Insulinaemia drug for oral administration and method thereof
(57) Abstract:The invention relates to medicine and concerns insulinosoderzhaschego medicines for oral use and method of its production. Being insulinaemia drug representing insulin immobilized on erythrocytes of fresh blood in the presence of a crosslinking agent at a ratio of 5-10:100 1250 content - 2000 E insulin per 1 g of dry mass. The method includes receiving a selection of erythrocytes from the blood of mammals under the influence of centrifugal forces, incubation of insulin with erythrocytes in the presence of a crosslinking agent when the pendulum swing of the mixture, subsequent washing of immobilized insulin saline solution in a few cycles when exposed to the slurry in each cycle by centrifugal forces, stabiliser and lyophilization. The advantage of the invention is to improve the content of insulin in oral dosage form. 2 C. and 10 C.p. f-crystals, 4 PL. The invention relates to the field of medicine and concerns insulinosoderzhaschego medicines for oral administration and how to obtain it.Diabetes mellitus is one of the most spread of the gastric glands - the insulin.Insulin is a polypeptide hormone with a molecular weight of 6000. It affects all types of metabolism in the body: it increases the penetration of glucose in the tissues of the body, promotes recycling, reduces the content of glycogen in the liver and increases its number in the muscles, increases the intensity of protein synthesis and slows the decay of the latter.The main method of administering insulin in the human body are subcutaneous or intramuscular injection. Attempts to introduce insulin is the most physiological and convenient for patients by oral proved unsuccessful, because the insulin is easily degraded under the action of digestive enzymes, which leads to loss of its biological activity.The main obstacle encountered on the road of oral form of insulin is its low resistance to the action of proteolytic enzymes of the gastrointestinal tract.In recent decades there have been many attempts to create an oral form of insulin, but still failed to create an effective drug that can compete on its activity with insulin, administered Inno-oil microemulsion, consisting of insulin, lipids and proteases inhibitor. The microemulsion is covered then the carboxymethyl cellulose  .A significant drawback of this drug, along with time-consuming and expensive manufacturing technology, is a medium - carboxymethylcellulose, which is exposed to microorganisms, and can absorb large quantities of insulin, resulting in a form does not meet the requirements of effective oral insulin product.Known solid insulinaemia medicinal product consisting of a nucleus containing insulin and an excipient, and a shell of biodegradable film-forming polymeric material .The tool is manufactured by introducing into hard gelatin capsule or a tablet of 1-40 mg of crystalline insulin and 200 mg stoichiometric mixture of 5-methoxysalicylaldehyde acid and sodium bicarbonate. Then the capsule (pill) is covered with a copolymer of hydroxyethylmethacrylate and styrene, crosslinked divinylbenzene. The shell is resistant to the environment of stomach and small intestine, but is broken down in the large intestine under the action of the present microorganisms. effect. Oral administration to rats of the specified products containing 1 unit of insulin leads to a decrease in the concentration of glucose in the blood by 20% after 9 hours after administration. At the same time, a subcutaneous injection of a solution of insulin in a dose of 0.1 or 1.0 unit reduces the level of glucose in the blood at 39 and 63%, respectively. The maximum hypoglycemic effect of individual animals is achieved in the period from 1 to 9 hours, and some animals have the effect of reducing the concentration of glucose is absent and 10 hours after administration of the drug.Known solid insulinaemia medicinal product consisting of a nucleus containing insulin, an inhibitor of proteolytic enzymes and auxiliary substances, and the gastro-resistant membranes [3 and 4].As an inhibitor of proteolytic enzymes medicine contains trypsin inhibitor from soy, as well as auxiliary substances - Holt sodium and lactose. Lactose is used as an inactive filler, and Holt sodium as compounds that increase the permeability of insulin across the intestinal wall.The disadvantage of this tool is the low efficiency. So, by oral administration of funds the wee animal is 18%, while using subcutaneous injections similar hypoglycemic effect can be achieved when the insulin dose is 10 times smaller. In addition, the medium containing trypsin inhibitor from soybeans, has a selective effect against various species of animals, in other words is not universal. Thus, the oral application it shows activity against dogs and not active against rats .Known insulinaemia medicinal product intended for the treatment of patients with diabetes through the mouth, consisting of a nucleus containing insulin, protein inhibitor of proteolytic enzymes, which represents a crosslinked hydrophilic polymer-modified ovomucoid, and auxiliary substances and gastro-resistant membranes .The product contains 10 IU of insulin per 1 tablet. The tool provides a statically reliable hypoglycemic effect on different types of mammals, including humans, i.e., has a universal character. Moreover, the dose required to achieve the desired therapeutic effects are comparable to the levels for