Each of compounds F57-66 represents and: nep.

In the following comments examples of preparing are examples synthesis of intermediate compounds, and examples are examples of synthesis of corresponding compounds according to the present invention.

Is an example of 1

2-g and-th (1-benzyl-6-oxo -1,6-dihydro-3- piridinil1amino1karbonilU-

cyclopentyl) metil1 -4- metoksibUtanovaya acid (F571

Mixture of benzyl ether from example of producing 1 (1/62) (850 mg, 1.64 mmol) and 5% palladium on carbon (250 mg) in 40% aqueous ethanol (21 ml) hydrogenated at 30 pounds/kv. inch (206.84 kPa ) and at room temperature for 30 minutes. This reaction mixture filtered through Hyflo ®, and filtrate evaporated at lower nnom pressure. Residual foam ochishchali by column chromatography on silica gel, using dichloromethane: methanol (97:3) as eluent, to obtain compound of, said in the heading of the, in the form of white foam, 550 mg, 79% ;^{1 n} nmr (dmso-b_6, 300 MGts ) b: 1.24-2,17 (T, 12n), 2.18-2,31 (T, 1h), 3.07 (s, value), 3.21 (t, 2h), 5.08 (s, 2h), 6.63 (d, 1h), 7.23-7,41 (T, 5h), 7.72 (d, 1h), 8.24 (s, 1h).

Analysis: show: with, 67.46 ; n, 7.18; N, 6.24. C24h30n2o5 require with, 67.58; n, 7.09 ; N, 6.57%.

Is an example of 2 2 - ([1 - ( 1GZ -(2-oxo-1- pirrolidinil1propil1aminokarboniltsiklopentil1metilM - fenilbutanovaya acid (F581

2- {[ 1 - ({[ 2 - (hydro ksimetil ) -2,3-dihydro -1 H- inden-2-il ] amino} carbonyl) cyclopentyl] methyl} - 4- fenilbutanovuyu acid ( WO 9110644) can be to purify using standard methods VEZhKh, using column ad (atmospheric pressure) and mixture of hexane: isopropanol: trifluoroacetic acid (70:30: 0.2) as eluent, to obtain compound of, said in the heading of the example 3,99.5% its (enantiomeric purity) ;_{d =} + 9,1 ° (c = 1,76 in ethanol).

Mixture of benzyl ether from example of preparing 4 (4/70) (187 mg, 0.39 mmol) and 10% palladium on carbon (80 mg) in ethanol (20 ml) hydrogenated at 60 pounds/kv. inch (413.69 kPa ) for 18 hours. Analysis of TSKh (small sloinaya chromatography) showed residual initial material, therefore a glucose additional 10% palladium on carbon (100 mg), and reaction continued for another 5 hours. Analysis of TSKh again showed residual initial material, therefore a glucose additional catalyst (100 mg), and hydrogenation of continued for 18 hours. This mixture filtered through Arbocel ®, filtrate consolidated at lower nnom pressure and subjected to azeotropic distillation with dichloromethane. Crude product ochishchali chromatography on silica gel, using column Biotage ® and dichloromethane: methanol (95:5) as eluent, to obtain compound of, said in the heading of the, in the form of a transparent oil, 80 mg, 53% ;^{1 n} nmr ( CDCI_3, 300 MGts ) 5 : 1.51-1,89 (m, 9h), 2.03 (T, 1h), 2.20 (T, 1h), 2.40 (T, 2h), 2.60 (T, 5h), 7.15-7,30 (T, 5Η ) ;LRMS (liquid stnaya chromatography-mass-spectrometry):

m/z 387.8 (min⁺⁾.

Is an example of 5 gshs -3-asaho ge-methoxyethoxy ^-gi-and ^- MFenilsulFonil ^ aminokarbonilUtsiklogeksil1amino1karbonil } iiklopentil ^ metil1propanovaya acid CF611

2- {[ and {[ 2 - (hydro ksimetil ) - 2,3-dihydro -1h- inden-2-il ] amino} carbonyl) tsikpopentil ] methyl} pentanoic acid ( WO 9110644) additionally ochishchaliVEZhKh, using column ad and mixture of hexane: isopropanol: trifluoroacetic acid (90:10: 0.1) as eluent, to obtain compound of, said in the heading of the example b, 99% of its ; [_and] = + 10.4 ° (with = 0,067, ethanol).

Is an example of 7 (+ ') -2-G (1- Sh5 -ethyl -1,3.4-thiadiazol-2- il1amino1karbonil } iiklopentil )-methyl! pentanoic acid (F63)

Acid from example of preparing 18 (18/πρ.4) (824 mg) additionally ochishchaliVEZhKh, using column ad and mixture of hexane: isopropanol: trifluoroacetic acid (85:15: 0.2) as eluent, to obtain compound of, said in the heading of the example 7, in the form of white foam, 386 mg, 99% of its;^{1 n} nmr ( CDCI_3, 400 MGts ) 5 : 0.90 (t, value), 1.38 (m, 6h), 1.50-1,79 (T, 9h), 2.19 (T, 1h), 2.30 (T, 1h), 2.44 (T, 1h), 2.60 (T, 1h), 2.98 (q, 2h), 12.10-12,27 (bs, 1Η ) ;LRMS : m/z 338 (min⁺⁾; and [α) ο = + 3,8^{0 (} with = 0,1, methanol).

Is an example of 8 2 - ((1 - [3- Benzilanilino1karbonil1iiklopentil > metil1pentanovaya acid ( em)

Mixture of gasoline ether from example of preparing 10 (10/53) (1.3 g, 2.47 mmol) and 5% palladium on carbon (130 mg) in water (10 ml) hydrogenated at 30 pounds/kv. inch (206.84 kPa ) and at room temperature for 2 hours. This reaction mixture filtered through Arbocel ®, filtrate consolidated at lower nnom pressure, and residue triturated with dichloromethane. Residual resin triturated with ether, then with hexane and was dried at 50° with to obtain compound of, said in the heading of the, in the form of hard substance, 0.79 g, 81% ;^{1 n} nmr ( CDCI_{3 >} 300 MGts ) b: 0.95 (t, value), 1.24-1,51 (m, value), 1.58-1,80 (T, 7h), 1.88 (dd, 1h), 2.15 (T, 2h), 2.24 (T, 1h), 2.48 (T, 1h), 4.00 (s, 2h), 6.98 (d, 1h), 7.24 (T, 6h), 7.40 (T, value); analytically show: with, 75.48; n, 7.76 ; N, 3.59. C_{2 5H3 iNO3 -0,25H2 o} require with, 75.44; n, 7.98 ; N, 3.51%.

Is an example of 9

2-f (1 - ([(1-benzyl-6-oxo -1,6-dihydro-3-pyridinyl) amino] carbonyl v iiklopentil1metil1pentanovaya acid ( F651

Is an example of 10 2 - ([1 - (( [(1p, 3 $, 4R ) - 4 - ( aminokarbonil1 -3- butiliiklogeksil1amino }-carbonyl) tsiklopentil1metil ) pentanoic acid (F66)

Compound of general formula ic, i.e. compound of general formula i, where g^{1 represents} propyl, obtained from corresponding tert butyl ester, following technique, similar technique, described in example of preparing 14 (14/ primer1 ).

(^{C w}:

Is an example of production of 1 (1/621

Benzilovyiefir2 -g/1 - {[ Y1-benzyl-6-oxo -1,6-dihydro-3- piridinil1amino1 -carbonyl} tsiklopentil1metil ] - 4- metoksibutanovoi acid

Oksalilkhlorid (0.26 ml, 3.00 mmol) a glucose to cooled in ice solution of 1 - {2 - [(benzyloxy) carbonyl] - 4-methoxybutyl} cyclopentane carboxylic acid (ep 274234) (1.0 g, 3.00 mmol) and n. n-dimethylformamide (2 drops) in dichloromethane (20 ml), and this reaction mixture stirring at room temperature for 2 hours. This solution consolidated at lower nnom pressure, and residue was subjected to azeotropic distillation with dichloromethane (3x10 ml). Product was dissolved in dichloromethane (20 ml), then was cooled in ice bath. A glucose amine from example of producing 2 (2/28) (600 mg, 3 mmol) and n-methylmorpholine (0.6 ml, 5.45 mmol), and this reaction mixture stirring at room temperature for 18 hours. Reaction mixture consolidated at lower nnom pressure and distribute between water and ether. Organic layer is washed with hydrochloric acid (2 n. ), sodium bicarbonate solution, water, was dried (MgS04) and evaporated at lower nnom pressure. Residual green solid substance ochishchali by column chromatography medium pressure on silica gel, using ethyl acetate: hexane (90:10) as eluent, to obtain compound of, said in the heading of the, 880 mg, 57%.^{1 n} nmr ( CDCI3, 300 MGts ) b: 1.37-2,28 (T, 12n), 2.46-2,64 (T, 1h), 3.20 (s, value), 3.31 (T, 2h), 4.97 (dd, 2h), 5.08 (dd, 2h), 6.57 (d, 1h), 7.12 (T, 1h), 7.18-7,48 (T, youn), 8.08 (d, 1h).

Is an example of preparing 2 (2/28)

5-amino-1-benzyl -2 (1 NUpiridinon

Mixture of 1-benzyl-5-nitro -1h-pyridine-2-one ( JustusLiebigs ann. Chem. 484 ; 1930 ; 52) (1.0 g, 4.35 mmol) and granulated of tin (3.5 g, 29.5 mmol) in concentrated hydrochloric acid (14 ml) was heated UP by at 90° with for 1.5 hours. Cooled solution diluted water, neutralized remnant, using solution of sodium carbonate, and extracted with ethyl acetate (the whole 250 ml). Combined organic extracts filtered, was dried ( MgS0₄₎ and evaporated at lower nnom pressure to obtain compound of, said in the heading of the, in the form of pale-of green of solid substance (with time stanovivshegosya blue), 440 mg, 51%.^{1 n} nmr ( CDCI_{3l 250}MGts ) δ: 4.12-4,47 (bs, 2h), 5.00 (s, 2h), 6.31 (d, 1h), 6.86 (s, 1h), 7.07 (T, 1h), 7.14-7,42 (T, 5h).

Is an example of preparing 3 (3/67)

Benzyl esir2-G (1 - ({[ 3 - (2-oxo-1-pyrrolidinyl) propil1amino ) karboniliiklopentilCh - metilM -phenylbutanic acid

1- (3-dimethylaminopropyl) - 3-ethylcarbodiimide hydro chloride (1.06 g, 5.53 mmol), 1-hydro ksibenzotriazola hydrate (0.60 g, 4.44 mmol) and 4-methylmorpholine (0.56 g, 5.54 mmol) in series a glucose to cooled solution of 1 - {2 - [(benzyloxy) carbonyl] - 4-phenylbutyl} cyclopentane carboxylic acid (ep 274234) (1.5 g, 3.94 mmol) in dry dichloromethane (15 ml) at room temperature, and then a glucose N( 3- aminolropil ) - 2-pyrrolidinone (0.56 g, 3.94 mmol), and this reaction mixture stirring at room temperature for 18 hours. This mixture is washed with water, 2h hydrochloric acid, saturated aqueous solution of sodium bicarbonate, and then was dried ( MgS0₄₎ and evaporated at lower nnom pressure. Residual yellow oil ochishchali by column chromatography on silica gel, using ethyl acetate: pentane (50:50) as eluent, to obtain compound of, said in the heading of the, in the form of transparent resin, 800 mg, 40% ;^{1 n} nmr ( CDCI_3, 300 MGts ) d: 1.37-2,20 (m, 16Η), 2.34-2,58 (m, 5Η), 2.92-3.46 (m, 6Η ), 5.07 (d, 1Η ), 5.18 (d, 1Η ), 6.98-7.47 (m, 10Η ).

Is an example of preparing 4 (4/70)

Benzyl ester of 2-K1 - (g (5-methyl -1.3 .4- tiadiazol-2-il ) amino1kaobonilUiiklopentil1metil1 -4-phenylbutanoic acid salts

Compound, specified in the heading of the, obtained in the form of a transparent oil with output of 74% from 1 - {2 - [(benzyloxy) carbonyl] - 4-phenylbutyl} cyclopentane carboxylic acid (ep 274234) and 2-amino-5-methyl -1, 3, 4-thiadiazole, following technique, similar technique, described in the example of producing^{5 (5/68);} 1 n nmr_{( CDCI} 3,400 MGts ) δ: 1.58-1,76 (m, 7h), 1.83-1.98 (T, value), 2.03 (T, 1h), 2.20 (T, 1h), 2.35 (T, 1h), 2.44 (T, value), 2.65 (s, value), 5.02 (dd, 2h), 7.00 (d, 2h), 7.15 (T, 1h), 7.19 (T, 2h), 7.35 (T, 5Η ) ;LRMS : m/z 478.7^(min +).

