FR2583982A1 - MEDICINAL PRODUCT OBTAINED FROM THYMUS AND METHOD OF OBTAINING THE SAME - Google Patents

Abstract

MEDICINAL PRODUCT OBTAINED FROM THYMUS CONTAINING POLYPEPTIDES WITH A MOLECULAR MASS OF 600 TO 6000 DALTON, AND THE COMPOSITION OF WHICH IS AS FOLLOWS (BY MASS): -POLYPEPTIDES AT ISOELECTRIC POINT 3,5-6,7: 80-90 -POLYPEPTIDES A ISOELECTRIC POINT 7-9: 20-10 AND MOLECULAR MASS FROM 2000 TO 4000. PROCESS FOR OBTAINING THE ABOVE MEDICINAL PRODUCT INCLUDING THE HOMOGENIZATION OF THE THYMUS TISSUE, THE EXTRACTION OF THE HOMOGENIZED PRODUCT OBTAINED BY AN AQUEOUS SOLUTION AT 1-10 D 'ACETIC ACID IN THE PRESENCE OF ZINC CHLORIDE DURING AT LEAST 24 HOURS, THE SEPARATION OF THE EXTRACT OBTAINED IN SEDIMENT AND SURNAGEANT LIQUID, THEN TREATMENT OF THE LATTER WITH ORGANIC SOLVENT WITH SUBSEQUENT ISOLATION OF THE CONCERNED PRODUCT.

Description

/ I

  MEDICINAL PRODUCT OBTAINED FROM THYMUS AND

ITS PROCESS OF OBTAINING

  The present invention relates to a medicament and to its method of obtcntion, and more particularly, it relates to a product

  The medicinal product obtained from the thymus and its method of production.

  : e-, prod.uis. It can be used to restore the disturbed processes of immunogenesis, regeneration, hematopoiesis. blood coagulation, as well as for the normalization of carbohydrate metabolism and lipid and protein metabolism in

the horme.

  It is well known that the thymus extracts of man and animals contain physiologically active substances which infe

  the growth and development of the organism, as well as cer-

  the United Nations, the United Nations and the United Nations.

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  According to the data of the technical literature, the thymus zu extract of calves (thtymosin) is used for therapeutic purposes for the restoration of the function of the immune system in humans during different oncological and immunological diseases. congenital suppressors (Goldstein A. et al., Transplant Proc., 7 (Supp.),

  681, 1975; Sakai et al., Cancer, 36, 3, 973, 1975).

  The administration of thymosin produces in patients

  restitution of the disturbed immunological reactivity of the organism.

  However, until now the therapeutic efficacy of physiologically active substances of the thymus and indications for their application in medical practice are not

sufficiently studied.

  At present, a drug product is known from thymus tissue-thymosin (Goldstein A. et al., Proc Nat Nat.

  Sci. USA, 69, 7, 1972), which consists of a complex (about 30

  acid polypeptides with a molecular weight of 1,000 to

  14,000 dalton (Hooper et al., Ann N.Y. Acad Sci., 249, 125, 1975).

  In order to obtain thymosin, the procedure is as follows (Hooper et al., 1975). The thymus of calves is homogenized in a 0.15M solution of NaCl, centrifuged at 14,000 daltons, the supernatant liquid is separated and heated to a temperature of 80 ° C., the precipitate is removed by filtration, and then 5 volumes of solution are added to the solution. acetone. The precipitate obtained is dried on a filter, then it is dissolved again and it is released with ammonium sulphate, initially for a saturation at 25%, then for a saturation at 50%. The precipitate obtained after the release is filtered, desalted in a column of Sefadex G-25 and lyophilized. The polypeptide complex obtained at this stage of purification of the drug product, has the highest biological activity, that is, ie the strongest ability to stimulate cellular immunity (Hooper

et al., 1975).

  The subsequent purification of the drug product leads to the

  reduction of its biological activity.

