BE566435A - - Google Patents

Description

       

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   This invention relates to cobalamin-peptide complexes, their preparation and their extraction.



   It is known that cyanocobalamin and other cobalamins in which the cyano radical is replaced by another radical such as, for example, hydroxocobalamin, have considerable therapeutic value in the treatment of pernicious anemia, particularly when administered. parenterally, and are much less active when taken orally.



   On the basis of this consideration it is accepted that the presence of another substance, often referred to as "intrinsic factor" is necessary in cases of pernicious anemia for.

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 allow the efficient uptake of these cobalamins by the belt or @@@ It is generally considered, in the literature devoted to this subject, that this factor is proteinaceous in nature and has a high molecular weight, and that to be effective, it should be capable of binding to cobalamin, which would thus prevent the destruction of the latter in the digestive system and therefore promote its absorption in the organism.



   Various attempts have been made to isolate from animal sources a suitable high molecular weight substance which would exhibit "intrinsic factor" activity; thus, for example, extracts from the gastric mucosa of pigs have been used with cyanocobalamin. However, the results of clinical trials are not entirely satisfactory.



  Other oral treatments for pernicious anemia are related to the use of a relatively large dose of crude liver extracts or cyanocobalamin itself. This is why the treatment currently used is almost exclusively a treatment by injections with all the dangers and all the difficulties that this can entail. In addition, the preparation of injectable solutions is relatively difficult and expensive.



   One of the objects of the present invention is to provide a process for the preparation of improved products containing co-balamine which are active in the treatment of pernicious anemia when they are administered orally.



   Thus, the present invention provides a process for preparing a cobalamin-peptide complex, which comprises the degradation of a microbial fermentation product containing cobalamin until this product becomes orally active in treatment of pernicious anemia.



   The term "cobalamin-peptide complex" as used herein denotes a substance containing a peptide group bonded to a cobalamin group, defined below, or a mixture thereof.

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   It is not necessary for the clinical use of the present invention that the complex of this invention be isolated, although it is preferred that it be so. This is why this invention comprises, in addition to the processes for preparing these complexes, the processes for manufacturing certain preparations which contain them.



   The cobalamin group of the complex may be cyano-cobalamin, i.e. vitamin B12, or a cobalamin which can be converted into cyanocobalamin by the action of cyanide ions, such as for example hydroxocobalamin, c ' i.e. vitamin B12b or still a cobalamin which is distinguished from the preceding b cobalamins by the substituents of the benzene ring of the benzimidazole moiety of cobalamin, such as for example the derivative of cobalamin having a 5-hydroxybenzimidazole group, c ' that is to say factor III, or else a cobalamin in which the benzimidazole part of the molecule is replaced by a naphthimidazole part. Cobalamin should be non-toxic and be active against pernicious anemia when administered parenterally.



   In order to show activity against pernicious anemia, in oral treatment, the cobalamin-peptide complex (or in general the various compounds of which the complex is a mixture) must have a molecular weight of less than about 15,000.



   The molecular weight can be determined by the usual techniques, such as, for example, determination of the sedimentation constant in an ultra-centrifuge, diffusion, dialysis, through semi-permeable membranes whose pore size is known, or measuring the ratio of the weight of the cobalamin part to the weight of the peptide part in the complex, such as for example the method which will be described below. As the molecular weight of the cyanocobalamin itself is 1350, the compounds Cobalamin-peptide plexes of the present invention exhibit a molecular weight generally greater than.

   Around 2000, although

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 lower molecular weight complexes, such as for example 1500, may also be effective in the oral treatment of pernicious anemia. The molecular weight range which is preferably chosen is between approximately 2000 and 11.0OC.
The complexes prepared according to this invention exhibit relatively low molecular weights, as evidenced by the dialysis test described below.



   By dialysing a certain quantity of the complex obtained from a culture of Streptomyces ATCC 11072, described in Example 1, it is observed that the product migrates through a “Cellophane” membrane having an average pore diameter of 24 Angstrom, which separates it from pure water, but that it does not migrate through the same membrane which separates it from an aqueous solution of ammonium sulfate containing 60 parts by weight of salt per 100 parts by weight of water. Cyanocobalamin itself, at approximately the same concentration, diffuses freely through the membrane which separates it from an aqueous solution of ammonium sulfate of the same concentration.



   The samples of cyanocobalamin-peptide complexes prepared by the methods described in Examples 1 to 5 are subjected to tests for determining the weights of cyanocobalamin and of peptide in these complexes. Assuming that the mole ratio of cyanocobalamin to peptide is 1/1 and the molecular weight of cyanocobalamin is 1350, the molecular weights of the complexes are those shown in the following table. :

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<tb> N <SEP> of <SEP> Prepared <SEP> as <SEP> in <SEP> Content <SEP> in <SEP> Content <SEP> in <SEP> Weight
<tb> com- <SEP> example <SEP> cyanoco- <SEP> peptide <SEP> moleplex <SEP> balamine <SEP>;

   <SEP> weight / <SEP> cular
<tb> HPP <SEP>% <SEP> weight / <SEP> weight <SEP> calculated
<tb> -weight
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<tb> 1 <SEP> 1 <SEP> (Streptomyces
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 ATCC 11072) ¯ ", r," 3¯7 6.) :. 5.illL ..-
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<tb> 4 <SEP> 3 <SEP> (Propionibact-
<tb>
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<tb> rium <SEP> freudenreichii) <SEP> 13.2 <SEP> 86.8 <SEP> 10. <SEP> 250
<tb>
<tb>
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<tb> 4A <SEP> 3 <SEP> (after <SEP> precipitated
<tb>
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<tb> tion <SEP> to <SEP> acetone) <SEP> 15.8 <SEP> 84.2 <SEP> 8.600
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<tb>
<tb>
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<tb> 5 <SEP> 4 <SEP> (Streptomyces
<tb>
<tb>
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<tb> ATCC <SEP> 11072) <SEP> 14.1 <SEP> 85.9 <SEP> 9.

