Polypeptide composition

 

(57) Abstract:

The invention relates to medicine, in particular to polypeptide compositions intended for the treatment of patients by oral ingestion of polypeptide drugs. The polypeptide composition comprises as active ingredient a polypeptide hormone of nature with molecular weight ranging from 1,000 to 21,000, an inhibitor of proteolytic enzymes such as trypsin inhibitor from soybean, pancreatic trypsin inhibitor, Aprotinin, ovomucoid of protein eggs of birds, and pharmaceutically usable water-soluble organic acid and a water soluble inert filler in the following ratio, wt.%: the polypeptide of 1.0-95,0; inhibitor of proteolytic enzymes of 0.02 to 10.0; organic acid 1,0-94,0; inert filler rest. To increase the effectiveness of this composition may further include known compounds, facilitating penetration of the polypeptide through the membrane the mucous membranes. The invention provides penetration into the bloodstream of polypeptides with molecular masses from 1000-21000, the polypeptides penetrate into the bloodstream without being destroyed. 1 C.p. f-crystals, 4 PL.

It is known that the vital substances polypeptide of nature (enzymes, inhibitors, hormones, etc.,) are synthesized inside the body, and not get into it with food. This is impossible due to the destruction of these substances in the digestive process, and also because of the limited absorption of drugs into the bloodstream through the mucous membrane of the intestine, due to the large size of their molecules.

The process of recycling proteins according to the modern view is the following. Upon contact with the acidic environment of the stomach denature proteins, losing tertiary structure resulting from the destruction of hydrogen bonds. This causes the unwinding of the polypeptide chain and increases the availability of protein for the action of proteolytic enzymes secreted by the glands of the stomach (pepsin). Gastric contents during digestion enters the duodenum, where it is mixed with bile and the secret of the pancreas. Contained in pancreatic secretion of proteolytic enzymes (trypsin and other) hydrolyzing the peptide bond with the formation of oligopeptides and amino acids. In h is absorbed in the intestine. It is possible that some hydrolytic processes (for example, in the case of dipeptides) fully completed in the intestinal wall. [R. Murray, D. Granner, P. Maes, W. Rodwell, Biochemistry, human, Moscow, Mir, 1993, S. 284-297]. However, even with the introduction of polypeptides directly into the small intestine (bypassing the stomach) about 75% of the drugs are destroyed by the action of proteases in the first half hour and the flow gets only 0.5% of the administered amount [Taki Y., Yamashita, S., Sakane T., Nadai So, Sezaki H., Langgath P., Amidon G. L. Gastrointestinal absorption of metkephamid. Quantitative evaluation of degradation and permeation. //Proceed. Intern.Symp.Control.Rel.Bioac.Mater. Nice. : Control.Rel.Soc.Inc. 1994. V. 21. P. 814-815].

Consequently, to eliminate the deficit in the body of any of the polypeptide it is introduced into the body in the form of injectable solution, for example, intramuscularly or intravenously, bypassing the digestive tract.

The task of creating polypeptide preparations for oral administration is to ensure a safe travel path of the polypeptide through the stomach and prevent the destruction of the polypeptide in the small intestine, where the absorption of the drug in the blood.

Known polypeptide composition comprising (wt.%) 0,001 -0,5 polypeptide, which is used as insulin, 1,0-30,0 EN N 2066551, MCI AND 61 TO 38/28, BI N 29, 1996]. Oral administration of this composition reduces the level of glucose in the blood of rabbits to 53% from baseline at the dose of insulin 10 units/kg

The disadvantages of this arrangement are the low efficiency, the impossibility of accurate dosing of the drug and low stability in storage (the biological activity of the drug is reduced by 70% when stored for 50 days at room temperature).

Known polypeptide composition comprising (wt.%) 0.01 to 0.4 polypeptide (insulin), 1-20 crosslinked hydrophilic polymer, a modified inhibitor of proteolytic enzymes, 2-50 sodium carboxymethylcellulose, 1-5 sodium lauryl sulphate, 0.05 to 1 calcium stearate and microcrystalline cellulose (100%) [Russian Federation Patent RU N 2117488, MCI AND 61 TO 38/28, BI N 23, 1998]. To prevent the destruction of the polypeptide in the stomach, i.e. with the purpose of the oral administration of the composition, its cover gastro-resistant shell, which does not dissolve in the stomach but dissolves in the small intestine with the release composition and containing polypeptide.