  Thymosin obtained by the above method contains unr.

  cuinulexe of acidic polypeptides with an isoelectric point of 3.5 to 6.7 and a molecular mass of 1,000 to 14,000 daltons with a clear preponderance of asparic and glycutic acids, which together make up 50% of rpn4d

of the total mass of thymoqin-

  2h The complexity of the process for obtaining thymnsin and the large number of technological steps it involves (10 steps) should be emphasized. Several operations raise certain difficulties for

  the use of said process under industrial conditions (ultracentriform

  trifugation, use of Sefadex). This method does not ensure a sufficient yield of medicinal product, namely thymosin. By

  In some patients, thymosin causes secondary effects in some patients.

  daries and allergic reactions. The therapeutic efficacy of thymosin and the possibilities of its use in practice

  are not yet sufficiently studied.

  It has been proposed by selection of appropriate technological operations to create a medicinal product from the thymus, comprising polypeptides of alkaline nature, and which has a wider range of pharmacological action than thymosin, and that in the absence of side effects and allergic reactions as a result of

its application.

  The present invention therefore consists of a medicinal product

  obtained from the thymus, comprising isoelective point polypeptides

  3.5 to 6.7, and, according to the invention, containing polypeptides with a molecular weight of from 600 to 6,000 daltons, of which 10 to 20% of the mass has an isoelectric point of 7 to 9 and a molecular mass of

2,000 to 4,100 dalton.

  The use of the medicinal product obtained from the thymus in medical practice makes it possible to reduce the reduced immunological reactivity of the organism, to stimulate the activity of the processes. IC and haematoxylin, as well as preventing the development of infectious and destructive processes in the body, which makes it possible to treat more effectively a whole range of pathologies and preventi it appears in Khoman. The use of the medicament product obtained from the thymus according to the invention under the conditions of hospitalization makes it possible to significantly reduce the time required for treatment. treatment

  patients suffering from chronic diseases and presenting disturbances

  bations of regeneration, which may be a determining factor in

  the effectiveness of the use of the invention in the medical profession

  cine. the difference of the known drug products obtained from

  thymuc, the product obtained from the thymus. according to the invention.

  The use of polyeptidepeptides of an alkaline nature stimulates the processes

  The use of the tissues l? es = n which is translated nar a rdlur.-

  delay of wounds. ulcers, as well as the 2 [hematopoietic process, which results in an increase in

  the content of white blood cells, platelets and red blood cells

iqu enr case of reduced content.

  The drug product obtained from the thymus, according to the invention

  tion, do not cause side effects and allergic reactions, while, according to the literature data, it is well known that

  Thymosin provides 20-30% of patients with allergic reactions.

  Another subject of the present invention is a process for obtaining

  the medicinal product obtained from the thymus, comprising the homogenization of the thymus tissue, the separation of the homogeneous product obtained in sediment and supernatant liquid, the treatment of the supernatant liquid with an organic solvent followed by a subsequent isolation of the targeted product, this process being characterized in that prior to separation, the homogeneous thymus tissue is extracted with an aqueous solution of 1-10% acetic acid in the presence of zinc chloride for at least

24 hours.

  The method according to the invention is characterized by a technology

  simple and it is easy to implement under the indus-

  -monthly. This process allows the hydrolysis of large

  teins and other macromolecular compounds, to achieve a sufficiently high yield of medicinal product obtained from the thymus with a high degree of purity, which, after its administration

  in the body, causes neither side effects nor allergic reactions.

  qiques. The use of prolonged extraction with acetic acid in the presence of catalytic amounts of zinc chloride promotes the release of alkaline polypeptides, the presence of which in the presence of

  The product ensures the widening of the range of its pharmacological action.

cal.

  It is advantageous, in order to extract as much as possible the

  with an isoelectric point of 7 to 9 and a molecular weight of 2,000 to 4,000 dalton, which have the highest biological activity,

  carry out extraction for 48-72 hours at pH = 2.5-4 and at a temperature of

  from 5 to 150 ° C., in the presence of zinc chloride at a rate of 0.2 to 2 g.

per liter of acetic acid.

  It is preferable, for a more complete extraction of substances

  these biologically active, namely polypeptides of alkaline nature

  lnc, and for better conservation of raw materials, dp

  to maintain the tissue of the thymus, prior to homoanization, at a temperature of

  from -30 to -50 C for at least 100 hours.

