ULTRASOUND CONTRAST MEDIUM CONTAINING HYDROGEN PEROXIDE-SENSITIVE NANOPARTICLES

The invention relates to nanoparticles to provide hydrogen peroxide sensitive bubbling induced ultrasonic contrast agents.

Or less, more detailed intended to 2000.

The present invention is represented by formula 1, oxalate (oxalate) 1, 4 - 1 showing cross protection on coupled 1 block; and oxalate and 4 - hydroxy benzyl alcohol (4 a-hydroxybenzyl alcohol) or a derivative thereof coupled 2 block; comprising nanoparticles consisting of ultrasound contrast media is fixed to the body.

[Formula 1]

In formula 1, R is H or OCH₃ And,

N is integer number of 10 to 50, m is an integer from 5 to 20.

The 4 - hydroxy benzyl alcohol (4 a-hydroxybenzyl alcohol) derivatives preferably neel reel handler does not limited to alcohol (vanillyl alcohol).

In the chemical formula 1 R is OCH₃ When, the nanoparticles (vanillyl alcohol provided containing copolyoxalate) represented by formula 2 PVAX are disclosed.

[Formula 2]

In the formula 2, n is integer number of 10 to 50, m is an integer from 5 to 20.

When R is H in formula 1, the nanoparticles represented by formula 3 (HBA provided incorporatred copolyoxalate) HPOX are disclosed.

[Formula 3]

In the formula 3, n is integer number of 10 to 50, m is an integer from 5 to 20.

The nanoparticles of the first molar ratio of 4:1 to 2:3 1 block 2 block is preferably, more preferably 2:3 are disclosed.

The 10,000 to 20,000 Daltons (Dalton) preferably has an average molecular weight of the nanoparticles in.

In one embodiment of the present invention, 1, 4 - cyclohexanedimethanol [heyk[heyk] It buys with the D methanol hydroxy benzyl alcohol or its derivative in a triethylamine mixing some excellent. The triethylamine is oxalate (oxalate) in polymer synthesis reagents as to make the HCl is removed to facilitate odorous acts as base (base) can be. (Oxalyl chloride) is added to the mixture after oil/oxalate reel chloride copolymers prepared nanoparticles by emulsion method (oil provided in-a water emulsion method).

The diameter of the nanoparticles can have a 100 nm to 2000 nm, encased material (e.g., capable to) sized according can be changed, not limited to.

The nanoparticle hydrogen peroxide (hydrogen peroxide, H₂ O₂ ) CO by₂ Generating a bubble effect which amplifies the ultrasonic frequency resonance to be implemented so as ultrasonic signal, density of hydrogen peroxide are useful as ultrasound contrast media can be pathological environment.

The the hydrogen peroxide is produced as active oxygen (Reactive Oxygen Species, ROS), trauma, damage pressure chest muscles (crush injury), radical number axis generated upon muscle damage by mechanical damage and can be, in particular specific cancer cells can be generated.

In one embodiment of the present invention, mouse calf muscles achilles tendon administrates the hydrogen peroxide is generated after confirming whether mechanical damage in the model, nanoparticles of the present invention injecting ultrasonic signal that is an amplification near the site of injury when a predetermined ultrasonic Image has been confirmed.

Known ultrasound contrast agents of the present invention having at least one effective ingredient of 1 can be further contains an amplification effect.

The commonly used imaging agents comprise an appropriate carrier, excipient and a diluent can be further comprises. Also, according to the conventional inhaled, granule, tablet, capsule, suspension, emulsion, syrup, aerosol such as oral formulations, xerosis, formulated in the form of a sterile injection solution or left system can be used. The known in the art (Remington's Pharmaceutical Science, recent, Mack Publishing Company, Easton PA) suitable preparations contain nucleotide to the disclosed of using preferably. For example pharmaceutical compositions can be included in the carrier, excipient and a diluent include lactose, dextran with [thu[thu] five [su[su], sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil etc.. The composition there is usually formulated using fillers, extenders, binders, wetting agent, disintegrating agent, surfactant such as diluent or excipient agent formulated as a substrate. Purification is a solid preparation for oral administration, annularity system, inhaled, granules, etc. included in a capsule, the composition at least one such solid formulations of one or more excipients for example, starch, calcium purification, sucrose, lactose, gelatin pressure is lowered formulated as a substrate. Also, in addition to simple excipients magnesium stearate, are also lubricant such as talc are used. For oral liquid formulations include suspension, content liquid, emulsion, syrup and the like commonly used diluent to a simple method for producing corresponding, in addition to the paraffin droplet various excipients, for example wetting agent, sweetener, fragrance, preservative can be like. For parental administration preparations contain a sterile aqueous solution, non-aqueous solvent, suspension, emulsion, freeze-dried preparation, left system multiple myelomas are included. Non-aqueous solvent, suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, an injectable the [ley[ley] which comes the [thu[thu] and ethyl ester is used as the alkali such as can be. Base include above [theyp[theyp] brushleft proposal, mark with goal, twin 61, carcass five fingers, the it soaked, is used as the preparing my Latin can be.

