Peptidase the drug and its production method

 

The invention relates to medicine, namely to the development of new dosage forms of the biologically active peptide compounds. The drug contains the active principle-peptide and protective environment consisting of sucrose, poliglyukina, cocoa powder, gelatin and ascorbic acid at the following ratio, wt. % dry substance: gelatin 16-20, sucrose 46-52, poliglyukin 6,0-10,0, ascorbic acid 2,5-3,5, cocoa powder 6,0-8,0, biologically active peptide - rest. As the peptide can be used interferon, insulin, interleukin. The method of obtaining the drug consists of mixing the peptide with a mixture of sucrose, poliglyukina, cocoa powder, gelatin and ascorbic acid, placing the mixture in a matrix and pelletizing by freeze drying at a temperature of from -25 to -35oAnd pressure of 8-15 PA for at least 48 hours proposed technology ensures lasting activity of the drug during storage and high efficiency for therapeutic use. 2 S. and 3 C.p. f-crystals, 2 tab. , 2 Il.

The invention relates to the field of biotechnology, and in particular to methods of obtaining new forms of biologically active peptide compounds of Tralach.

Currently, the majority of peptide drugs, such as interferon, interleukins, insulin, produced in vials containing solution or a dry product intended for intravenous or intramuscular injection. However, this form of introduction is awkward in everyday practice when prescribing long-term courses of treatment that raises the question of the need to develop other forms of drug use, in particular, the use of aerosols, suppositories or tablets containing MAPS.

So, there are some preparations for rectal administration, containing a mixture of interferon and antioxidant - alpha-tocopherol acetate (U.S. Pat. RF 2057544, 1996, class a 61 K 38/21), aerosols, containing a mixture of BAPS, dextrans and buffer solutions (U.S. Pat. RF 2095081, 1997, class a 61 K 38/21), and so on,

The most convenient form of application are tablets for oral administration, however, in the case of use as an active start BAPS, the latter, as a rule, lose a significant portion of activity during tabletting and storage, which limits the range of application of BAPS.

One of the most promising forms of introduction of peptide drugs is sublingual introduction, during which they are absorbed into the blood naibu currently have not been clearly established.

Currently for dry biologics in the form of tablets are mainly used methods of compaction due to high productivity and relatively low cost (Technology of medicinal forms./Ed. by L. A. Ivanova, M., Medicine, so 2, S. 134). The process includes obtaining current bioagent, its sublimation dehydration and grinding, mixing the active agent with excipients (fillers, disintegrating agents, binders, and so on), hydration and granulating the resulting mixture and compressing the tablets under pressure 25-250 MPa.

The disadvantages of this method include its multi-stage, as well as a significant inactivation of the active agent.

The prototype of the proposed product and method of its production technology is getting dry preparation on the basis of thymopentin (U.S. patent 5036049, 1990 , CL And 61 To 37/02). The drug is produced by mixing solutions thymopentin with protective environment consisting of carbohydrates (lactose, sucrose, raffinose) and amino acids (glycine, lysine, aspartic acid, followed by freeze-drying the mixture in capsules and their subsequent sealing. Obtained after freeze-drying biological product had access to the With without a noticeable decrease in their initial biological characteristics.

Lack of preparation and the production technology is the need for preliminary rehydration preparation before use, inability storage of the drug in the presence of air without significant loss of activity, inability to use and storage of the drug in tablet form.

The problem to be solved by the authors was to develop a tablet containing peptides as active principle, providing stability MAPS during storage and the production technology, which can be used sublingual.

Technical solution to this problem was found on the basis of obtaining tablets with modified trituration method used to get nitroglycerin tablets, etc. especially dangerous chemicals.

The essence triturating way of getting of pills is to obtain fine powders of the active agent and excipients, for example lactose or glucose, mixing them with solutions of ethanol to education plastic mass, which is then rubbed into the matrix and dried in air (Technology of medicinal forms./Ed. by L. A. Ivanova, M., Medicine, so 2, S. 291).

The method is effective for nitroglycerin and t is unestami obtain fine powders while maintaining the activity of the biological agent, and relatively low productivity. Additionally, the tablets of this type can have only limited application due to insufficient strength and high hydroscopicity because of their high porosity.

The method allows the cooking process is finished dosage forms in the most sustainable manner, preserving the biological activity of its constituent ingredients.

However, this property of tablets provides fast absorption BAS cells and allows you to use the tablet in a different way, in particular the introduction of biologically active substances in the body sublingually.