injecting insulin. However, the tool has NEA on 1 g of dry tablets. A method of obtaining insulinaemia polymer hydrogels, including immobilization of insulin in the amount of crosslinked polymer modified inhibitor of proteolytic enzymes - ovomucoid .The method allows to obtain the tool, with activity, defined as 60 - 70% of the activity of preparation of insulin after subcutaneous administration. However, the content of insulin in 1 g of the hydrogel is low.Closest to the proposed invention is a method for insulinosoderzhaschego medicines for oral administration, comprising the incubation of insulin with erythrocytes in the ratio of 1 - 4:100 in the presence of a crosslinking agent at a final concentration of 0.15 to 0.25% .The result is a drug containing 1,000 U/1 g dry weight with a period of storage in the lyophilized form to several years.The task of the invention is to create insulinosoderzhaschego tools for oral administration, i.e., resistant to the action of proteolytic enzymes in the gastrointestinal tract with a high content of insulin in 1 g of dry matter, which expands the possibility of using funds in a variety of dosage forms.The specified tool as excipients may contain gelatin in an amount of from 1 to 2.5%.The tool includes as red blood cells by immobilization of insulin to cells isolated from fresh blood pigs, or cattle, or horses, or humans.This tool as a cross-linking agent may include glutaric dialdehyde.The method of obtaining insulinosoderzhaschego medicines for oral administration includes the allocation of erythrocytes from the blood of mammals, their incubation with insulin in a ratio, wt.%: insulin:the erythrocytes from the blood of mammals 5-10:100 when the final content of the crosslinking agent 0,05 - 0,35% for 4 - 6 hours 4 - 8oC, while in the process of separation of erythrocytes in the blood is affected by centrifugal forces value 350 - 1100 g for 15-30 minutes, and the incubation of insulin from ericr is carried out in several cycles when exposed in each cycle of the centrifugal forces of the order of 350 - 1100 g for 0.5-10 minutes.These conditions allow immobilization to improve the content of insulin in the immobilized product to 1250 - 2000 E. insulin in 1 g of dry matter.The technical result of the invention is that despite sustained hypoglycemic effect of increased activity and persistence derived medicinal forms not only in dried form, but in liquid form.The invention is implemented as follows.Example 1. Erythrocytes were isolated from fresh blood by adding 1/10 volume of 3.8% sodium citrate under the influence of centrifugal forces 400 g for 30 minutes at 4oC. the Erythrocytes were washed twice fourfold volume of 0.15 M sodium chloride solution. To 20 ml of the suspension of erythrocytes was added 10 ml of 0.1 M phosphate buffer solution pH of 6.8, containing 0.15 M sodium chloride, 50 ml of 1% solution of crystalline insulin and 1% solution of glutaric dialdehyde to a final concentration in solution of 0.05% and incubated the mixture at a pendulum swinging with a frequency of 0.5 Hz with a 6oC for 6 hours, the ratio of insulin: erythrocytes 5:100. Then the suspension was washed from unbound insulin and glutaric dialdehyde ten RA is Uchenie 5 minutes. After the last wash to the precipitate was added as a stabilizer solution of gelatin to a final concentration of 2.5%, are thoroughly mixed for 10 minutes at room temperature and freeze-dried.Received 2 g of the finished product, a powder brown with content 1250 E. insulin per 1 g of dry product.Example 2. Getting insulinosoderzhaschego drugs as in example 1, except that was added 100 mg of 1% aqueous solution of crystalline insulin, and then guitarby of dialdehyde to a final concentration of 0.35%. The ratio of insulin: erythrocytes 10:100. The mixture is incubated for 4 hours. The ratio of insulin:erythrocytes 10:100. Before lyophilization was added gelatin to a final concentration of 1%.Received a finished product containing insulin 2000 E 1 g of dry product. Before use, the drug was emulsiable in water to the required concentration.Example 3. Test insulinosoderzhaschego medicines were performed on rats with experimental diabetes induced by streptozotocin. Rats-males were injected intraperitoneally streptozotocin the rate of 120 mg/kg weight of the animal. Streptozotocin was dissolved in citrate buffer the frame means, immobilized using glutaraldehyde is dialdehyde at the rate of 15 - 20 units of insulin preparation (per animal), prepared according to example 1, and after 3 hours the animals were determined in blood glucose. Served as control animals with streptozotocin diabetes not receiving insulinosoderzhaschego medicines.As can be seen from table 1, animals with streptozotocin diabetes after 3 hours after injection insulinosoderzhaschego medicines glucose level in the blood was decreased on average by 65% compared with diabetic animals not receiving insulinosoderzhaschego medicines.Example 4. Insulinaemia drug obtained in example 1, was administered to adult mice-males weighing 20 grams through the probe in a volume of 0.2 ml of Animals received 2.0 to 2.5 units of insulin in insulinaemia medicine. The glucose content in the blood was determined by glucose oxydase method. The results are shown in table 2.The data show that insulinaemia drug when administered to mice reduces the level of glucose in the blood in an average of 55% compared to animals not receiving insulinaemia drug Trsteno