JPH0247966B2 - - Google Patents

Description

[Detailed description of the invention]

FIELD OF INDUSTRIAL APPLICATION The present invention relates to a method for the production of thymus products, which are used for the restoration, regeneration, hematopoiesis, blood clotting, and normalization of carbohydrate, fat and protein metabolism of disturbed immune processes in humans. It is useful for PRIOR ART It is known that in extracts of human and animal thymic tissue there are physiologically active substances that have a significant influence on the growth and development of the organism, as well as on several immune and metabolic processes. (Comsa T.,
Amer.J.Med.Sci.250, 79, 1965; Goldstein A.
et al., J. Immunol., 140, 359, 1970; Soviet Inventor's Certificate Application No. 459227; U.S. Patent No. 4120951). According to data available in the literature, extracts from calf thymus (thymosin) have been shown to be effective for restoring the functions of the human immune system in certain cases of neoplastic and congenital immunodeficiency. [Goldstein A. et al., Transplant
Proc. 7, 1 (Suppl. 1), 681, 1975; and Sakai et al., Cancer 36, 3, 97, 3, 1975]. By administering a thymus product to a patient, the organism's perturbed immune response is restored. To date, the therapeutic effects of physiologically active substances obtained from thymus tissue and their use in medical practice have been investigated, but to an insufficient extent. (Hooper et al., Ann. NYAcad. Soi., 249,
125, 1975) Pharmaceutical products from thymus tissue, namely thymosin (Goldstein A. et al., Pro.
Nat. Acad. Sci. USA, 69, 7, 1800, 1972) is known in the art. The following method is used for the production of thymosin (Hooper et al., 1975). Calf thymus was homogenized in 0.15 M NaCl solution, centrifuged at 14,000 G, and the supernatant was separated.
Then, heat at a temperature of 80°C to remove the formed precipitate, and add 5 volumes of acetone to the remaining solution. The precipitate obtained is dried on a filter, then redissolved and salted out with ammonium sulfate, first at 25% saturation and then at 50% saturation. The precipitate obtained by the salting-out operation was filtered and
Desalt in a -25 column and lyophilize.
The resulting complex of polypeptides contained in the preparation at this stage of purification has the most significant biological activity, namely the ability to stimulate cellular immunity (Hooper et al., 1975). Further purification of this preparation reduces its biological activity. Thymosin produced by this method is 3.5~
It has an isoelectric point of 6.7 and a molecular weight of 1000-14000 daltons, and contains substantially predominant amounts of aspartic acid and glutamic acid (the total amount of thymosin
Contains complexes of acidic polypeptides (up to 50% by weight). However, it should be noted that this method for thymosin production is rather complex and multistep (10 steps in total). The number of operations poses some difficulties when applying this method industrially (ultrafiltration, use of security packs)
related to By this method it is not possible to obtain a pharmaceutical preparation, namely thymosin, in high yields. Thymosin also produces some side effects and allergic reactions in many patients. The therapeutic efficacy of thymosin and the possibility of applying it in medical practice have not been well studied. (Problem to be Solved by the Invention) The present invention is based on a thymus gland that contains an alkaline polypeptide and has a broad spectrum of pharmacological action, on the other hand side effects and allergic reactions to the application are eliminated. The aim is to provide pharmaceutical formulations that Another object of the invention is to provide a method for the production of pharmaceutical preparations from the thymus, which ensures high yields by a simple method, comprising the separation of alkaline polypeptides, which guarantees a high clinical efficacy in cases of immunodeficiency. It is to be. (Means for solving the problems) These objects are a thymus product comprising a polypeptide with a molecular weight of 600 to 6000 daltons, of which 10 to 20% by weight has an isoelectric point of 7 to 9 and
