Process of purification of the hydroxocobalamine

AS AFRICAN AND MALAGASY

INDUSTRIAL PROPERTY

P. 887

Yaounde (Cameroon) Patent International Patent Classification: c07 - a61 no. 00146

o.C. Æ P.I..

THE V *

Delivered 15 March 1966, published at olficiel ballot no. 2 of 1966. Priority: Patent application deposited Monaco under the no. 252.62.320 22 September 1960 and in France under the PV n° 869,812 (1.346.257) 2 august 1961 to names of requesters.

Method of purifying rhydroxocobalamïne.

The present invention relates to a method for

the separation and purification of hydroxocobalamin

(also called vitamin b 126) from mixtures storytelling -

gym this substance.

It is known that the vitamin b 126 differs from the Vita -

12 b-lead (or cyanocobalamin) by the fact that in the

first cobalt carries a hydroxy group which is EMR -

located in the second with a cyano group.

For pharmaceutical use, the attempt today

to prefer hydroxocobalamin to cyanocobalamin, because

the first having basic properties between more

easily in the metabolism of individuals.

For making the hydroxocobalamin, it is customary

from the cyanocobalamin, which is caused to undergo

various physical or chemical treatments.

Thus, a known technique provides to reduce the

cyanocobalamin by catalytic hydrogenation which

the converts to cobalamin. Then the solution is oxidized

converting it into hydroxocobalamin.

According to a method described in the French Patent

no. 1.346.256 deposited by the same of 28 July 1961 -

it can be also the claimant party hydrogenation without - Advanced

lysor, using hydrogen nascent obtained " in vivo

in situ " by contact with an acidic solution of the cyanocoba -

laminate with granulated zinc, after which the oxidation is

performed by blowing air into the liquid obtained diluted

water.

By these various methods must undergo

purifications are primary before being used. Thus,

in the case of the last method aimed, solution is processed

obtained after the oxidation with a mineral base such as

the soda, which precipitates the zinc salts in the form of a HY -

droxyde. Is filtered to remove the latter and evaporation

the filtrate under vacuum. The residue is taken up by a small

amount of methyl alcohol. Is filtered and evaporation of the

filtrate: the result is hydroxocobalamin raw.

The crude product from the product technically

pure, it is known to proceed in this way: redissolved

the hydroxocobalamin raw water, free of co2,

and the pH is adjusted to between 8.5 and 9.5.

Then added an organic solvent-soluble skin, belonging to the group formed by acetone, dioxane and tetrahydrofuran. These solvents are abbreviated herein as "preferred solvents". These bodies are used in an amount of about 20% to 80% of solvent of aqueous solution. And then allowed to stand at low temperature (5 to 10 °c for example), and is obtained by crystallization of crystals of hydroxocobalamin to 70% purity about, which constitute the hydroxocobalamin technology.

The 30% impurities consist of moisture, by cyanocobalamin unreacted, and by salts entrained during the preceding crystallization.

It is known also that can increase the purity technical by subjecting the same to a chromatographic separation, mainly on an alumina column. This can eliminate a substantial portion of the cyanocobalamin unreacted.

By elution, collected and a hydroxocobalamin enriched (to 80% purity for example). Cyanocobalamin was thus separated and an amount of salts, absorbed together with the hydroxocobalamin with alumina.

However these impurities can compromise the stability of I ' hydroxocobalamin which can degrade over time. Instability of hydroxocobalamin is are known by the literature, and it has been suggested the addition in solutions of hydroxocobalamin tampons or stabilizers to counter this, but these bodies might reduce its therapeutic efficacy.

The process which is the object of the present invention overcomes the deficiencies of prior methods of purification, allowing to achieve a product very rich (more than 95% purity) and hence a high stability. The aqueous solutions of hydroxocobalamin thereby established may have a pH around 8, yet remain stable, for several months, when stored at room temperature but to the exclusion of light.

According to the invention, the method is mainly characterized in that after carrying out, in a manner known in itself, a treatment chromatograpMgue to hydroxocobalamin technical purity, and after elution of the chromatography column using a water-soluble organic solvent, is Iraite eluate thus obtained by a strong acid to form an acid salt of hydroxocobalamin and subjecting this salt to a passage successively on two ion exchange resins, the first being neutral resin retain impurities in saline to pass through hydroxocobalamin, the second being a basic resin, which fixes the acid by regenerating the hydroxocobalamin.

