The complex analogue of insulin and protamine, the production method, pharmaceutical composition and method for the treatment of diabetes

 

(57) Abstract:

The invention discloses various parenteral pharmaceutical compositions that include a Monomeric insulin analog, zinc, Protamine, and a phenolic derivative. Composition similar provide a prolonged action. Also described is a method of obtaining the composition of the insulin analog-Protamine. The invention also includes a method of treatment of diabetes with the help of insulin and Protamine, and also containing pharmaceutical compositions. The technical result of the invention is to provide means for the treatment of diabetes c prolonged action. 8 C. and 23 C.p. f-crystals, 1 table. 3 Il.

The invention relates to Monomeric analogues of human insulin. In particular, the present invention relates to various parenteral compositions that include similar Monomeric insulin, zinc, Protamine, and a phenolic derivative. Compositions provide the prolonging effect. Also describes a method of producing compositions insulin analog-Protamine.

Since the introduction of insulin in 20-ies of XX century. continuously taking steps to improve the treatment of diabetes. The main successes achieved have Developed various compositions with different times of the actions. Now, generally speaking, there are seven commercially available insulin compositions: regular insulin, the insulin semilente, globin insulin, isophane insulin suspension zinc insulin, Protamine-zinc-insulin, insulin, ultralente.

Despite the many suitable compositions for the treatment via subcutaneous injection, the patient still needs a convenient regulation and normalized glycemic control. Frequent transitions from normal blood glucose levels during the life of patients lead to Hyper - or hypoglycemia and distant complications, including retinopathy, neuropathy, nephropathy, and micro - and macroangioapthy.

To help avoid extreme blood glucose levels, diabetics often practiced therapy using multiple injections through the introduction of insulin before each meal. However, this therapy is still not optimal. Most commercially available fast-acting insulin provides peak too late after injection and lasts too long to optimal glucose levels. Therefore, considerable efforts are aimed at creating insulin compositions and compositions of the insulin analogue that alter the kinetics process insulin in coassociative condition and mostly in the review of the form, I believe that stage, limiting the rate of absorption of insulin from the depot after subcutaneous injection into the bloodstream, represents the dissociation camogregorian hexamer insulin. Recently developed Monomeric insulin analogs, which are less prone to Association with the formation of higher molecular weight than human insulin. This loss of self due to modifications of the amino acid sequence of human insulin, which reduce the Association using mainly the destruction of dimer formation (see, for example, Brems et al., Protein Engineering 5: 6, 527-533 (1992) and Brange et al., Nature, 333:679-682 (1988)). In accordance with this Monomeric insulin analogs have a relatively more rapid onset of activity while maintaining the biological activity of native human insulin. These insulin analogues provide rapid absorption, the settling time of injection and the peak action of insulin in greater intimacy to occur after ingestion of change of glucose level associated with the response to a meal.

Physical properties and characteristics of Monomeric analogues are not similar to insulin. For example, Brems et al. reveals that different Monomeric analogues have is a set of entities with a higher molecular weight. This is very different from insulin, which is almost exclusively an orderly review of the conformation in the presence of zinc. Brange et al., Diabetes 13:923-954 (1990) high-speed characteristics analogues are attributed to the lack of Association. As analogues have a lower tendency to associate, it is quite surprising that the Monomeric insulin analogue can be prepared in such a way as to ensure the average duration of action.

The present invention provides a composition comprising a Monomeric insulin analog, which has when using the average duration of action. In addition, the invention provides a new proteinemia crystal, called insulin NPD-analogue. The present invention also provides a mixture of insulin NPD analogue and soluble Monomeric insulin analog. This mix provides a quick onset of action and intermediate duration of action. In accordance with this mixture has the advantages as before insulin and Monomeric analogue. In addition, the present invention provides a method of obtaining a homogeneous crystals insulin analog-NPD.

This invention is exposed to the exhaust gas, Protamine, zinc, and phenol derivative.