Is an example of preparing 5 (5/681

Benzyl ester of 2 - ( G1 -{ GZ -( metilaminoUZ - oksopropil1aminokarbonilUtsiklopentil1metil14 - senilbutanovoi acid

1- (3-dimethylaminopropyl) - 3-ethylcarbodiimide hydro chloride (122 mg, 0.64 mmol), 1-hydro ksibenzotriazola hydrate (86 mg, 0.64 mmol) and 4-methylmorpholine (173 mcl, 1.59 mmol) in series a glucose to cooled _AOE280A2AO> solution of 1 - {2 - [(benzyloxy) carbonyl] - 4-phenylbutyl} cyclopentane carboxylic acid (ep 274234) (202 mg, 0.53 mmol) in n. n-dimethylformamide (5 ml) at room temperature, and then a glucose amine hydro chloride from example of preparing 6 (6/23) (146 mg, 1.06 mmol), and this reaction mixture stirring at 90° with for 18 hours. Cooled solution consolidated at lower nnom pressure, and residue distribute between water (20 ml) and ethyl acetate (100 ml). Layers shared, organic phase is washed with water (3x30 ml), brine (25 ml), was dried ( MgS0₄₎ and evaporated at lower nnom pressure to obtain transparent oil. This crude product ochishchali by column chromatography on silica gel, using dichloromethane: methanol (98:2) as eluent, to obtain compound of, said in the heading of the, in the form of colourless oil, 162 mg, 67% ;^{1 n} nmr ( CDCI_3, 400 MGts ) 6 : 1.38-1,53 (m, 2h), 1.53-1,96 (T, 8h), 2.02 (T, 2h), 2.27 (t, 2h), 2.46 (T, value), 2.76 (d, value), 3.44 (T, 2h), 5.13 (s, 2h), 5.79 (bs, 1h), 6.38 (T, 1h), 7.06 (d, 2h), 7.18 (T, 1h), 7.22 (T, 2h), 7.38 (T, 5h); LRMS : m/z 465.5 (min⁺⁾.

Mixture benzilkarbamata from example of preparing 7 (7/13) (7.92 g, 33.5 mmol) and 5% of palladium on carbon (800 mg) in ethanol (300 ml) hydrogenated at 50 pound/kv. inch (344.74 kPa ) and at room temperature for 4 hours. This reaction mixture filtered through Arbocel ®, washing with ethanol, and to the resulting filtrate a glucose 1h hydrochloric acid (36.9 ml, 36.9 mmol). This solution evaporated at lower nnom pressure, and residue was subjected to azeotropic distillation with dichloromethane to obtain compound of, said in the heading of the, in the form of colorless foam, 4.66 g.^{1 n} nmr ( flMCO -d_{6) 300}MGts ) 6 : 2.46 (t, 2h), 2.60 (s, value), 2.95 (m, 2h), 7.98-8.16 (T, 2h).

Is an example of preparing 7/7/13)

Mixture of [m-[ (benzyloxy) carbonyl]-p-alanine (10 g, 44.8 mmol), methylamine hydro chloride (3.33 g, 49.28 mmol), 1-hydro ksibenzotriazola hydrate (6.05 g, 44.8 mmol), 1 - (3-dimethylaminopropyl) - 3-ethylcarbodiimide hydro chloride (10.3 g, 53.76 mmol) and n-methyl morpholine (11.33 ml, 103 mmol) in dichloromethane (200 ml) stirring at room temperature for 18 hours. Obtained sediment filtered to obtain desired product in the form of colorless foam, and filtrate evaporated at lower nnom pressure. Residue ochishchali by column chromatography, using gradient elution with mixture of ethyl acetate: hexane (with 90:10 to 100:0) to produce additional product, 7.96 g, total output of 75% ;^{1 n} nmr ( CDCI_3, 300 MGts ) δ: 2.42 (t, 2h), 2.80 (s, value), 3.50 (m, 2Η ), 5.21 (s, 2Η ), 5.49 (bs, 1Η ), 5.63 (bs, 1Η ), 7.36 (m, 5Η ) ; analysis: show: with, 60.68 ; n, 7.00; N, 11.95. C_{12 h16 n2 03 require} with, 61.00; n, 6.83 ; N, 11.86%.

Is an example of preparing 8 (8/66)

gshs - lyuet - Butilovyiefir3 -(2-μ ℮τοκοη3τοκοη1 -2-γ (1 {g ( Fenilsulsonil1 - aminokarbonilk1iklogeksil ) amino1karbonilU

iiklopentil1metil1propanovoi acid

Is an example of preparing 9 (9/631

4- ([and-(3 - / iii > el7 -butoxy-2- K2 - metoksietoksi1metil1 -3- oksopropilU

tsiklopentilkarbonil1aminok ! iklogeksankarbonovaya acid

where

obtained from chloroanhydride of acid from example of preparing 11 (11/3) and corresponding amine, following technique, similar technique, described in example of preparing 12 (12/52).

Is an example of preparing 11 (11/31

Benzyl ester 2-ff1 - ( khlorkarbonil1tsikpopentil1metil ) pentanoic acid

Oksalilkhlorid (1.15 ml, 13.2 mmol) a glucose to cooled in ice solution of 1 - {2 - [(benzyloxy) carbonyl] pentyl} cyclopentane carboxylic acid (ep 274234) (2.0 g, 6.3 mmol) in dry dichloromethane (20 ml), and this solution stirring at room temperature for 2 hours. This reaction mixture consolidated at lower nnom pressure, and residue was subjected to azeotropic distillation with dichloromethane (zkh dye printing paste) to obtain compound of, said in the heading of the, in the form of golden oil, 2.1 g,^{1 n} nmr ( CDCI_3, 300

Is an example of preparing 12 (12/52)

Benzyl ester of 2 - ((1 - [(3- piridinilaminokarbonil1iiklopentilU methyl) pentanoic acid

Triethylamine (0.11 ml, 0.78 mmol) a glucose to mixture chloroanhydride of acid from example of preparing 11 (11/3) (200 mg, 0.60 mmol) and 2-aminopyridine (61 mg, 0.65 mmol) in dichloromethane (3 ml), and this reaction stirring at room temperature for 16 hours. This mixture evaporated at lower nnom pressure, residue distribute between solution of sodium bicarbonate (5 ml) and ethyl acetate (20 ml) and shared layers. Organic phase was dried (MgS04) and evaporated at lower nnom pressure to produce resin. This crude product ochishchali by column chromatography on silica gel, using ethyl acetate as eluent, to obtain compound of, said in the heading of the, 130 mg;^{1 n} nmr ( CDCI3, 400 MGts ) d: 0.82 (t, value), 1.21 (m, value), 1.40 (m, 1Η ), 1.43-1,72 (m, 6Η), 1.81 (d, 1Η ), 1.98 (m, 1Η ), 2.18 (m, 1Η ), 2.24 (m, 1Η ), 2.46 (m, 1Η ), 4.98 (m, 2Η ), 7.20-7,38 (m, 6Η), 7.42 (s, 1Η ), 8.06 (d, 1Η ), 8.35 (d, 1Η ), 8.56 (s, 1Η ).

Is an example of preparing 13 (13/561

The following compound:

where

obtained from chloroanhydride of acid from example of preparing 11 (11/3) and corresponding amine, following technique, similar technique, described in example of preparing 12 (12/52).

U rimer of preparing 14 (14/is an example of 1) 2 - ({1 - [(1.3-benzodioxol-5-ylamino) karbonil1tsiklopentil ) bromide pentanoic acid

Is an example of preparing 15 (15/34)

The following compound:

obtained from acid from example of producing 16 (16/1) and corresponding amine compound, following technique, similar technique, described in example of preparing 17 (17/33).

Mixture of 1 - [2 - (thread? -butoxycarbonyl) - 4- pentenil ]-cyclopentane carboxylic acid (ep 274234) (23 g, 81.5 mmol) and 10% of palladium on carbon (2 g) in dry ethanol (200 ml) hydrogenated at 30 pounds/kv. inch (206.84 kPa ) and at room temperature for 18 hours. This reaction mixture filtered through Arbocel ®, and filtrate evaporated at lower nnom pressure to obtain yellow oil. This crude product ochishchali by column chromatography on silica gel, using etilatsetag. pentane (40:60) as eluent, to obtain the desired product in the form of a transparent oil, 21 g, 91% ;^{1 n} nmr ( CDCI₃₎: 0.86 (t, value), 1.22-1,58 (m, 15n), 1.64 (T, 4h), 1.78 (dd, 1h), 2.00-2,18 (T, value), 2.24 (T, 1h); LRMS : m/z 283 (m-n) '.

Is an example of preparing 17 (17/331

yayuet -butyl ester of 2 - ([1 - ((and-(hydro ksimetil ) iiklopentil1aminoU carbonyl ') iiklopentil1metil ] lentanovoi acid

Is an example of production of 18 (18/is an example of 4)

Compound mentioned below of the formula obtained from corresponding mpem -butyl ester, following technique, similar technique, described in example of preparing 14 (14/is an example of 1).

3 = perekristallizovano from ester

Is an example of preparing 19 (19/is an example 211 2 - (( 1-G (3- Benzilanilino ) karbonil1iiklopentil ) methyl) pentanoic acid

Mixture of benzyl ether from example of preparing 10 (10/53) (1.3 g, 2.47 mmol) and 5% of palladium on carbon (130 mg) in water (10 ml) and ethanol (40 ml) hydrogenated at 30 pounds/kv. inch (206.84 kPa ) and at room temperature for 2 hours. This reaction mixture filtered through Arbocel ®, filtrate consolidated at lower nnom pressure, and residue triturated with dichloromethane. Residual resin triturated with ether, then with hexane and was dried at 50° with to obtain compound of, said in the heading of the, in the form of hard substance, 0.79 g, 81% ;^{1 n} nmr ( CDCb, 300 MGts ) δ: 0.95 (t, value), 1.24-1,51 (m, value), 1.58-1,80 (T, 7h), 1.88 (dd, 1h), 2.15 (T, 2h), 2.24 (T, 1h), 2.48 (T, 1h), 4.00 (s, 2h), 6.98 (d, 1h), 7.24 (T, 6h), 7.40 (T, value); analysis: show: with, 75.48 ; n, 7.76; N, 3.59. C25H_{3 iNO3} -0,25H_{2 o} require with, 75.44; n, 7.98 ; N, 3.51%.

Analysis of apf

PRODUCTION AND ANALYSIS OF SOLUBLE ANGIOTENSIN-CONVERTING ENZYME (APF) FROM OF CORTICAL SUBSTANCE PIG KIDNEY AND HUMAN.

Soluble apf obtained from of cortical substance kidney and analyzed by measuring speed of splitting substrate apf Abz -gly-p- nitro -phe-pro-oh with creation of its fluorescent product Abz -gly.

1. MATERIALS

Entire water twice deionizirovana.

1.1 kidney human us ( Pennsylvania. U.s.a. ) or uk

Human Tissue Bank (Uk NTV )

1.2 kidney pig Sigma (Α2580)

1.3 buffer homogenization of -1

100 mm mannitol and 20 mm tris n ph 7.1

2.42 g tris ( Fisher t/ R630 /60) is diluted in 1 liter of water and is brought ph to 7.1, using 6m HCI at room temperature. To this is added 18.22 g mannitol ( Sigma m -9546).

1.4 buffer homogenization of -2

100 mm mannitol, 20 mm tris-ph 7.1 and 10 mm MdS ^- bIgO ( FisherM0600 /53)

To 500 ml of buffer homogenization of -1 (1.3) is added 1.017 g MgCI_2.

1.5 tris buffer (apf buffer)

50 mm tris and 300 mm NaCI n ph 7.4

50 ml of 50 mm tris, ph 7.4, ( SigmaT2663 ) and 17.52 g NaCI ( Fisher S/3160/60) diluted to 1000 ml in water.

1.6 substrate ( Abz -d-gly-p- nitro -phe-pro-oh) ( Bachem m -1100)

Substrate apf is stored in the form of powder at -20 ° with. 2 mm initial solution is prepared, resuspension substrate in apf buffer; its because mixing on vortex or process ultrasound.

Aliquots by 400 mcl of this 2 mm initial solution stored at -20 ° with up to one month.

1.7 total product

Samples, corresponding to 100% conversion of substrate in product, include plotting board, determination % conversion of substrate (cm. computing). Total product is formed incubation 1 ml of 2 mm substrate with 20 mcl initial solution enzyme in for 24 hours at 37° with.

1.8 feet-solution

For preparation of 2 mm solution of 0,5m EDTA ( Promega cas [ 6081/92/6 ]) is diluted 1:250 in apf buffer.

1.9. Dimethylsulfoxide (Dmso).

1.10. Magnesium chloride- MgCl2.6H_{2 0} ( Fisher Μ0600/53).

1.11. Black 96 well flat bottom analytical boards ( Costar 3915 or Packard ).

1.12. Topseal a ( Packard 6005185).

1.13. Centrifuge test tubes.

2.1. Centrifuge Sorvall RC-5B (rotor SS34 GSA, preliminarily cooled to 4° with).

2.2. Mixer mini homogenizer Braun.

2.3. Centrifuge Beckman CS-6R.

2.4. BmgFluostar Galaxy.

2.5. Jointing incubator Wesbart 1589.

3. PROCEDURES

3.1. PREPARING THE PREPARATION OF TISSUE

3.2. Apf human is produced from of cortical substance kidney, using adapted method Booth, A.G. & Kenny, A.J. (1974) Biochem. j. 142, 575-581.

