Process for the selective acylation of proinsulin, insulin or an insulin analogue, with freeand-amino

 

(57) Abstract:

The invention relates to the acylation of proteins and can be used in chemical and medical industries. The invention is described single-stage process for the selective acylation of the amino group of proinsulin, insulin or an insulin analog in the presence of free amino groups with soluble activated complex fatty acid ester in a polar solvent at a pH of 9.5 to 11.5. The technical result - the high yield of the target product and the simplification of obtaining derivatives of insulin. 4 C.p. f-crystals, 2 tab.

This invention relates to the acylation of proteins. In particular, the invention relates to a one-step method for the selective acylation of the amino group of proinsulin, insulin or an insulin analog in the presence of free amino groups.

Acylation of amino groups is one of the most common methods used for the chemical modification of proteins. Conventional methods of acylation described in Methods of Enzymology, 25: 494-499 (1972), and they include the use of activated esters, acid chlorides or anhydrides of the acids. The use of activated esters, the hour is for free amino acids fatty acid. Lapidot et al., J. of Lipid Res. 8: 142-145 (1967). Lapidot et al. describe the production of esters of N-hydroxysuccinimide and their use to obtain the N-lauroyl-glycine, N-lauroyl-L-serine and N-lauroyl-L-glutamic acid.

Early studies of selective acylation of the amino groups of insulin are described Lindsay et al., Biochem.J. 121: 737-745 (1971). Lindsay et al., describe the reactivity of insulin with N-Succinimidyl acetate at low concentrations of the reagent and near neutral pH, with the receipt of two monosubstituted products, PheB1-acetyl-insulin and GlyA1-acetyl insulin. At pH 8.5 the amount PheB1-acetyl-insulin decreases and, in addition, it turns LysB29-acetyl-insulin. Thus, Lindsay et al. concluded that at pH 6,9 order of reactivity (the amino groups of insulin) Glycine (A1)= Phenylalanine (B1) >> Lysine (B29) and at pH 8.5 Glycine (A1) > Phenylalanine = Lysine (B29).

Lindsay et al., Pat. USA 3 869 437, reveal the acylation B1amino acid acyl group containing up to seven carbon, and optional blocking A1and/or B29- amino acyl group with up to four carbons. Esters of N-hydroxysuccinimide described as particularly useful alli the creation Alliluyeva agent is relatively low (no more than one to two molar equivalents Alliluyeva agent). In addition, the maximum output monosubstituted B1the product is obtained at a pH of 7 or about pH 7. At a pH of 8.5 to 9.0, the desired output of the B1-acylated product drops significantly in favor of additional substitution in the provisions of the A1and B29.

D. G. Smyth, in U.S. Pat. USA 3 868 356 and Smyth et al., in Pat. USA 3 868 357, discloses N-acylated, O-substituted derivatives of insulin, in which at least one of the A1B1or B29amino acid amino groups in turn blocked amino group. The acylation is carried out at a relatively small excess Alliluyeva agent, for example from 2 to 3 mol of the amino group at neutral or moderately alkaline pH, for example 7,8. The reaction proceeds with very high yield with the formation of the disubstituted derivative, resulting in the interaction of A1and B1-amino groups. In the presence of excess Alliluyeva agent, for example, up to 10 molar, the reaction proceeds further at B29the amino groups with the formation trisemester derived.

To selectively allievate insulin, Muranishi and Kiso, in the patent application of Japan 1-254 699, open platitudinal synthesis of obtaining derivatives of insulin fatty acids. Stage one get the carbonyl azide (pMZ); stage three insulin-pMZ is subjected to interaction with a complex ester of fatty acid; at stage four acylated insulin is removed protection; and stage five acylated insulin is isolated and purified. The most remarkable thing is that the selective acylation of the amino group can be achieved only when using pMZ blocking group, which protects the other amino group. Using this methodology, Muranishi and Kiso, obtained the following compounds: LysB29-Palmitoyl insulin (allyawan-amino group), PheB1-Palmitoyl insulin (allerban N-terminal-amino group of the B chain), and PheB1, LysB29-dipalmitoyl insulin (etilirovany both the amino and N-terminal-amino group).

Similarly, Hashimoto et al. in Pharmaceutical Research 6: 171-176 (1989), describes chetyrehstoronny synthesis of obtaining N-Palmitoyl insulin. The synthesis includes the protection and unprotect N-terminal A1-glycine and the amino group of the B29-lysine using the pMZ. Under the conditions described in the link, get two main acylated product, B1-monopalmitate insulin and B1B29-dipalmitoyl insulin.