The disadvantage of this arrangement is the difficulty of its application (neo the concentration of glucose in the blood after oral administration of the composition is 47% from baseline.

The closest in technical essence and the achieved result to the claimed solution is a polypeptide composition comprising the polypeptide and inhibitor of proteolytic enzymes in the following ratio of components (wt.%):

The polypeptide - of 14.28

Inhibitor of proteolytic enzymes - 85,72

[M Kidron, N. Bar-On, E. M. Berry, E. Ziv, The absorption of insulin from various regions of the rat intestine, Life Science, 1982, V. 31, p. 2837-2841].

As the polypeptide composition comprises insulin, and as an inhibitor of proteolytic enzymes - trypsin inhibitor from soybeans. The composition is applied by dissolving in physiological solution (0.9% sodium chloride solution) to achieve a concentration of the polypeptide of 0.05 wt.% (5 mg in 10 ml) followed by the introduction of 1 ml of a solution directly into the small intestine of the animal.

The disadvantage of this polypeptide composition is the inability to use oral route of administration of the composition (of its type in the small intestine directly) and its low efficiency. So, with the introduction of the composition containing 12 units of insulin directly into the small intestine of rats, the concentration of glucose in the blood is reduced to 69 5.0 and 85 8.1% of the original after 1 and 2 hours after administration sootvetsvenno the introduction of the polypeptide with the effectiveness of drug action close to the efficiency of the polypeptide, introduced by injection.

The solution of the set goal is achieved by the fact that the polypeptide composition comprising the polypeptide and inhibitor of proteolytic enzymes, additionally contains an organic acid and an inert filler in the following ratio of components (wt.%):

Polypeptide - 1,0 - 95,0,

Inhibitor of proteolytic enzymes - 0,02 - 10,0,

Organic acid - 1,0 - 94,0,

The inert filler is 0.2 - 98,8;

As the polypeptide composition comprises a polypeptide with a molecular mass of 1000-21000, as an inhibitor of proteolytic enzymes - trypsin inhibitor from soybean, pancreatic trypsin inhibitor, ovomucoid of protein duck eggs as organic acids citric, ascorbic, salicylic acid and so on, and as the inert filler is lactose, cellulose and its derivatives, starch, sucrose, polyvinylpyrrolidone, gelatin, polyethylene glycol, sorbitol, etc.

The composition is used by dissolving in water or saline solution to achieve a concentration of polypeptide 0,000004 - of 0.015 wt.% (0,004 -15 mg per 100 ml) followed by oral administration of a solution in an amount necessary to achieve the desired physiological E. the local connection, accelerating the process of absorption in the amount of 10 to 50 times greater than the number of polypeptide.

A known solution for oral administration of polypeptides containing polypeptide, water and at least two connections, accelerating the absorption of the polypeptide, in quantities of 1-10 wt.% [WO 96/36352, PCT/CA/00305, A 61 K 38/28, 1996]. This solution may also contain a protective polymer, antioxidant, inhibitor of proteolytic enzymes and inorganic salt.

The disadvantage of this solution is the impossibility of direct use for oral administration polypeptide drug. The composition of the solution does not protect the contained polypeptide from the ravages of the acidic environment of the stomach. Therefore, before orally administered solution of an animal injected with sodium bicarbonate for the neutralization of the acid of the stomach. Although physiological effect known solution is high enough (for example, oral administration to rats with diabetes of 10 units of insulin in the composition of the known solution containing 0.02% insulin, leads to a decrease in the concentration of glucose in the blood of animals to 70-80% after 2 hours after injection), the need to fully neutralize the acidic environment of the stomach primenenie aqueous solution of the polypeptide (insulin) for its oral administration [M Saffran, W. Pansky, G. C. Budd, F. E. Williams, Insulin and the gastrointestinal tract, Journal of Controlled release, 1997, V. 46, 89-98] . When using such a solution with a concentration of insulin of about 0.4 wt.% (up to 400 mg per 100 ml) instead of drinking water, the concentration of glucose in the blood of rats was reduced by 40-60%.

The disadvantage of this solution is the need to use huge amounts of insulin, which lowers the digestion process. Animals lose weight, and in their digestive tract find a large amount of undigested food.

Example 1. The composition contains 15 mg of insulin (3.0 wt.%), 0.1 mg (0.02 wt. %) pancreatic trypsin inhibitor, 470 mg (94 wt.%) citric acid and 14.9 mg (2.98 wt.%) the lactose. The molecular weight of insulin 6500. The composition is dissolved in 100 ml of physiological solution (concentration of insulin is 0,015 wt.%) and 6.7 ml of the obtained solution (25 units of insulin) orally administered to the rabbit. Blood samples are taken before the test and after 30, 60, 90, 120 and 150 minutes after administration of the drug. The results are presented in table. 1.