The terms "administration" are used in the invention any suitable method subject of the present invention means that the composition of the substrate.

Pharmaceutical composition of the present invention the preferred dosage of an individual state and body weight, degree of disease, forms, according to the stated time routes of different but, can be appropriately selected by one skilled. Said composition may be administered once per day administration, may be administered tracks formed between the disapproval.

The invention of contrast agent to a subject can be administered by a variety of routes. All of the schemas of unexpected administration can be, for example, oral, rectal or intravenous, muscle, subcutaneous, intrauterine epidural or cerebrovascular in such as administration by injection can be.

Ultrasound contrast agents of the present invention amplification on its own, or can be used in combination with drugs, is in docetaxel (Docetaxel), cisplatin (cis-a platin), cam toe tax new (camptothecin), paclitaxel (paclitaxel), tamoxifen (Tamoxifen), drug the [su[su] it will doze with the reel (Anasterozole), chronic myeloid leukemia (Gleevec), 5 - fluoro uracil (5 a-FU), a flocked shoe [tin[tin] (Floxuridine), type pro leading (Leuprolide), polyimide (Flutamide) flow other, with the red which will doze four the [thu[thu] (Zoledronate), doxorubicin (Doxorubicin), vincristine (Vincristine), gemcitabine (Gemcitabine), impaired (Streptozocin), [phul[phul] Latin (Carboplatin) carbonate, (Topotecan) irinotecan topotecan, irinotecan (Belotecan) bellows, irinotecan (Irinotecan), non-[leyl[leyl] Bin (Vinorelbine), atlas hour thunder Oh (hydroxyurea), foot baserain shoes (Valrubicin), retinoic roh [ik[ik] it buys (retinoic acid) sequence, maul small [thu[thu] rack tax [thu[thu] (Methotrexate), maul claw [ley[ley] it burns it pushed (Meclorethamine), claw [lam[lam] faithlessness (Chlorambucil), it will break plate (Busulfan), killing by poison[...][...] (Doxifluridine), bin [pul[pul] [su[su] (Vinblastin), e toe feed (Mitomycin), [phu[phu] red [ni[ni] hand (Prednisone), testosterone (Testosterone), american toe acid oestrone (Mitoxantron), aspirin (aspirin), salicylate (salicylates), ibuprofen (ibuprofen), my pro it counted (naproxen), lung Roh pro pen (fenoprofen), indomethacin (indomethacin), phenyl father hit zone (phenyltazone), polyimide (cyclophosphamide) cyclo-phosphatase, the maul claw eta it pushed (mechlorethamine), dexamethasone promotes (dexamethasone), tree sitcom roh theory (triamcinolone), [phu[phu] red [ni[ni]brush theory (prednisolone), celecoxib (celecoxib), valdecoxib (valdecoxib), [ni[ni] maul [syul[syul] leading (nimesulide), or corticosteroid (corticosteroid) be a drug such as nose [lu[lu] mote hand (cortisone).

The invention also relates to a method of information acquisition ultrasonic images using the ultrasonic contrast agents.

Or less, to aid in the understanding of the present invention preferred embodiments, experiments and examples example aaeap. However of the following embodiments, the present invention easily understand than experiment example and embodiments provided for ephemeral, embodiments, and restricted if an example process by the experiments are not correct.

Embodiments 1. PVAX Of making nanoparticles

1, 4 - cyclohexanedimethanol (1, 4 a-cyclohexanedimethanol) (-10. 98 mmol) and 4 - neel reel alcohol (4 a-vanillyl alcohol) (-16. 47 mmol) dissolved in 20 ml of tetrahydrofuran (Tetrahydrofuran, THF) well dried nitrogen filling and 4 °C (triethylamine) in triethylamine (60 mmol) was added to a drip. The, 60% based on the molar amount of alcohol neel reel was included in the mixture. The mixture obtained by dissolving dried 25 ml THF oxalate reel chloride (oxalyl chloride) (-27. 45 mmol) was added in a 4 °C leak in. The reactants at room temperature nitrogen-containing 6 to hold time, dichloro methane (DCM) precipitating the polymer synthesized using cold hexane to adapted to extract PVAX copolymer finally obtained.