This is achieved by providing a product characterized in that it contains a polypeptide having a molecular weight of 2000 to 4000 daltons and furthermore an isoelectric point of 3.5 to 6.7. The use of the thymus products of this invention in medical practice makes it possible to stimulate the reduced immune reactivity of the organism, enhance the activity of repair processes and hematopoietic activity, and prevent the progression of infectious and destructive processes in the organism. This makes it possible to treat many diseases more effectively or to prevent their development in humans. By using the thymus product of the invention in hospitals, it will be possible to shorten the treatment period for patients with chronic diseases and disturbed regenerative processes. This will be a decisive factor relating to the effectiveness of the use of this invention in medicine. The thymus product of the present invention, unlike known thymus-based products, stimulates the regeneration process of damaged tissues due to the presence of alkaline polypeptides in it, which stimulates the regeneration process of damaged tissues, which is effective during the healing period of wounds, ulcers. or, in stimulation of hematopoiesis, by an increase in their number in cases where the content of leukocytes, thrombocytes and red blood cells in the peripheral blood is reduced. Although the thymus products of this invention do not cause any side effects or allergic reactions, it is known from the literature that administration of thymosin causes allergic reactions in 20-30% of patients. The invention also relates to a method for manufacturing a thymus product,
The method comprises homogenizing the thymus tissue, separating the resulting homogenate into a precipitate and a supernatant, treating the latter with an organic solvent, and then separating the desired product, in which case Prior to separation, the thymus tissue homogenate is extracted with a 1-10% aqueous acetic acid solution in the presence of zinc chloride for at least 24 hours. The method of the invention is characterized by a simple method and is easily reproduced on a commercial scale. This method allows for sufficiently high yields in the hydrolysis of large proteins and other macromolecular compounds, as well as a high degree of purification of the product from the thymus gland, and this product does not cause any side effects or allergies when introduced into humans. Does not cause a reaction. The use of a somewhat prolonged extraction with acetic acid in the presence of catalytic amounts of zinc chloride contributes to the recovery of the alkaline polypeptide, which, when present in the product, has an expanded range of pharmacological properties. Guaranteed effectiveness. Has the greatest biological activity, with an isoelectric point of 7-9 and
The extraction, with a view to ensuring maximum recovery of polypeptides with molecular weights between 2000 and 4000 daltons,
~72 hours at 5℃ ~ PH2.5~4
0.2 to 2 per acetic acid at a temperature in the range of 15°C
Preferably it is carried out in the presence of zinc chloride in an amount of g. The thymus is kept at a temperature of -30°C to -50°C for at least 100 hours prior to homogenization for sufficient recovery of biologically active substances, i.e. alkaline polypeptides, and for preservation of the starting material. It is preferable to do so. The inventors characterized the properties of the products from the thymus by using ion-exchange chromatography on ^Biocarb (NNKuznetsova et al., High-molecular
Comp., 18a, 2, 235, 1976), gel filtration (Figh et al., T. Biol. Chem. 244, 4989,
1969), zone electrophoresis on paper or in polyacrylamide (according to conventional methods), and amino acid analysis (Stein and Arm method, T.Biol.
Chem. 176, 307, 1948).
By ion-exchange chromatography, it was found that the recovered compound contained three fractions in specific proportions that differed in their electrochemical properties. The physicochemical properties of the fractions contained in the thymus product of this invention are shown in Table 1 below. The fractions, and, comprise a number of components identified by zonal electrophoresis. The molecular weight of each fraction was determined by gel filtration using Sephadex G-25.

[Table] To determine the amino acid composition of the peptides in the above fractions, an automatic amino acid analyzer “LKB-3201” was used.
(Sweden) was used. The measurement method is
Spackman DH et al., Anal.Chem.1958, 30, 1190
~1206. The amino acid composition of the thymus product of this invention is shown in Table 2.

【table】

[Table] The biological activity and wide-ranging pharmacological effects of the thymus products of this invention with a molecular weight of 600-6000 daltons include an isoelectric point of 7-9 and an isoelectric point of 2000-4000.