The chromatography is carried out preferably by sending on an alumina column dissolution of hydroxocobalamin technically pure in an aqueous solution of a preferred solvent (acetone, dioxan, tetrahydrofuran), the solvent concentration is between 75% and 85%, advantageously 80%.

The elution is carried out using an aqueous solution of the same solvent for chromatography, but with EMI water content higher, for example comprised between 40 and 80%.

The hydroxocobalamin obtained after elution with a pH of about 7.

It is acidified with a strong acid such as HCl or H₂ S0₄ so that the pH of the solution falls between 1.2 and 3.

This forms a salt of hydroxocobalamin (hydrochloride, sulphate for example). This salt is separated by crystallization and the crystals thus obtained are combined in aqueous solution. This creates a solution of hydrochloride or sulfate of hydroxocobalamin whose pH is about 3.5 to 4.

This solution is then sent on the resins provide breath freshening.

The first ion-exchange resin used is of the type. It is obtainable by polymerizing an anionic monomer in the pores of an anionic exchange resin (or basic) or by polymerization of a cationic monomer in a cationic exchange resin (or acid).

The linear polymer is locked inside the resulting from the cation exchange resin is being crosslinked. A stable product is produced, physically and chemically, anionic and cationic exchangers, mixed at the molecular scale.

Resins of this type are generally designated by the expression "ion retardation resins", because the flow of ions in the filtering tower is delayed due to ionic absorption properties of the resin.

In the case of the invention the resin secures the neutral salts entrained by the hydroxocobalamin and it can be regenerated by simple washing with purified water.

The basic resin layer is preferably anionic or comprises an active group of strong base. It can be prepared, for example, from aromatic amines and phenol. Also suitable are polystyrene-type resins quaternary ammonium. These resins can be regenerated by passing countercurrent to a dilute solution of fresh basic compounds (soda, ammonia, and the like) and subsequent washing.

The fact of having a strong basic resin after the resin is contrary to the practice of using known laboratory filtration on the resin and at the end of operations.

The resins are preferably inserted in columns superimposed sheets which are successively traversed by the solution to be purified. Each of these columns has a porous bottom for retaining the grains of the resin.

The procedure is controlled by the pH of the solution collected after the last column qpii must be at least equal to 7. To avoid the retention of too much product in the columns, can push the solution after its introduction, by sending a result of demineralized water.

The liquid obtained from the output columns can be re-crystallized by addition of a preferred solvent. The crystals formed are collected by filtration, washed by a preferred solvent or ether, then dried. In this manner a product concentration at least equal to 95%.

We now describe in more detail certain modalities of practical method. It will be assumed, for securing the ideas, that hydroxocobalamin was produced using nascent hydrogen obtained by contacting an acidic solution of cyanocobalamin with granulated zinc.

The aqueous solution of hydroxocobalamin raw must first be purged of soluble salts of zinc that it contains. For this purpose, by converting these salts in zinc hydroxide insoluble by action of a mineral base such as NaOH, nh4oh, of Ba (OH-) 2, by bringing the pH of the solution to a value greater than or equal to 8.5. Is filtered to remove zinc hydroxide which is precipitated, and evaporation of the filtrate under vacuum to dry. The residue, taken up by a small amount of methyl alcohol, gives a red solution. Is filtered, and then evaporation of the filtrate to obtain raw hydroxocobalamin.

Which is then diluted with the aqueous solution obtained, by adding one of the following organic solvents, soluble in water: tetrahydrofuran, dioxane or acetone.

The amount of solvent added is such that the resulting solution contains 75 to 85% of organic solvent for 25 to 15% water. Typically used a mixture 80/20. This mixture dissolved sufficiently cyanocobalamin or Phydroxocobalamine to accommodate the preparation of the solution to be used.

This solution is then subjected to chromatography on an alumina column after adjusting the pH of the solution and that of the column 7 to the same value. Alumina retains more strongly hydroxocobalamin that cyanocobalamin, removed from the solution and the fixed at a different level of the cyanocobalamin.

To elute, it is to say for then removing hydroxocobalamin that has been attached to the alumina, a solution comprising the same solvent, but whose water content has been increased. The invention thus makes benefit greater solubility of hydroxocobalamin, the adsorption of the product on the alumina becomes non sufficient to retain the product attached.

Is used, in general, for the elution, solutions containing 50 % solvent and 50 % water, but may be used solutions containing 20 to 60% of solvent, and thus 80 to 40% water.

The greater the water content is low, more product extraction of the alumina column is long, but instead the separations between the various products adsorbed are clearer.