3.3. Frozen pochkam is allowed to thaw at room temperature, and korkovoe substance cut off from brain substance.

3.4. Korkovoe substance finely cut and homogenized approximately in 10 volumes of buffer homogenization of 1 (1.3), using mini homogenizer Braun (2.2).

3.5. To gomogenatu is added magnesium chloride (1.11) (20.3 mg/g of tissue) and stirred in ice bath for 15 minutes.

3.6 homogenate is centrifuged at 1500 g (3820 rev/min) for 12 minutes in centrifuge Beckman (2.3), after which the supernatant are transferred in pure centrifuge test tube, and precipitate discarded.

3.7 supernatant centrifuged at 15000 g (12100 rev/min) for 12 minutes in centrifuge Sorvall (2.1), and supernatant discarded.

3.8 pale-pink layer on the surface of the residue is extracted and is resuspended in buffer homogenization of 2 (1.4) (5 ml buffer on 1 g of tissue).

3.9. This suspension is centrifuged at 2200 g (4630 rev/min) for 12 minutes in centrifuge Beckman, and residue discarded.

3.10. Supernatant centrifuged at 15000 g (12100 rev/min) for 12 minutes, using centrifuge Sorvall, and supernatant discarded.

3.11. Final residue is resuspended in buffer homogenization of 2 (0.5 ml buffer on 1 g of tissue). Homogeneous suspension is obtained, using mini homogenizer Braun. Then it is frozen in aliquots by 100 mcl, to to analyze on activity apf.

4. DETERMINATION OF ACTIVITY OF APF

Activity apf, preliminarily divided into aliquots, is determined by its ability to cleave specificity to apf peptide substrate.

Pork apf (1.2) defrosted and is resuspended in apf buffer (1.6) at 0.004 U./ mcl, and then is frozen in aliquots at 50 mcl.

4.1. Is prepared 4% solution dmso/apf buffer (4 ml dmso in 96 ml apf buffer).

4.2. Substrate (1.7), total product (1.8) and enzyme (1.1, 1.2, 1.3) is left on ice to thawing.

4.3. In each is added 50 mcl 4% solution of dmso/apf buffer.

4.4 . 2 mm initial solution substrate diluted 1:100 to obtain 20 mcm of solution. In each is added 100 mcl 20 mcm substrate (final concentration in analysis of 10 mcm).

4.5. For initiating reaction is added 50 mcl dilution series of enzyme (used is usually 1:100,1:200,1:400,1:800,1:1600 and 1:3200). In pure holes is added 50 mcl apf buffer.

4.6 . 2 mm total product is diluted 1:200 to make 10 mcm of solution. In the first four holes new board is 200 mcl 10 mcm product.

4.7. Boards are incubated at 37° with in shaking incubator for 60 minutes.

4.8. Enzymatic reaction is stopped addition of 100 mcl 2 mm EDTA in apf buffer and incubated at 37° with in shaking incubator for 20 minutes until red color on BMGFluorstar Galaxy ( ℮χ320 / ℮ γ U 420).

5. ANALYSIS ON INHIBITION APF

5.1. Initial solutions substrate, total product and enzyme is left on ice to thawing.

5.2. Is prepared initial solutions of compound in 100% dmso and diluted with 1:25 in apf buffer with produce 4% dmso of solution. All further dilution is carried out in solution of 4% dmso/apf buffer (4 ml dmso in 96 ml apf buffer).

5.3. In 96-alveolar plotting board is 50 mcl compound in two repetitions and 50 mcl 4% dmso/apf buffer in control and pure holes.

5.4. Stage 5.2 and 5.3 can be carried out either manually, or using multiprobnye robots Packard.

5.5 . 2 mm initial solution substrate diluted 1:100 in apf buffer with produce 20 mcm solution of (10 mcm final concentration in analysis of) (110 mcl 2 mm substrate, added to 10.89 ml buffer, is sufficient for 1 plate).

5.6. Initial solution of enzyme is diluted in apf buffer, as defined tests on activity (4. Q).

5.7 . 2 mm initial solution total product is diluted 1:200 in apf buffer with produce 10 mcm of solution. In the first four holes separate board is 200 mcl.

5.8 . 0.5 m initial solution EDTA diluted 1:250 to obtain 2 mm initial solution (44 mcl EDTA on 10.96 ml apf buffer).

5.9. In each well of 96 - ; board is added the following reagents:

5.10 . 50 mcl the highest concentration of each compound, used in analysis, is added in two repetitions in the same 96-alveolar plotting board, that and of summary (5.7). 150 mcl apf buffer is added for determining fluorescence of any compound.

5.11. Reaction is initiated addition of apf of enzyme, and then are incubated at 37° with for 1 hour in shaking incubator.

5.12. Reaction is stopped addition of 100 mcl 2 mm EDTA and incubated at 37° with in shaking incubator for 20 minutes until red color on BMGFluorstar Galaxy ( ℮χ320 / ℮γπ420 ).

6. CALCULATION OF

Activity apf of enzyme is determined in the presence and absence compounds and is expressed in percents.

Fu = unit fluorescence

(1) control activity in % (conversion of enzyme)

Average fu testing runs of-average fu pure testing runs khYuO average fu summary-average fu pure testing runs

Average fu compound-average fu pure testing runs khYuO average fu summary-average fu pure testing runs

% Activity with inhibitor khYuO

% Control activity

or

Average fu compound-average fu pure testing runs khYuO average fu testing runs of-average fu pure testing runs

(4) % of inhibition = 100% control

(5) for fluorescent compounds average fu pure testing runs, containing compound (5.10), is subtracted from middle fu from values of compound, used for calculating % activity.

Is constructed sigmoidalnuyu curve of dose-response at points % of activities of (%) against concentration of compound, and value of IC50 is calculated, using LabStats curves of correspondence in Excel.

Authors-developed animal model, which displays the physiological reaction of excitation, compute observed during sexual excitation of woman, and directly shows clinical data, obtained in people-Volunteers. In this model are used laser Doppler technology for recording small changes in vaginal and klitornom circulation, induced by stimulation of pelvic nerve or vazoaktivnymineiromediatorami. During sexual excitation of has place amplification of genital blood flow, which is-innervation from the pelvic nerve. This induced pelvic nerve amplification of vaginal and kpitornogo blood flow, observed in animal model, represents endogenous vascular effects, observed during sexual excitation of woman-i.e. overflow blood. Consequently, this model can be used for, in-first, for detecting mechanisms, involved in regulation of vaginal and klitornogo circulation and, in-second, of new approaches to intensifying genital blood flow.

In this examination, for show to, that vip mediate genital blood flow, and identify camp as mediator/secondary the intermediary of, regulating genitalnuyuvazorelaksatsiyu (and relaxation vaginal wall), was successfully used combination of in vivo, in vitro and biochemical techniques. Using this animal model, authors invention demonstrated, that infusion infusion of vip enhances vaginal and klitornyi blood flow. Using inhibitor of vip metabolism (for example inhibitor of nep EC 3.4.24.11), authors invention also demonstrated, that amplification of genital blood flow, observed during stimulation of pelvic nerve (i.e. sexual excitation of) indirectly vip. Authors of the invention shown, that mediated vip amplification of genital blood flow is a result of diffusion condensate tissue camp, while previously was shown, that VIR enhances vaginal blood flow in healthy Volunteers, but cell mechanism was not identified. Besides, authors invention demonstrated, that genital blood flow can be enhanced directly mimetic camp or indirectly diffusion savings concentrations of camp inhibitor of FDEtsamf type 2 or antagonist receptor ΝΡΥ Y1.

Main reason for RPVZh is attenuated genital blood flow, and this itself alone appears in the form of lower nnogo overfilling blood vagina, lips and of the clitoris. Treatment of women with RPVZh achievable by reduction of normal reaction sexual of excitation. Achieve by enhancement of genital blood flow. Approach of authors of the invention to treatment RPVZh consists in amplification of genital blood flow and by this potentsirovanii overfilling blood/lubrication of vagina and overfilling blood/sensitivity of the clitoris by means of either direct, or indirect potentiation of endogenous camp signaling, for example inhibitor of nep (EC 3.4.24.11), inhibitor of pde, hydro lizuyushchei camp, or antagonist npy receptor. As a whole this will act on reduction or taking antilogarithms normal reaction sexual of excitation without cardio-vascular adverse effects. Floor excitation/overflow blood will rather be increased, than simply induced in the absence of sexual attraction, it in case of with some exogenous added vazoaktivnymi agents, for example vip.

So, in transcribed the present invention among other things-to the next:

Pharmaceutical composition for use (or when it is used) in treatment of ZhSD, preferably RPVZh, containing agent, capable of potentiate camp in the sexual genitalia of a woman, affected ZhSD, preferably RPVZh, which possibly mixed with pharmaceutically acceptable carrier, diluent or excipient.

Use of an agent in manufacturing of medicine for treatment ZhSD, preferably RPVZh ; where this agent is able potentiate amf in sexual genitalia of a woman, affected ZhSD, preferably RPVZh.

Method of treatment of a woman (such as female experts, suffers ZhSD, preferably RPVZh ), at which this woman is introduced agent, which can potentiate camp in sexual genitalia, in such amount, to cause taking antilogarithms camp in sexual genitalia this woman; at that agent possibly mixed with pharmaceutically acceptable carrier, diluent or excipient.

In especially preferred embodiments the present invention among other things-to the next:

Pharmaceutical composition for use (or when it is used) in treatment of ZhSD, preferably RPVZh, containing agent, capable of potentiate camp in the sexual genitalia of a woman, affected ZhSD, preferably RPVZh, which possibly mixed with pharmaceutically acceptable carrier, diluent or excipient; and which is administered orally.

Use of an agent in manufacturing of medicine for treatment ZhSD, preferably RPVZh ; where this agent is able potentiate camp in the sexual genitalia of a woman, affected ZhSD, preferably RPVZh ;

and where said introduced perorally.

Method of treatment of a woman (for example of a woman, affected ZhSD, preferably RPVZh ), at which this woman is introduced agent, which can potentiate camp in sexual genitalia, in such amount, to cause taking antilogarithms camp in sexual genitalia this woman; at that agent possibly mixed with pharmaceutically acceptable carrier, diluent or excipient; and said agent is administered orally.

In additional especially preferred embodiments the present invention among other things-to the next:

Pharmaceutical composition for use (or when it is used) in treatment of ZhSD, preferably RPVZh, containing agent, capable of potentiate camp in the sexual genitalia of a woman, affected ZhSD, preferably RPVZh, which possibly mixed with pharmaceutically acceptable carrier, diluent or excipient, and which-potentiated synthesis of endogenous camp.

Use of an agent in manufacturing of medicine for treatment ZhSD, preferably RPVZh ; where this agent is able potentiate camp in the sexual genitalia of a woman, affected ZhSD, preferably RPVZh ;

and where said agent-potentiated synthesis of endogenous camp.

Method of treatment of a woman (for example of a woman, affected ZhSD, preferably RPVZh ), at which this woman is introduced agent, which can potentiate camp in sexual genitalia, in such amount, to cause taking antilogarithms camp in sexual genitalia this woman; at that agent possibly mixed with pharmaceutically acceptable carrier, diluent or excipient; and said agent-potentiated synthesis of endogenous camp.

In one more especially preferred embodiments the present invention among other things-to the next:

Pharmaceutical composition for use (or when it is used) in treatment of ZhSD, preferably RPVZh, containing agent, capable of potentiate camp in the sexual genitalia of a woman, affected ZhSD, preferably RPVZh, which possibly mixed with pharmaceutically acceptable carrier, diluent or excipient;

which is introduced orally; and which-potentiated synthesis of endogenous camp.

Use of an agent in manufacturing of medicine for treatment ZhSD, preferably RPVZh ; where this agent is able potentiate camp in the sexual genitalia of a woman, affected ZhSD, preferably RPVZh ;

where said agent is added orally; and where said agent-potentiated synthesis of endogenous camp.

Method of treatment of a woman (for example of a woman, affected ZhSD, preferably RPVZh ), at which this woman is introduced agent, which can potentiate camp in sexual genitalia, in such amount, to cause taking antilogarithms camp in sexual genitalia this woman; at that agent possibly mixed with pharmaceutically acceptable carrier, diluent or excipient; said agent introduced perorally; and said agent-potentiated synthesis of endogenous camp.

All publication, mentioned in given above description, are connected in this document a link on them. Various modification and variants described methods and combination according to the present invention without deviation from volume and essence of the present invention should be obvious for specialists in this area. At the present invention described with respect to specific preferred ways to make your, should have in form, that this invention, it stated, should not unduly limited to such specific embodiments. An unconditional, different modification describes methods of invention, which obvious professionals in biochemistry and biotechnology or related fields, are in volume of the next below the formula of invention.