However, when included in the composition of the tablets peptides have to simultaneously solve several problems: - to maintain the porosity of the tablets and the possibility of sublingual application, which virtually eliminates the possibility of using a protective sheath or capsule; - to preserve the activity of the peptides during storage in terms of their active contact with oxygen and moisture in the external environment.

To overcome this problem was developed formulation of a biological product on the basis of BAPS ensuring the stability of the spatial configuration of the peptide and thereby snii cocoa powder when introduced into a mixture of gelatin and sucrose to create a viscous environment spatial mesh polyfunctional structure, the nodes of which, apparently, are bipolar molecules peptides are additionally protected from exposure to adverse environmental factors included in the protective shell of the antioxidant ascorbic acid.

This structure allows relatively easy dehydration of the mixture, which allowed us to use the technology of freeze drying.

The resulting product has the following composition of the final product, wt.%: Gelatin - 16-20 Sucrose - 46-52 Poliglyukin - 6,0-10,0 Ascorbic acid 2,5-3,5 cocoa Powder - 6,0-8,0 Biologically active peptide - rest as peptides it contains interferon, interleukins, insulin, etc. connections.

The introduction of lower concentrations of the antioxidant does not guarantee the complete protection of the peptide from the action of oxygen, administration of high concentrations of economically impractical.

When the concentrations poliglyukina, gelatin, sucrose and cocoa powder for the claimed parameters violated the internal structure of the drug and either does not form a tablet or drying significantly increased inactivation of BAPS.

The peculiarity of the technology used for obtaining tablets is
introduction in a mixture of BAPS with sugar the matrix biomaterial method of freeze-drying at a temperature of from -25 to -35oAnd pressure of 8-15 PA for at least 48 hours. The resulting freeze-dehydration dry biological product (tablets) are extracted from the matrix and subjected to biological testing.

Tablets were homogeneous throughout the volume of the structure and a light-brown color. The diameter of the tablets - 19.5 mm, height - 6.6 mm, weight - 0.27, If dropped on a hard, horizontal surface from a height of 1 m tablets is not mechanically damaged, while fully retaining its original shape. Out of line tablets had a sweet taste with a touch of cocoa. Complete dissolution of the tablets in water is 30 seconds.

During this task, managed to get a dry tablet having high porosity, providing for its dissolution in the oral cavity of the person for not more than 30 seconds with subsequent absorption of the beginning of the current through the oral mucosa, which allows you to use it as a sublingual drug.

Sublingual method of using a drug excludes the possibility of its destruction by digestive enzymes, as a complete dissolution and absorption components of the tablet occurs through the mucous membrane of the oral cavity. P. the biological activity of the claimed us tablets sublingual preparations showed during the time of contact of the prisoner in dry tablet acting early inactivation of the drug were found.

The structure of the new tablets is illustrated by the electron micrograph of the cross-section of the edge of the tablet obtained by scanning the surface of the fault (Fig. 1). Tablet contains protective components described in example 1, description, and interferon concentration of 3 million ME on the pill.

As can be seen from the electron micrograph, the drug is characterized by a high degree of porosity, and the main pores are aimed not only vertically (from the substrate to the surface of the tablet), but also horizontally (from the periphery of the tablet to the center). The emergence of the tablet horizontal porosity, due to its composition and production technology and allows, in the radial shrinkage of the drug, extract, tablet, formed in the drying process of biological solutions simple vytryahivanie. The possibility of such extraction arises due to the fact that when the radial shrinkage of the tablet between its outer side surfaces and the inner surface forms occurs clearance for a smooth output pill form.

Avon used excipients and technology of reception of tablets. The nature of the used peptide active principle in this case is secondary, although it provides achieved after absorption of biologically active substances of medicinal effect. Achieved in the use of tablets effects are being studied in medical centers in S. Petersburg. Along with achievable with the use of insulin effects on blood sugar and improving the immune status of drug use with interleukin, there has been a great influence sublingual use of interferon in the treatment of hepatitis C, exceeding the level of use interferonsource drugs introduced into the body by other means, primarily by injection. The latest results were obtained by the Department of infectious diseases, S. Petersburg medical Academy of postgraduate education for comparative testing of conventional drugs, administered injectable and declare tablets, administered sublingually in the treatment of patients with viral hepatitis C.

The evaluation was carried out in a pilot study on human volunteers, patients with viral hepatitis C. Before the beginning of the study all patients had received the random numbers were divided into two groups: the subjects (group 1) and control (group 2). Group 1 consisted of 19 persons, in group 2 - 21 people. In the study group patients received the drug, and in the control and placebo.