According to the invention, the fraction of eluate containing hydroxocobalamin at the outlet of the alumina column, is acidified by excess appropriately chosen strong acid (HCl, h2so.1, and so on) so that the pH of the solution is raised to between 1.2 and 3. The solution was added with 9 volumes of the solvent used for the previous operation. In the presence of acid, hydroxocobalamin having basic properties, forms a salt (hydrochloride or sulfate) is allowed to crystallize at a temperature between 5 and 10 °c, and red crystals of the salt of hydroxocobalamin, which are isolated by filtration, and washed with acetone and ether. They are then redissolved in distilled water, and this new solution is then passed over a column of neutral resin capable of binding the impurities, while passing the salt of hydroxocobalamin.

For example, the resin may be any of those known under the trademark Retardion and sold by The Dow Rheology Index of Chemical Company analysis, the Midland, Mich.

Such a resin consists of very fine grain, which can be placed on a layer of about 10 to 50 cm high. The filtration is carried out at room temperature with a flow rate of about 3 ml/min.

The resin retains salts entrained within the hydroxocobalamin but without fully securing thereof, thereby regenerating the resin by further washing with water. It is verified the absence of salts by samples of filtrate. For example, if the acidification was performed by hosohoso.j, checked the absence of chlorides free in the solution.

The solution to this first purification by neutral resin is then passed over a column containing a basic ion exchange resin, which consequently stops the acid ion. This last column retains the chlorine ion or sulfate combined to cobalamin and regenerated thereby Phydroxocobalamine which remains in solution at the column outlet.

It can use a strongly basic anion exchange resin, consisting for example of spherical particles of 300 to 900 microns of a copolymer of styrene and divinylbenzene having quaternary ammonium moieties. The hourly flow rate for such a resin is preferably between 0.5 and 1.5 M.³ by m² resin.

This resin can be regenerated by 50 to 80 g of sodium hydroxide in solution at 5% per liter of resin.

Strong basic resins of this type are conventional industrial products.

The hydroxocobalamin may be identified by conventional assays: chromatography, partition coefficient between benzyl alcohol and water, acidimetric titration curves or spectrography.

Particularly methods which it will issue further may be used to monitor the purity of hydroxocobalamin obtained after treatment according to the invention.

Example 1: example witness. - 0.20 G of cyanocobalamin is dissolved in 50 cm³ a solution decinormal hydrochloric acid. This solution is passed over a column of 40 g of metallic zinc granules (500 to 1,400 micron). When the entire solution has been introduced and passed over the column, it is washed twice with 10 cm³ of distilled water.

Collecting the liquid in a vial where bubbles through an air flow throughout the operation.

Air oxidation is continued for 10 min of the last wash water. The solution is then ruby red.

The pH of this solution is adjusted to 8.5 by addition of a solution of caustic soda to precipitate decinormal zinc. Is filtered. Returning the solution pH to 7. This represents about 70 cc of solution, to which is added 280 cc of tetrahydrofuran, and extends over an alumina column chromatography of 30 mm in diameter and 120 mm high. The period of time of 1 hour is. This alumina has been previously buffered to pH 7. The elution is performed using a mixture of equal parts water and tetrahydrofuran.

The colored portion is collected, either 50 cc, rich hydroxocobal amino.

This fraction is added tetrahydrofuran so that there will be only 10% of water, and then initiates the crystallization with a few crystals of hydroxocobalamin, and allowed to crystallize between 5 and 10 °c during 24 hours. The crystals are collected by filtration, and washed with acetone and ether, and dried under vacuum at ambient temperature. This gives 0.15 g of crystals representing substantially 0.12 g of hydroxocobalamin be pure a purity of 80%.

Example 2.' - Operation is carried out as for the example 1 until the output of the alumina column.

The eluate rich is acidified to a pH of 1.2 by means of an excess of hydrochloric acid, then added tetrahydrofuran stem so that not more than 10% water in the solution. Allowed to crystallize between 5 and 10 °c.

The crystals of red chlorocobalamine formed are collected by filtration and washed with acetone and ether.

Such crystals is dissolved in distilled water at a rate of 50 cm³ water for 0.20 g of crystals. The solution so obtained is superimposed on two columns. First of all, on a first resin column named "Retardion" by Dow of Chemical Company analysis (Retardion 11 has 8) of 25 mm in diameter and 100 mm in height. The output of the first column, the liquid passes into the second, of the same size, which contains a strong basic resin, such as that designated "Dowex 2" of Dow.

The throughflow rate on these columns 2 cc/min is set.