means, obtained according to example 2 of the calculation of 10-15% And after 3 to 6 hours was determined glucose level. Served as control rats not receiving insulinosoderzhaschego medicines. The data are shown in table 3.As can be seen from table 3, in rats treated insulinaemia drug, after 3 and 6 hours there is a significant reduction of the glucose level in the blood, constituting 52% and 53% respectively to the original level, while the control animals the level of glucose in blood in the same time interval was not significantly changed.According to the invention insulinaemia drug can be successfully used not only in the treatment of diabetes, but also in other types of pathology, accompanied by hyperglycemia (extensive surgical wounds, thermal damage, septic conditions, hemorrhagic shock, anesthesia, and other), as well as in pathological conditions characterized by increased protein degradation and decreased its synthesis (various stages of burn disease, nephropathy, and so on).The drug is in the course of manufacture or in the finished form can be processed gelatin or any other inert compound, is to, secure any inert with respect thereto connection and also be applied in the form of a suspension, remaining at +4oC for at least 3 months.SOURCES OF INFORMATION
1. Cho Y. W., Flynm M., Lancet, 1989, # 30, p.1518 Saffran, M., Kumar, G. S.2. Savarlar C., et al., A new approach to the oral administration of insulinand other peptide drugs, Science, 1986, v.233, pp.1081-1084.3. Ehud Ziv, Miriam Kidron, Itamar Raz et al., Oral administration of insulin of solid form to nonbiabetic and diabetic dogs. Journal of Pharmaceutical Science 1994, x.83, # 6 pp.792-794.4. Kidron, M., Krausz, M., Raz I et al., The absorbtion of insulin: from the intestine in dogs, Nenside. Surfactants. Deterg. 1989, v.26, #5, pp. 352-354.5. EN N 2117488 C1, 20.08.98.6. EN N 2066551 C1, 20.09.96.7. EN N 2058788 C1, 20.04.96. 1. Insulinaemia drug for oral administration containing insulin and excipient, characterized in that it contains insulin immobilized on the erythrocytes of the blood of mammals in the presence of a crosslinking agent at a ratio, wt.%:
Insulin is a 5 - 10
Erythrocytes isolated from fresh blood of mammals - 100
and is a freeze-dried form content of 1250-2000 E. insulin per 1 g of dry matter.2. Means under item 1, characterized in that as auxiliary substances it contains gelatin.< 4. A tool according to any one of paragraphs.1 to 3, characterized in that the quality of red blood cells using red blood cells isolated from fresh blood pigs, or cattle, or horses.5. A tool according to any one of paragraphs.1 to 3, characterized in that it contains erythrocytes isolated from fresh human blood.6. A tool according to any one of paragraphs.1 to 5, characterized in that as the cross-linking agent it contains glutaric dialdehyde.7. The method of obtaining insulinosoderzhaschego medicines for oral administration, including the selection of erythrocytes from the blood of mammals, their incubation with insulin in the presence of a crosslinking agent, washing the immobilized insulin saline solution, adding a stabilizer and lyophilization, characterized in that the incubation of erythrocytes with insulin is carried out at a ratio of insulin : erythrocytes 5 to 10 : 100, and when the content of the crosslinking agent at a final concentration of 0.05 - 0.35% of 4 - 8oC for 4 to 6 hours, while in the process of separation of erythrocytes in the blood is affected by centrifugal forces value 350 - 1100 g for 15 - 30 minutes, and during the incubation of insulin with erythrocytes produce pendulum swing mix with a frequency of 0.1 - 0.5 Hz, and the author forces of the order of 350 - 1100 g for 0.5 to 10.0 min.8. The method according to p. 7, characterized in that the stabilizer is used gelatin.9. The method according to p. 8, characterized in that the stabilizer is used gelatin in the amount of 1 - 2.5 wt.%.10. The method according to any of paragraphs. 7 to 9, characterized in that the quality of red blood cells using red blood cells isolated from fresh blood pigs, or cattle, or horses.11. The method according to any of paragraphs.7 to 9, characterized in that it contains erythrocytes isolated from fresh human blood.12. The method according to any of paragraphs.7 to 11, characterized in that as the cross-linking agent it contains glutaric dialdehyde.
SUBSTANCE: method involves taking lavage fluid samples from injured bronchi in preoperative period in making fiber-optic bronchoscopy examination. Microflora colonizing bronchial mucous membrane and its sensitivity to antibiotics is determined. Therapeutic dose of appropriate antibiotic and therapeutic dose of immunomodulator agent like leykinferon is introduced in endolymphatic way 40-60 min before operation. Smears are taken from outlying bronchi in doing operation. Sputum or fluid in retained pleural cavity are taken in 1-2 days after the operation. Prophylaxis effectiveness is determined on basis of bacteriological study data. Therapeutic dose of antibiotics and leykinferon are introduced in 6-8 and 20-24 h after the operation in endolymphatic way. The preparations are introduced at the same doses in endolymphatic way making pauses depending on selected antibiotic elimination half-time once or twice a day until the drains are removed mostly during 48-72 h after operation.
EFFECT: enhanced effectiveness of antibacterial protection; high reliability of antibiotic prophylaxis.