   <SEP> 550
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<tb>
<tb>
<tb> 5A <SEP> 4 <SEP> (after <SEP> precipitated
<tb>
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<tb> tion <SEP> to <SEP> acetone) <SEP> 29.3 <SEP> 70.7 <SEP> 4.600
<tb>
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<tb>
<tb>
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<tb> 7 <SEP> 5 <SEP> (Propionibact-
<tb>
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<tb> rium <SEP> freudenreichii) <SEP> 13.4 <SEP> 67.5 <SEP> 8.100
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The behavior in acidic or alkaline hydrolysis and the absorption spectrum in the visible and ultra-violet are other characteristic properties of the complexes of this invention. These properties are described below.



   After hydrolysis in 6N hydrochloric acid in a sealed tube for 24 hours of the cyanocobalamin-peptide complex obtained from a culture of Streptomyces ATCC 11072 as described in Example 1, a hydrolyzed mixture is obtained, the analysis of which by chromatography on paper, using the phenol / ammonia and water / butanol / acetic acid systems as developing solvents, reveals the presence of the following amino acids: glutamic and aspartic acids, glycine, valine, proline, arginine, cysteine, or cystine, serine , alanine, leucine, isoleucine, phenylalanine, lysine, hystidine, and threonine. After alkaline hydrolysis in the presence of barium hydroxide, the presence of tryptophan can be demonstrated.



   The remainders of the same amino acids can be demonstrated in the cobalamin-peptide complex derived from Propionibacterium freudenreichii as described in Example 2, by the same method.



   The examination of the cyanocobalamin-peptide complexes prepared as described in Examples 1 to 5 was supplemented by a spectroscopic study using light having a wavelength

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 of the visible or ultra-violet domain. The absorption spectra of a few of these complexes are shown in Figures 1 to 3 of the drawings which accompany this patent application.



   When making preparations which contain the complexes of the present invention from the fermentation broth or culture broth, it is desirable to incorporate in the process one or more operations of concentration and purification of the product, and it is even preferred to isolate the complex in the form of a pure substance. If we consider the clinical efficacy of the complex which manifests itself in the fact that an amount of about 10 micrograms of cobalamin constitutes a suitable dose, it will be understood that not only the pure product but also the compositions which contain the complex in a moderately concentrated form, will contain too much complex to be a convenient medication for oral administration.

   Therefore, it is preferred to prepare these complexes by mixing the pure complex or a concentrated purified product containing this complex with non-toxic liquid or solid diluents. It is clear that the term non-toxic qualifies a substance which is non-toxic in amounts which will be used in clinical use.



   Mannitol has been shown to be a suitable solid diluent, although other non-toxic solids such as starch, or dibasic calcium phosphate can be used if. The complex can be dissolved or suspended in water or other liquids containing, if desired, stabilizing agents such as, for example, buffering agents.



   When the preparation is in liquid form, it is advantageous to incorporate therein a sweetening agent or a flavoring agent so that its taste is more pleasant. However, suitably purified products can be used without such additions being necessary, and solid preparations can be in the form of tablets or cachets, eliminating the need for flavoring. In any case it is -

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 it is possible to incorporate into the preparations other substances of recognized therapeutic activity, provided of course that they are chemically compatible with cobalamins and with peptides.



   Any suitable substance can be used for flavoring, or in general for making preparations more palatable, since these are primarily intended for. oral treatment. Among these substances there may be mentioned sugar or other sweetening agents and fruit flavors.



   The clinical results obtained by oral treatment using the complexes described in Examples 1 to 5 are shown graphically in Figures 4 to. 10 of the drawings which accompany this patent application. These results show that after an initial dose of complex corresponding to about 100 micrograms of cyanocobalamin daily for a period of 7 to. 8 days, patients suffering from pernicious anemia are maintained in good health without recurrence of symptoms of the disease, throughout the period of testing by a daily oral dose of the complex containing approximately 10 to 20 micrograms of cyanocobalamin.



   Clinical results show that the cobalamine-peptide complexes of this invention are extremely effective in the oral treatment of pernicious anemia, particularly at doses which contain the same amount of cyanocobalamin as those which would produce the same results in pernicious anemia. parenteral therapy. Daily doses of complex containing about 10 micrograms of cobalamin have been shown to be fully effective in permanent maintenance therapy by the oral route.



   This discovery is quite surprising since it is opposed to the prevailing opinion which asserts that only substances of high molecular weight like true proteins, which are dond

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 not dialyzable, can fulfill the role of intrinsic factor ".



   In addition to the clinical results observed by oral administration of the complexes of the present invention, these products have also been shown to activate the maturation of megaloblast cells, i.e., unripe red cells. of the marrow. long bones of patients with pernicious anemia, in normoblasts, ie fully formed and mature cells, in vitro. Pure, crystallized cyanocobalamin requires the addition of some normal gastric juice to be effective as a maturation agent for megaloblastic cells from these sources.



  (Callendrer and Lajtha, Blood (1951) 6, p.1234).



   The present invention includes methods of preparing the complexes described above and methods of making preparations which contain these complexes, either with or without intermediate isolation of these complexes.



   As stated above, the cobalamin-peptide complexes are extracted from cultures in fermentation and they can be produced by various microorganisms cultivated in a suitable medium. To the extent of current knowledge, all the organisms which produce cobalamins by fermentation produce * these substances in a form from which the complexes of the present invention can be isolated. Different organisms that make cobalamins already are. well known. Suitable known organisms belong to the Fungi group which is divided into subgroups of Myxomycetes, Schizomycetes and Eumycetes described for example in "Industrial Mycolog by Smith and Raistrick, London, Arnold & C.