Thus, until the present invention the selective acylation B29-N-amino group of insulin was carried out by the step-wise synthesis of acylation of the amino group of proinsulin, insulin and insulin analogues. This invention enables selectively allroute-amino group in the one-step method with high yield. Thus, this invention eliminates the need for protection of other amino groups of the protein and then removing them from the protection. This invention provides a more efficient and less costly method of obtaining derivatives of insulin acylated on the amino group.

This invention provides a process for the selective acylation of proinsulin or insulin analog having a free-amino group and the free-amino group, fatty acid, and this method comprises the interaction of an amino group with soluble activated complex fatty acid ester in a polar solvent under alkaline conditions at a pH of 9.5 to 11.5.

All materials used in this manual abbreviations for amino acids are abbreviations accepted for the office of the United States patent and trademark office and set forth in the Code of Federal Regulations U.S. 37 C. F. R. $ 1.822 (B) (2).

As indicated above, this invention provides a highly selective one-step acylation of the amino group of proinsulin, insulin or an insulin analogue. This invention prednizalona-amino group is obtained with less than 5% yield.

Used herein, the term "insulin" refers to human insulin, porcine insulin or bovine insulin. Insulin has three free amino groups: if B1-phenylalanine, A1-glycine and B29-lysine. Free amino groups in the provisions of the A1and B1are amino groups. The free amino group in position B29a-amino group.

Used herein, the term "proinsulin", respectively, is crosslinked protein formula:

B-C-A

where:

A is A chain of insulin or a functional derivative thereof;

B is the B chain of insulin or its functional derivatives with the amino group; and

C is a connecting peptide of proinsulin.

Preferably proinsulin is A chain of human insulin B chain of human insulin and C represents a natural binding peptide. When proinsulin is a natural sequence, proinsulin has three free amino groups: B1-Phenylalanine (amino group), C64-Lysine (amino group) and B29-Lysine (amino group).

Used herein, the term "insulin analog", respectively, is a crosslinked protein formula:

Preferred insulin analogs include insulin, where:

amino acid residue in position B29is Asp, Lys, Leu, Val or Ala;

amino acid residue in position B29is Lys or Pro;

amino acid residue in position B10is His, or Asp;

amino acid residue in position B1is Phe, Asp or he removed alone or in combination with removal of the residue in position B2;

amino acid residue in position B30is Thr, Ala or deleted; and

amino acid residue in position B9is Ser or Asp;

provided that any provision of B28or B29represents Lys.

In standard biochemical terms known to the person skilled in the technical field preferred insulin analogs represent LysB28ProB29-human insulin (B28is Lys; B29is Pro; AspB28-human insulin (B28is Asp; AspB1-human insulin, ArgB31,B32-human insulin, AspB10-human insulin, ArgA0-human insulin, AspB1GluB13-human insulin, AlaB26-people who tion" means the introduction of one or more acyl groups, covalently linked to free amino groups of the protein.

The term "selective acylation" means the preferred acylation of the amino group(s) compared to the amino groups. Usually selective acylation leads to the ratio of the number of product monoarylamino-amino group to the number of product monoarylamino-amino group, greater than about 5. Preferably the ratio is greater than about 10, and most preferably greater than about 50.

The term "fatty acid" means a saturated or unsaturated C6-C21fatty acid. The term "ester activated fatty acid" means a fatty acid, which is activated using the General techniques described in Methods of Enzymology, 25, 494-499 (1972) and Lapidot et al., in J. Of Lipid Res. 8: 142-145 (1967). Preferred fatty acids are saturated and these include myristic acid (C14), pentadactyla acid (C15), palmitic acid (C16), heptacellular acid (C17) and stearic acid (C18). The most preferred fatty acid is palmitic acid. Ester activated fatty acids include derivatives of ofanim complex ester is N-Succinimidyl palmiet.

The term "soluble" indicates that the liquid phase is present in a sufficient amount of ester to allievate insulin analogue insulin or proinsulin. Preferably in the liquid phase is about 1-4 molar equivalents of an activated complex of ester per mole of insulin.

