DE102018005078A1 - Process for the preparation and composition of stabilized cob (II) alamine and cob (II) inamide solutions as starting preparations for the production of medicaments, medical products, food supplements and cosmetics - Google Patents

Abstract

The invention includes a process for the preparation and composition of liquid, stabilized cob (II) alamin and cob (II) inamide solutions. These physiological forms of the vitamin B12 group have not hitherto been available in stable solutions for direct use as medicaments, dietary supplements or available for cosmetics. According to the invention, cobalamines and cobinamides of oxidation level III are dissolved in alcohols or aqueous sugar solutions and reduced to oxidation levels II and I with an excess of reducing agents. The reaction takes place by heating these solutions for several minutes and with the exclusion of oxygen and light. The stabilization of the reduced forms is achieved by the excess of reducing agents, the selection of solvents, the reduced water content in these solvents and the exclusion of light and oxygen Manufactured and assembled liquid preparations are suitable as a starting product for the production of medicinal products, medical devices, food supplements and cosmetics.

Description

Several compounds are assigned to the vitamin B 12 group, for example cyanocobalamin, methylcobalamin or hydroxocobalamin. Numerous patents have been registered for the stability of pharmaceutical preparations (e.g. DE102007012644A1 ). Physiologically intermediate compounds from this group are cob (II) alamin (also called vitamin B12r) and cob (I) alamin (B12s). There they serve, among other things, as an electrophile for positively charged methyl groups of N-methyltransferase and play a role in the transfer of methyl groups to DANN (1), among others. They are also used physiologically to reduce dehydro-ascorbic acid to ascorbic acid (2).

Cob (II) alamin (CAS No. 14463-33-3) represents the reduced form of hydroxocobalamin i.e. In contrast to all other compounds in the group, the central atom cobalt is not in oxidation state III but in oxidation state II. Cob (I) alamin (CAS No. 18534-66-2) is the compound that is further reduced to oxidation level I. With a redox potential of - 740mV or - 1.45 V, the complexed cobalt compounds are in oxidation levels II and I. the strongest known nucleophiles in aqueous solution (3,4). In the case of cobinamides, the central structure of a cobalt cation complex-bound in a corrin is identical to that of the cobalamines. The statements on the oxidation levels and redox potentials are therefore transferable. Corresponding stable preparations of these physiological forms of the vitamin B12 group have not yet been described.

Cob (II) alamin can be prepared in vitro from hydroxocobalamin or cyanocobalamin by reduction with ascorbic acid (5), nascent hydrogen, borohydride or other reducing agents. In some cases there is a further reduction to oxidation level I or symmetry to oxidation levels I and III. Isolated cob (II) alamine is quickly oxidized back to the trivalent state with oxidizing compounds, especially atmospheric oxygen from its surroundings, or under the influence of light (4).

Cob (II) alamin is used for the synthesis of methylcobalamin. Here the methylcobalamin is produced from the intermediate cob (II) alamine with positively charged methyl groups (eg dimethyl sulfate or methyl iodide) (numerous patents, for example in the patents US3928320A and EP 1 236 737 B1 )

Haloalkanes and alkylating cytostatics exert their toxic effects via intermediately formed, positively charged three-ring or carbenium compounds. These highly reactive intermediates can react with nucleophilic compounds and thus be detoxified (6). Epoxies are oxygen tri-rings with a high ring tension, which can also be opened by nucleophiles. Inflammatory reactions include characterized by a high concentration of reactive oxygen species, which often lead to additional tissue damage. Inactivating these oxygen compounds can also significantly improve wound healing. Reactive oxygen compounds can also be inactivated by nucleophiles (7,8). Oxides of nitrogen, nitrous gases and nitrites are metabolized to nitrosamines, which in turn are metabolized to carbenium ions, which leads to DNA adducts, comparable to alkylating agents. Reactions with nucleophiles can effectively deactivate them. Nitric oxide can be bound by nucleophiles and thereby a shock caused by endotoxin will be revised (9,10,11). Ionizing rays form radicals in biological tissues, which have a damaging effect on the surrounding biomolecules. Radicals lose their reactivity when they react with nucleophiles (12).