The effectiveness of this form of the drug and the method of application was evaluated by the following indicators: activity of Alat, the frequency of positive reactions PCR in serum and the level of endogenous interferon was determined by induced activity). The course of IFN therapy was 4 months of daily use of the drug.

In its original records both groups had no differences. So the activity of Alat before the start of the study in group 1 was equal to 120 u/lthe 5.25 u/l (N=40 u/l), in the group 2-115 u/l4,5 u/l, reaction PCR in group 1 was positive in 19 persons, in group 2 - 21 indicators induced activity of IFN-in the 1st group was equal to 10of 0.5 PG/ml and 12.5of 0.5 PG/ml, respectively, in the 2nd group. The results of the study showed good tolerance of the drug, no side reactions that occur after subcutaneous administration of IFN. The activity of Alat after a course of therapy in patients of the 1st group was 452,5 u/l, in group 2 - 51of 0.5 PG/ml to 20of 1.2 PG/ml, and group 2 - from 12.5of 0.5 PG/ml to 14.51 PG/ml

. As the equipment can be used sublimation installation chamber type firms "Hoch Vacuum" (Germany), VIR-Tis (USA ) and similar equipment.

The obtained dry biological product can be stored for several months at a temperature of +4 - +40oWith almost no loss of activity.

The essence of the proposed technology is illustrated by the following examples.

Example 1. The substances containing the human interferon activity from 0.9108ME/mg protein was added to the protective environment of the following composition ( wt.% calculated on dry substance): gelatin - 16, saccharose - 50, poliglyukin - 8, ascorbic acid 2,5, cocoa powder - 8. The ratio of amounts of substance of biological testing on cells L-68, infected with vesicular stomatitis virus and after checking activity was poured into sterile aluminum forms with a diameter of 20 mm and a height of 7 mm Dosing is carried out using the dispenser 2 see cubic in every form of error5%.

Then shape with spilled material in them were placed in pre-cooled to minus 25oFrom the shelf of the freeze camera installation TG-5 (Germany) and conducted preliminary freezing of the material to a temperature of -252oWith the vacuum sublimation chamber to a pressure of 10 PA and freeze dehydration of the drug interferon.

After the material the final moisture content of 3.2% in the chamber was injected argon until the normal pressure carried out the unloading of the finished tablets interferon.

The obtained tablets were plunged repeated testing (result - 0.7106IU/mg protein) and was placed in storage. When the initial activity of interferon in the tablet 0.7106when stored for 12 months at +4oSince she was 0.8105and during storage for 3 months at +40oWith - 0.6105.

the tree was conducted tableting of interferon with the introduction of the original substance of the protective environment, containing the following concentrations of substances-protectors ( wt.% calculated on dry substance): gelatin - 20, sucrose - 46, poliglyukin - 7, ascorbic acid 3.5, cocoa powder - 8.

In the sublimation tabletting interferon activity tablets - 3.0107IU/mg protein; storage 12 months at + 4oSince she was 3.0107IU/mg protein, and the storage for 3 months at + 40oWith -3.0107IU/mg protein, i.e., preservation of protein - 100%
Example 3. In the conditions of example 1 with the activity of the parent interferon 0.8104IU/mg of protein was carried out tableting of interferon with the introduction of the original substance protective environment containing the following concentrations of substances-protectors ( wt.% calculated on dry substance): gelatin - 18, sucrose - 46, poliglyukin - 10, ascorbic acid - 3.0, cocoa powder - 7.5.

In the sublimation tabletting interferon activity tablets - 0.8104IU/mg protein; storage 12 months at +4oSince she was 0.8104IU/mg protein, and the storage for 3 months at +40oWith - 0.6

The resulting solution was tested for biological activity in animals to reduce the level of glucose in the blood and the content of conformationally active insulin enzyme-linked immunosorbent assay (ELISA) according to the standard procedure described in "Clinical biochemistry", Minsk, 1976, S. 117-120.

After checking the source of the biological characteristics of the solution is frozen and freeze-dried in tablet form according to the technology described in examples 1-3. The results are given in table 1.

Example 5. To the substance of interleukin-8 from the calculation of 0.3 mg per pill add a protective environment, containing in wt.% calculated on the dry product: 50 sucrose, 18 gelatin 8 poliglyukina, 2.8 ascorbic acid at the ratio between the amount of substance of interleukin-8 and protective environment is 1:9.

The resulting solution was tested for biological activity by the method of degranulation of leukocytes in human blood and the content of conformationally active interleukin enzyme-linked immunosorbent assay (ELISA).

After verification IP is technology, described in example 1. The results are given in table 2.