  The study of the properties of the drug product obtained from the thymls according to the invention was obtained by ion exchange chromatography in protonated "Biocarb" hydroxyl cation exchangers (NN Kuznetsova et al., "Macromolecular compounds", 18a, 2, 355, 1976), gel filtration (Fish et al., J. Binl Chem., 244, 4989, 1969), zonal paper electrophoresis and polyacrylamide gel (by generally adopted methods) and amino analysis. acids (method of Stein and Moore, J. Biol Chem., 176, 307, 1948). It has been established by ion exchange chromatography that the substances obtained contain three fractions in a ratio (in percent) determined and

  which are distinguished by their electrochemical properties.

  Table 1, which presents the parameters, is given below.

  physico-chemical properties of the fractions contained in the medicinal product

from the thymus.

TABLE 1

  Component Content Molecular weight Isoelectric point% mass dalLonr pH unit Fraction I 40-70 600-1,000 3,5-5,0 Fraction II 20-40 5,000-6,000 5,5-6,7 Fraction III 10-20 2,000-4,000 7.0-9.0 Fractions 1, I, III have different components, detected by zonal electrophoresis. The molecular mass of each fraction has

  was determined by gel filtration on Sefadex G-25.

  For the determination of the amino acid composition of

  peptides of the abovementioned fractions, was used the automatic amino acid analyzer "LKB-3201" (Sweden) according to the method of

  Spackman D.H. et al. (Anal Chem, 1958, 30, 1190-1206).

  Table 2 gives the composition of amino

  Acids of the drug product obtained from the thymus according to the invention.

TABLE 2

  Amino acid content in fractions,% mole Amino acids of the product Amilno-acid (, according to the invention Fraction Fraction vention% moc I TI III i 2 3 4 5 Aspartic acid 5.8 6, 3. 0.6 4.2 Threonine 5.7 6.3 6.4 5.8 Serine 5.7 6.5 7.5 10.4 Glutamic 6.2 8.6 14.1 6.2 Proline 10 1 10.8 13, 2 9.5 Glycine 8.6 10.4 11.7 10.1 Alanine 10.7 12.6 8.7 18.6 Cysteine 3.4 2.6 1.0 0.9 Valine 8.8 5.2 6.5 3.9

Methionine trace traces - -

  Isoleucine 3.9 2.3 4.9 1.3 Leucine 6.7 6.2 5.8 3.2 Tyrosine 2.4 1.2 2.4 0.88 Phenylalanine 3.2 1.7 3.3 0 , 9 Histidine 2.9 2.9 2.2 0.5 Lysine 7.0 8.5 7.1 21.6 Arginine 8.5 7.8 4.6 2.1 Tryptophan traces traces traces traces

  Biological activity and the wide range of pharmacological action

  of the product according to the ivention obtained from the thymus with a molecular weight of 600 to 6,000 daltons are due to the presence in the latter of 10 to 20% by weight of isoelectric point polypeptides of 7 to 9 and of molecular mass of 2,000 to 4,000 dalton, which are characterized by their content of basic amino acid residues, for example in

  lysine. Because of its physicochemical properties and its amino

  acid, the medicinal product according to the invention obtained from the thymus differs substantially from thymosin which consists of a complex of acidic polypeptides (with an isoelectric point of 3.5 to 6.7) of

  molecular weight of 1,000 to 14,000 daltons, containing a strong predominance

  of aspartic acid and glutamic acid, which form 50% of the

  mass of product n (Hooper et al., Ann Y. Acad Sci., 149, 125, 1975).

  The drug product according to the invention obtained is obtained at

from the thymus, comne follows.

  Thymus tissue is cleared of blood vessels, blood clots,

  the adipose cell, it is washed with water and subjected to

  généisation. In order to better conserve the raw material and to increase the yield of biologically active substances, that is to say in isoelectric points of solubility of 3.5 to 6.7 and 7 to 9, it is recommended to keep To homogenize the thymus tissue at a melting point of -50 to -30 C, or to treat with an organic solvent such as ac.tonc. chloroform, Vthanol, at a temperature of -20 to

-1 C.