In addition, the invention provides a complex of crystalline insulin analogue and Protamine. This complex is defined as an insulin analogue-NRD. LysB28ProB29-human insulin-NRD includes LysB28ProB29-human insulin, 0,27-0,32 mg Protamine/100 Units insulin analogue, 0,35-0,9% zinc by weight and a phenolic derivative.

This invention also provides a method of producing complex LysB28ProB29-human insulin-NRD, which includes:

the mixture of aqueous solution of LysB28ProB29-human insulin in a review of the associated condition and solution of Protamine at a temperature from about 8 to about 22oC;

specified aqueous solution includes from 0.35 to 0.9% zinc by weight, LysB28ProB29-human insulin and phenolic derivative at a pH of from about 7.1 to about 7.6;

the specified solution includes Protamine Protamine at a pH of from 7.1 to 7.6, such that the final concentration of Protamine is about 0.27 to about 0.32 mg Protamine/100 E insulin analogue.

The invention also provides a composition having both fast and medium action. The compositions presented the new ratio of the two components is about 1-99:99-1.

Finally, the invention provides a method of treating a patient suffering from diabetes mellitus, which includes the introduction of the indicated patient a pharmaceutical composition containing the crystal of the complex of the insulin analog-Protamine.

Fig. 1 is a graphical representation of the profile actions LysB28ProB29-hI-NPD and human insulin-NPH. The graph represents the dependence of mked/ml from the time of infusion. Fig. 1 demonstrates the advantages of the present invention.

Fig. 2 represents the image of crystals AspB28-human insulin-Protamine of the present invention. The picture is at 1000x magnification c differential phase contrast.

Fig. 3 represents the image of crystals LysB28ProB29-hI-Protamine of the present invention. The picture is at 1000x magnification with differential phase contrast.

As noted above, the invention provides various compositions of Monomeric insulin analog. Used herein, the term "Monomeric insulin analog or an insulin analogue" means a fast-acting insulin analog that is less prone to dimerization or famouscomet on Asp, Lys, Leu, Val or Ala and Lys at position B29 is lysine or Proline; des (B28-B30); or des (B27). Monomeric insulin analogs described by Chance et al., EPO ublication 383472 and Brange et al., EPO publication 214826, which are here referred to.

The person skilled in the art should be aware that other modifications are available for Monomeric insulin analog. These modifications are widely known in this area and include the substitution of his-tag residue at position B10 on aspartic acid; substitution of the phenylalanine residue in position B1 on aspartic acid; replacement of a residue of threonine in position B30 to alanine; the substitution of the serine residue at position B9 on aspartic acid; the loss of amino acids only in the position B1 or in combination with loss in position B2, and the loss of threonine in position B30.

Used in this manual abbreviations of all amino acids are the abbreviations adopted by the United States Patent & Trademark Office as specified in 37 CFR § 1.822(b) (2). Particularly preferred Monomeric insulin analogs are LysB28ProB29-human insulin (B28 is Lys; B29 is Pro) and AspB28-human insulin (B28 is Asp).

The term "Monomeric insulin analog-NPD" or "insulin analogue-ND" means suramin according to De Felippis. Composition was prepared in accordance with the inventive method described herein. Related term "crystals insulin analog-NPD", "crystalline insulin analog-NPD or crystals LysB28Pro29-human insulin-Protamine refers to the crystals insulin analog-Protamine in NPD composition.

Used herein, the term "treatment" refers to the assistance and care of the patient with the purpose of combating a disease, condition, or disorder and includes the introduction of compounds of the present invention to prevent the beginning of the disease or complications, alleviating the symptoms or complications, or eliminating the disease, condition or violation.

The term "isotonic agent" refers to an agent that is physiologically tolerant and which gives a suitable tone of the composition, to prevent the net flow of water across the cell membrane. Compounds such as glycerin, usually used for such purposes in known concentrations. The concentration of the isotonic agent is in the range known in the field of application for the compositions of insulin.