It is associated with the presence of 10-20% by weight of a polypeptide having a molecular weight of With regard to its physicochemical properties and amino acid composition, the thymus product of the invention has a molecular weight of 1000 to 14000 daltons and contains fairly predominant glutamic and aspartic acids (the sum of which amounts to 50% by weight of the product). (Hooper et al., Ann. NYAcad.
Sci., 249, 125, 1975) It differs from thymosin, which consists of a polypeptide complex (isoelectric point 3.5-6.7). The thymus product of this invention is manufactured as follows. The thymus is cleaned of blood vessels, blood clots, and adipose tissue, washed with water, and homogenized. Prior to homogenization, the thymic tissue was incubated at -50°C to -30°C to ensure conversion of the starting material and increase the yield of biologically active substances, i.e. polypeptides with isoelectric points of 3.5-6.7 and 7-9. Preferably, it is maintained at a temperature in the range of 0.degree. C. or treated with an organic solvent such as acetone, chloroform or ethanol at a temperature of -20.degree. C. to -1.degree. Homogenates of thymus tissue are extracted with 1-10% aqueous acetic acid in the presence of zinc chloride for at least 24 hours. Extraction under such conditions requires 2000~
Contributes to the most complete recovery of polypeptides with a molecular weight of 4000 Dt (Dalton) and an isoelectric point of 7 to 9, and removes proteins and macromolecular compounds that cause allergic reactions when applying thymus products to patients. Assure. The optimal conditions for extraction are
PH is 2.5 to 4, temperature is 5℃ to 15℃, time required is 48
~72 hours, and the concentration of zinc chloride per acetic acid
It is 0.2-2g. After the extraction is completed, the obtained substrate is separated into precipitate and supernatant, and the supernatant is treated with an organic solvent, such as acetone, chloroform, ethanol, and then
Collect the generated precipitate. The latter is dried into powder. This powder contains biologically active substances, ie polypeptides. The powder is dissolved in acetic acid, the solution is sterilized by filtration and poured into a 1 ml sterile flask, which is sealed with a rubber stopper. One flask contains 10 mg of material obtained from the thymus. The thymus product of the invention thus obtained contains biologically active substances, i.e. the ratio between them isoelectric point 3.5.
It contains polypeptides with a molecular weight of 600-6000 Dt, 90-80% by weight of polypeptides with an isoelectric point of ~6.7 and 10-20% by weight of polypeptides with an isoelectric point of 7-9. The thymus product of this invention is a white powder or a yellowish white powder, soluble in 5% acetic acid solution and water, and substantially insoluble in alcohol. It contains virtually no protein and the pH of a 1% aqueous solution is 5-5.8. Thymus products are
Stored for 2 years at a temperature below 20°C in a dry, shaded area. Thymus products are used for purulent inflammatory diseases of bone and soft tissue, chronic traumatic and hematopoietic osteomyelitis, bone fractures, burn lesions,
Trophic ulcers, pressure sores, radionecrosis of tissues, atherosclerosis obliterans, chronic pneumonia, gastric and duodenal ulcers, and hypofunction of the thymus, respectively, inhibition of immunity and hematopoiesis after radiotherapy and chemotherapy in cancer patients. It can be used to treat symptoms associated with. The thymus product of this invention can also be used for the prevention of infectious complications, during the course of radiotherapy or chemotherapy with large doses of antibiotics, after trauma and after surgery, inhibition of immunohematopoiesis, regenerative processes. can. It can also be used in cases of intolerance to antibacterial therapy or lack of effect by this therapy and as a normalizing agent for functional disturbances of carbohydrate, fat and protein metabolism in the organism. The inventors have suggested a simple and inexpensive method of manufacturing the thymus products of this invention. This process can be easily carried out on a commercial scale and provides high quality products, for example in 5 or higher yields of thymosin. Thymus is commonly obtained from calves. However, other young mammals such as horses, pigs, sheep,
It can also be obtained from whales, etc. Next, to explain this invention in more detail,
Give an example. Example 1 500 g of bovine thymus tissue was cleaned of blood vessels, blood clots, and adipose tissue, then washed with water and acetone, and then placed in a 4:1 weight ratio of acetone to thymus - 1 Hold at ℃ for 24 hours and homogenize. The obtained thymus homogenate is added to a 3% aqueous acetic acid solution in a weight ratio of 1:6. Extraction is carried out in the presence of 1 g of zinc chloride per acetic acid at PH 3.2 and a temperature of 10° C. for 72 hours.