Used herein, the term "alkaline conditions" refers to the basicity of the reaction. The reaction should be carried out with all the free amino groups, mainly deprotonirovannymi. In an aqueous solvent or water-solvent mixture, the term "alkaline conditions" means that the reaction is performed at a pH higher than 9.0 in. In the non-aqueous organic solvent, the reaction is carried out in the presence of alkaline compounds with a basicity equivalent to the value of the pKain the water, which may exceed or be equal 10,75.

The term "crosslinked" refers to the formation of disulfide bonds between cysteine residues. Properly stitched proinsulin, insulin or an insulin analogue contains three disulfide bridge. The first disulfide bridge is formed between the cysteine residues in positions 6 and 11 of the A-chain. The second disulfide bridge connects the cysteine residue in position 7 of the A-chain cysteine at position 7 of the B-chain. The tar of the present invention a specialist in this area was selectively utilireal-amino group through the use of protecting groups in a multi-stage synthesis. Muranishi and Kiso, application for U.S. Pat. Japan 1-254, 699, open platitudinal synthesis of receipt of acylated derivatives of insulin. Similarly, Hashimoto et al., in Pharmaceutical Research 6: 171-176 (1989) reported chetyrehletiem synthesis of obtaining N-Palmitoyl insulin. For the selective acylation of insulin both links point to the use of pMZ protecting group.

This invention enables to obtain the N-acylated proinsulin, insulin or an insulin analog by one-step synthesis with high yield. The reaction allows obtaining N-acylated proteins without the use of amino-protecting groups. The acylation is accomplished by the interaction of ester activated fatty acid-amino group of a protein under alkaline conditions in a polar solvent. In weakly alkaline conditions, all free amino groups are not deprotonirovannymi and the result is a significant acylation of N-terminal amino groups. In an aqueous solvent or water-solvent mixture alkaline conditions mean that the reaction is carried out at a pH of more than 9,0. As the degradation of the protein takes place in the region of pH over 12,0, the pH of the reaction mixture preferably is in the range from 9.5 to 11.5 the body is equal to the pH of the aqueous phase before mixing.

The data in Table 1 demonstrate the effect of the basicity of the reaction mixture at the reaction selectivity. The data presented in Table 1, correspond to the human insulin acylated two molar equivalents of N-succinimidylester 50% mixture of CH3CN/water.

Table 1 shows that the acylation of the amino group depends on the basicity of the reaction. At pH higher than 9.0 in the reaction selectively allerede-amino group of B29-lysine.

In the nonaqueous solvent, the reaction is carried out in the presence of alkaline compounds with a basicity equivalent to the value of the pKathat is greater than or equal 10,75 in water, in order to sufficiently deprotonate-amino group(s). I.e. alkaline compound should be at least as strong as triethylamine. Preferably, it was tetramethylguanidine (TMG), diisopropylethylamine or tetrabutylammonium hydroxide.

The choice of the polar solvent depends on the solubility of proinsulin, insulin or insulin analogue, and complex ester of fatty acid. Most preferably, when the solvent is completely organic. Usually acceptable organic solvents include DMSO, DMF, etc. indicators is limited only by the solubility of the reagents. The preferred solvents and solvent system are DMSO; DMF; acetonitrile and water, acetone and water; ethanol and water; isopropyl alcohol and water; isopropyl alcohol, ethanol and water; and ethanol, propanol and water. The preferred solvent is a mixture of acetonitrile and water; the most preferred 50% acetonitrile. For the person skilled in the art it is obvious that acceptable and also other polar solvents.

The ratio of reactants is not crucial. Usually prefer the activated ester of the fatty acid was in molar excess. Preferably the reaction is carried out with 1-4 molar equivalents, most preferably 1-2 molar equivalents of ester. However, for the specialist in this field should be obvious that at very high levels of activated complex ester bis - or tri-acylated product will be obtained in a considerable amount.

The reaction temperature is also not critical. The reaction is carried out at a temperature in the range of 0-40 degrees Celsius and usually it ends in a period from 15 minutes to 24 hours.

After the acylation reaction is quenched and the product was then purified using standard methods, one is using standard methods, such as drying, freezing or crystallization.