The term "phenolic derivative" denotes m-cresol, phenol, or preferably a mixture of m-cu is osili. The calculation of the free base adjusts the water content and salt content of Protamine, commercially acceptable and commonly used in parenteral compositions. Preferred Protamine, Protamine sulfate, approximately 80% of Protamine.

The term "IU" or "U" means international unit.

The term "safanova ratio" means the equilibrium amount of Protamine required to kompleksowe with insulin analogue set Krayenbuhl and Rosenberg, Stent Memorial Hospital Report (Copenhagen), 1:60 (1946). Safanova ratio determined by titration according to the method well known in the art and described Krayenbbuhl, et el.

The present invention provides a composition insulin analog-Protamine, which includes Monomeric insulin analog, Protamine, zinc, and phenol derivative. The concentration of Protamine is preferably from 0.2 to 1.5 mg of Protamine per 100 units of insulin analogue in the calculation of the free base. Most preferably, the range of Protamine was from 0.27 to 0.35 mg/100 Units. The concentration of zinc is from 0.35 to 0.9% by weight. Preferably the concentration of zinc is about 0.7%.

Phenolic derivative Presse m-cresol and phenol. The concentration of the phenol derivative known to the person skilled in the art. The concentration should be sufficient to maintain the efficiency of conservation, i.e. to slow down microbial growth. In General, the concentration of the phenol derivative, for example, is in the range from 1.0 to 6.0 mg/ml, preferably higher than about 2.5 mg/ml the Most preferred concentration of about 3 mg/ml the presence of the phenolic derivative is significant because it affects the complexation of similar, Protamine and zinc in addition to functioning as a preservative. However, I believe that in the crystal structure of molecule insulin analogue has only one molecule of phenol.

Preferably isotonic agent added to the composition. Preferred isotonic agent is glycerin. The concentration of the isotonic agent is, for example, from 14 to 18 mg/ml, preferably about 18 mg/ml

the pH of the composition can be installed using a physiologically tolerable buffer, preferably a phosphate buffer, such as Dooney sodium phosphate. Other physiologically tolerant buffers include TRIS, sodium acetate, or citrate natrc about to about 5.0 mg/ml, preferably 3.8 mg/ml

In addition, the present invention provides specific conditions under which insulin analog-Protamine exists in the form of a stable crystal. The composition of these crystals is defined as insulin analog-NPD. Insulin analog-NPD is a formulated suspension of crystals insulin analog-NPD, and he is using shows an intermediate step. The activity profile of an insulin analog-NPD quite unexpected from the point of view of lack of self Monomeric analogue.

The ability of the composition with Monomeric analogue to be interim action is demonstrated in Fig. 1. Fig. 1 illustrates the profile of actions for LysB28ProB29-hI-NPD and human insulin-NPH. NPD profile similar to the profile of the insulin-NPH. The duration of action for NPD composition and insulin-NPH composition is approximately the same. But most importantly, the present composition improves more quickly and remains stable over a longer period than the insulin-NPH. This difference is quite unexpected from the point of view of a high-speed profile Monomeric analogue.

Particularly preferred composition insulin is insulin, from 0.27 to 0.32 mg Protamine/100 Units insulin analogue, from 0.35 to 0.9% zinc by weight and a phenolic derivative. The concentration of Protamine is preferably 0.3 mg/100 Units per free base.

The invention also provides a method of obtaining LysB28ProB29-human insulin-Protamine crystals, which includes:

the mixture of aqueous solution of LysB28ProB29-human insulin in the status review of associate and solution of Protamine at a temperature from about 8 to about 22oC;

specified aqueous solution includes from 0.35 to 0.9% zinc by weight, LysB28ProB29-human insulin and phenolic derivative at a pH of from 7.1 to about 7.6;

the specified solution includes Protamine Protamine at a pH of from 7.1 to 7.6 so that its final concentration ranged from 0.27 to 0.32 mg Protamine/100 Units insulin analogue.