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ZhSD = zhenskaya floor dysfunction

RPVZh = disorder sexual excitation of women

camp = cyclic adenosine -3 ', 5' -monophosphate

cgmp = cyclic guanosine 3 ', 5' -monophosphate

Ptsamf = potentsiator camp

Ptsgmf = potentsiator cgmp

Atsamf = activator of camp

Atsgmf = activator cgmp

NMtsamf = negative modulator camp

NMtsgmf = negative modulator cgmp

Itsamf = inhibitor of camp

Itsgmf = inhibitor of cgmp

And: Itsamf = inhibitor of inhibitor of camp

And: Itsgmf = inhibitor of inhibitor of cgmp

And: NMtsamf = inhibitor of negative modulator of camp and: nm_{centner gmp} = inhibitor of negative modulator cgmp etc: Atsdmf = positive regulator of activator camp etc: Atsgmf = positive regulator of activator cgmp

ATs = adenylate cyclase

And: ATs = activator ATs

Nep = neutral endopeptidaza

And: nep = inhibitor of nep

Vir = vazoaktivnyiintestinalnyi peptide ( vasoactiveintestinalpeptide ) vip_{r =} receptor vip (may be radius as VIPR ) νΐΡ U = subtype of receptor vip (such as VIPR1, VIPR2 ) a: vip_{r =} activator vip_r

A: vip_{n =} activator vi p_U

M: vip_{r =} inhibitor of vip_r

H: vip_{n =} inhibitor of vip_n

H:h: vip_{r =} inhibitor of inhibitor of vip_r

H:h: VIPn = inhibitor of inhibitor of vip_n

Pde = fosfodiesteraza

Pde_{U =} family of pde (for example FDE1, FDE2 and T. d. ) FDEtsdmf = pde, hydro lizuyushchaya camp

FDEtsgmf = pde, hydro lizuyushchaya cgmp

And: pde = inhibitor of pde

FDEtsamf1 = inhibitor of pde, hydro lizuyushchei camp FDEtsgmf ! = inhibitor of pde, hydro lizuyushchei cgmp and: FDEtsdmf = inhibitor of pde, hydro lizuyushchei camp and: FDEtsgmf = inhibitor of pde, hydro lizuyushchei cgmp and: FDEtsamf = inhibitor of family of camp-hydro lizuyushchikh pde

Npy = neuropeptide y

Npy_{r =} receptor npy (may be radius as NPYR ) npy y_{n =} y_{n subtype of} receptor npy (for example npy Υ·,) (for example NPYR1 )

H: npy = inhibitor of npy

And: npy y_{n =} inhibitor of npy y_n

kd = kDa

u. about. = base pair

t. u. about. = thousand pairs of

1. NEP (EC 3

LOCUS DETERMINATION OF ACCESS

NID

VERSION KEY WORD SOURCE OF ORGANISM

REFERENCE AUTHORS NAME

THE LOG MEDLINE SIGNS

CDS

mi sc. number of bases a. 4.24.11)

hsmrnaen 3181 U. about. mrna pri 12 September 1993 human mrna enkefalinazy (EC 3.4.24.11)

Kh07166

d34757

Χ07166 .1 GI:34757

Enkefalinaza ; metalloprotein ; neutral endopeptidaza

man

Homo sapiens

Eukaryota ; Metazoa ; Chordata ; vertebrata ; Mammalia ; Eutheria ; Primates ;

Catarrhini ; Hominidae ; Homo

1 (of base 1-3181)

Malfroy, Β ., Kuang, w. i., seeburg, ρ. η ., Mason, a. 3. and Schofield, P.R.Molecularcloning and amino acid sequence care human enkephalinase ( neutralendopeptidase )

FebsLett. 229 (1), 206-210 (1988)

88152222

Location/ Qualifiers

1.. 3181

/ organism = ' homo sapiens"

/ db_xr℮ f = ' taxon : 9606 *

/ tissue_type = " placenta *

/ clone_lib = ' lambdagtlO"

/ clone = * lambda H7"

18.. 2249

/ note = * enkephalinase (ΑΑ 1-743)"

/ codon_sCart = l

/ protein_id = "CAA30157.1"

/ db_xref = "PID:g347S8"

/ db_xref = * gi: 34758 *

/ db_xref = ' swiss- PROT : Ρ08473 *

/ translation = " MDITDINTPKPKKKQRWTPLEISLSVLVLLLTIIAVTMIALYATYDDGICKSSDCIKSAARLIQNMDATTEPCTDFFKYACGGWLKRNVIPETSSRYGNFDILRDELEWLKDVLQEPKTEDIVAVQKAKALYRSCINESAIDSRGGEPLLKLLPDIYGWPVATENWEQKYGASWTAEKAIAQLNSKYGKKVLINLFVGTDDKNSVNHVIHIDQPRLGLPSRDYYECTGIYKEACTAYVDFMISVARLIRQEERLPIDENQLALEMNKVMELEKEIANATAKPEDRNDPMLLYNKMTLAQIQNNFSLEINGKPFSWLNFTNEIMSTVNISITNEEDVWYAPEYLTKLKPILTKYSARDLQNLMSWRFIMDLVSSLSRTYKESRNAFRKALYGTTSETATWRRCANYVNGNMENAVGRLYVEAAFAGESKHWEDLIAQIREVFIQTLDDLTWMDAETKKRAEEKALAIKERIGYPDDIVSNDNKLNNEYLELNYKEDEYFENIIQNLKFSQSKQLKKLREKVDKDEWISGAAWNAFYSSGRNQIVFPAGILQFPFFSAQQSNSLNYGGIGMVIGHEITHGFDDNGRNFNKDGDLVDWWTQQSASNFKEQSQCMVYQYGNFSWDLAGGQHLNGINTLGENIADNGGLGQAYRAYQNYIKKNGEEKLLPGLDLNHKQLFFLNFAQVWCGTYRPEYAVNSIKTDVHSPGNFRIIGTLQNSAEFSEAFHCRKNSYMNPEKKCRVW"

. feature 3073. .3078

/ note = " poly a signal"

1055 a 582 c 657 g 887 t

1 gcaagtcaaaaagtcagatggatataactgatatcaacactccaaagccaaagaagaaac 61 agcgatggactccactggagatcagcctctcggtccttgtcctgctcctcaccatcatag 121 ctgtgacaatgaccgcactctatgcaacctacgatgatggtatttgcaagtcatcagact 181 gcataaaatcagctgctcgactgatccaaaacatggatgccaccactgagccttgtacag

241 actttttcaaatatgcttgcggaggctggttgaaacgtaatgtcattcccgagaccagct 301 cccgttacggcaactttgacattttaagagatgaactagaagccgttttgaaagatgtcc 361 ctcaagaacccaaaactgaagatatagtagcagtgcagaaagcaaaagcaCtgtacaggt 421 ctcgtataaatgaatctgctattgatagcagaggtggagaacctctactcaaactgttac 481 cagacatatatgggcggccagtagcaacagaaaactgggagcaaaaatacggtgcttctt 541 ggacagctgaaaaagctactgcacaaccgaattctaaatacgggaaaaaagtccttatCa 601 atttgtttgttggcactgatgacaagaactctgtgaatcatgtaattcacattgaccaac 661 ctcgacttggcctcccttctagagattactatgaatgcactggaatctataaagaggctt 721 gtacagcatacgtggaccttacgacccccgcggccagatcgattcgtcaggaagaaagat 781 tgcccatcgatgaaaaccagctcgctetggaaatgaataaagttatggaattggaaaaag 841 aaatcgccaatgctacggctaaacctgaagatcgaaatgaCccaacgcttctgtataaca 901 agatgacattggcccagatccaaaataacctttcactagagatcaatgggaagccattca 961 gctggttgaatttcacaaacgaaatcatgccaactgtgaaCattagtattacaaatgagg 1021 aagatgtggttgtttatgccccagaatatttaaccaaacttaagcccattcttaccaaat 1081 attctgccagagatcttcaaaatctaatgtcctggagactcataatggatcttgtaagca 1141 gcctcagccgaacctacaaggagCccagaaatgctttccgcaaggccctttatggtacaa 1201 cctcagaaacagcaacttggagacgttgtgcaaactatgtcaatgggaatatggaaaatg 1261 ctgtggggaggctttatgtggaagcagcattcgccggagagagtaaacatgtggccgagg 1321 atttgattgcacagatccgagaagtttttatccagaccttagatgacctcacctggatgg 1381 aCgccgagacaaaaaagagagctgaagaaaaggccttagcaattaaagaaaggatcggct 1441 atcctgatgacattgtttcaaatgataacaaactgaataatgagtacctcgagttgaact 1501 acaaagaagatgaatacttcgagaacataattcaaaatttgaaattcagccaaagtaaac 1561 aaccgaagaagctccgagaaaaggtggacaaagatgagtggataagtggagcagctgtag 1621 tcaatgcattttactcttcaggaagaaatcagatagtcttcccagccggcattctgcagc 1681 cccccttctttagtgcccagcagtccaactcattgaactatgggggcatcggcatggtca 1741 taggacacgaaatcacccaCggcttcgatgacaatggcagaaaccccaacaaagacggag 1801 accccgttgactggtggactcaacagtctgcaagtaacttCaaggagcaatcccagtgca 1861 cggcgcatcagcatggaaacctctcctgggacctggcaggtggacagcaccttaatggaa 1921 ttaatacactgggagaaaacattgctgataatggaggtcttggtcaagcaCacagagcct 1981 atcagaattacatcaaaaagaatggcgaagaaaaattacttcctggacttgaccCaaatc 2041 acaaacaactatttttcttgaacttcgcacaggtgcggtgtggaacctataggccagagt 2101 atgcggttaactccattaaaacagatgtgcacagtccaggcaatttcaggattattggga 2161 ctttgcagaactctgcagagttttcagaagcctttcactgccgcaagaattcatacatga 2221 atccagaaaagaagtgccgggtttggtgatcttcaaaagaagcattgcagcccttggcta 2281 gacttgccaacaccacagaaatggggaatcctctaatcgaaagaaaatgggccctagggg 2341 tcactgtaccgacttgagggtgattaacagagagggcaccaccacaatacagacaacatt 2401 aggttgtcctagaaagggtgtggagggaggaagggggcccaaggtctaccaagtcaatca 2461 tttctcactgtgtacataatgctCaatttctaaagataatattactgtttatttctgttt 2521 ctcatatggtctaccagtttgctgatgtccctagaaaacaatgcaaaacccctgaggtag 2581 accaggatttctaatcaaaagggaaaagaagatgttgaagaatacagttaggcaccagaa 2641 gaacagtaggtgacactatagcttaaaacacactgcccaactactagtttttacctttat 2701 ttgcaacatttacagtccttcaaaatccttccaaagaattcteatacacattggggcctt 2761 ggagcttacatagctttaaactcatttttgccaCacatcagccattcaccctgtgatcat 2821 ttattttaagcactcttaaagcaaaaaatgaacgcccaaaactgtcccccgttgcacctg 2881 ctttgactgatgccgagatccttcaggcctcccgcaaccttccaagcaatttcttgctct 2941 atctctcaaaactcggcatttttcagagatttatataaatgtaaaaataataatttttat 3001 atttaattattaactacatttatgagcaactatCatCataggtaatcaatgaatattgaa 3061 gtttcagcttaaaataaacagttgtgaaccaagatctataaagcgatatacagatgaaaa 3121 tttgagactatttaaacttataaatcatattgatgaaaagatttaagcacaaactttagg 3181 g

2. PDE TYPE OF 1

LOCUS DETERMINATION

ACCESS

NID

VERSION KEY WORD SOURCE OF ORGANISM

REFERENCE AUTHORS

NAME

THE LOG MEDLINE REFERENCE AUTHORS

NAME LOG

Signs of source

gene cds HSPDE1A3A 2008 U. about. mrna pri 12 April 1996 dna-mrna 3 ', 5' phosphodiesterase cyclic nucleotides (HSPDEA3A), complete cds.

U40370

gll51108

U40370.1 Gi:1151108

kalmodulin - stimuliruemayafosfodiesteraza

Man

Homo sapiens

Eukaryota ; Metazoa ; Chordata ; vertebrata ; Mammalia ; Eutheria ; Primates ;

Catarrhini ; Hominidae ; Homo

1 (of base 1-2008)

Loughney, Κ ., Martins, T. E ., Harris, Ε. Α ., Sadhu, Κ ., Hicks, 3. Β ., Sonnenburg, w. k., Beavo, J.A. and Ferguson, Κ.

Isolation and characterization care cDNAs corresponding to two human calcium, calmodulin - regulated, 3 ', 5' -cyclic nucleotidephoshodiesterases 1st Biol. Chem. 271 (2), 796-806 (1996)

96132810

2 (of base 1-2008)

Loughney, κ ., Martins, t. j., Harris, E.A. S., Sadhu, κ ., Hicks, 1. Β ., Sonnenburg, w. k., Beavo, J.A. and Ferguson, Κ.