For the use of nucleophilic substances in the field of medicinal products, medical products, food supplements and cosmetics, compounds are to be used which are well tolerated on the one hand, which are stable and which do not lead to any toxic secondary products.

Numerous active substances from the field of nucleophilic compounds are used pharmaceutically or in food supplements such as ascorbic acid, N-acetylcysteine, tocopherol, ubiquinone, nicotinamide or glutathione. The substances are administered orally and externally, but are also partially used as injections. The effect on the processes mentioned under [0005] is partly absent or very limited in the substances used to date. In particular, sufficient detoxification of the alkylating cytostatics is not observed. In the patent US8524774B1 describes a topical preparation for the treatment of psoriasis according to this principle, which however only contains cobalamines of oxidation level III. The effect described in this patent is presumably due to the fact that the reactive reduced forms of the cobalamins are formed physiologically by reduction potentials in the skin.

The reduced forms of cobalamines and cobinamides react with the compounds mentioned under [0005] to give the corresponding organocobalt compounds (for example methylcobalamin, ) respectively. Nitritocobalamin (NO2-Cbl, CAS-Nr.20623-13-6), Nitrosocobalamin (NO-Cbl) and Sulfitocobalamin (S03-Cbl). Here they again take on oxidation level III. These compounds are chemically stable. Physiological organocobalt compounds from the vitamin B12 group are methylcobalamin and adenosylcobalamin. Nitritocobalamin, nitrosocobalamin and sulfitocobalamin are also physiological.

In the patent US 2016/0000820 A1 "COMPOSITIONS AND METHODS FOR TREATING CARBON MONOXIDE AND / OR CYANIDE POISONING" describes an in-situ production of cob (II) alamine as a solution for infusion. Here, cob (II) alamine is partially formed by mixing hydroxocobalamin with ascorbic acid in a patented two-chamber solution, which is then applied immediately. The patented procedure requires a clear lead time, does not represent a controlled and complete implementation of the reduced forms and is only suitable for immediate use. The patent treats the treatment of cyanide and carbon monoxide poisoning as a development goal. This principle of therapy is based on a detoxification mechanism that is completely different from point [0008]: cyanide and carbon monoxide are bound in a complex manner to the central atom cobalt and oxidized to carbon dioxide in further intermediate steps (13).

The solution principle according to the invention includes the preparation of cob (II) alamine or reduced forms of cobinamides in aqueous-organic solution and their stabilization in oxidation state II over a longer period of time. The solutions stabilized in this way can be used in preparations for external use on the skin, on mucous membranes, in wounds or in the eye. The solution can also be used orally and could be used in the mouth and gastrointestinal mucosa in the form of liquid capsules. The passage of the cob (II) alamin into the bloodstream can also take place via the oral mucosa. In-situ dilution with physiological saline or Aqua pro injectione and use as an infusion or inhalation solution is also possible.

The preparation of cob (II) alamine according to the invention is carried out from hydroxocobalamin or cyanocobalamin in a mixture consisting of water and water-miscible organic solvents (e.g. polyethylene glycol, propylene glycol, glycerol) and / or reducing sugars (e.g. glucose, lactose, lactulose) at pH 4 to 8 is reduced with an excess of reducing agent (preferably ascorbic acid) to cob (II) alamine ( ). For this, the solution is heated to 25 to 40 ° C for about an hour. The reducing agent is added in a molar excess of at least 1:10. The water content of the solvent may not exceed 40%, the proportions of the other organic components mentioned can be varied in any ratio to one another. The viscosity of the solution can be increased by using higher molecular weight polyethylene glycol (up to 10,000 MW). This enables the production of appropriate polyethylene glycol ointments. When used orally, a predominance of the glycerol or sugar content is advantageous. The pH range mentioned can be achieved by a buffer and / or by the proportionate use of sodium ascorbate. The solution is additionally stabilized by adding nicotinamide, since this is also a nucleophile. In all manufacturing processes, oxygen contact and exposure to light must be excluded. This can be achieved by working under protective gas (nitrogen or argon) and / or under vacuum. Filling and storage must also be carried out with the exclusion of oxygen and exposure to light. This can be done using protective gas and / or mechanical barriers (gas-impermeable packaging materials).