Prior to the invention it was known that Monomeric insulin analogs are less prone to the Association and the formation of hexamers. The conditions necessary to cause Association of Monomeric insulin analogues with Protamine to form crystals, which were previously not known in this area. Early studies treated sofan insulin compositions according Krayenbuhl and Rosenberg, Sterd Memorial Hospital Report (Copenhagen), 1:60 (1946) is not relevant from the point of view of differences in the properties of the Monomeric insulin analogs. In fact, a commercial method of obtaining Hunilin-NTM(insulin-NPH), acid-neutral way, not result in crystalline insulin analog-NPD.

Most important to establish what the parameters of the present invention, namely the crystallization temperature and education review of complex insulin analogue, zinc, and phenol derivative, are essential characteristics for the formation of stable LysB28ProB29-hI-NPD crystals.

The crystallization temperature must be from about 8 to about 22oC, preferably from 13 to 17oC. If the temperature is outside this range, it is mainly amorphous composition insulin analog-Protamine.

It is also significant that the insulin analogue is transformed into a review of the state before crystallization. Crystallization leads to amorphous product when the process is carried out in the Monomeric state Association. The crystals formed without stirring for 5 to 30-60 hours Crystals high cachexy associat when suspendirovanie solid Monomeric analogue in the solvent, containing phenol derivative, and adding zinc to until the concentration of zinc will not be from 0.35 to 0.9% by weight. The zinc is preferably added in the form of a salt. To illustrate examples of zinc salts include zinc acetate, zinc bromide, zinc chloride, zinc fluoride, zinc iodide and zinc sulfate. The person skilled in the art should know that there are other zinc salts, which can also be used in the method of the present invention. It is preferable to use zinc acetate and zinc chloride.

The dissolution of the insulin analogue as a solvent, as is well known, can contribute to the acidification. When the acidic solution to increase the solubility of similar pH is reduced to about 3-3,5 using a physiologically tolerated acid, preferably HCl. Other physiologically tolerant acids include acetic acid, citric acid and phosphoric acid. pH then fail physiologically tolerant base, preferably NaOH, to about 7.1 and 7.6 for crystallization. Other physiologically tolerant grounds are KOH and ammonium hydrochloride.

Most importantly, the method of obtaining LysB28ProB29-hI-NPD complex sensitive to the are mixed with an amorphous product. Thus, preferably, in order to avoid the formation of ions, salts, Monomeric analogue to dissolve at neutral pH. Finally, the analogue can be dissolved in the solvent at acidic pH before adding the buffer. This reduces the concentration of salts generated as a result of summing up the pH. However, the order in which the components are added, is not essential for the formation of hexamer or amorphous education.

As indicated above, the compositions of the present invention can be added to an isotonic agent. Isotonic agent can be added to the solution similar to the solution of Protamine or final insulin analog-NPD composition. Similarly, the addition of a physiologically tolerable buffer can be performed by solution similar to the solution of Protamine, or the final insulin analog-NPD composition. However, preferably, as a solution similar and the solution of Protamine contained isotonic agent and a buffer before mixing the aqueous solution and Protamine. Due to the influence of NaCl on the method of obtaining crystalline insulin analog-NPD glycerin is the preferred isotonic agent.

The invention also provides compositions insulin analogues the new analog. These compounds get in the range of from about 1: 99 to 99:1 by volume suspended insulin analog-NPD to soluble insulin analogue. Soluble insulin analogue is a Monomeric insulin analog, soluble in an aqueous solvent containing zinc, phenol derivative, an isotonic agent and a buffer. Concentration in the solvent are the same as previously specified. Preferably, the ratio of insulin analog-NPD to soluble insulin analogue was 25:75 to 75:25 and more preferably 50:50. Mixtures are easily obtained by mixing the individual components.