After the extraction is complete, centrifuge the substrate at 3000 rpm for 20 minutes, separate the supernatant, and add acetone at a weight ratio of 1:5. The precipitate formed is collected by filtration, washed with acetone and dried on a filter. The obtained 5 g of white powder contains biologically active substances, i.e. 80% by weight of polypeptides with a molecular weight of 600-6000 Dt and an isoelectric point of 3.5-6.7 and a polypeptide with an isoelectric point of 7-9. It contained a polypeptide consisting of 20% by weight of peptide. The product thus obtained from the thymus is a white powder soluble in 5% acetic acid and water, and the pH of the 1% aqueous solution is
It is 5.5. To prepare the thymus product medicament according to the invention, the powder prepared as described above is dissolved in 500 ml of 1% acetic acid. A solution of this product is sterilized by filtration and poured into 1 ml sterile flasks, lyophilized and sealed with rubber stoppers. As a result, one flask contains 10 mg obtained from the thymus.
Contains the following substances. Example 2 500 g of bovine thymus was cleaned by removing blood vessels, blood clots, adipose tissue, washed with water and chloroform, and placed in chloroform at a weight ratio of 8:1 to thymus tissue at -10°C. At temperature 48
Hold for an hour and then homogenize. The obtained homogenate of thymus tissue is added to a 1% aqueous acetic acid solution in a weight ratio of 1:10. Extraction is carried out in the presence of 2 g of zinc chloride per acetic acid at PH 4 and a temperature of 15° C. for 48 hours. After extraction is complete, remove the substrate
After 24 hours, the supernatant is separated and chloroform is added in a weight ratio of 1:8. The precipitate formed is collected by filtration and washed with chloroform, then dried on a filter. 3g thus obtained
The white powder contains biologically active substances, i.e. 600
It has a molecular weight of ~6000 Dt and contains a polypeptide consisting of 90% by weight of a polypeptide with an isoelectric point of 3.5-6.7 and 10% by weight of a polypeptide with an isoelectric point of 7-9. The resulting thymus tissue product is a white powder soluble in 5% acetic acid and water, with a PH of 5.8 in a 1% aqueous solution. The solution of this product was sterilized by filtration, and
ml into sterile flasks, then lyophilized and sealed with rubber stoppers. As a result, one flask contains 10 mg of material obtained from the thymus. Example 3 500 g of bovine thymus tissue is cleaned by removing blood vessels, blood clots, adipose tissue, washed with water, and homogenized. The resulting homogenate of thymus is placed in a 10% aqueous acetic acid solution in a weight ratio of 1:4. Extraction is carried out in the presence of 0.2 g of zinc chloride per acetic acid at a pH of 2.5 and a temperature of 5° C. for 24 hours. After the extraction is completed, the substrate is heated at 5000 rpm for 10
Centrifuge for a minute, separate the supernatant, and add ethanol to it in a weight ratio of 1:10. The precipitate formed is separated by filtration and washed with ethanol and then dried on a filter. 4 g of white powder thus obtained contains 87% by weight of biologically active substances, i.e. polypeptides with a molecular weight of 600-66000 Dt and an isoelectric point of 3.5-6.7 and an isoelectric of 7-9. Polypeptide with points 13
% by weight of the polypeptide. The thymus product thus obtained is a white powder soluble in 5% acetic acid, and the pH of the 1% aqueous solution is 5.3.
It is. In order to manufacture this thymus product medicine, the powder is
Dissolve in 400 ml of 3% acetic acid. The solution is sterilized by filtration and poured into 1 ml sterile flasks, which are then lyophilized and sealed with rubber stoppers.