Proinsulin, insulin or insulin analogs can be obtained using any of a number of known peptide synthesis techniques including classical (solution) methods, solid phase methods, semi-synthetic methods, and more recent methods using recombinant DNA. For example, Chance et al. , application for U.S. Pat. USA N 07/388 201, EPO publication number 383 472, Brange et al. , EPO 214 826 and Belagaje et al. Pat.USA 5 304 473, disclose various proinsulin and insulin analogs, which are here referred to. A and B chain insulin analogues of this invention can be obtained through such proinsulin molecule predecessor, using the technique of recombinant DNA. See Frank et al., Peptides: Synthesis-Structure-Function, Proc. Seventh Am. Pept. Symp., Eds. D. Rich and E. Gross (1981), which is here referred to.

The following examples are intended only to further illustrate the present invention. The scope of this invention should not be construed as only consisting of the following examples.

Example 1

Acylation of insulin, using N-succinimidylester in DMSO.

Crystals biosynthetic human insulin (BCI, BHI) (71,9 mg) is dissolved in 6,58 ml Shalem observation. The activated solution of ester (N-succinimidylester) is obtained by adding 20 mg of solid activated complex ester to 2 ml of DMSO and vigorous mixing until until all the particles of the activated complex ester is dissolved by visual observation. At this time, 1,1,3,3-tetramethylguanidine (26,8 μl) are added to 5 ml BCI (BHI) solution, and then add DMSO (94,4 ml) and the previously obtained solution of the activated complex ether (400 ml). Allow to proceed the reaction at room temperature (20 to 25oC) for about 60 minutes. A sample taken after 15 minutes, diluted with 20 times 1 N acetic acid and analyzed HPLC (HPLC). The yield of the reaction, calculated as the number of B29-N-Palmitoyl human insulin repaid in the sample divided by the initial number of BCI amounted to 67.1 per cent.

Example 2

Acylation of insulin, using N-Succinimidyl-palmitate in a mixture of acetonitrile/water

Crystals biosynthetic human insulin (BCI, BHI) (199,5 mg) was dissolved in 20 ml of 50 mm boric acid solution at a pH of 2.5. the pH re-adjusted to 2.5 using 10% HCl, and the solution is stirred until until the crystals are completely dissolved PR is actor activated complex ether (N-succinimidylester) is produced by adding 24 g of solid activated complex ether to 2.4 liters of acetonitrile, preheated to approximately the 50oC, and vigorous stirring until until all the particles of the activated complex ester is dissolved by visual observation. At this time, the pH BCI (BHI) solution adjusted to approximately 10,22 by adding 10% NaOH. Acetonitrile (18 l) was added to pH brought BCI solution. Allow to proceed the reaction at room temperature (20 to 25oC) for 110 minutes, then quenched by addition of water (123 l) and bringing the pH of the resulting solution to 2.1 using 10% HCl and 10% NaOH. The yield of the reaction, calculated as the number of B29-N-Palmitoyl human insulin in quenched reaction divided by the initial number of BCI (BHI) was 73%.

Example 3

The acylation LysB28-ProB29-human insulin, using N-succinimidylester in a mixture of acetonitrile/water

Crystals LysB28ProB29-human insulin (2,22 g) dissolved in 100 ml of 50 mm boric acid solution at a pH of 2.5. the pH re-adjusted to 2.5 using 10% HCl, and the solution is stirred until the crystals are completely dissolved by visual observation. The activated solution of ester (N-succinimidylester) get dobavlenno 50oC, and vigorous stirring until until all the particles of the activated complex ester is dissolved by visual observation. the pH of the solution adjusted to approximately 10,22 by adding 10% NaOH and the solution is allowed the opportunity to mix at 4oC for 15 minutes. To pH-driven solution was added acetonitrile (73 ml), and then pre-obtained solution of the activated complex ester. Allow to proceed the reaction at 4oC for 85 minutes and quenched by addition of 1 N acetic acid (600 ml), resulting in the pH 2,85. The yield of the reaction, calculated as the number of B28-N-Palmitoyl LysB28ProB29-human insulin in the quenched reaction divided by the initial number LysB28ProB29-human insulin, was 72,5%.

Example 4

The acylation of BCI using N-succinimidylester in a mixture of acetonitrile/water

Crystals biosynthetic human insulin (BCI, BHI) (3 g) dissolved in 300 ml of 50 mm boric acid solution at a pH of 2.5. the pH re-adjusted, if necessary, up to 2.5 using 10% HCl, and the solution is stirred until until the crystals are completely dissolved by visual observation. The solution aktivirovaniya to 40 ml of acetonitrile and vigorous stirring. At this time, the pH of the solution, BCI (BHI) crystals is brought to approximately 10,2 by adding 10% NaOH. Acetonitrile (240 ml) are added to BCI solution, and then add the previously obtained solution of the activated complex ester. Reactions give the opportunity to proceed at room temperature (20 to 25oC) for about 90 minutes, then quenched by addition of water (1800 ml) and bringing the pH of the resulting diluted to approximately 2.5, using 10% HCl. The yield of the reaction, calculated as the number of B29-N-Palmitoyl human insulin, obtained in the reaction divided by the initial number of BCI(BHI), 75.7%.