¹ Using the chemical structures of copolymer H NMR PVAX is confirmed, the result also 1 precursor, has been identified and is as follows.

¹ H NMR: 7. 0 - 7. 3 (m, 3H, Ar), 5. 3 (m, 2H OCH₂ - PhO provided CH₃ ), 4. 1 - 4. 2 (m, 4H, COOCH₂ CH), 3, 8 (m, 3H, OCH₃ ), 2. 2 (m, 2H, C (CH₂ ) ₃ HO), 1. 0 - 1. 8 (m, 8H, Cyclic CH₂ ).

50 mg of the 5 ml of 10% DCM 500 μL PVAX copolymer of poly (vinyl alcohol) (PVA) (w/v) after dissolving some excellent solution. The reaction mixture is ultrasonic crusher (Sonicator, Fisher Scientific, Sonic Dismembrator 500) using 30 seconds when the ultrasonic treatment, oil/water emulsions for 2 minutes to form a homogeneous stored in the reactants (PRO Scientific, PRO 200 a-homogenizer). 20 ml of the PVA emulsion (1 w/w %) adding solution, stored in the further homogeneous 1 minutes. The remaining solvent is removed using a rotary evaporator, 4 °C, 11,000 rpm in 5 minutes centrifuging finally PVAX nanoparticles obtained. Deionized water (deionized water) washed and twice to same, then experiments using the air. PVAX nano particle produced dynamic scattering method using nano particle analyzer (Brookheaven instrument corp. , Holtsville, NY) by using a have, in the form of nanoparticles (SUPRA 40VP, Carl Zeiss, Germany) scanning electron microscope observation by using a transparent conductive layer, each of the 2A and 2B shown and also to.

Embodiments 2. HPOX Of making nanoparticles

1, 4 - cyclohexanedimethanol (1, 4 a-cyclohexanedimethanol) (-10. 98 mmol) and 4 - hydroxy benzyl alcohol (4 a-hydroxybenzyl alcohol) (-16. 47 mmol) dissolved in 20 ml of tetrahydrofuran (Tetrahydrofuran, THF) well dried nitrogen filling and 4 °C (triethylamine) in triethylamine (60 mmol) was added to a drip. The, 60% based on the molar amount of 4 - hydroxy benzyl alcohol was included in the mixture. The mixture obtained by dissolving dried 25 ml THF oxalate reel chloride (oxalyl chloride) (-27. 45 mmol) was added in a 4 °C leak in. The reactants at room temperature nitrogen-containing 6 to hold time, dichloro methane (DCM) precipitating the polymer synthesized using cold hexane to adapted to extract HPOX copolymer finally obtained.

¹ H NMR HPOX identifying chemical structures of copolymer using a transparent conductive layer, as a result that has been identified as follows.

¹ H NMR: 7. 2 - 7. 3 (m, 2H, Ar), 7. 5 - 7. 6 (m, 2H, Ar), 5. 3 (m, 2H OCH₂ - PhO), 4. 1 - 4. 3 (m, 4H, COOCH₂ CH), 1. 8 - 2. 0 (m, 2H, C (CH₂ ) ₃ HO), 1. 0 - 1. 8 (m, 8H, Cyclic CH₂ ).

50 mg of the 5 ml of 10% DCM 500 μL HPOX copolymer of poly (vinyl alcohol) (PVA) (w/v) after dissolving some excellent solution. The reaction mixture is ultrasonic crusher (Sonicator, Fisher Scientific, Sonic Dismembrator 500) using 30 seconds when the ultrasonic treatment, oil/water emulsions for 2 minutes to form a homogeneous stored in the reactants (PRO Scientific, PRO 200 a-homogenizer). 20 ml of the PVA emulsion (1 w/w %) adding solution, stored in the further homogeneous 1 minutes. The remaining solvent is removed using a rotary evaporator, 4 °C, 11,000 rpm in 5 minutes centrifuging finally HPOX nanoparticles obtained. Deionized water (deionized water) washed and twice to same, then experiments using the air.

Embodiments 3. Preparing animal model

For the manufacture of mechanical damage model for experiment 8 week zero[lays[lays] Sprague Dawley (male, Orient BIO, Korea) have been used in the Committee effective jeollabuk-do experiments all animals university abstract is been.

Before damage, [khey[khey] otherit pushed withrum penny (8:1) and system for detecting BAX was implanted in a selected from anesthesia. The anesthesia for detecting BAX (lateral decubitus position) which faces the opposite knee in the bent state to the armrests of side and above safe fixing has been, 3 also showed the same.

Then the damage shown in fig. 4 for detecting BAX constant seal properties (TMS provided PRO texture analyzer, FTC corp. , Sterling, VA) the VIP, through 5 mm thickness at a rate of 20 mm/min until the the back of the armrests 160N compressing force of his.