   (1946) on pages 1 to 3.



   It has been shown that, in particular, members of the genera Streptomyces and Propionibacterium (the latter is described in the work "Manual of Determinative Bacteriology", by Bergey, 6th Edition, Waverley Press, Baltimore), provide good yields of these complexes. , and that among these Streptomyces ATCC 11072, Streptomyces griseus and Propionibacterium freudenreichii are

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 are found to be particularly suitable. These organisms are already widely used in industrial fermentations.



    Propionibacterium shermanii and P.technicum also give satisfactory yields of the complexes. It is possible to ensure that a particular microorganism is capable of making cobalamins by fermenting a suitable nutrient medium containing sources of nitrogen, assimilable carbon and cobalt such as, for example, corn steeping liquid, glucose and cobalt chloride, respectively, and examining the fermentation product for cobalamins.

   If it is observed that cobalamins have formed, the extent to which the microorganism is suitable for the purpose of the present invention should be determined by preparing a cobalamin complex from the fermentation product by the methods described in section 4.2. present invention and by subjecting the complex thus prepared to clinical trials according to the usual technique. On the other hand, the complex can be subjected to tests relating to the other properties mentioned above in order to determine whether the microorganism is suitable for the purpose pursued without having to carry out the clinical tests.



   In order to manufacture these complexes on a relatively large scale, it is preferred to carry out the fermentation with selected microorganisms immersed in a liquid medium. In the case of aerophilic organisms such as Streptomyces it is necessary to provide aeration of the fermentation medium, while when using certain species of Propionibacterium such as for example P.freudenreichii, it has been shown that it is advantageous to observe anaerobic fermentation conditions of the nutrient medium during the first period of fermentation, and then to admit a certain amount of air, which is also advantageous.



   If desired, certain precursors can be added, to the fermentation medium, preferably after development 4, A

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 certain amount of microorganisms, in order to prepare cobalamin-peptide complexes which contain cobalamins other than cyanocobalamin. For example, 5-hydroxybenzimidazole can be added to produce a complex containing the corresponding cobalamin, also known as factor III.



   The preparation process may include stages related to transformation of the type of cobalamin present: hydroxyco-balamine, for example, can be converted into cyanocobalamin according to known methods; but these stages do not form part of the present invention and will not be mentioned further below.



   It is known that cobalamins, for example cyanocobalamin and hydroxocobalamin, occur largely in the fermentation product in a "bound" form. This means that cobalamins are linked to compounds of high molecular weight and of a protein nature, and that in this form cobalamin is not available for most microorganisms, as for example for the protozoan Ochromonas malhamensis, of which frequent use is made for the microbiological determination of cobalamins. In this form cobalamins are also without effect against pernicious anemia when ingested orally.



   The different steps of the known process for the isolation of cyanocobalamin from fermentation cultures lead to a degradation of the cyanocobalamin-protein compound, which cannot be determined by microbiological means, and the cobalamin group of which is qualified as "bound" to form a cyanocobalamin complex. -peptide of lower molecular weight, which is microbiologically determinable and whose cobalamin group is qualified as "free" and finally as combined linon cyanocobalamin "of molecular weight 1350.



   In order to obtain the cobalamin-peptide complexes of the desired molecular weight for the present invention the degradation of the "bound" cobalamines can be effected by the action of cyanu ions.

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 res proteolytic enzymes, acids or heat, for example. The degradation treatment can be carried out at any convenient time after the end of the fermentation, for example on the fermentation culture or on the extracts or concentrates derived therefrom.



   Fermentation cultures can be concentrated and purified by the methods used to effect concentration and purification in "uncombined" cyanocobalamin manufacturing processes, but it is desirable to obtain a good yield of the complex to treat the cells or the substance. mycelium of the organism used at an acidic pH value, in order to release the products containing cobalamin from the cell contents - For example, the cells or the mycelium can be suspended in the fermented culture liquid after adjusting that here at an acidic pH value, for example in the pH range of 1 to 3, for an appropriate period of time, then filtering and thus removing cells or mycelium.



   On the other hand, the cells or the mycelium can be separated from the fermented liquid by filtration or by centrifugation and the cells or the mycelium can be resuspended in a liquid of favorable pH until all of the product containing the cobalamine is released from the cells, after which the insoluble cellular product is filtered and removed.



   The "bound" and "free" cobalamins thus obtained can be purified and concentrated by absorption on a suitable ion exchange resin. They are then eluted from the resin, for example by means of acidified isopropanol, and the eluate. can be further concentrated and purified by known methods such as, for example, by repeated extractions from an aqueous solution in a phenol / benzene solution and re-extractions from this mixture of solvents in water, by the addition n-butanol or butyl acetate.



   It has been observed that it is desirable to purify and concentrate the solution until it contains a concentration.

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 total cyanocobalamin concentration between 500 and 1500 micrograms per milliliter, such as for example an aqueous solution containing approximately 1200 micrograms of cobalamins per milliliter.



  Total cobalamins can be determined by microbiological assay after an aliquot of the solution has been subjected to the action of heat in aqueous solution and. in an acidic medium until all "bound" cobalamin has been liberated or by spectrophotometric assay, for example using light having a wavelength of 550 millimicrons.



   The solution thus obtained normally contains "bound" and "free" cyanocobalamin, unless the fermentation has been carried out in the presence of a precursor, such as for example a benzimidazole, such as 5-hydroxybenzimidazole and in this case the corresponding cobalamin (factor III) will be obtained in "bound" and "free" form, as well as cyanocobalamin. In order to increase the yield of "free" cyanocobalamin the solution may be subjected to the partial degradation necessary to obtain a desired molecular weight product.