The proportion of reduced cobalamin achieved during production can be determined photometrically via the ratio of the absorption at 350 nm to the absorption at 440 nm ( ). This relationship is determined at the beginning of the implementation and during and after the completion of the implementation. The percentage content of the not yet reduced form of content ox is calculated as follows: $$\begin{array}{l}\text{salary}-\text{Ox}\left(\text{in \%}\right)=100*[(\text{Absorption before reduction at 350 nm / absorption before reduction at}\\ 440\text{nm})-(\text{Absorption after reduction at 350 nm /}\left(\text{Absorption after reduction at 440 nm}\right)-\\ 1.1]\end{array}$$

In addition to the ingredients mentioned under [0011], further emulsifiers and lipids can be added in a proportion of up to 10%.

The high molar excess of reducing agents, the selection of other additives and solvents, and the exclusion of light and atmospheric oxygen during preparation, further processing, filling and storage mean that stabilization of at least 12 months is achieved.

Exemplary embodiment: skin ointment with 0.5 mmol cob (II) alamin / 100g (details refer to 100g ointment)

Preparation of the ointment base

The following ingredients are heated to 50 ° C in the absence of light and oxygen, mixed and stirred cold: Miglyol Ph.Eur. 5 g Tergitol TMN 10 5 g PBS buffer pH 7.2 20 g PEG 200 20 g PEG 4000 33 g

Add hydroxocobalamin

The solution cooled to room temperature is added and dissolved by stirring brought: Hydoxocobalamin 673 mg

The solution is now intensely colored red-violet

Add ascorbic acid

The solution is added and dissolved by stirring at 35 ° C. for one hour: ascorbic acid 1.3 g PEG 10,000 15 g

The solution turns brown and solidifies on cooling.

Exemplary embodiment: adhesive wound and mucous membrane solution with 0.5 mmol cob (II) alamin / 100g (details refer to 100g solution)

Hydroxocobalamin solution

The following ingredients are mixed to the exclusion of light and oxygen and stirred at room temperature until a homogeneous solution is reached: hydroxocobalamin 673 mg Tergitol TMN 10 5 g PBS buffer pH 7.2 20 g Glycerin Ph.Eur. 73 g

The solution is now intensely colored red-violet.

Add ascorbic acid

The solution is added and dissolved by stirring at 35 ° C. for one hour: Sodium ascorbate 1.3 g

The solution turns brown.

Exemplary embodiment: Oral application form with 0.5 mmol cob (II) alamin / 100g (details refer to 100g solution)

Hydroxocobalamin solution

The following ingredients are mixed and stirred at room temperature until a homogeneous solution is reached: hydroxocobalamin 670 mg ascorbic acid 1.3 g Lactulose solution 66% 96 g

The solution is now intensely colored red-violet.

alkalization

The solution is mixed with 1 g of nicotinamide and adjusted to pH 7.2 with sodium hydroxide solution 10 N and then stirred at 35 ° C. for 1 hour. The solution turns brown.

Exemplary embodiment: concentrate for the preparation of 500 ml of infusion solution with 0.5 mmol cob (II) alamin / 10 ml (details refer to 10 ml concentrate)

Hydroxocobalamin solution

The following ingredients are mixed and stirred at room temperature with the exclusion of light and oxygen until a homogeneous solution is reached: hydroxocobalamin 670 mg ascorbic acid 0.9 g Glycerin 80% 10 g

The solution is now intensely colored red-violet.

alkalization

The solution is adjusted to pH 7.2 with sodium hydroxide solution 10 N and then stirred for about 1 hour with the exclusion of light and oxygen at 35 ° C. until a conversion rate of> 95% has been reached. The solution turns brown.