3 - 1. Muscle damage model of

(Gastrocnemius, GCM) for detecting BAX of pivoted near the fixed for impairing the, downward compression of the upper knee joint 1 cm (triceps surae muscle) of his knee. (Soleus muscle) (gastrocnemius muscle) which is composed of the upper and the knee that muscle of family spleen near, the whole experiment for detecting BAX of damaged knee joint in order to spleen musclewith that muscle of family downward in a similar 0. With one 5 cm height, the same 5A also shown.

Upon detection and ultrasonic inspection after damage, muscle damage sites for accuracy of damaged skin marker display when the Devon, ultrasonic after confirmation muscle skin suture (skin suture) was variously.

3 - 2. (Achilles tendon) achilles tendon damage model of

For impairing of the fixed achilles tendon for detecting BAX, achilles tendon of insertion in the heel bone (calcaneus) (insertion) in compression when the vessel immediately above, 5B also showed the same.

After ultrasonic inspection and damage upon detection, a Devon achilles tendon skin marker have been designated for accuracy of damaged site of injury.

Experiment example 1. Agar with [su[su] gel Phantom using ultrasound images inspection

Ultrasonic (Z. One ultra, Zonare medical systems, San Farancisco, USA) is a linear transducer utilizing probe model of 5 - 14 MHz (transducer length=62x10 mm, viewing width=55 mm) was used. Ultrasonic inspection of musculoskeletal ultrasonic Image diagnosis experience was a pseudo acoustic performance. First, agar with [su[su] gel (agarose gel phantom) 3 mg/mL of hydrogen peroxide after the addition of the nanoparticles HPOX PVAX or phantom to measuring changes in ultrasound imaging signal according to a transparent conductive layer, each of the same and also shown to also 6A 6B.

As also shown in also 6A and 6B, but does not require such a hydrogen peroxide-free under the ultrasonic signal amplification, hydrogen peroxide (5 mm) in the presence of over time ultrasonic signal is increased and sustained 30 min or Image has been confirmed. This hydrogen peroxide PVAX and HPOX CO by nano-particles₂ Ultrasound imaging signal amplifying and exhibits a bubble.

Experiment example 2. Animal model using ultrasound images inspection

Ultrasonic (Z. One ultra, Zonare medical systems, San Farancisco, USA) is a linear transducer utilizing probe model of 5 - 14 MHz (transducer length=62x10 mm, viewing width=55 mm) was used. Ultrasonic inspection of musculoskeletal ultrasonic Image diagnosis experience have a pseudo acoustic performance, immediately after achilles tendon of damaged muscles, enforcing his long axe PVAX to three times after the injection.

PVAX scanning is 1 mg/mL concentration after sprayed PBS PVAX nanoparticles to have legs animal model 300 μL right scanning, site of injury is generated H PVAX both muscle and achilles tendon damage model₂ O₂ In response to view whether damaged site without direct injection, the muscles of his scanning near the groin of the heat on the site of injury.

1 - 1. Muscle damage model

3 - 1 of the upper knee in the model always embodiments of pivoted near damage when identifying the processor immediately after scanning PVAX scanning and PLGA and facia, 7 also showed the same.

As also shown in 7A, after identifying the upper knee of ultrasonically site of injury when, intramuscular portion identifying a free area of all holes by local that echothat echo region uses a local damage RAT when stop, clear the compromised position obtain information transmitted to the wanted. While, the scanning ultrasonic waves while identifying result PVAX site of injury, site of injury CO along after the injection₂ After 20 - 30 seconds generates particles began to tell on and around the site of injury by echo characteristicecho characteristic grain cluster is not less than making sure that the facia, damaged and size was similar.

Also, as shown in also 7B, PLGA identifying site of injury while scanning ultrasonic waves result, fluid does not exhibit any echo characteristic provided only diffusion of particles has been confirmed.

1 - 2. (Achilles tendon) achilles tendon damage model

3 - 2 always embodiments of damage in the model when identifying achilles tendonachilles tendon PVAX and damage when the ultrasound scanning immediately after, the same 8 also shown.

As shown in fig. 8, after identifying site of injury when ultrasonically achilles tendon damage, tendon fiber sample is input to a shaped line up. However detecting BAX achilles tendon itself to identify a site of injury through ultrasonic insertion of very difficult. While, the scanning ultrasonic waves while identifying result PVAX site of injury, site of injury CO along₂ By echo characteristic particles generates up clusters has been confirmed.

This PVAX nanoparticles caused hydrogen peroxide exhibits that ultrasonic signal of the site of injury.