   On the other hand, it is also possible to carry out the degradation operation of the "bound" product without having previously carried out the concentration of the product or even when the product is in the form of a suspension (particularly of a resuspension. ) fermenting cells, and in this case the complex solution thus obtained can be separated and packaged for clinical use without appreciable concentration or dilution.



   The "bound" cobalamins can very easily be broken down into a cobalamin-peptide complex of a desired molecular weight by treating the concentrated aqueous solutions with cyanide ions, such as, for example, by adding a solution of potassium cyanide to the mixture. 2% weight / volume approximately, for a determined time, such as for example for 1 to 2 hours at the temperature of the chamber.



   While the use of cyanide ions to effect partial degradation of the "bound" cobalamin is convenient, in that the degradation is thus controllable, the process

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 of this invention is not limited only to this method.

   Other methods can also be used, for example acid treatment or treatment with proteolytic enzymes such as pepsin, trypsin, chrymotrypsin or papa.ine. Partial degradation can be effected if desired by prolonged treatment of the cells or mycelium to an acidic pH value, described above, which releases the "bound" cobalamin from the cellular material which is sufficient to cause the. degradation of at least part of the "bound" cobalamin into a cobale complex, mine-peptide of the desired molecular weight. Here also the necessary degradation can be effected by heating the "bound" cobalamin preferably in aqueous solution.

   The degradation can be carried out at. a suitably high temperature, for example greater than approximately 50 ° C. and preferably greater than approximately 80 C. It is even possible to obtain the necessary degree of degradation of a part of the product containing the cobalamin by allowing the fermentation to continue until the stage of autolysis by the fermenting organisms, but this process is inefficient, and d 'difficult to control, which is why we prefer other processes.



   In order to be able to establish when the degradation is sufficiently advanced, dialysis tests are carried out from time to time using membranes such as "cellophane" which have an appropriate pore size, after removal of unwanted cobalamin. "linked". for example by absorption on active carbon, as described below.



   In addition, tests can be carried out with suitable microorganisms and observed whether their growth is stimulated.



   The aqueous solution which contains the complex of the desired molecular weight can, if desired, be further concentrated or it can be treated at this stage with an organic or inorganic precipitating agent, which makes it possible to obtain the cobala- mine complex. -peptide desired in solid form.



   On the other hand, this solution can be purified according to the

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 needs and used as such or after subsequent concentration in the manufacture of liquid preparations for clinical use.



  For this purpose, the solution is diluted until the concentration of cobalamin present in the form of a complex is less than 100 micrograms per milliliter and preferably until the concentration is less than 10 micrograms per milliliter.



     The precipitating agent that is added to. the concentrated aqueous solution of the cobalamin-peptide complex may be either of the suitable agents which are used to release organic compounds from their aqueous solutions, such as, for example, inorganic salts such as sodium sulphates. ammonium, sodium or potassium. The release agent is added in an amount such that an aqueous solution is obtained, the salt release concentration of which is between that of a 25% saturated solution and that of a fully saturated solution, and preferably in the case of ammonium sulphate between that of a 50% saturated solution and that of a 70% saturated solution.

   The complex can also be precipitated by adding organic precipitating agents such as acetone, propanol or isopropanol.



   If it is desired to obtain the cobalamin-peptide complex in its pure state, the "unbound" cobalamins will be removed after the isolation operation so that these "unbound" cobalamins formed from the complex during the concentration and isolation operations, as well as those that have formed previously, are removed together in a single extraction.



  If desired, however, "unbound" cobalamins can be removed prior to the isolation operation, and the impure complex will however be susceptible to clinical application.



  However, it is preferred to isolate the complex in the pure state (or at least a solid containing the complex free from "unbound cobalamin) and prepare the clinical preparations from it, so as to simplify the standardization and the dosage of the compounds. preparations, which are made more complicated by the presence

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 of "unbound" cobalamin. The presence of large amounts of "bound" cobalamin presents the same drawbacks but it will be understood that the method of preparation which has been described avoids the risk of significant contamination by the. bound cobalamin.



   Treatment of a solution which contains "unbound" cobalamins with activated carbon is a suitable method for the removal of these substances. The amount of activated carbon which should be used is to some extent a function of the amount of unbound cobalamins present in the solution, but it has been shown that the addition of an amount of activated carbon equal to about 2% by weight based on the volume of the solution is well suited. The cobalamin is adsorbed on the activated carbon while the complex remains in solution. This treatment also removes colored impurities and the appearance of the complex is present. thus improved.



   After precipitation, the solid complex can be further purified, for example by redissolution in water and washing this aqueous solution with organic solvents such as butanol and / or 'benzene, followed by removal of the organic solvents by distillation. The complex can then be reprecipitated and isolated. This can be repeated until the solid cobalamin complex exhibits the desired degree of purity.



   The following examples illustrate the methods of preparing the cobalamin-peptide complex from fermentation cultures according to this invention. In the examples, the terms "part by weight" and "part by volume" are related to each other as are grams to milliliters.
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  EXI ":.". 1: PLE l.-
The culture broth obtained by the fermentation of a nutrient medium containing. soybean flour, glucose, monopotassium phosphate and cobalt chloride by a microorganism streptomyces strain ATCC 11072 is acidified for one hour to a pH of 2, then is filtered. The filtrate is passed through an ion exchange resin which absorbs linon cobalamins

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 bound "consisting of cyanocobalamin with some hydroxocobalamin and any peptide complex which may have formed. These absorbed substances are referred to below as" cobalamins ".