Mixed composition of the present invention is particularly suitable for the treatment of diabetes due to a combination of rapid early action and prolonged action. These compounds allow for "fine control" by varying the amount of each individual component based on the needs, diet and physical activity of the patient. The advantage of mixtures suspended insulin analog-NPD and soluble insulin analogue is that they are homogeneous, i.e., for any equilibrium exchange between the suspended crystals and soluble insulin is the same time of any of a number of recognized technologies for the synthesis of peptides including classical (solution) methods, solid phase methods, semi-synthetic methods and more modern methods using recombinant DNA. For example, Chance et al., EPO 214826, disclose various Monomeric analogues.

The following examples are given to further illustrate receiving insulin and analogs of the present invention. Scope of the invention is not limited to the following examples.

Example 1

Getting LysB28ProB29- hI-NPD

The solution LysB28ProB29-human insulin (LysB28ProB29-hI) with a concentration of 200 IU/ml (E200) obtained by dissolving zinc-containing crystals LysB28ProB29-hI in the system preservative/buffer containing 1.6 mg/ml m-cresol, 0.73 mg/ml phenol (equivalent to 0.65 mg/ml phenol, calculated as 89%), 16 mg/ml glycerol and of 3.78 mg/ml dibasic sodium phosphate buffer. Endogenous organism) level of zinc crystals complement by adding the appropriate volume of acid solution ZnO (10 mg/ml) to achieve a final concentration of 0.025 mg/100 ME (0,7%). Dissolution LysB28ProB29carried out at ambient temperature by lowering the pH to about 3 μl CLASS="ptx2">

The solution of Protamine is produced by dissolving solid enough Protamine sulfate in solution preservative/buffer to achieve a final concentration of 0.6 mg/100 ME per free base. the pH of this solution was adjusted to 7.5 and stabilize at the 15oC.

Both solution diluted to the final concentration with water for injection and filtered. 5 ml aliquots LysB28ProB29-hI component fill in a separate clean glass vials, and the samples incubated in water bath at 15oC. After appropriate stabilisation time (15 min), causing precipitation by rapid addition of 5 ml of Protamine to LysB28ProB29hI samples. Crystallization was carried out for about 24 h at 15oC.

Example 2

Getting LysB28ProB29-hI-NPD

The method is identical to example 1, except that the dissolution LysB28ProB29-hI occurs at neutral pH. The method is carried out so that the final pH was 7.4.

Example 3

Getting LysB28ProB29hI NPD

Insulin analog-NPD receive by way similar to example 1, but the dissolution in the acidic environment LysB28ProB29-hI perform in the presence of all fillers, except for actor insulin analogue return to a pH of 7.4. Adding dibasic sodium phosphate leads to OSVETLENIE solution.

Example 4

Getting mixed compositions insulin analog

A mixture of LysB28ProB29hI compositions intermediate and fast action is obtained as follows. The suspension composition of the intermediate steps is obtained using the methods described in example 3, and this song serves as a component of the intermediate steps to the mixture. A separate solution of LysB28ProB29-hI (100 ME) is produced by dissolving zinc-containing LysB28ProB29hI crystals at ambient temperature in a solvent described in example 1. Endogenous levels of zinc LysB28ProB29-hI, this solution is complemented by adding an acidic solution of ZnO to match the level of the suspension component (i.e., 0.025 mg/100 ME (0,7%)). After pH was adjusted to 7.4 using 10% solutions of HCl and/or NaOH, to dilute the solution to a final concentration of use water for injections. This solution is a high-performance component mixtures. The final mixture obtained by mixing appropriate volumes of the components of the intermediate and fast action, getting the required relationship. 50/50 Astia volume.