Example 5

Acylation of proinsulin N-succinimidylester in a mixture of acetonitrile/water

An aqueous solution of human proinsulin (CPI) (28,2 mg/ml) diluted with 50 mm solution of boric acid to a final volume of 100 ml at 16.2 mg/ml CPI. A solution of ester activated at any given time by dissolving 150 mg of N-Succinimidyl-palmitate in 15 ml of acetonitrile (atsn, ACN) with rapid stirring. pH CPI solution was adjusted to 10.2 10% NaOH, and then add 88 ml atsn. The reaction is initiated by adding 12 ml of the activated complex ether (2 x molar the t able to proceed at room temperature (20 to 25oC) for about 60 minutes, then quenched by addition of an equivalent volume (200 ml), 50 mm glycine, pH of 10.0.

The exact relationship aminoacridine derivatives to aminoacridone derived't counted the amount of all-aminoacridine derivatives calculated by the chromatogram was 87-90% of the total area, while the sum of all related substances (which would presumably include any-aminoacridine derivatives) were less than 7% of the total area, for any given point in time.

Example 6

The acylation ArgB31, ArgB32human insulin complex ether hexanoyl-N-hydroxy-succinimide

ArgB31, ArgB32human insulin (1.3 mg) was dissolved in 200 μl of 200 mm (3-[Cyclohexylamino] -1-propanesulfonate) buffer at a pH of 10.4. Then to the solution was added ester hexanoyl-N-hydroxysuccinimide (0.3 mmol) dissolved in N,N-dimethylformamide (DMF) and stirred. The reaction mixture was stirred at ambient temperature (20 to 25oC) for about four hours, then quenched by bringing the pH to about 2.5, using a 0.1 N HCl. Gelatinase particles are removed by passing the mixture through a 0,45 micro is coy HPLC column with C4reversed phase. The yield of the reaction, calculated as the number of B29-N-hexanoyl-ArgB31, ArgB32-human insulin in the quenched reaction divided by the initial number ArgB31, ArgB32-human insulin was $ 69.4%.

Example 7

The acylation LeuB26-human insulin N-succinimidylester in DMSO.

LeuB26-human insulin (1.0 mg) was dissolved in 1 ml of 95% dimethyl sulfoxide (DMSO), 5% triethylamine (tea). Then the solution is added N-Succinimidyl palmitate (0.7 mmol) dissolved in N,N-dimethylformamide (DMF) and stirred. The reaction mixture was stirred at ambient temperature (20 to 25oC) for approximately ninety minutes, then quenched by dilution of the sample to 0.2 mg/ml of 0.1 N HCl. Gelatinase particles are removed by passing the mixture through the 0.45 micron filter prior to HPLC (HPLC) analysis. The Department named product from the original substances reach on an analytical HPLC column with C4reversed phase. The yield of the reaction, calculated as the number of N-Palmitoyl-LeuB26human insulin suppressed the reaction divided by the initial number LeuB26human insulin, with the t in dimethyl sulfoxide (DMSO)

A solution of insulin get complete dissolution of crystals biosynthetic human insulin (1 g, 0,17 mmol) in 20 ml of DMSO at room temperature. At the same time receive the activated solution of ester by dissolving N-succinimidylester (0,0817 g, 0.23 mmol) in 3 ml of DMSO at 50oC. To a solution of insulin, which is vigorously stirred, add first 1,1,3,3-tetramethyl-guanidine (0,432 ml, 3.4 mmol) and then the entire active solution of ester. After 30 minutes the reaction is quenched with 120 ml of 0.05 M HCl, pre-cooled to 0oC. the pH of the mixture was approximately 1.8. Analysis of the quenched mixture by HPLC (HPLC) with reversed phase shows that B29-N-Palmitoyl insulin was 72.2% of the total lirovannomu protein and represents 95% of the total monoarylamino insulin.