Direct submission

Submitted (07-NOV-1995) kate Loughney, ICOS, 22021 20^{th Αν}℮. s. ε ., Bothell, wa 98021, USA

Location/ Qualifiers

1. .2008

/ organism *' homo sapiens"

/ db_xref = " taxon : 9606"

85. .1692

/ gene = ' PDElA"

85. .1692

/ gene = "PDE1A"

/ note = " PDE1A3 ; type i phosphodiesterase *

/ codon_start = l

/ product = "3", 5 "cyclic nucleotidephosphodiesterase */ protein_id =" AAC50436.1"

/ db_xref = "PID:gl151109"

/ db_xref = "GI:1151109"

/ translations " MGSSATEIEELENTTFKYLTGEQTEKMWQRLKGILRCLVKQLERGDVNWDLKKNIEYAASVLEAVYIDETRRLLDTEDELSDIQTDSVPSEVRDWLASTFTRKMGMTKKKPEEKPKFRSIVHAVQAGIFVERMYRKTYHMVGLAYPAAVIVTLKDVDKWSFDVFALNEASGEHSLKFMIYELFTRYDLINRFKIPVSCLITFAEALEVGYSKYKNPYHNLIHAADVTQTVHYIMLHTGIMHWLTELEILAMVFAAAIHDYEHTGTTNNFHIQTRSDVAILYNDRSVLENHHVSAAYRLMQEEEMNILINLSKDDWRDLRNLVIEMVLSTDMSGHFQQIKNIRNSLQQPEGIDRAKTMSLILHAADISHPAKSWKLHYRWTMALMEEFFLQGDKEAELGLPFSPLCDRKSTMVAQSQIGFIDFIVEPTFSLLTDSTEKIVIPLIEEASKAETSSYVASSSTTIVGLHIADALRRSNTKGSMSDGSYSPDYSLAAVDLKSFKNNLVDIIQQNKERWKELAAQEARTSSQKCEFIHQ *

627 a 400 c 437 g 544 t

1 gaattctgatgtgcttcagcgcacagaacagtaacagatgagctgcttttggggagagct 61 tgagtactcagtcggagcatcatcatggggtctagtgccacagagattgaagaattggaa 121 aacaccacttttaagtatcttacaggagaacagactgaaaaaatgtggcagcgcctgaaa 181 ggaatactaagatgcttggtgaagcagctggaaagaggtgatgttaacgtcgtcgactta 241 aagaagaatattgaatatgcggcatctgtgctggaagcagtttatatcgatgaaacaaga 301 agacttctggatactgaagatgagctcagtgacattcagactgactcagtcccatctgaa number of bases a

361 gtccgggactggctggcttctacctttacacggaaaatggggatgacaaaaaagaaacct 421 gaggaaaaaccaaaatctcggagcattgtgcacgctgttcaagctggaatctttgtggaa 481 agaatgtaccgaaaaacatatcatacggttggtttggcatatccagcagctgtcaccgca 541 acattaaaggatgttgataaatggtctttcgatgtatttgccctaaatgaagcaagtgga 601 gagcatagtctgaagtctatgatctatgaactgtttaccagatatgatcttatcaaccgt 661 ttcaagattcctgtttctcgcctaatcacctttgcagaagctctagaagtCggttacagc 721 aagtacaaaaacccatatcacaaettgattcatgcagctgacgccacccaaactgtgcat 781 tacacaatgcttcatacaggtatcatgcactggctcactgaactggaaattttagcaatg 841 gtctttgctgctgccattcatgactacgagcatacagggacaacaaacaaccctcacatc 901 cagacaaggtcagatgtcgccattttgtataatgatcgctctgtccttgagaatcaccac 961 gcgagtgcagcttaccgactcatgcaagaagaagaaatgaaCatcttgataaatttatcc 1021 aaagatgactggagggatcttcggaacctagcgattgaaatggttttatctacagacatg 1081 tcaggtcacttccagcaaactaaaaatacaagaaacagtttgcagcagcccgaagggatt 1141 gacagagccaaaaccatgtccctgattccccacgcagcagacatcagccacccagccaaa, 1201 tcctggaagctgcatcatcggtggaccatggccctaatggaggagtttttcctgcaggga 1261 gataaagaagctgaaccagggcctccatttcccccaccccgtgatcggaagtcaaccaCg 1321 gtggcccagtcacaaataggtctcatcgacttcatagtagagccaacattttctcttctg 1381 acagactcaacagagaaaatcgttattcctcttatagaggaagcctcaaaagccgaaact 1441 ccttcctatgtggcaagcagctcaaccaccaccgcggggttacacactgctgatgcacca 1501 agacgatcaaatacaaaaggccccatgagcgatgggtcctattccccagactactccctt 1561 gcagcagtggacctgaagagcttcaagaacaacctggtggacatcattcagcagaacaaa 1621 gagaggtggaaagagtcagccgcacaagaagcaagaaccagttcacagaagcgtgagctt 1681 attcatcagtaaacacctccaagtaaaaccccgtgcatggtggcagctctaatttgacca 1741 aaagacttggagactttgattatgcttgctggaaatccaccctgtcctgcgtgagacagg 1801 aaatctatttttgcagattgctcaataagcatcatgagccacataaataacagctgtaaa 1861 ctccttaattcaccgggctcaactgctaccgaacagattcatctagtggctacatcagca 1921 ccttgtgctttcagacatccgtttcaatggcatcttgtggcatttgtctttaccgagtgc 1981 caataaattttctttgagcaaaaaaaaa

3. PDE TYPE OF 2

LOCUS DETERMINATION

ACCESS NID VERSION KEY WORD SOURCE OF ORGANISM

REFERENCE AUTHORS

NAME

THE LOG MEDLINE REFERENCE AUTHORS

NAME LOG

Signs of source

gene cds HSU67733 4240 l. about. mrna pri 21 May 1997 human mrna cgmp-stimulating 3 ', 5' phosphodiesterase cyclic nucleotides (HSPDEA3A), complete cds.

1) 67733

g2108051

U67733.1 Gl:2108051

Man

Homo sapiens

Eukaryota ; Metazoa ; Chordata ; vertebrata ; Mammalia ; Eutheria ; primates ;

Catarrhini ; Hominidae ; Homo

1 (of base 1-4240)

Rosman, G.3 ., Martins, τ.3 ., Sonnenburg, w. k., Beavo, 3. Α ., Ferguson, κ. and Loughney, κ.

isolation and characterization care human cDNAs encoding a cgmp- stimulated 3 ', 5' -cyclic nucleotidephoshodiesterases

Gene 191 (1), 89-95 (1997)

97354299

2 (of base 1-4240)

Rosman, G.3 ., Martins, T. E ., Sonnenburg, w. k., Beavo, 3. Α ., Ferguson, Κ. and Loughney, κ.

Direct Submission

Submitted (21-AUG-1996), Icos corporation, 22021 20^{th Αν}℮. s. ε ., Bothell, wa 98021, USA

Location/ Qualifiers

1.. 4240

/ organism = " homo sapiens'

/ db_xref = ' taxon : 9606"

162.. 2987

/ gene = "PDE2A"

162. .2987

/ gene = ' PDE2A"

/ function = " cgmp- stimulated 3 ', 5' -cyclic nucleotidephosphodiesterase'

/ note = "PDE2 family; splice accents 3"

/ codon_start = l

/ product^{3 "}PDE2A3"

/ protein_id = 'AAC51320.1'

/ db_xref = "PID:g2108052"

/ db_xref = ' GI:2108052"

/ translation^3'MGQACGHSILCRSQQYPAARPAEPRGQQVFLKPDEPPPPPQPCADSLQDALLSLGSVIDISGLQRAVKEALSAVLPRVETVYTYLLDGESQLVCEDPPHELPQEGKVREAIISQKRLGCNGLGFSDLPGKPLARLVAPLAPDTQVLVMPLADKEAGAVAAVILVHCGQLSDNEEWSLQAVEKHTLVALRRVQVLQQRGPREAPRAVQNPPEGTAEDQKGGAAYTDRDRKILQLCGELYDLDASSLQLKVLQYLQQETRASRCCLLLVSEDNLQLSCKVIGDKVLGEEVSFPLTGCLGQWEDKKSIQLKDLTSEDVQQLQSMLGCELQAMLCVPVISRATDQWALACAFNKLEGDLFTDEDEHVIQHCFHYTSTVLTSTLAFQKEQKLKCECQALLQVAKNLFTHLDDVSVLLQEIITEARNLSNAEICSVFLLDQNELVAKVFDGGWDDESYEIRIPADQGIAGHVATTGQILNIPDAYAHPLFYRGVDDSTGFRTRNILCFPIKNENQEVIGVAELVNKINGPWFSKFDEDLATAFSIYCGISIAHSLLYKKVNEAQYRSHLANEMMMYHMKVSDDEYTKLLHDGIQPVAAIDSNFASFTYTPRSLPEDDTSMAILSMLQDMNFINNYKIDCPTLARFCLMVKKGYRDPPYHNWMHAFSVSHFCYLLYKNLELTNYLE di el FALFISCMCHDLDHRGTNNSFQVASKSVLAALYSSEGSVMERHHFAQAIAILNT

^{: HGCNIFDHFSRKDYORMLDLMRDIIL}ATDLAHHLRIFKDLQKMAEVGYDRNNKQHHRLLLCLLMTSCDLSDQTKGWKTTRKIAELIYKEFFSQGDLEKAMGNRPMEMMDREKAYIPELQ1SFMEHIARPIYKLLQDLFPKAAELYERVASNREHWTKVSHKFTIRGLPSNNSLDFLDEEYEVPDLDGTRAPINGCCSLDAE"

902 and 1260 with 1202 g 876 t

1 cagcagagctggatcggggtgctgagtccaggctgagtagggggcagcccactgctcctg 61 gtccctgtgcctgctgggggtgccccgccctgaactccaggcagcggggacagggcgagg 121 tgccacctcagtctggctggggaggcggacgatgaggagtgatggggcaggcatgcggcc 1S1 actccaccctctgcaggagccagcagtacccggcagcgcgaccggctgagccgcggggcc 241 agcaggtcttccccaagccggacgagccgccgccgccgccgcagccatgcgccgacagcc 301 tgcaggacgccttgccgagtctgggctctgtcatcgacatttcaggcctgcaacgtgccg 361 tcaaggaggccctgtcagctgtgctcccccgagtggaaactgtctacacctacctactgg 421 atggtgagtcccagctggtgcgtgaggaccccccacatgagctgccccaggaggggaaag 481 tccgggaggctatcatctcccagaagcggccgggctgcaatgggccgggcttctcagacc 541 tgccagggaagcccttggccaggctggtggctccactggctcctgatacccaagtgctgg 601 tcaegccgccagcggacaaggaggccggggccgtggcagctgtcatcttggtgcactgtg 661 gccagctgagtgacaatgaggaatggagcctgcaggcggtggagaagcatacccCggtcg 721 ccctgcggagggtgcaggtcctgcagcagcgcgggcccagggaggctccccgagccgtcc 781 agaaccccccggaggggacggcggaagaccagaagggcggggcggcgtacaccgaccgcg 841 accgcaagatcctccaactgtgcggggaactctacgacctggatgcctcttccccgcagc 901 tcaaagtgctccaacacctgcagcaggagacccgggcatcccgctgctgcctcctgctgg 961 tgtcggaggacaatctccagccttcttgcaaggtcatcggagacaaagtgctcggggaag 1021 aggccagctttcccccgacaggacgcctgggccaggtggtggaagacaagaagtccatcc 1081 agctgaaggacctcacctccgaggaCgtacaacagctgcagagcatgttgggctgcgagc 1141 tgcaggccatgctctgtgtccctgtcatcagccgggccactgaccaggtggtggccttgg 1201 cctgcgccttcaacaagctagaaggagacttgttcaccgacgaggacgagcatgtgatcc 1261 agcactgcttccactacaccagcaccgtgctcaccagcaccctggccttccagaaggaac 1321 agaaactcaagtgtgagtgccaggctcttctccaagtggcaaagaacctcttcacccacc 1381 tggatgacgtctctgtcctgctccaggagatcatcacggaggccagaaacctcagcaacg 1441 cagagatctgctctgtgttcctgctggatcagaatgagctggtggccaaggtgttcgacg 1501 ggggcgtggtggatgatgagagctatgagatccgcatcccggccgatcagggcatcgcgg 1561 gacacgtggcgaccacgggccagatcctgaacatcccCgacgcatacgcccatccgcttt 1621 tctaccgcggcgtggacgacagcaccggcttccgcacgcgcaacatcctctgcttcccca 1681 tcaagaacgagaaccaggaggccatcggtgtggccgagctggtgaacaagatcaatgggc 1741 catggttcagcaagttcgacgaggacctggcgacggccttctccatctactgcggcatca 1801 gcatcgcccattctctcctatacaaaaaagtgaatgaggctcagtatcgcagccacctgg 1861 ccaatgagatgatgatgtaccacatgaaggtctccgacgatgagtataccaaacttctcc 1921 atgatgggatccagcctgtggctgccattgactccaattttgcaagtttcacctataccc 1981 ctcgttccctgcccgaggatgacacgtccacggccatcctgagcatgctgcaggacatga 2041 atttcatcaacaactacaaaattgactgcccgaccctggcccggttctgtttgatggtga 2101 agaagggctaccgggatcccccctaccacaactggatgcacgccttttctgtctcccact 2161 tctgctacctgctctacaagaacctggagctcaccaactacctcgaggacatcgagatct 2221 Ctgccttgtttatctcctgcatgtgtcatgacctggaccacagaggcacaaacaactctt 2281 tccaggtggcctcgaaatctgtgceggctgcgctctacagcCctgagggctccgtcatgg 2341 agaggcaccactctgctcaggccatcgccaCccccaacacccacggctgcaacatctttg 2401 atcattcctcccggaaggaccatcagcgcatgceggatctgatgcgggacaccatcctgg 2461 ccacagacctggcccaccatctccgcatcttcaaggacctccagaagatggctgaggtgg 2521 gctacgaccgaaacaacaagcagcaccacagacttcccctctgcctcctcatgacctcct 2581 gtgacctctctgaccagaccaagggctggaagactacgagaaagatcgcggagctgatct 2641 acaaagaattcttctcccagggagacctggagaaggccatgggcaacaggccgatggaga 2701 tgatggaccgggagaaggcctatatccctgagctgcaaatcagcttcatggagcacattg 2761 caatgcccacctacaagctgttgcaggacctgttccccaaagcggcagagctgtacgagc 2821 gcgtggcctccaaccgcgagcactggaccaaggtgtcccacaagctcaccatccgcggcc 2881 tcccaagcaacaactcgctggacttcctggatgaggagtacgaggcgcctgatctggacg 2941 gcactagggcccccatcaatagctgctgcagccccgatgctgagtgatcccctccaggac 3001 acttccctgcccaggccacctcccacagccctccactggcctggccagatgcactgggaa 3061 cagagccacgggtcctgggtcctagaccaggacttcctgtgtgaccctggacaagtacta 3121 ccttcctgggccccagctttctcgtctgtataaCggaagcaagacttccaaccccacgga 3181 gacctcgcaatttgcccccccgagagcacaggggtgaccaatgagcagcgggccctactc 3241 tgcacctcegaccacaccccggcaagtcccccccaagccattctttgtcCgagcagcttg 3301 acggtttccccctgccccacctctgccccaccagacctctgctcctttccctttgaggac 3361 ecccaccctttgggtctccaggatcctcatggaaggggaaggcgagacacccgagcgagc 3421 agagtgtggcatctcggaaacagtccttagttctgtgggaggaccagaaacagccgcggc 3481 gaaggccccctgaggaccactactatactgacggtgggattgggacctgggggatacagg 3541 ggccccaggaagaagctggccagaggggcagctcagcgctccgcagagaggggccctggg 3601 gagaagcaggatgggatcgatgggcaggagggatccccgcactgggagacaggcccaggt 3661 atgaatgagccagccatgcttcctcctgcccgtgCgacgctgggcgagtctcttcccctg 3721 cctgggccaaacagggagcgggtaagacaatccacgctccaagatccattttagatcaat 3781 gtctaaaatagctccatggctccgcggagtcccagcagaggctacggaatgtttccgcaa 3841 ccctaaggcacagagagccaaccctgagcgtctcagaggccccctgagtgttccccttgg 3,901 cctgagccccttacccactcctgcagccagtgagagacctggcctcagcctggcagcgct 3961 ctcttcaaggccatatccacctgtgccctggggcttgggagaccccataggccgggactc 4021 ctgggtcagcccgccactggctcctctctctttctccgttccattctgcgtgcgttgtgg 4081 ggtgggggagggggtccacccgccttaccttectgagttgcctttagagagatgcgtttt 4141 tctaggaccctgtgcaactgccgtatatggtcccgcgggctgaccgctttgtacatgaga 4201 ataaatctatttctttctaccaaaaaaaaaaaaaaaaaaa