  The homo-eneived thymus tissue is extracted with 1-10% aqueous acetic acid in the presence of zinc chloride for at least 24 hours. Performing the extraction under such a regime favors a better yield of polypeptides with a molecular weight of 2,000 to 4,000 daltons and an isoelectric point of 7 to 9,

  while ensuring the elimination of proteins and macromolecular compounds

  the allergic reactions that occur after administration to patients of the drug product obtained from the thymus. The optimal conditions for extraction are: 35. pH .........

  .................. 2,5-4,0 temperature ................... 5-15 C duration from the extraction .......... 48-72 hours the zinc chloride content then being 0.2 to 2 g in 1 liter..DTD: acetic acid.

  At the end of the extraction, the substrate obtained is separated into sediment

  and supernatant liquid, and the latter is then treated with an organic solvent.

  such as acetone, chloroform or ethanol with subsequent extraction of the sediment that forms. The sediment is dried until it turns into a powder, which contains the biologically responsible substances.

  active agents, in particular the polypeptides.

  The resulting powder is dissolved in acetic acid, sterilized by filtration and poured into sterile vials.

  because of i ml, then lyophilizes and closes the fldcons with

  sterile rubber chews. Each vial contains 10 mg of substances

extracted from the thymus.

  The medicinal product obtained according to the invention from the thymus thus prepared contains biologically active substances, in particular polypeptides with a molecular mass of 600 to 6,000 dalton in the following ratio (% by weight): polypeptides with an isoelectric point 3,5 -6.7 ... The product obtained according to the invention, from the thymus is an amorphous powder of white or yellowish-white color, soluble in the solution it contains 5% acetic acid and is virtually insoluble in water and contains no protein and the pH of its solution.

  I% aqueous is 5 to 8. We keep the drug product oh ..

  to use the Lhymus in a dry room. away from light, and at a

  temperature not exceeding + 200C for a period of two years.

  The mMdicanient product obtained from the thymus is administered by intramuscular injection. For this purpose, just before the injection, it is necessary to dissolve the contents of the vial in 1-2 milliliters of physiological saline or novocaine solution. Duration of the cure from 5 to 20 days, right

from 10 to 30 mg of product per day.

  The drug product according to the invention obtained from the

  The thymus is subjected to a large therapeutic trial on various mala-

  This test demonstrates that this product has a wider range of pharmacological action than the product obtained from the thymus already known from the technical literature, while producing no side effects or allergic reactions. is recommended to use the drug product according to the invention obtained from the thymus in the treatment of purulent and inflammatory chronic diseases of bone and soft tissue, posttraumatic and hematogenous chronic osteomyelitis, bone fractures, burns, trophic ulcers of decubitus, necrosis

  radiotherapy, atherosclerosis obliterans, chronic pneumonias,

  gastric and duodenal ulcerative diseases, as well as for the therapy of thymus hypofunction, the inhibition of immunity and haematopoiesis as a result of radiotherapy and chemotherapy of cancer patients. It is also advisable to use the medicinal product obtained from the thymus to prevent the infectious complications of the inhibition of immunity and hematopoiisis, processes of regeneration in the post-traumatic period. and post-operative, in radiotherapy and in

  chemotherapy, as well as following the administration of dosess mss-

  with antibiotics. it is also recommended to use this medicinal product when intolerance to antibacterial therapy or as a result of the lack of effect to this therapy, and also as a normalizing agent during functional disturbances of the metabolism of carbohydrates and lipid and protein metabolisms.

  It is used to obtain the drug product according to the invention.

  vention obtained from the thymus technology simple and good marnhé, easy to achieve under industrial conditions, and to obtain a product of high quality and with a good performance that is

  higher, for example, than that of thymosin 5 times.

  We give below concrete examples of obtaining the product

  drug obtained according to the invention from the thymus.

EXAMPLE 1

  500 g of thymus tissue, vessels,

  blood lots and adipose cell, washed with water and acetone, and then maintained in acetone, taken in a proportion by weight of 4 / i relative to the thymus tissue, at the temperature of - 1 C for 24 hours, after which it undergoes homogenization. The homogenized thymus tissue is placed in a 3% aqueous solution of acetic acid at a ratio of 1/6 respectively. The extraction is carried out under conditions of pH 3.2, a temperature of 10 C for 72 hours, in the presence of zinc chloride, used at a rate of 1 g per liter of acetic acid mentioned. At the end of the extraction, the substrate is centrifuged at 3000 rpm for 20 minutes, the supernatant liquid is removed and the acetone is added in a weight ratio.