The entire reaction mixture was applied on preparative Vydac column with C4reversed phase (5 x 25 cm), previously equilibrated with a mixture solvent containing 0.1% triperoxonane acid, 20% acetonitrile in water. After application, the column is first washed with 500 ml of the same solvent and then set the flow rate of 4 ml/min and washed with a solvent system consisting of 0.1% t the 29-N-Palmitoyl insulin was elyuirovaniya this solvent system consisting of approximately 53% of acetonitrile. After removal of the solvent by lyophilization output N-Palmitoyl insulin was 414 mg (0,0684 mmol) or 40.2 per cent in the calculation of the original substance.

Example 9

The acylation LysB28-ProB29-human insulin complex ester 1-octanoyl-N-hydroxysuccinimide

Crystals Lys(B28)Pro(B29) human insulin (EOC) (2.0 g) is dissolved in 200 ml of 50 mm boric acid buffer at a pH of 2.5. the pH re-adjusted to 2.5 using 10% HCl, and the solution is stirred until until the crystals are completely dissolved by visual observation. The activated solution of ester (ester 1-octanoyl-N - hydroxysuccinimide) is produced by adding 175 mg of solid activated complex ether to 25,62 ml of acetonitrile, and vigorous stirring until until all the particles of the activated complex ester is dissolved by visual observation. pH EOC solution was adjusted to approximately 10,4 by adding 10% NaOH, and the solution is allowed the opportunity to mix at ambient temperature for about 5 minutes. To pH-driven EOC solution was added acetonitrile (176 ml), the AI at ambient temperature for 90 minutes and quenched by adding 5.5 ml of 10% HCl (2.75% V/V) and three volumes (1200 ml) of cold H2O, receiving the result, the final pH 2,70. The yield of the reaction, calculated as the number of LysB29(C8)KPB repaid in the reaction divided by the initial number of BCI, was 75.5%. This solution is divided into two 800 ml aliquots for cleaning hydrophobic chromatography (SP20SS). For column chromatography followed by ultrafiltration and lyophilization.

The data in Table 2 demonstrate the selective acylation of insulin, insulin analogues and proinsulin. Experiments performed at room temperature with esters of N-hydroxysuccinimide fatty acids. In Table 2 TMG and tea are tetramethylguanidine and triethylamine, respectively. ND indicates no data.

1. Process for the selective acylation of proinsulin, insulin or an insulin analog having a free - and-amino group using an activated complex fatty acid ester, characterized in that the interact-amino group with soluble activated complex fatty acid ester in a polar solvent at a pH of 9.5 to 11.5.

2. Process for the selective acylation under item 1, characterized in that acelerou human insulin, insulin analog or LysB28ProB29human insociety is N-hydroxysuccinimidyl ether C6-C18- fatty acids.

4. Process for the selective acylation under item 1, characterized in that the activated fatty acid ester is N-hydroxysuccinimidyl ether C14-C18fatty acid.

5. Process for the selective acylation according to any one of paragraphs.1 to 4, characterized in that the activated fatty acid ester is N-hydroxysuccinimidyl ester of palmitic acid.

 

Same patents:

The invention relates to novel analogues of human insulin with rapid onset desired effect after subcutaneous injection, and the solutions of insulin for injection containing insulin analogs and to methods for new insulin analogues

The invention relates to medical biochemistry, in particular to a new fragment having biological activity of insulin

The invention relates to the field of biotechnology for the production of recombinant human insulin, which can be applied as a drug with anti-diabetic action, specifically to an improved method of allocating a hybrid protein containing the sequence of human proinsulin

The invention relates to medicine and concerns insulinosoderzhaschego medicines for oral use and method of its production
The invention relates to medicine, namely to cleaning used in the treatment of diabetes insulin secreted from animal products or derived through biotechnology (for example, genetically engineered human insulin)

The invention relates to medicine, namely to endocrinology and purulent surgery

The invention relates to pharmaceutical industry and relates to a powdery product containing medical useful polypeptides

The invention relates to the preparation of insulin, including the suspension of crystals of human insulin, ultralente and zinc in a total concentration in a preparation of 0.5-20 mg per 100 units of insulin
Antidiabetic drug // 2134122
The invention relates to medicine and can be used in the treatment of diabetes
The invention relates to medicine and can be used to restore the natural reactivity to drugs and endogenous substances produced in the body, or toxic substances

FIELD: medicine.

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.

Up!