4. ΝΡΥ

LOCUS DETERMINATION OF ACCESS

NID

VERSION KEY WORD

SOURCE OF ORGANISM

REFERENCE AUTHORS NAME

THE LOG MEDLINE COMMENTS

Signs of source

mRNA

gene

Humnpy 551 π. ο. ΜΡΗΚ pri 07 January 1995 dna-mrna neuropeptide Υ ( νρυ ), complete cds.

κ01911

gl89273

κ019 11.1 ci:189273

neuropeptide Υ

human feokhromotsitoma, cdna-mrna, clone ρΝΡΥ3 -75

Homo sapiens

Eukaryota ; Metazoa ; chordata ; vertebrata ; Mammalia ; Eutheria ; PrimatesCatarrhini ; Hominidae ; homo

1 (of base 1-551)

Minth, s. d., bloom, s. roption., Polak, J.M. and Dixon, D.E.

Cloning, characterization and dna sequence care a human cDNA encoding neuropeptidetyrosine

Proc. Natl. Acad. Sci. USA 81 (14), 4577-4581 (1984)

84272678

neuropeptide Υ ( ΝΡΥ ) is one of the most abundant peptides in nervous system mammals, and its wide distribution of allows call role of neuromediator or-of modulator, ΝΡΥ also is detected in some khromaffinnykh cells of brain substance adrenal.

Location/ Qualifiers

1. .551

/ organism = * homo sapiens'

/ db_xref = " taxon : 9606 *

/ 1issue_type = ' pheochromocytoma'

/ map = " 7 pter -q 22'

<1. .551

7gene = ' npy"

/ note = " GOO-119-456'

1. .551

/ gene = ' npy"

sig_peptide 87. .170

/ gene = 'npy'

/ note = "GOO-119-456"

CDS 87. .380

/ gene = "npy"

/ codon_start = l

/ db_xref = * gdb: G00 -119-456"

/ product = " neuropeptide y"

/ protein_id = "AAA59944.1"

/ db_xref = * pid: gl89274"

/ db_xref = 'GI:189274'

/ translate οη = " MLGNKRLGLSGLTLALSLLVCLGALAEAYPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRYGKRSSPETLISDL. LMRESTENVPRTRLEDPAMW" mat_peptide 171. .278

/ gene = " npy'

/ note = " G00-119-456'

/ product = ' neuropeptide y"

number of bases 131 a 171 c 129 g 120 t

a 51 π. ο. no ascending from site Rsal

1 accccatccgctggctctcaccccecggag 61 ccagccacgcccgcgcgccagccaccacgc 121 tgaccctcgccctgtccctgctcgtgtgcc 181 agccggacaacccgggcgaggacgcaccag 241 tgcgacactacatcaacctcatcaccaggc 301 cactgatttcagacctcttgatgagagaaa 361 aagaccctgcaatgtggtgatgggaaatga 421 gcccatatttcatcgtgtaaaacgagaatc 481 tgctgaatcctgcaatgttttcctttgtca 541 atcatgcacc c

acgctcgcccgacagcatagtacttgccgccaggtaacaagcgactggggctgtccggactgggtgcgctggccgaggcgtacccctccacggaggacatggccagatacCactcggcgcagagatatggaaaacgatccagcccagagagcacagaaaatgttcccagaactcggcttggacttgctctctggccttttcctattttcacacccatcctaccaatgcatgcagccaccgtcatcgtatatatgtgtgtttaaataaagt 5. Receptor ΝΡΥYl

Α26481 2624 U. about. mrna RAT 17 October 1995

to DNA of human receptor ΝΡΥYl

A26481

gl247452

Α26481 .1 61:1247452

man

Homo sapiens

Eukaryota ; Metazoa ; Chordata ; Vertebrata ; Mammalia ; Eutheria ; Primatescatarrhini ; Hominidae ; homo

1 (of base 1-2624)

Patent: WO9309227 -a 3 13- ΜΑΥ -1993

Location/ Qualifiers

1.. 2624

/ organism = * homo sapiens*

/ db_xref = * taxon : 9606 *

152.. 1306

/ codon_start = l

/ product = " neuropeptideΥYlreceptor */ protein_id = * CAAC18 19.1*

/ db_x' re £= * pid: e205126 *

/ db_xref = " PID:gl247453*

/ db_xref = * gi: 1247453 *

/ db_xre £= * SW1 ss- PROT : Ρ25929 */ translation = * MNSTLFSQVENHSVHSNFSEKNAQLLAFENDDCHLPLAMIFTLALAYGAVIILGVSGNLALI11ILKQKEMRNVTNILIVNLSFSDLLVAIMCLPFTFVYTLMDHWVFGEAMCKLNPFVQCVSITVSIFSLVLIAVERHQLIINPRGWRPNNRHAYVGIAVIWVLAVASSLPFLIYQVMTDEPFQNVTLDAYKDKYVCFDQFPSDSHRLSYTTLLLVLQYFGPLCFIFICYFKIYIRLKRRNNMMDKMRDNKYRSSETKRINIMLLSIWAFAVCWLPLTIFNTVFDWNHQIIATCNHNLLFLLCHLTAMISTCVNPIFYGFLNKNFQRDLQFFFNFCDFRSRDDDYETIAMSTMHTDVSKTSLKQASPVAFKKINNNDDNEKI"

Number of bases 791 a 479 c 473 g 878 Τ 3 other

0 RIDZhIN

1 attgttcagttcaagggaatgaagaattcagaataattttggtaaatggattccaatatc 61 gggaataagaataagctgaacagttgacctgctttgaagaaacatactgtccatttgtct 121 aaaataatctataacaaccaaaccaatcaaaatgaattcaacattattttcccaggttga 181 aaatcattcagtccactctaatttctcagagaagaatgcccagcttctggcttttgaaaa 241 tgatgattgtcatctgcccttggccatgatatttaccttagctcttgcttatggagctgt 301 gatcattcttggtgtctctggaaacctggccttgatcataatcatcttgaaacaaaagga 361 gatgagaaatgttaccaacatcctgattgtgaacctttccttctcagacttgcttgttgc 421 catcatgtgtctcccctttacatttgtctacacattaatggaccactgggtctttggtga 481 ggcgatgtgtaagttgaatccttttgtgcaatgtgtttcaatcactgtgtccattttctc 541 tctggttctcattgctgtggaacgacatcagctgataatcaaccctcgagggtggagacc 601 aaataatagacatgcttatgtaggtattgctgtgatttgggtccttgctgtggcttcttc 661 tttgcctttcctgatctaccaagtaatgactgatgagccgttccaaaatgtaacacttga 721 tgcgtacaaagacaaatacgtgtgctttgatcaatttccatcggactctcataggttgtc 781 ttataccactctcctcttggtgctgcagtactttggtccactttgttttatatttatttg 841 ctacttcaagatatatatacgcctaaaaaggagaaacaacatgatggacaagatgagaga 901 caataagtacaggtccagtgaaaccaaaagaatcaatatcatgctgctctccattgtggt 961 agcatttgcagtctgctggctccctcttaccatctttaacactgtgtttgattggaatca 1021 tcagatcattgctacctgcaaccacaatctgttattcctgctctgccacctcacagcaat locus determination of access

NID

VERSION KEY WORD SOURCE OF ORGANISM

Reference authors name the log signs of source

CDS

1081 gatatccacttgtgtcaaccccacactccacgggttcctgaacaaaaacttccagagaga 1141 cttgcagttcttcetcaacttctgcgatetccggtctcgggatgatgatcacgaaacaac 1201 agccatgtccacgatgcacacagacgtttccaaaacttcCttgaagcaagcaagcccagc 1261 cgcattcaaaaaaatcaacaacaacgacgataatgaaaaaatccgaaaccacttatagcc 1321 tatggtcccggatgacatctgtctaaaaacaagcacaacctgcaacatactttgattacc 1381 tgttcccccaaggaatggggttgaaaccattcgaaaatgactaagacccccttgtctcgc 1441 ttttttactgcttttgttgtagtgtcacaattacatttggaacaaaaggtgtgggccccg 1501 gggtcttctggaaatagttttgaccagacatcCCtgaagtgctctttgtgaatttatgca 1561 tataatataaagactcttacactgtacCtattggaatgaaatttctttaaagtattacga 1621 tnnnctgacttcagaagtacctgccatccaacacggtcattagattgggtcatctCgatt 1681 agaCCagattagactagactgccaacagattgggccatccctaccttatgataggcatca 1741 ccctagtgcgttacaatagtaacagcacgcaaaagcagcattcaggagccgaaagatagt 1801 ctCgaagccattcagaagtggrctgaggtttctgttttctggcggttttcgtttgtcttc 1861 ttettttttcaccttaagggaggccctcatttcctcccgactgattgecacttaaatcaa 1921 aacttaaaaatgaataaaaacacacaccccccagccgcaaatatcacggagaaccgggca 1981 cccacaggaatgaagagagaaagcagctccccaacttcaaaaccactttggtacctgaca 2041 acaagagcattttagagtaattaatttaataaagtaaattagtattgctgcaaatagcta 2101 aattatatccatctgaattgarggtcaagagaccttccattttttttacagactgCtcag 2161 tgtttgtcaagcttccggtccaacacgcactcgaaagactttccgcttacaatctgtaga 2221 aacacaaatatcgttctccatacagcagcgcctatatagCgactgatttcaactttcaat 2281 gcccatctttcaaaggaagtaacaccaaggcacaatgttaaaggaatattcactttacct 2341 agcagggaaaaatacacaaaaactgcagatacttcatatagcccattttaacttgtacaa 2401 actgtgtgacttgtggcgtctcataaacaacgcactgtaaagattactgaatagttgtgt 2461 caCgttaatgtgcctaattccacgtatcttgtaatcacgattgagcctcagaatcatttg 2521 gagaaaccacaccttaaagaacaagacataccccaacgtattatacagataaagtattac 2581 atgtgtttgattctaaaagggcggacattttattaaaatcaagg

6. RECEPTOR ΝΡΥΥ2

LOCUS DETERMINATION OF ACCESS

N10

VERSION KEY WORD

SOURCE OF ORGANISM

REFERENCE AUTHORS

NAME

THE LOG MEDLINE REFERENCE AUTHORS NAME LOG

Signs of source

HSU36269 1200 U. about. mrna pri 14 November 1995

mrna of human receptor neuropeptide y/ leptidaυυ Y2, complete cds.