9 2583982

  of 1/5 respectively. The sediment formed is filtered off and washed with acetone and dried on a filter. White powder

  obtained in the quantity of 5 9 contains biological substances

  polypeptides having a molecular weight of from 600 to 6,000 daltons in the following ratio (% by weight): polypeptides with isoelectric point 3, 5-6,7 .... 80 polypeptides with an isoelectric point 7-9. The medicinal product thus obtained from the thymus is a cheek powder. white, soluble in 5% acid solution

  acetic acid and water, the pH of the 1% aqueous solution being 5.5.

  To obtain the drug form of the product from the thymus, the powder obtained is dissolved in 500 ml of 1% solution of dicylic acid. The solution of the product is sterilized by filtration and poured at a rate of 1 ml into sterile vials, lyophilized and closed with rubber stoppers. Each bottle

  contains 10 mg substances extracted from the thymus.

EXAMPLE 2

  500 g of thymus, vessels, and blood clots are removed.

  quins and adipose tissue, wash it with water and chloroform, and then stick it in the chloroform employed in a proportion by weight of 8/1 with respect to the weight of the thymus, at a temperature of -10 C during: eurel apr, subject to homogenization. Homogenous hrymus tissue is placed in a 1X aqueous solution of acetic acid in a weight ratio of 1: 1. respectively, extraction is carried out under the conditions of pH = 4, of temperature C for 48 hours in the presence of zinc chloride, used at a rate of 2 g per liter of acid

  acetic. At the end of the extraction, the substrate is subjected to filtration.

  for 24 hours, takes the supernatant liquid and adds chloroform in a weight ratio of 1/8 respectively. The sediment formed is filtered off and washed with chloroform and dried on a filter. The 3 g of white powder thus obtained contain biologically active substances-polypeptides with a molecular weight of from 600 to 6,000 daltons in the following ratio (% by weight): polypeptides with isoelectric point 3,5-6,7 .... 90 isoelectric point polypeptides 7-9 ........ The drug product obtained from the thymus thus prepared is a white powder, soluble in a 5% solution of acetic acid, the pH of the 1% aqueous solution of said product being

of 5.8.

2583982

  To obtain the drug form of the product obtained from the thymus, the powder obtained is dissolved in 300 ml of 5% acetic acid solution. The solution of the product is sterilized by filtration and poured at a rate of 1 ml into sterile flasks, then freeze-dried and closed the flasks with rubber stoppers. Each

  vial contains 10 mg of extraneous substances from the thymus tissue.

EXAMPLE 3

  500 g of the thymus tissue, vessels, clots

  blood cells and the fat cell, wash with water and then

  Neise. The homogenized thymus tissue is placed in an aqueous solution

  to 10% acetic acid for a weight ratio of 1/4 respectively.

  The extraction is carried out under the conditions of pH 2.5, of temperature C for 24 hours, in the presence of zinc chloride, used rationally

  0.2 g per liter of the above-mentioned acetic acid. At the end of the

  The substrate is centrifuged at 5,000 rpm for 10 minutes, the supernatant liquid is removed and ethanol is added at a weight ratio of 1/10, respectively. The sediment formed is filtered off, washed with ethanol and dried on a filter. The

  white powder thus obtained in a quantity of 4 g contains

  Biologically active polypeptides - polypeptides having a molecular weight of from 600 to 6,000 daltons having the following ratio (% by weight): Polypeptides with an isoelectric point 3,5-6,7. The drug product obtained from the thymus thus prepared is a white powder, soluble in 5% acetic acid,

  the pH of the 1% aqueous solution of said product being 5.3.

  For obtaining the drug form of the product obtained from the thymus, the powder obtained is dissolved in 400 ml of 3% acetic acid. The solution of the product is sterilized by filtration and poured at a rate of 1 ml into sterile flasks, then freeze-dried and closed the flasks with rubber stoppers. Each bottle

  contains 10 mg of substances extracted from the thymus tissue.