   The absorbed cobalamins are eluted from the resin by acidic isopropyl alcohol and the eluate is neutralized. This is then concentrated by distillation under reduced pressure in order to remove the isopropanol and the pH of the concentrated solution obtained is adjusted to a value of 7%. 7.5. A 2% w / v solution of potassium cyanide is then added and the mixture is allowed to stand. The mixture thus obtained is treated with a mixture of phenol and benzene (30/70% v / v) to extract the cobalamins. The solution thus obtained of cobalamins in the phenol / benzene mixture is then treated with water and n-butanol, which causes the cobalamins to return to aqueous solution.



   The aqueous solution contains approximately 1260 micrograms of total oobalamins, ie, "bound", "unbound" and "free" cobalamins, (as a complex with a peptide), per millimeter.



   2.5 parts by weight of sulfate are added. of ammonium to 3.2 parts by volume of the solutions thus obtained and the mixture is left to stand. An oily layer and a precipitate separate and are decanted and mixed with water. The mixture, which forms an emulsion, is then washed with benzene to remove traces of organic solvents such as phenol or butanol. The washed emulsion is then treated with activated charcoal to remove certain impurities and unbound cyanocobalamin. ”The charcoal is then removed by filtration and washed with water, which is added to the filtrate.

   The purified aqueous solution of cyanocobalamin-peptide compound is washed with n-butanol then with benzene and the complex is precipitated from the washed aqueous solution by adding a sufficient quantity of ammonium sulfate so that the concentration is 60 grams per 100 ml of solution. The precipitate is again dissolved and precipitated twice in the same manner and the solid cyanocobalamin-peptide complex finally obtained is dried in air.

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   On spectroscopic examination using light of a wavelength in the visible and ultra-violet range, the spectrum of Figure 1 is obtained. For comparison purposes, the same figure also shows the spectra of cyanocobalamin and of a concentrated aqueous solution containing cyanocobalamin mainly in the "bound" form.



   The cyanocobalamin content of the complex is calculated after spectrophotometric examination using a light ray having a wavelength of 550 millimicrons. The ammonium sulfate content can also be estimated and, by difference, the peptide balance is calculated. In this way, it is found that the compound contains 5.5% cyanocobalamin and 37.6% peptide.

   Based on these figures and assuming that in the compound the cyanocobalamin / peptide molecular ratio is 1: 1 and the molecular weight of cyanocobalamin is 1350, the molecular weight of the complex is calculated to be about 10,500. The behavior of a sample of the complex is examined in an ultra-centrifuge operated for 320 minutes at 5.98 x 104 rpm and it is observed that the uncorrected sedimentation constant is 0.44 x 10-3 Svedberg units which indicates that the molecular weight is approximately of the same order of magnitude as that which is calculated (. by the spectrophotometric method).



  EXAMPLE 2. -
A culture broth containing cells obtained by fermentation of a medium containing corn steeping liquid, glucose and cobalt chloride using a strain of Propionibacterium freudenreichii is acidified with sulfuric acid to, pH 2, then left to stand for 6 hours: to-separate the cobalamins in the form of a cyanocobalamin-peptide complex of the 'insoluble cellular product'-The insoluble product is removed by filtration and: the filtrate is treated with a phenol / mixture. benzene to extract the complex. The solution thus obtained is then treated with butyl acetate and. water and the complex

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 is extracted as an aqueous solution.

   This operation is repeated until a concentrated aqueous solution is obtained containing a total of 620 micrograms of cyanocobalamin, mainly in the form of a peptide complex, per milliliter of solution.



   This concentrated solution is then treated with activated charcoal lice to remove the impurities and the "unbound" cyanocobalamin itself. After removal of the charcoal by filtration, the filtrate is extracted with butanol and washed with toluene to remove the dissolved butanol. The concentrated aqueous solution is distilled under reduced pressure to remove traces of toluene, then reprocessed with activated carbon which is then removed by filtration.



   We add to. 9.4 parts of this concentrated aqueous solution 7.15 parts of ammonium sulfate and the precipitate is filtered off. The precipitate is dissolved again in water and reprecipitated twice under similar conditions and then finally air dried.



   A portion of the complex is then dissolved again in water and subjected to ultra-filtration through an Oxoid membrane whose pores have a size of 5000 to 1000 Angstroms.



  'A certain amount of impurity with a high molecular weight remains in the filter.



  EXAMPLE 3.-
Potassium cyanide is added to an aqueous solution of the complex prepared according to the method of Example 2 and containing 500 micrograms of cyanocobalamin per milliliter to obtain a concentration of 2% weight / volume at a pH of 7 to 7.5. Ammonium sulfate is then added to obtain a concentration of 12% w / v and the complex is extracted with normal butanol. Toluene and water are then added and the complex is re-extracted into aqueous solution. This is treated with a mixture of phenol and toluene until all the complex is extracted into the organic phase. The complex is then concentrated by re-extraction in aqueous solution by addition

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 acetic acid, butanol and water.

   The aqueous solution is then separated and then concentrated by distillation under reduced pressure, thus removing the excess solvent and the cyanide.



   Analysis of the complex obtained indicates that it contains 13.2% cyanocobalamin and 86.8% peptide, which gives a molecular weight of 10,250 as indicated in the table on page 5 of this specification.



   Chromatographic and electrophoretic examination of the complex indicates that virtually all of the. cyanocobalamin occurs as a peptide complex. It shows the absorption spectrum of Figure 2.



   Mixed with mannitol, the complex was found to be of a pleasant flavor and very effective in treating the symptoms of pernicious anemia, as described in Example 7.