Example 5

The influence of ionic strength on the crystallization LysB28ProB29hI Protamine

The influence of ionic strength on the crystallization estimate by adding NaCl to LysB28ProB29-hI component before mixing with Protamine. NaCl added so that the total concentration was 20, 30 and 40 mm (1.2; 1.8; 2.3 mg/ml). The distribution of the particles by volume demonstrates a multimodal behavior (additional peaks for particles of small size) with increasing NaCl concentration. The average volumetric particle size decreases with increasing NaCl concentration, indicating an increase of amorphous substances. The results of particle size depending on the concentration of NaCl is as follows:

[NaCl] - Average particle size (µm)

13 mm - 3,9

20 mm - 3.5

30 mm - 3,3

40 mm - 3,2

Analysis using microscopy showed that all samples contained a mixture of amorphous and crystalline substances. The sample containing 40 mm NaCl, were essentially amorphous substance and contained very few crystals.

Example 6

Comparative dynamics LysB28ProB29-hI-NPD and human insulin

This study conducted on the model of conscious dogs. Before the study make llivan the frequent monitoring of plasma glucose and inject a variable amount of glucose (20%), in order to maintain glycemia near normal. The samples were taken all the time and analyzed for immunoreactive insulin (Linco antibody) and glucose. The results are illustrated in Fig. 1.

Example 7

Obtaining crystals Asp (B28) analog-Protamine

Component Asp (B28)-hI at a concentration of 200 IU/ml (E 200) obtained by dissolving lyophilized unit (95% purity) in the system preservative/buffer containing: 1.6 mg/ml m-cresol, 0.73 mg/ml phenol (equivalent to 0.65 mg/ml phenol, calculated as 89%), 16 mg/ml glycerol and of 3.78 mg/ml of dibasic sodium phosphate. To the system add zinc, using the appropriate amount of acid ZnO solution (10 mg/ml) to give a final concentration of 0.025 mg/100 ME. Dissolution Asp (B28) is carried out at ambient temperature under neutral pH conditions. The final pH component equal to 7.4.

Crystallization was carried out as described in example 2. Get final concentration of Protamine of 0.3, 0.35 and 0.4 mg/100E. These concentrations of Protamine correspond to 2.9, 9.3 and 10.5 percent, respectively, in calculating the weight/weight. The incubation temperature is 5oC (0.3 mg/100E only), 15 and 22oC. After aging for 24 h at these temperatures, the samples will be analyzed by the formation of crystals. Results Opredelenie 8

Obtaining crystals Asp (B28) analog-Protamine

Crystallization Asp (B28) Protamine were carried out as described in example 7, except that the protein is dissolved in the first solvent-free buffer. Add sour ZnO mixture was sufficient to acidify the sample to pH 2.0 to 2.5. After the solution will lighten, pH adjusted to approximately 7 with Microlitre amounts of 5N NaOH. Sodium phosphate dibasic add, using a concentrated solution mixture at 47,25 mg/ml, obtaining a final concentration of 3.78 mg/ml This component is brought to a pH of 7.4. using Microlitre amount of HCl.

Crystallization was initiated by combining the components of the Asp (B28) and Protamine, as described in the previous examples. Receive a final concentration of Protamine of 0.3, 0.35 and 0.4 mg/100E. The incubation temperature is 15 and 22oC. After aging for 24 h at these temperatures the samples examined for the formation of crystals. Results identified using microscopy, give the mixture of crystals and amorphous product.

Example 9

Obtaining crystals Leu (B28) Pro(V) analog-Protamine

Component of Leu (B28) Pro (W) (93% purity) at a concentration of 200 IU/ml (E 200) are obtained as described in example 8, and is as described above. Explore the final concentration of Protamine of 0.3, 0.35 and 0.4 mg/100E. The incubation temperature include 5, 15, and 22oC. After 24 h at these temperatures, all samples contain some crystals, but mostly they were amorphous, as determined by microscopy.