U36269

G1063633

U36269.1 GI:1063633

man

nosho sapiens

Eukaryota ; Metazoa ; Chordata ; vertebrata ; Mammalia ; Eutheria ; Primates ;

Catarrhini ; Hominidae ; Homo

1 (of base 1-1200)

Gerald, s., walker, m. w., vaysse, Ρ.3 ., he, s.,. Branchek, Τ. Α. and weinshank, r.l.

Expression cloning and pharmacologicalcharacterization care a human hippocampalneuropeptide y/ peptideΥΥΥ2receptor subtype page

3. Biol. Chem. 270 (45), 26758-26761 (1995)

96070760

2 (of base 1-1200)

Gerald, C.A.

Direct Submission

Submitted (13-SEP-1995) Christope a. Gerald, synaptic Pharmaceutical corporation, Molecular biology, 215 college road, Paramus, N3 07652, USA location/ Qualifiers

1. .1200

/ organism = "homo sapiens"

/ db_xref = ' taxon : 9606"

/ clone = * hhY2"

/ sex = " male"

/ tissue_type = " brainhippocampus"

/ dev_stage = " adult*

5 * UTR 1..20

CDS 21. .1166

/ note = "npy/pyy Υ2receptor"

/ codon_start = l

/ product = " neuropeptide y/ peptideΥΥΥ2receptor"/ protein_id = "AAC50281.1"

/ db_xref = "PID:gl063634"

/ db_xref = "gi.- 1063634"

/ translations ·MGPIGAEADENQTVEEMKVEQYGPQTTPRGELVPDPEPELIDSTKLIEVQWLILAYCSIILLGVIGNSLVIHWIKFKSMRTVTNFFIANLAVADLLVNTLCLPFTLTYTLMGEWKMGPVLCHLVPYAQGLAVQVSTITLTVIALDRHRCIVYHLESKISKRISFLIIGLAWGISALLASPLAIFREYSLI el IPDFEXVACTEKWPGEEKS x YGTVYSLSSLLILYVLPLGIISFSYTRIWSKLKNHVSPGAANDHYHQRHQKTTKMLVCWWFAVSWLPLHAFQLAVDIDSQVLDLKEYKLIFTVFHIIAMCSTFANPLLYGWMNSNYRKAFLSAFRCEQRLDAIHSEVSVTFKAKKNLEVRKNSGPNDSFTEATNV"

3' UTR 1164. .1200

number of bases 292 a 295 c 299 g 314 t

A

1 caagtggacctgtactgaaaatgggtccaataggtgcagaggctgatgagaaccagacag 61 tggaagaaatgaaggtggaacaatacgggccacaaacaactcctagaggtgaactggtcc 121 ctgaccctgagccagagcttatagatagtaccaagctgattgaggtacaagttgttctca 181 tattggcctactgctccatcatcttgcttggggtaattggcaactccttggtgatccatg 241 tggtgatcaaattcaagagcatgcgcacagtaaccaactttttcattgccaatctggctg

301 tggcagatctCttggtgaacactctgtgtctaccgttcactcttacctataccttaatgg 361 gggagtggaaaatgggtcctgtcctgtgccacctggtgccctatgcccagggcctggcag 421 tacaagtatccacaatcaccttgacagtaattgccctggaccggcacaggtgcatcgtct 481 accacctagagagcaagatccccaagcgaatcagcttcctgattattggcttggcctggg 541 gcatcagtgccctgctggcaagtcccctggccatcttccgggagtattcgctgattgaga 601 tcatcccggactttgagattgtggcctgtactgaaaagtggcctggcgaggagaagagca 661 tctatggcactgtctatagtctttcttccttgctgatcttgtatgttttgcctctgggca 721 ttatatcatttccccacacccgcatttggagtaaattgaagaaccatgccagtcccggag 781 ctgcaaatgaccactaccatcagcgaaggcaaaaaaccaccaaaatgctggtgtgtgtgg 841 tggtggtgtttgcggtcagctggctgcctctccatgccttccagcttgccgttgacattg 901 acagccaggtcctggacctgaaggagtacaaactcatcttcacagtgttccacatcatcg 961 ccatgtgctccacttttgooaatccccttctctatggctggatgaacagcaactacagaa 1021 aggctttcctctcggccttccgctgtgagcagcggttggatgccattcactctgaggtgc 1081 ccgtgacattcaaggctaaaaagaacctggaggtcagaaagaacagtggccccaacgact 1141 ccttcacagaggctaccaatgtctaaggaagctgtggtgtgaaaatgtatggatgaattc 7. RECEPTOR ΝΡΥΥ5

LOCUS DETERMINATION OF ACCESS

NID

VERSION KEY WORD SOURCE OF ORGANISM

REFERENCE AUTHORS

NAME

THE LOG MEDLINE REFERENCE AUTHORS

NAME LOG

Signs of source

HSU66275 1370 U. about. mrna pri 18 October 1996 mrna of human receptor neuropeptide Υ5 ( NPYR5 ), complete cds.

υ66275

G1620655

U66275.1 GI:1620655

Man

Homo sapiens

Eukaryota ; Metazoa ; chordata ; vertebrata ; Mammalia ; Eutheria ; Primates ;

catarrhini ; Hominidae ; homo

1 (of base 1-1370)

nor, γ ., Bloomquist, Β. τ ., cornfield, l. j., Decarr, l. b., Flores - Riveros, 3. R., Friedman, l., liang, ρ ., lewis- Higgins, l., Sadlowski, γ ., Schaefer, 3 ., Velazquez, n., and McCaleb, m.l.

Identification of a novelhypothalamicneuropeptide γ receptor associated with feedingbehavior

3. Biol. Chem. 271 (42), 26315-26319 (1996)

96421636

2 (of base 1-1370)

Hu, γ ., Bloomquist, β. τ ., cornfield, l. j., oecarr, l. b., Flores - Riveros, j. roption., Friedman, l., liang, ρ ., lewis- Higgins, l., Sadlowski, γ ., Schaefer, 3 ., Velazquez, n., and McCaleb, M.L.

Direct Submission

Submitted (06-AUG-1996) Metabolic disorder mixes, Bayer corporation, 400 MorganLane, west Haven, ct 06516, USA

Location/ Qualifiers

1. .1370

/ organisms"homo sapiens"

/ db_xref = " taxon : 9606"

gene 18. .1355

/ gene = " NPYR5"

CDS 18. .1355

Number of bases a/gene = " NPYR5"

/ functions"g protein - coupledreceptor"

/ codon_start = l

/ products " neuropeptideΥ5receptor"/ protein_id = "AAC50 ' 741,1"

/ db_xref = "PID:gl620656"

/ db_xref = " GI:1620656'

/ translations _AOE296A0AO> MDLELDEYYNKTLATENNTAATRNSDFPVWDDYKSSVDDLQYFLIGLYTFVSLLGFMGNLLILMALMKKRNQKTTVNFLIGNLAFSDILWLFCSPFTLTSVLLDQWMFGKVMCHIMPFLQCVSVLVSTLI li si AIVRYHMIKHPISNNLTANHGYFLIATVWTLGFAICSPLPVFHSLVELQETFGSALLSSRYLCVESWPSDSYRIAFTISLLLVQYILPLVCLTVSHTSVCRSISCGLSNKENRLEENEMINLTLHPSKKSGPQVKLSGSHKWSYSFIKKHRRRYSKKTACVLPAPERPSQENHSRILPENFGSVRSQLSSSSKFIPGVPTCFEIKPEENSDVHELRVKRSVTRIKKRSRSVFYRLTILILVFAVSWMPLHLFHWTDFNDNLISNRHFKLVYCICHLLGMMSCCLNPILYGFLNNGIKADLVSLIHCLHM'

392 a 263 c 257 g 458 t

1 ccaagcaggactataatatggatttagagctcgacgagtattataacaagacacttgcca 61 cagagaataatactgctgccactcggaattctgatttcccagtctgggatgactataaaa 121 gcagtgtagatgacttacagtattttctgattgggctctatacatttgtaagtcttcttg 181 gctttatggggaatctacttattttaatggctctcatgaaaaagcgtaatcagaagacta 241 cggtaaacttcctcataggcaatctggccttttctgatatcttggttgtgctgttttgct

301 cacctttcacactgacgtctgtcttgctggatcagcggatgtttggcaaagtcatgtgcc 361 atattatgccttctcttcaatgtgtgtcagttttggtttcaactetaattttaatatcaa 421 ttgccattgtcaggtatcatatgacaaaacatcccacatctaataatttaacagcaaacc 481 atggctactttccgatagctactgtctggacactaggttttgccatcCgttctccccttc 541 cagtgtttcacagtcttgtggaacttcaagaaacatttggttcagcattgctgagcagca 601 ggtatccacgtgCtgagtcatggccatctgattcatacagaattgcctttactatctctt 661 taetgctagttcagtatatcctgccctcagtttgtcttactgtaagtcatacaagtgtct 721 gcagaagcacaagctgcggattgcccaacaaagaaaacagacttgaagaaaatgagatga 781 tcaacttaactcttcatccatccaaaaagagtgggcctcaggtgaaactctctggcagcc 841 ataaatggagttattcactcatcaaaaaacacagaagaagatatagcaagaagacagcat 901 gtgtgttacctgctccagaaagaccttctcaagagaaccactccagaatacttccagaaa 961 actttggccctgtaagaagtcagctcccttcatccagtaagttcacaccaggggtcccca 1021 cttgctttgagataaaacccgaagaaaactcagatgttcaCgaattgagagtaaaacgtc 1081 ctgtcacaagaataaaaaagagaCctcgaagegttttctacagactgaccaeactgatat 1141 tagtatttgctgttagctggatgccactacacccttcccatgtggtaactgattttaaCg 1201 acaatcccacttcaaaeaggcatctcaagtcggcgcattgcattCgccatttgctgggca 1261 cgatgtcctgttgtcttaatccaactccatacgggctccctaacaatgggactaaagccg 1321 acctagtgtcccttatacaccgcctccacatgtaataattctcactgttt

8. VIP

LOCUS DETERMINATION OF ACCESS N10 VERSION KEY WORD

SOURCE OF ORGANISM

REFERENCE AUTHORS

NAME

THE LOG MEDLINE COMMENTS

Signs of source

Vip 1511 U. about. Mrna pri 19 March 1999

mrna vasoactive intestinal peptide (VIP) homo sapiens ΝΜ003381

g4507896

ΝΜ003381.1 GI:4507896

man

Homo sapiens

Eukaryota ; Metazoa ; Chordata ; vertebrata ; Mammalia ; Eutheria ; Primates ;

Catarrhini ; Hominidae ; Homo

1 (of base 1-1511)

Delamarter, j. f., Buell, g. n., Kawashima, ε ., Polak, 1. Μ ., and bloom, S.R.

Vasoactiveintestinalpeptide : expression care the prohormone in bacterial cell s

Peptides 6 Suppl 1,95-102 (1985)

86016352

Refseq : this reference sequence has been received from M36634.

provisionalREFSEQ ; this recording represents temporary background sequence, which still has not been object observation by a person.

Final recommended reference sequence can a certain way differ from this.