From the above examples that the proposed technology allows to obtain the pill, stable during storage in a wide range of temperatures.


Claims

1. Peptidase preparation comprising the active agent is a peptide and a protective environment containing carbohydrates, characterized in that the carbohydrate it contains sucrose, and the protective environment impose additional poliglyukin, cocoa powder, gelatin and ascorbic acid in the following ratio of ingredients, wt. % dry matter:
Gelatin - 16 - 20
Sucrose - 46 - 52
Poliglyukin - 6,0 - 10,0
Ascorbic acid is 2.5 - 3.5
Cocoa powder - 6,0 - 8,0
Biologically active peptide - Rest
2. Peptidase drug under item 1, characterized in that it contains peptide interferon.

3. Peptidase drug under item 1, characterized in that it contains peptide insulin.

4. Peptidase drug under item 1, characterized in that it contains peptide interleukin.

5. The method of obtaining peptidases preparation, including the preparation of the active agent, its mixture of cocoa powder, gelatin and ascorbic acid, after which the resulting mixture is placed in a matrix and tabletirujut, subjecting to freeze-drying at a temperature of from -25 to -35oAnd pressure of 8-15 PA for at least 48 hours

 

Same patents:

The invention relates to the use of certain salts monoalkyl esters of fumaric acid individually or in combination with dialkylamino to obtain microtablets for the treatment of psoriatic arthritis, neurodermatitis, psoriasis and regional enteritis (Crohn's disease)
Psychotropic drug // 2210360
The invention relates to medicine, namely the creation of medicines used in psychiatric and neurological practice for the treatment of neurotic and neurosis-like States, as well as chronic alcoholism

The invention relates to pharmaceutical preparations 8-chloro-6,11-dihydro-11-(4-piperidylidene)-5H-benzo[5,6] cyclohepta[1,2-b] pyridine, known as descarboethoxyloratadine (DCL)
The invention relates to medicine, more precisely to the pharmacy, and can be used for the preparation of solid pharmaceutical compositions, the active compound which is enalapril maleate

The invention relates to the field of pharmacology, and relates to a composition containing diphosphonic acid
The invention relates to medicine, more precisely to the pharmacy, and can be used for the preparation of solid pharmaceutical compositions, the active compound which is Asparaginate

The invention relates to medicine, more specifically to pharmaceutical compositions for the treatment of diseases of the pancreas and digestive disorders associated with liver disease

The invention relates to the medical industry and relates to a derivative of insulin or its physiologically acceptable salt with accelerated compared with human insulin onset of action, in which the asparagine (Asn) at position B3 chain replaced by natural basic amino acid residue and at least one amino acid residue in positions B27, B28 or W chain replaced by other natural neutral or acidic amino acid residue, and asparagine (Asn) at position A21 chain And may be substituted for Asp, Gly, Ser, Thr or Ala, and the phenylalanine (Phe) in position B1 chain and the amino acid residue in position B30 chain may be missing

The invention relates to the field of medicine and refers to fragments of C-peptide human proinsulin

The invention relates to a method for producing the precursor of insulin or insulin derivatives with properly connected cystine bridges in the presence of cysteine or testingground and chaotropes excipients, characterized in that the following steps performed in sequence: (a) mixing the aqueous slurry of the precursor of insulin or insulin derivatives with a number of cysteine or testingground, which gives up to 15 SH-cysteine residues or testingground one cysteine residue of the precursor, (b) the introduction of cysteine or containsignorecase suspension predecessor in 4-9-molar solution chaotropes excipients when the pH value of 8 to 11.5 and a temperature of 15 - 55oWith, keeping the mixture for 10-60 min at this temperature and (b) introducing the mixture at a pH value of 8 to 11.5 and a temperature of 5 to 30oWith the amount of water, which gives a dilution of the concentration of cysteine or testingground in a mixture of 1-5 mm and chaotropes excipients 0.2-1.0 M

The invention relates to medicine

The invention relates to medicine and the medical industry and for the production of preparations of rapid-acting insulin
The invention relates to medicine, in particular to endocrinology and sexologist, and can be used for the correction of erectile dysfunction in men with diabetes

The invention relates to zinc-containing crystals of insulin, which has a diameter less than 10 microns, suitable for introduction through the lungs

The invention relates to the field of medicine may be used for the treatment of late diabetic complications, namely diabetic polyneuropathy
The invention relates to medicine, in particular to obstetrics

The invention relates to medicine, namely to Phthisiology, and can be used for treatment of tuberculosis predominantly productive type of tissue reaction
Up!