   On purifying a portion of the complex by the addition of 25 volumes of acetone to an aqueous solution of the complex exhibiting the above analysis, a pink powder precipitates and is allowed to air dry.



   The precipitate thus obtained contains 15.8% cyanocobalamin and 84.2% peptide, which gives a molecular weight of approximately 8,600 according to the process described above.



   The absorption spectrum of. this product is also reproduced in figure 2.



   Elemental analysis gives the following results: carbon 49.8%; hydrogen 6.9% nitrogen 12.5%; sulfur 0.96%; phosphorus 0.62%; cobalt 0.60%.



  EXAMPLE 4. -
An aqueous solution containing cyanocobalamin-peptide complex is prepared starting from a culture broth obtained by fermentation of a nutrient medium by a streptomyces microorganism strain ATCC 11072, by acidification to pH 2, followed by purification and concentration. by extraction and re-extraction

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 in organic solvents and in water and by treatment with activated carbon. To 9.4 parts by volume of this aqueous solution is slowly added with stirring 6 parts of ammonium sulfate and the precipitate which separates is collected by filtration. The precipitate is then redissolved in water and reprecipitated by adding 70 g of ammonium sulfate per 100 milliliters of solution.



   Purification of the complex is continued by redissolving in water and treating the solution thus obtained with 2% potassium cyanide at pH 7 for one hour, after which the dicyanocobalamin-peptide complex which has formed. is extracted with butanol in the presence of 12% ammonium sulfate. By adding water, and toluene, the dicyano- complex is then re-extracted into water. The aqueous solution enriched with the dicyano- complex is treated with a phenol / toluene mixture (30% / 70% v / v) to extract this complex which is then re-extracted into water after acidification with glacial acetic acid. The aqueous solution of cyanocobalamin-peptide complex thus obtained is distilled under reduced pressure to remove the cyanide ions and the solvents.



   Analysis of the solution thus prepared indicates that the complex contains 14.1% cyanocobalamin and 85.9% peptide which gives a molecular weight of approximately 9.550.



   Mixing part of the complex with mannitol was found to be of a pleasant flavor and to be very effective in combating the symptoms of pernicious anemia, as described in Example 8.



   The remainder of the complex in aqueous solution is precipitated by adding 25 volumes of acetone per volume of aqueous solution.



    After air-drying, the precipitated pink powder exhibits the following analysis: cyanocobalamin 29.3%; peptide 70.7% which gives a molecular weight of 4,600.



   Elemental analysis of the product precipitated with acetone gives the following results: carbon 45.3%; hydrogen 7.1%; nitrogen 12.55%; sulfur 1.43%; phosphorus 0.33%; cobalt 1.37%. The absorption spe @ tres are shown in figure 3.

   feel

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 EXAMPLE 5.-
A culture broth obtained from the fermentation of a nutrient medium containing corn steep liquor, glucose and cobalt chloride by a strain of Propionibacterium freudenreichii is adjusted to pH 2 and allowed to stand for 6 hours, after which it is purified and concentrated by extraction in organic solution, then by re-extraction in aqueous solution, to obtain an aqueous solution of "bound" "free" and "unbound" cobalamins.

   The pH of the aqueous solution is then adjusted to 1% ne value between 7 and 7.5, 2% weight / volume of potassium cyanide is added and the solution is left to stand for three hours after which the cobalamin-peptide complex is added. extracted in organic solution and re-extracted in aqueous solution. The pH thereof is then adjusted to. a value of 4.5 and the solution is concentrated to remove cyanide ions and solvents. The volume of the enriched aqueous solution thus obtained is limited to 40 gallons, containing
500 micrograms of peptide complex and uncombined cyanocobalamin per milliliter.



   Ammonium sulfate is added with stirring to obtain a concentration of 60 g per 100 milliliters of solution and the precipitate which forms is left to stand for 12 hours, after which it is filtered and dried in air. The precipitate weighs 766 g.



   Amalysis shows that 1 product contains 13.4% co-balamines. and 67.5% de-peptide,., s, 19.1% olde, being ammonium sulfate, to give a. molecular weight of about 8,100.



    EXAMPLE 6.-
The cyanocobalamin-peptide complex prepared according to the method of Example 1 and which contains approximately 13% by weight of cyanocobalamin is mixed with mannitol, at a concentration of 0.16% of complex in the mixture, and doses, of this preparation are administered orally to six patients with pernicious anemia. The patients were not subjected to other treatments.

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   The normal number of red blood cells in healthy people varies between 4.5 and 5.0 x 106 per cubic millimeter, and the hemoglobin level is between 90 and 100.



  Case n 1.



   During the first eight days of treatment, a patient is administered orally a daily dose of a preparation containing 780 micrograms of complex (containing 100 micrograms of cyanocobalamin), after which the daily dose is reduced by half over the next 14 days. The daily dose is then reduced to an amount of preparation containing: 78 micrograms of complex (corresponding to 10 micrograms of cyanocobalamin), during the remainder of the treatment (263 days). The patient was maintained in good health with this daily dose, and did not show symptoms of pernicious anemia.



   Throughout the treatment, red blood cell counts and hemoglobin estimates were taken, and the values are shown in Figure 4.



  Case n 2.



   During the first eight days of treatment a patient is administered a daily dose of a preparation containing 780 micrograms of complex, after which the dose is reduced to half this value for the following 14 days.



   Considering the patient's age (72 years), a quantity of preparation containing 156 micrograms of complex (corresponding to 20 micrograms of cyanocobalamin) was administered to him daily for the following 77 days, after which the dose was reduced by half during the remainder of treatment (155 days).



   The patient is maintained in good health and has no symptoms of pernicious anemia.



   Figure 5 shows the results of red blood cell count readings and hemoglobin level estimates made during treatment.