Location/ Qualifiers

1. .1511

/ organism"' homo sapiens*

/ db_xref = * taxon : 9606"

/ map = " 6q24-q27*

/ tissue_type = " pancreatictumor *

gene 1. .1511

/ gene "" vip"

/ db_xref = "MIM:192320"

/ db_xref = " LocusID : 7432 *

sig_peptide 67. .129

/ product "" vasoactiveintestinalpeptide *

CDS 67. .579

/ gene = * vip*

/ codon_start = l

/ product = * vasoactiveintestinalpeptide */ protein_id = * NP_003372.1'

/ db_xref = * pid: g4507897 *

/ db.xref = " GI:4507897*

/ translation "" MDTRNKAQLLVLLTLLSVLFSQTSAWPLYRAPSALRLGDRIPFEGANEPDQVSLKEDIDMLQNALAENDTPYYDVSRNARHADGVFTSDFSKLLGQLSAKKYLESLMGKRVSSNISEDPVPVKRHSDAVFTDNYTRLRKQMAVKKYLNSILNGKRSSEGE

mat_peptide

mat_peptide

polyA_signal

SPDFPEELEK *

307.. 387

/ product = " vasoactiveintestinalpeptide"

439.. 522

/ product "* vasoactiveintestinalpeptide"

1326.. 1331

NUMBER OF BASES 541 A 255 C 276 G 439 Τ

A

1 ggtcagccccaaaacaatccggaacggccagctccgggggagcacgaccgggcgagaggc 61 acagaaatggacaccagaaataaggcccagctccctgtgctcctgacecttctcagtgtg 121 cccctctcacagactccggcatggcctcctcacagggcaccttccgctctcaggctgggt 181 gacagaataccctttgagggagcaaacgaacccgatcaagtttcactaaaagaagacatc 241 gacatgttgcaaaatgcactagctgaaaacgacacaccctattatgacgtatccagaaat 301 gccaggcacgccgatggagcccccaccagcgaccccagtaaactcttgggtcaactttcc 361 gccaaaaagtaccttgagtctcctatgggaaaacgtgttagcagtaacatctcagaagac 421 cctgtaccagtcaaacgtcactcagatgcagccttcactgacaactatacccgccttaga 481 aaacaaatggctgcaaagaaatacttgaactcaattctgaatggaaagaggagcagtgag 541 ggagaatctcccgactCtccagaagagttagaaaaatgatgaaaaagacctttggagcaa 601 agctgatgacaacttcccagtgaattcccgaaggaaaatgatacgcaacataactaaact 661 ttagaccccacataagtaacccaagaaaacaacttcaatatccaaaccaaataaaaatat 721 tgtgttgtgaacgctgtgatgtactccagccaatgCaataactgtgaagCttacatcgta 781 aatagtatttgagagttctaaatcttgtctctaactcaCaaaaagcccgcaacctcatat 841 gctgtatatcctttccaacaaaaaaatacactccaatgataagtaatgctaggttaatcc 901 aaetacaegagacgcttttggaagagtagcaacagagcaaaattgatgcgtttatctata 961 gagtgtactcaactaCtcaggagagtagaacagacaaccagtgtgtctaaatttgaacgt 1021 taagcagatggaatgctgtgtcaaataaacctcaaaatgtctaagatagtaacaatgaag 1081 acaaaaagacattctCccaaaaagaCtttcagaaaatattatgtgtttccataccttata 1141 ggcaacccctatttttaatggtgtcttaaaaaatctcaaatttggattgctaatcaccaa 1201 aggctctctcctgatagCcttccagtcaaggagaacgacccctgcccctgacactgaaac 1261 ttccctctctgcttgtgttaagtatgtgcaaaacgtgaagtgaaCgaaacactcagttgt 1321 tcaataacaaatatttttgccataatgaetcagaatattgctttggtcatatgagcttcc 1381 ttctgtgaaatacattttggagacacaactatttctccaaaataattttaagaaatcaaa 1441 gagagaaaataaagaccttgcttatgattgcagataaaaaaaaaaaaaaaaaaaaaaaaa 1501 aaaaaaaaaa a

9. Receptor VPACl

LOCUS DETERMINATION OF ACCESS

NID

VERSION KEY WORD SOURCE OF ORGANISM

REFERENCE AUTHORS

NAME

THE LOG MEDLINE COMMENTS

Signs of source

Vip 1511 U. about. Mrna pri 19 March 1999

mrna vasoactive intestinal peptide (vip) homo sapiens nm_003381

g4507896

NM_003381.1 Gl:4507896

Man

Homo sapiens

Eukaryota ; Metazoa ; chordata ; vertebrata ; Mammalia ; Eutheria ; Primates ;

Catarrhini ; Hominidae ; Homo

1 (of base 1-1511)

Delamarter, 3. F., Buell, g. n., Kawashima, ε ., Polak, J.M., and bloom, S.R.

vasoactiveintestinalpeptide : expression care the prohormone in bacterialcells

Peptides 6 Suppl 1,95-102 (1985)

86016352

refseq : this reference sequence has been received from M36634.

provisionalrefseq : this recording represents temporary background sequence, which still has not been object observation by a person.

Final recommended reference sequence can a certain way differ from this.

Location/ Qualifiers

1. .1511

/ organisjn = " homo sapiens'

/ db_xref = " taxon : 9606"

/ map = '6q24-q27'

gene

sig_peptide

CDS

mat_peptide

mat_peptide

polyA_signal

/ tissue_type = ' pancreatictumor'

1. .1511

/ gene = "vip"

/ db_xref = "MIM:192320"

/ db_xref ≈ " LOCUSID : 7432"

67. .129

/ product = " vasoactiveintestinalpeptide'

67.. 579

/ gene = " vip'

/ codon_start = l

/ products " vasoactiveintestinalpeptide"/ protein_id = ' NP_003372.1"

/ db_xref = "PID:g4507897"

/ db_xref = "GI:4507897"

/ 1 rans la t i οη = " MDTRNKAQLLVLLTLLSVLFSQTSAWPLYRAPSALRLGDRIPFEGANEPDQVSLKEDIDMLQNALAENDTPYYDVSRNARHADGVFTSDFSKLLGQLSAKKYLESLMGKRVSSNISEDPVPVKRHSDAVFTDNYTRLRKQMAVKKYLNSILNGKRSSEGESPDFPEELEK"

307.. 387

/ products " vasoactiveintestinalpeptide'

439.. 522

/ product " ' vasoactiveintestinalpeptide'

1326.. 1331

541 A

255 C

276 G 439 Τ

1 ggtcagctccaaaacaatccggaacggccagctccgggggagcacgactgggcgagaggc 61 acagaaaeggacaccagaaataaggcccagctccttgtgctcctgactcttctcagtgtg 121 ctcttctcacagactteggcatggcctctttacagggcaccttctgctctcaggttgggt 181 gacagaataccctttgagggagcaaatgaacctgatcaagtttcattaaaagaagacatt 241 gacatgttgcaaaatgcattagctgaaaatgacacaccctattatgatgtatccagaaat 301 gccaggcatgctgatggagttttcaccagtgacttcagtaaactcttgggtcaactttct 361 gccaaaaagtaccttgagtctcttatgggaaaacgtgttagcagtaacatctcagaagac 421 cctgtaccagtcaaacgtcacccagatgcagtcttcactgacaactatacccgccttaga 481 aaacaaatggccgtaaagaaatatttgaactcaattctgaacggaaagaggagcagtgag 541 ggagaatctcccgactttccagaagagttagaaaaatgatgaaaaagacctttggagcaa 601 agctgatgacaacttcccagtgaactcttgaaggaaaatgacacgcaacacaattaaatt 661 ttagattctacataagtaattcaagaaaacaacttcaatatccaaaccaaataaaaatac 721 tgtgttgtgaatgttgtgatgtattctagctaatgtaataactgtgaagtttacaetgta 781 aatagtatttgagagttctaaattttgtctttaactcataaaaagcctgcaatttcatat 841 gctgtatatcctttctaacaaaaaaatatattttaatgataagtaatgctagtrttaatcc 901 aattatacgagacgttttcggaagagtagtaatagagcaaaattgatgtgtttatccaca 961 gagtgtacttaactattcaggagagcagaacagataatcagcgtgtctaaatttgaatgt 1021 taagcagatggaatgctgtgttaaataaacctcaaaatgtctaagatageaacaatgaag 1081 ataaaaagacattcttccaaaaagattttcagaaaatattatgtgtttccatattctata 1141 ggcaacctttatttttaatggtgttttaaaaaatctcaaatttggaetgctaatcaccaa 1201 aggctctctcctgatagtcttccagttaaggagaacgacccctgcttctgacactgaaac 1261 ttccctttctgcttgtgttaagtatgtgtaaaatgtgaagtgaatgaaacactcagttgt 1321 tcaataacaaatattcttgccacaatgactcagaatattgctttggtcatatgagcttcc 1381 ttccgtgaaatacattttggagacacaactatttttccaaaataattttaagaaatcaaa 1441 gagagaaaataaagacctcgctcacgaccgcagataaaaaaaaaaaaaaaaaaaaaaaaa 1501 aaaaaaaaaa a

10. RECEPTOR OF DETERMINATION OF ACCESS

VERSION KEY WORD SOURCE OF ORGANISM

REFERENCE AUTHORS

NAME

THE LOG MEDLINE SIGNS VPAC2

humhvrp 1640 U. about. mrna pri 16 February 1995

mrna human khelodermin - predpochitayushchego receptor vip (receptor VIP2 / PACAP ), complete cds.

LB6566

g550477

LB6566.1 GI:S50477

receptor pacap; receptor vip; khelodermin - predpochitayushchii receptor cdna-mrna homo sapiens

Homo sapiens

Eukaryota ; Metazoa ; Chordata ; vertebrate ; Mammalia ; Eutheria ;

Primates ; Catarrhini ; Hominidae ; Homo

1 (of base 1-1640)

Svoboda, Μ ., Tastenoy, μ ., van Rampelbergh, 3 ., Goossens, 3. F., de Neef, ρ ., waelbroeck, Μ ., and Robberecht, ρ.

Molecularcloning and functional characterization care a human vip receptor from subclause sup-t1 lymphoblasts

Biochem. Biophys. res. Commun. 205 (3), 1617-1624 (1994) 95110300

Location/ Qualifiers

source 1. .1640

/ organism = ' homo sapiens"

/ db_xref = " taxon : 9606"

/ cell_line = ' sup Τ1 lymphoblast"

/ clone_lib = " lamda zap ii"

sig_peptide 163. .231

/ note = " putative ; putative"

CDS 163. .1419

/ note ^ 'human VIP2 receptorwas previously called _AOE296A0AO> helodermin - preferring vip receptor' ; transmembranedomains are located at positions : 637-696 ; 772-844;

880-948 ; 1003-1071 ; 1147-1209 ; 1243-1302. ;Potentialglycosylation sites are located at: 334-336 ; 424-426;

436-438. "

/ codon_start = l

/ functions"vip and PACAPreceptor"

/ protein_id = * AAC37569.1'

/ db_xref = ' PID:g550478"

/ db_xref = * gi: 550478^-

/ translations " MRTLLPPALLTCWLLAPVNSIHPECRFHLEIQEEETKCTELLRSQTEKHKACSGVWDNITCWRPANVGETVTVPCPKVFSNFYSKAGNISKNCTSDGWSETFPDFVDACGYSDPEDESKITFYILVKAIYTLGYSVSLMSLATGSIILCLFRKLHCTRNYIHLNLFLSFILRAISVLVKDDVLYSSSGTLHCPDQPSSWVGCKLSLVFLQYCIMANFFWLLVEGLYLHTLLVAMLPPRRCFLAYLLIGWGLPTVCIGAWTAARLYLEDTGCWDTNDHSVPWWIRIPILISIIVNFVLFISIIRILLQKLTSPDVGGNDQSQYKRLAKSTLLLIPLFGVHYMVFAVFPISISSKYQILFELCLGSFQGLWAVLYCFLNSEVQCELKRKWRSRCPTPSASRDYRVCGSSFSHNGSEGALQFHRASRAQSFLQTETSVI" mat_peptide 232. .1476

/ functions"vip and PACAPreceptor"

NUMBER OF BASES

315 A 512 C

461 G 352 Τ

gggcggcccctcgcccccggcgctgggcggtgacctgctgaaacacaggaaagcctgcagccgtcacggcgcaaaaactggccacagcgaataccctgggcccccaggaatcctgagagccgcaccgcccagcacegcacccccggcggcgcceccccacgtcgccgggactcccaccactaacaccccocgctcctgccgcatctcccccggcggccgcggcgaagccgCcccccacaatcccgcaaacgcccacggccgcaccgtgccccacacccggcgcgcCcgggcccacgctggcccccggcacgccgctcgccggaagaaacatggcgtctgggcccegcccacacgagtgaccccggaggatctacagcgccgccgcacCgccaccccagcgcgaccagccacacggccaaccacgctcccccgcccgcacctacaaacgaccgccaactccagacgtcggceaecccgcegcaaataccagcceceactgegcgcccgacccggcccggagggagaccccgcggggcccccgggcaggccagcgcccggctaggcggcggagccgagctcgcccgtgaacaaaatgtacaggacaacatcaaaagccctcaggacggtcaggagagcaagacccctgacgcaccaggaatcccggccaaggecccccegggctcccccggccceagaaggcggtgcacggacacagcgcgcgcccttcccaggcaacgacceccggcgcccacaccgcttgtccccgaacaccgcccgcgaggcgccccgcgccacccagcgcggggccgagcgcggccccacagctgcggacccgggggaggatgcggacgcattcacccagceecegagcgtgcCggcggcaatttttaagacgtCccctcacgettcactctCgcaacacacccacctacgacgttcccgggctgcaaCgctggtggagcttcccggcctgcggccagcctggtgggttttagcactacagccccagtaaccacaCggtagccgtgcctgtgaggtgcagccgggaCtaagCtccaccgcccacccccggacgccggctgactccgtcaggcccgaggtcctaggacgggctgctgcctagaacgccgagtcCcaaacagcctgccaatcagcaaagcaagatcccgtctatcctggtgaggaagcatagaacccgcccctaccccagcgccgagcccgggggccctacctacctcccggctccacccacatacgaacaacgaactccgcaagaggccggtttgccgcgcgggccgctcgcgcgagccgcagggtccgccgcgCcccgacctgtcggaccccCccceccagccggccgc