EXAMPLE 4

  500 g of the thymus tissue, vessels, clots

  blood cells and the fat cell, wash with water and then

  Neise. The homogenized thymus tissue is placed in an aqueous 5% acetic acid solution at a weight ratio of 1/5 respectively. The extraction is carried out under the conditions of pH = 4 and temperature 12 C for 56 hours, in the presence of zinc chloride, used with good reason.

  0.5 g per liter of the above-mentioned acetic acid. At the end of the

  The substrate is centrifuged at 4000 rpm for 20 minutes, the supernatant is removed and acetone is added for a weight ratio of 1/6, respectively. Filtration is separated

  sediment deposited and lp washed with acetone. then dry on filter.

  The yellowish-white powder thus obtained in an amount of 5.5 g contains biologically active substances, namely the polypeptides of molecular weight of from 600 to 6,000 danton in the following ratio (% by mass IC): polypeptides with isoelectric point 3, 5-6.7 .... 82 polypeptides with isoelectric point 7-9 ...... 18 the drug product obtained from the thymus thus prepared is a white-yellowish puffer, soluble in 5% acetic acid O, the

  oH of the 1% aqueous solution of said product being 5.2.

  To obtain the drug form of the product obtained from the thymus, the powder obtained is dissolved in 550 ml of 4% acetic acid. The product solution was sterilized by filtration and poured into sterile vials at 1 ml, lyophilized and sealed with rubber stoppers. Each bottle

  contains 0 mg of substances extracted from the thymus tissue.

  ryrMPLE 5 O ': ibeera 500 q thymus tissue, vessels. clots anguins and adipose cell, washed with water, maintained for 10 hours at -50 C, and then homogenized. The homogenized thymus tissue is placed in an aqueous 2% acidic solution at a weight ratio of 1/7, respectively, and the extraction is carried out under conditions of pH 3.0 and 8 C. During hours in the presence of zinc chloride, employed at a rate of 1.5 g per liter of the above-mentioned acetic acid, the substrate is centrifuged at 6,000 rpm for 10 minutes and the liquid is withdrawn. supernatant and ethanol is added in a weight ratio of 1/10 respectively The deposited sediment is filtered off, washed with ethanol and dried on a filter The white powder thus obtained in an amount of 5, 6 g contains biologically active substances, namely polypeptides with a molecular weight of 600 to 6,000 daltons in the following ratio (% by weight):

12 2583982

  isoelectric point polypeptides 3,5-6,7 .... 80 polypeptides with isoelectric point 7-9 ........ The drug product obtained from the thymus thus prepared is a white powder, soluble in water. 5% acetic acid and in water, the pH of the aqueous solution at 1% of said product being 5.4. To obtain the drug form of the product obtained from the thymus, the powder obtained is dissolved in 570 ml of 2% acetic acid. The product solution is sterilized by filtration and poured into sterile vials at 1 ml, then lyophilized and sealed with rubber stoppers. Each bottle

  contains 10 mg of substances extracted from the thymus tissue.

MU

Claims (4)

  1. Product obtained from the thymus containing polypeptides with isoelectric point 3.5-6.7, characterized in that it contains polypeptides with a molecular weight of 600 to 6,000 daltons, of which 10 to 20% of mass point isoelectric 7-9 and molecular weight 2,000- 4.000 dalton.   2. A process for obtaining the product obtained from the thymus according to claim 1, comprising the homogenization of the thymus tissue, the   separation of the homogenized product obtained in sediment and supernatural liquidation   in that it is treated by organic solvent with subsequent isolation of the desired product, characterized in that, prior to separation, extraction of the homogenized thymus tissue is carried out with an aqueous solution of -ih acetic acid in the presence of zinc during at least 24 hours.   3. Production process according to claim 2, characterized in that   the extraction is carried out for 48-72 hours.   4. Production process according to claims 2 and 3, characterized   in that zinc chloride is used for   0.2-2 g per liter of acetic acid.   2. Method of obtaining according to claims 2 to 4, characterized   in that the extraction is carried out under the conditions of J 2.5-_and P tfpmnArature 5-15 C.   The embodiment according to claims 2 to 5, characterized in that   ri96 Pn which. before homogenization, the thymic tissue is maintained   at a temperature of -50 to -30 C for at least 100 hours.