Example 2. The composition contains 10 mg of insulin (2.0 wt.%), 20 mg (4.0 wt. %) ovomucoid of protein duck eggs, 469 mg (93,8 wt.%) ascorbic acid and 1.0 mg (0.2 wt.%) polyvinylpyrrolidone. Composition Rast is Diniz insulin) orally administered to the rabbit. The results are presented in table. 1.

Example 3. The composition contains 10 mg of insulin (2.0 wt.%), 0.1 mg (0.02 wt. %) of trypsin inhibitor from soybean, 1.0 mg (0.2 wt.%) citric acid and 488,9 mg (of 97.78 wt.%) sodium carboxymethylcellulose. The composition is dissolved in 100 ml of physiological solution (concentration of insulin is equal to 0.01 wt.%) and 10 ml of the obtained solution (25 units of insulin) orally administered to the rabbit. The results are presented in table. 1.

Example 4. The composition comprises 10,98 mg of insulin (42,23 wt.%), 0.01 mg (0,04 wt. %) ovomucoid of protein duck eggs, 15 mg (57,69 wt.%) citric acid and 0.01 mg (0,04 wt.%) polyvinylpyrrolidone. The composition is dissolved in 100 ml of physiological solution (concentration of insulin is equal to 0.011 wt.%) and 9.1 ml of the obtained solution (25 units of insulin) orally administered to the rabbit. The results are presented in table. 1.

Example 5. The composition comprises of 14.5 mg of insulin (15.3 wt.%), 0.2 mg (0.3 wt.%) ovomucoid of protein duck eggs, 40 mg (42,2 wt.%) citric acid and 40 mg (42,2 wt.%) the lactose. The composition is dissolved in 100 ml of physiological solution (concentration of insulin is equal to 0,0145 wt.%) and 6.9 ml of the obtained solution (25 units of insulin) orally administered to the rabbit. The results are presented in table. 1.

Example 6 (cons.%) citric acid and 0.04 mg (0.2 wt.%) the lactose. The composition is dissolved in 100 ml of physiological solution (concentration of insulin is equal to 0,0205 wt.%) and 4.9 ml of the obtained solution (25 units of insulin) orally administered to the rabbit. The results are presented in table. 1.

From table. 1 shows that the increase in the concentration of the polypeptide in the composition of the above stated limits leads to a sharp decrease in the effectiveness of the drug. Thus, oral administration of 1 mg of insulin in the solution compositions of the claimed composition leads to a decrease in the concentration of glucose in the blood of rabbits by 40-60%. Oral administration of the same amount of insulin (1 mg) in a solution of a composition containing insulin higher than the stated limits, does not change the concentration of glucose in the blood of animals. This suggests that in the latter case, insulin is not secreted into the blood, and probably either destroyed in the digestive system, or is not absorbed in these conditions from the intestinal lumen.

Example 7. Carried out as in example 1, but as an experimental animal use white rat. The results are presented in table. 1.

Example 8. Conducted according to example 2, but as an experimental animal use white rat. The results are presented in table. 1.

Example 9 (cDNA, as in the case of the use of animals of other species (rats instead of rabbits) the excess content of insulin above stated limits leads to the complete disappearance of its physiological action.

Example 10 (control). White rats are placed in a cell canisters for collection of urine and 120 minutes of record urine output in natural urination. Without the introduction of water load rats emit 120 minutes 0,40,04 ml of urine per 100 g of body weight. 15 rats in the morning before feeding introducing the probe into the stomach 5 ml of water per 100 g of body weight. These rats for 120 minutes to produce 4,420,19 ml of urine per 100 g of body weight.

Example 11. The composition contains 0.004 mg (95 wt.%) desmopressina - polypeptide (molecular weight 1069), less urination, 0,000042 mg (1 wt.%) ovomucoid of protein duck eggs, 0,000042 mg (1 wt.%) citric acid and 0,00013 (3 wt.%) sucrose.

The composition is dissolved in 100 ml of water (concentration desmopressina equal 0,000004 wt.%) and this solution is injected probe into the stomach of 11 rats at the rate of 5 ml per 100 g body weight. Within 120 minutes urination in rats is 1,110,41 ml of urine per 100 g of body weight, i.e. it decreases 4 times after administration of the composition.