As a result, one flask contains 100 mg of material obtained from the thymus. Example 4 500 g of bovine thymus tissue is cleaned by removing blood vessels, blood clots, adipose tissue, washed with water, kept at -30°C for 200 hours, and then homogenized. The obtained homogenate of thymus is placed in a 5% aqueous acetic acid solution in a weight ratio of 1:5. Extraction is carried out in the presence of 0.5 g of zinc chloride per acetic acid at PH 4 and a temperature of 12° C. for 56 hours. After the extraction is complete,
The substrate is centrifuged at 4000 rpm for 20 minutes, the supernatant is separated and acetone is added to it in a weight ratio of 1:6. The precipitate formed is centrifuged and washed with acetone and dried on a filter. 5.5
g of the obtained yellowish white powder has a biologically active substance, i.e. a molecular weight of 600-6000;
82% by weight polypeptide with isoelectric point between 3.5 and 6.7
and 18% by weight of a polypeptide with an isoelectric point of 7-9. This product, produced from thymus gland, is a yellowish-white powder, soluble in 5% acetic acid, and a PH of 1% aqueous solution is 5.2. To prepare the thymus product medicine, the powder obtained is dissolved in 550 ml of 4% acetic acid. A solution of this product is sterilized by filtration and poured into 1 ml sterile flasks, lyophilized and sealed with rubber stoppers. As a result, one flask contains 10 mg of material obtained from the thymus. Example 5 500 g of bovine thymus tissue was cleaned by removing blood vessels, blood clots, and adipose tissue, washed with water,
Hold at -50°C for 100 hours, then homogenize. The resulting homogenate of thymus is placed in a 2% aqueous acetic acid solution in a weight ratio of 1:7. per acetic acid
Extraction is carried out in the presence of 1.5 g of zinc chloride at pH 3.0 and a temperature of 8° C. for 60 hours. After the extraction is complete, the substrate is centrifuged at 6000 rpm for 10 minutes, the supernatant is separated and ethanol is added to it in a weight ratio of 1:10. The resulting residue is collected by filtration and washed with ethanol and dried on a filter. The obtained 5.5 g of white powder is
Biologically active substance, i.e. a polypeptide having a molecular weight of 600-6000 Dt and consisting of 80% by weight of a polypeptide with an isoelectric point of 3.5-6.7 and 20% by weight of a polypeptide with an isoelectric point of 7-9. Contains. The white powder obtained is soluble in 5% acetic acid and water, and the pH of a 1% aqueous solution is 5.4. To prepare the thymus product medicine, the powder produced as described above is dissolved in 570 ml of 2% acetic acid. A solution of this product is sterilized by filtration and poured into 1 ml sterile flasks, which are then lyophilized and sealed with rubber stoppers. As a result, one flask contains 10 mg of material from the thymus.

Claims (1)

[Scope of Claims] 1. A thymus product comprising a polypeptide having a molecular weight of 600 to 6000 daltons, wherein 10 to 20
% by weight is a polypeptide having an isoelectric point of 7 to 9 and a molecular weight of 2000 to 4000 Daltons, and further
To produce a product containing a polypeptide with an isoelectric point of 3.5 to 6.7, thymus tissue is homogenized, the resulting homogenate is separated into a precipitate and a supernatant, which is treated with an organic solvent, and then A method comprising isolating the desired product, characterized in that, prior to separation, the thymus tissue homogenate is extracted with a 1-10% aqueous solution of acetic acid in the presence of zinc chloride for at least 24 hours. 2. The method according to claim 1, wherein the extraction is carried out for 48 to 72 hours. 3. Zinc chloride 0.2-2g per acetic acid for extraction
3. A method according to claim 1 or 2, characterized in that it is used in an amount of . 4. Claims 1 to 4, characterized in that the extraction is carried out at a pH of 2.5 to 4 and a temperature of 5 to 15°C.
The method according to any one of paragraph 3.