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   Due to the age of the patient, judging by the red blood cell count readings and the hemoglobin estimate, the response to treatment is slower than in the other cases described herein.



  Case n 3.



   A patient takes orally for 8 days a daily dose of preparation containing 780 micrograms of complex after which the daily dose is reduced to one fifth of this amount for 22 days, then during the following 41 days this last amount of half. After that the treatment is stopped.



   After 35 days without treatment, the patient's health deteriorates, as shown by a drop in the number of red blood cells and hemoglobin level.



   A single dose of the preparation containing 1560 micrograms of complex (corresponding to 200 micrograms of cyanocobalamin) is administered on the sixtieth day after discontinuation of treatment and 14 days later the patient is started to be administered daily with a dose of. maintenance containing 78 micrograms of complex. The patient is thus maintained in good health and shows no symptoms of pernicious anemia during the remainder of treatment (105 days).



   We will find at. Figure 6 shows red blood cell counts and estimates of hemoglobin level.



  Case n 4.



   For 7 days a patient is administered a daily dose of the preparation containing 780 micrograms of the complex, after which the daily dose is reduced to one tenth of that amount for the remainder of the treatment (207 days).



   The patient remains healthy and has no symptoms of pernicious anemia.



   Figure 7 gives readings of red blood cell counts and estimates of hemoglobin level.

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  Case
For 8 days, a patient is administered orally a daily dose containing 780 micrograms of complex, after which the daily dose is halved for 7 days and then during the following five days it is reduced to one-fifth of the initial dose * After this period, the amount of preparation administered is reduced to. a daily dose containing 78 micrograms of complex and .ce during the rest of the treatment (204 days).



   The patient is maintained in good health and has no symptoms of pernicious anemia. Figure 8 shows the readings of the number of red blood cells and the estimated hemoglobin level.



  EXAMPLE 7.-
A patient is treated with a preparation which consists of a complex produced according to the method described in Example 3 mixed with mannitol in the proportion of about 1 to 600.



  For 7 days the patient takes orally a daily dose of preparation containing 760 micrograms of the complex (corresponding to 100 micrograms of cyanocobalamin), after which the daily dose is reduced by half for 5 days. Then finally, it is reduced to an amount of preparation containing 76 micrograms of the complex and it is maintained at this level for the remainder of the treatment (80 days).



   The patient is maintained in good health and has no symptoms of pernicious anemia.



   Figure 9 shows the readings of the number of red blood cells and the estimated hemoglobin level.



    EXAMPLE 8.-
A patient is treated with a preparation administered orally which consists of a complex prepared according to the method described in Example 4, mixed with mannitol. For 7 days the patient takes a daily oral dose

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 of the preparation containing 710 micrograms of complex (corresponding to. 100 micrograms of cyanocobalamin), after which, during the following 11 days the daily dose is reduced to an amount corresponding to. 142 micrograms of complex- Then finally it is reduced to 71 micrograms and this during the remainder of the treatment (82 days).



   Figure 10 gives readings of red blood cells and estimates of hemoglobin level.



   The reaction to the treatment is similar to that described in Example 7.



    EXAMPLE 9.-
A 52-year-old patient who underwent treatment with a specialty for the oral treatment of pernicious anemia for 16 months, a. meanwhile reacted favorably to treatment at. judging by the readings of red blood cells and estimates of hemoglobin level, and thus is maintained in good health.



   At the end of this period, despite continuing treatment, she began to experience difficulty standing and walking, accompanied by torpor of the foot and lack of sensation in both ankles. Reflex movements in the knees and ankles are indolent, and reflexes in the sole of the foot are not clearly visible. The patient is no longer able to detect the sensation of the contact of cotton wool under the foot.



   These symptoms persist despite the fact that the red blood cell count is 4.3 x 106 red blood cells per cubic millimeter and the hemoglobin level is 94%, and they are diagnosed as resulting from degeneration. subacute of the spinal cord, which, as is known, occurs in a certain proportion of patients with pernicious anemia.



   The patient's treatment was changed and a preparation comprising the complex was administered orally.

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 cyanocobalamin-peptide as prepared in Example 1, mixed with mannitol ...



   During the first seven days, the patient takes daily amounts of preparation containing an amount of complex containing 300 micrograms of cyanocobalamin. After that, the daily dose is reduced to an amount of preparation containing 200 micrograms of cyanocobalamin and this for 8 weeks.



   During treatment walking improves, and sensory symptoms disappear, while the red blood cell content and hemoglobin level remain constant.



    EXAMPLE 10.-
It is known that crystalline cyanocobalamin alone cannot cause in vitro maturation of megaloblastic (unripe) red blood cells in the bone marrow of patients with pain. - pernicious anemia in normoblasts (mature cells), see for example, Callender and Lajtha, Blood (1951) 6 p. 1234.



   It has been found, however, that the cyanocobalamine-peptide complexes which are the subject of the present invention cause the in vitro maturation of such cells.



   Assays are performed on cell cultures taken from the bone marrow of patients with pernicious anemia, following the method described by Callender and Lajtha, "Blood" (1951) 6, p. 1234, except that Ringer's solution was used instead of Gey's solution and 50 units of penicillin are added to avoid infection during the test.