Sterilization and delivery

The solution is sterile filtered and transferred to a 500 ml glass infusion bottle under sterile conditions. In this case, Aqua p. inj. a solution for infusion can be prepared on site.

patent citations

Publication number Priority date Publication date Assignee title DE102007012644A1 2007-03-16 2008-09-18 Bayer HealthCare AG Stabilization of vitamin B12 US8524774B1 2012-08-28 2013-09-03 Cymbiotics Inc Topical two step polytherapy for treatment of psoriasis and other skin disorders US20160000820A1 2014-07-02 2016-01-07 Virginia Commonwealth University Compositions and methods for treating carbon monoxide and / or cyanide poisoning US3928320A 1971-11-10 1975-12-23 Jean Boige Process for the preparation of methylcobalamine EP 1 236 737 B1 12/09/1999 JP 35068399 04.09.2002 Eisai Co., Ltd. Tokyo 112-8088 (JP) PROCESS FOR PRODUCTION OF METHYLCOBALAMINE

QUOTES INCLUDE IN THE DESCRIPTION

This list of documents listed by the applicant has been generated automatically and is only included for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Patent literature cited

• DE 102007012644 A1 [0001, 0034]
• US 3928320 A [0004]
• EP 1236737 B1 [0004]
• US 8524774 B1 [0007, 0034]
• US 2016/0000820 A1 [0009]
• US 20160000820 A1 [0034]

Claims (10)

Manufacturing process and composition of stable cosmetic and / or pharmaceutical liquid or viscous preparations as well as liquid preparations of food supplements or liquid or viscous preparations of medical devices, as well as preparations that serve as a preliminary product for this (a) Cob (II) alamin (CAS No. 14463-33-3) or Cob (I) alamin (CAS No. 18534-66-2) or the forms of cobinamides reduced to oxidation states II and I (CAS No. 13497-85-3) and mixtures thereof in any proportions as well as in small proportions the starting products from in-situ production (b) Reducing agents for the in situ production of the reduced forms (c) solvents suitable for stabilizing the reduced forms (d) other stabilizing and synergistic additives Preparations after Claim 1 , characterized in that component (a) by adding component (b) and component (c) and component (d) and by dissolving, mixing and heating the mixture in situ from hydroxocobalamin (CAS No. 13422-51-0) or cyanocobalamin (CAS No. 68-19-9) or cobinamide (CAS No. 13497-85-3). Manufacturing process according to Claim 2 , characterized in that from hydroxocobalamin or cyanocobalamin or cobinamides at least 50%, preferably at least 95% are converted to the reduced forms. Manufacturing process according to Claim 2 , characterized in that the mixture is adjusted to a pH of 4.0 to 8.0, preferably pH 7.2 before the start of the reaction phase and for 20 to 60 minutes with stirring to 25 ° to 38 ° C until the desired implementation rate Claim 3 is heated and the production takes place in the absence of light and oxygen, preferably with nitrogen or argon fumigation. Manufacturing process according to Claim 2 , characterized in that the mixture after formation of component (a) by sterile filtration or other sterilizing processes and sterile filling in infusion bottles or injection containers is present as a concentrate for the production of infusion solutions or inhalation solutions. Manufacturing process according to Claim 2 , characterized in that the mixture after the formation of component (a) by low-germ filling or other physical germ-reducing processes as a pharmaceutical or cosmetic preparation or as a food supplement or medical product or as a concentrate for the preparation of these preparations. Preparations after Claim 1 , which are characterized in that component (a) is present in the finished product in a concentration of 1 mmol to 100 mmol per liter. Preparations after Claim 1 , characterized in that they contain as component (b) a reducing agent or mixtures of reducing agents which are selected from the group consisting of ascorbic acid, sodium ascorbate, potassium ascorbate, ascorbic acid esters with fatty acids, sodium sulfite, potassium sulfite, nascent hydrogen ( formed from metallic zinc and an acid) and sodium borohydride. Preparations after Claim 1 , characterized in that they contain, as component (c), demineralized water in a proportion of up to 40% and water-miscible organic substances which are selected from the group consisting of ethanol, glycerol, propylene glycol, polyethylene glycol (MW 200 to 10,000), Sucrose, fructose, glucose, mannose, galactose, lactose, lactulose, lactitol, sorbitol, erythritol and dimethyl sulfoxide is formed. Preparations after Claim 1 , which are characterized in that they contain, as component (d) in concentrations up to 1%, the following further stabilizing or synergistic substances: nicotinic acid, nicotinic acid amide, nicotinic acid methyl ester, nicotinic acid diethylamide.

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