Example 12. Composol acid and 0,00086 (2 wt.%) sucrose.

The composition is dissolved in 100 ml of water (concentration desmopressina equal 0,00004 wt. %) and the test of 9 rats in example 7. Within 120 minutes urination in rats completely suppressed.

Example 13. The composition contains 10 mg of glucagon (80 wt%), 1.25 mg of trypsin inhibitor from soybean (10 wt%), 0.2 mg of citric acid (1.6 wt.%) and 1.05 mg polyvinylpyrrolidone (8.4 wt.%). The molecular mass of glucagon is equal to 3500. The composition is dissolved in 100 ml of water (concentration of glucagon is 0.01 wt. %) and 10 ml orally administered to the rabbit. The concentration of glucose in the blood is measured after 5, 10, 20, 30 and 60 minutes. The results are shown in table. 2.

Example 14 (control). The composition contains 100 mg of glucagon (98 wt. %), of 1.02 mg of trypsin inhibitor from soybean (1 wt.%), 0,51 mg citric acid (0.5 wt. %) and 0.51 mg polyvinylpyrrolidone (0.5 wt%). The composition is dissolved in 100 ml of water (concentration of glucagon 0.1 wt.%) and 1 ml orally administered to the rabbit. The results are shown in table. 2.

Shows that oral administration of a solution of the claimed composition provides a manifestation of glucagon its physiological effect - the increased concentration of glucose in the blood. Thus, the introduction of 1 mg of glucagon leads to increased concentration gtid not accompanied by changes in glucose concentration in the blood of animals.

Example 15. The composition comprises 15,0 mg (80 wt.%) growth hormone (a protein with molecular weight of 21000), 1,58 mg (8.4 wt.%) ovomucoid of protein duck eggs, 1.3 mg (6.9 wt.%) ascorbic acid and 0.88 mg (4.7 wt.%) polyvinylpyrrolidone. The composition is dissolved in 100 ml of physiological solution (concentration of growth hormone is 0,015 wt.%) and 6.7 ml of the resulting solution (1 mg hormone) orally administered to the rabbit. The results are presented in table. 3.

Example 16. The composition comprises 15,0 mg (80 wt.%) growth hormone was 1.58 mg (8.4 wt.%) pancreatic trypsin inhibitor, 0,88 mg (4.7 wt.%) citric acid and 1.3 mg (6.9 wt.%) the lactose. The composition is dissolved in 100 ml of physiological solution (concentration of growth hormone is 0,015 wt.%) and 6.7 ml of the resulting solution (1 mg hormone) orally administered to the rabbit. The results are presented in table. 3.

Example 17 (control). The composition contains 100 mg (98 wt.%) growth hormone 0,51 mg (0.5 wt.%) ovomucoid of protein duck eggs, 0,51 mg (0.5 wt. %) of ascorbic acid and of 1.02 mg (1.0 wt.%) polyvinylpyrrolidone. The composition is dissolved in 100 ml of physiological solution (concentration of growth hormone is equal to 0.1 wt.%) and 1.0 ml of the resulting solution (1 mg hormone) orally administered to the rabbit. The results are presented in table. 3.

Example 18. The composition of example 1 optionally contains 150 mg of sodium lauryl sulphate. The test results of this composition are shown in table. 4.

Example 19. The composition of example 1 optionally contains 750 mg of sodium lauryl sulphate. The test results of this composition are shown in table. 4.

It is seen that the introduction of the compositions of known compounds that accelerate the process of absorption in the small intestine, increases the efficiency of action of drugs.

Thus, the claimed composition provides pronikshie. Thus polypeptides penetrate into the bloodstream in the native state, which ensures the manifestation of their inherent physiological activity.

1. Polypeptide composition for oral administration containing the hormone polypeptide of nature and the inhibitor proteoliticheskikh enzymes, characterized in that it further comprises a water-soluble organic acid and a water soluble inert filler in the following ratio of components, wt.%:

Hormone polypeptide of nature with a molecular mass of 1000-21000 - 1,0 - 95,0

Inhibitor of proteolytic enzymes - 0,02 - 10,0

Water-soluble organic acid is 1.0 - 94,0

Water soluble inert filler is 0.2 - 98,8 (rest)

2. The composition according to p. 1, characterized in that it additionally contains compounds that accelerate the absorption process, in the amount of 10 to 50 times greater than the number of polypeptide.

 

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