   In this test, 7.8 micrograms of cyanocobalamin-peptide complex prepared according to the method described in Example 1 and containing 1 microgram of cyanocobalamin are used, and the results shown in the following table are obtained:

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 EMI27.1
 
<tb> Patient <SEP> Initial <SEP> count <SEP> <SEP> count after <SEP> a <SEP> culture
<tb>
<tb>
<tb> n <SEP> of the <SEP> cells <SEP> of <SEP> the <SEP> of <SEP> 18 <SEP> hours <SEP> in <SEP> of the <SEP> serum, <SEP> in
<tb>
<tb>
<tb>
<tb> marrow <SEP> of <SEP> bones <SEP> long <SEP> presence <SEP> of <SEP> complex <SEP> cyano-
<tb>
<tb>
<tb>
<tb> cobalamin-peptide.
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>



  Megalo- <SEP> Normo- <SEP> Megalo- <SEP> Normo-
<tb>
<tb>
<tb> blasts <SEP> blasts <SEP> blasts <SEP> blasts <SEP> ¯¯¯¯
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1 <SEP> 94% <SEP> 6% <SEP> 55% <SEP> 46%
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 2 <SEP> 90% <SEP> 10% <SEP> 35% <SEP> 55%
<tb>



    

Claims (1)

CLAIMS 1.- Process for the production of a cobalamin-peptide complex, characterized in that a microbial fermentation product-containing cobalamin is degraded until it becomes effective in the treatment of anemia pernicious,, by oral administration. 2. - Process for the production of a cobalamin-peptide complex, characterized in that a microbial fermentation product containing cobalamin is degraded until it dialyzes through a "Cellophane" membrane whose average pore diameter is about 24 Angström units, in pure water and stopping degradation before the product is degraded to such an extent that it can dialyze through a similar membrane in a solution of 60 grams of ammonium sulfate per 100 grams of water. 3. - Process for the production of a cobalamin-peptide complex, characterized in that a microbial fermentation product containing cobalamin is degraded until its molecular weight is less than 15,000 and it is effective. in the treatment of pernicious anemia, by the oral route. 4. A method according to claim 3, characterized in that the microbial fermentation product containing cobalamin is degraded until its molecular weight is between 2000 and 11000. 5. - Process for the production of the cobalamin-peptide complex, characterized in that a microbial fermentation product containing cobalamin is degraded until it is effective in promoting the development of Ochromonas malhamensis, and that it is effective by oral administration in the treatment of pernicious anemia. 6. - Process for the production of a cobalamin-peptide complex, characterized in that a fermenta- <Desc / Clms Page number 29> microbial treatment until a complex is obtained having an absorption spectrum substantially identical to the spectra of the peptide complexes shown in Figs. from 1 to 3 of the accompanying drawings. 7. - A method of producing a cobalamin-peptide complex, according to either of the preceding claims, characterized in that a suitable nutrient medium is subjected to fermentation by means of a microorganism producing cobalamin, EMI29.1 of the genus 9ILionbacteri, for example Pronionibacterium freudenreichii, and that a fermentation product containing "bound" cobalamin according to the definition given above, is then subjected to a treatment which results in the degradation of the "bound" cobalamin, in at least one cobalamin-peptide complex having a molecular weight of less than 15000, and in that the complex is collected in the form of a non-toxic product, effective in the treatment of pernicious anemia, when it is administered orally . 8. A process for the production of a complex according to any one of the preceding claims, characterized in that the degradation comprises treatment with cyanide ions. 9. A process for the production of a complex according to any one of claims 1 to 7, characterized in that the degradation comprises treatment with a proteolytic enzyme such as pepsin, trypsin, chymotrypsin or papain. . 10. - A method of producing a complex according to any one of claims 1 to 7, characterized in that the degradation treatment comprises maintaining the pH at an acid value, for example between 1 and 3. He. - A method of producing a complex according to any one of claims 1 to 7, characterized in that the degradation treatment comprises heating the product of the fermentation. 12.- Production process of a complex following a <Desc / Clms Page number 30> any of the preceding claims, characterized in that the fermentation product is purified and concentrated to obtain an aqueous solution containing a total cobalamin concentration of between 500 and 1500 micrograms per milliliter, and in that the complex is then isolated from this solution. 13. A method according to claim 12, characterized in that the complex is collected by adding to the solution a product causing precipitation, such as ammonium sulfate or acetone, and then is purified. 14. A process according to claims 12 and 13, characterized in that the degradation is carried out in such an aqueous solution. - 15. - Process for the production of a preparation containing a complex produced according to any one of the preceding claims, characterized in that it comprises the purification and the concentration of the fermentation product to obtain an aqueous solution containing a concentration of total cobalamins greater than 500 micrograms per milliliter and then diluting the product to a cobalamin concentration as a complex of less than 100 micrograms per milliliter. 16.- A process for the production of a preparation containing a complex prepared according to any one of claims 1 to 14, characterized in that it comprises the degradation of the cobala- mine "bound" contained in the fermentation product in cobalamin complex -peptide, the removal of the "unbound" cobalamin and the addition to the complex thus obtained of a non-toxic diluent. 17. - Manufacturing process according to claims 15 or 16, characterized in that a sweetening agent and / or another 'flavoring is also added. 18. - A method of manufacturing a preparation containing a complex prepared according to any one of claims 1 to 14, characterized in that it comprises the addition to the complex as well <Desc / Clms Page number 31> obtained from a non-toxic diluent and / or a sweetening agent and / or other flavoring agent. 19.- A method of manufacturing a preparation containing a complex prepared according to any one of claims 1 to 14, characterized in that it comprises the addition to the complex of a non-toxic solid diluent and the formation of tablets or capsules containing the product thus obtained. 20. - A method of manufacturing a preparation according to claims 15, 16, 17 and 18, characterized in that the non-toxic diluent is water or an aqueous liquid. 21.- A method of making a cobalamin-peptide complex, substantially as described in Examples 1 to 5. 22. - Cobalamin-peptide complexes prepared by the methods according to any one of claims 1 to 14. EMI31.1 236 Preparations containing cobalamin-peptide complexes prepared by the methods of any one of claims 15 to 20.