Example 10

Des (B27) hI-Protamine crystals

Component Thr Des (B27) (97,37% purity) at a concentration of 200 ME/ml (E200) are obtained as described in example 8, using acid dissolution of the block and then bringing the pH with 5N NaOH to 7.4. Crystallization was carried out as described in example 8. Explore the final concentration of Protamine of 0.3, 0.35 and 0.4 mg/100E. The incubation temperature include 15 and 22oC. After 24 h at these temperatures, all samples were mainly amorphous, as determined by microscopy. Qualitatively observed crystals.

Example 11

Des (B28-B30) hI-Protamine

Component Des (28-30) (96,3% purity) at a concentration of 200 IU/ml (E200) are obtained as described in example 8, using acid dissolution of the block and then bringing the pH with 5N NaOH to 7.4. Crystallization was carried out using method a neutral/neutral mixture of protein and profenofos components, as described above. Explore the ultimate con the arts all samples were mainly amorphous, as determined by microscopy. Qualitatively observed crystals. These crystals are well defined.

Example 12

Asp (B28) analog-Protamine

A solution of insulin Asp (B28)-human insulin analog produced by dissolution of 16.6 mg protein in 1 ml of a solution containing 3.2 mg/ml m-cresol, 1.3 mg/ml phenol and 32 mg/ml of glycerol. Add 14.4V-µl aliquot of the solution mixture was acidified zinc (10 mg/ml Zn2+obtained by dissolving 0,311 g of zinc oxide in 5 ml of 10% HCl and diluted to 25 ml with water). the pH of the solution was 2.3, which ensured the complete dissolution of the protein. Add a 10-ál aliquot of 10 NaOH to bring the pH to 7,06. To the solution add 100 ál of 0.28 M dibasic phosphate, pH 7.0, which increases the pH of the solution to 7.27. To the solution was added 870-ál aliquot of water for injection. Additionally, add 10% HCl (1 ml) and NaOH (0,7 mm), and the final volume of the solution was adjusted to 2 ml with water for injection, receiving the final pH 7,26. The solution is filtered through an 0.2 µm SuporRAcrodiscR13, Gelman Sciences) filter before use.

Protamine mixed solution obtained by dissolution of Protamine sulfate in a solution containing 1.6 mg/ml m-cresol, 0.65 mg/ml phenol, 16 mg/ml glycerol and 14 mm dvukhosnovnogo/100E in the calculation of the free base. Both solution is filtered through a 0,22 µm (Millipore SterivexTM-GV) filter element prior to use.

Crystallization is achieved by mixing the solution of Asp (B28)-human insulin in a ratio of 1:1 at a controlled temperature indicated in the table. End conditions of the mixture were of 3.94 mg/ml Asp (B28)-human insulin, 0,0359 mg/ml (0,9%) zinc ions, 1.6 mg/ml m-cresol, 0.65 mg/ml phenol, 16 mg/ml glycerol, 14 mm dibasic phosphate and 0.30 mg/100E Protamine at a pH of 7.3. In particular, 50-200-μl portion of the solution AspB28-human insulin transferred into glass ampoules, and the temperature of the samples was adjusted to 4, 8, 15 or 23oC (ambient temperature). Part of both prolinovyh solutions also lead up to these temperatures. After 15-20 min the equivalent amount of any proteinovogo solution is transferred with a pipette into the sample Asp (B28)-human insulin. The mixture is gently shaken at rotation, cover and then left stationary at a controlled temperature during crystallization. All samples examined by microscopy after 24 h, and, as discovered, they are mostly amorphous. After 48 h, the samples containing 0.30 mg/100E Protamine and incubated at 15oC, shows the Example 13

A solution of insulin Asp (B28)-human insulin analog produced by dissolution to 10.62 mg protein of 0.71 ml of a solution containing 3.2 mg/ml m-cresol, 1.3 mg/ml phenol and 32 mg/ml of glycerol. Add a 10.2-ál aliquot of mixed acidic solution of zinc (10 mg/ml Zn2+obtained by dissolving 0,311 g of zinc oxide in 5 ml of 10% HCl and diluted to 25 ml with water). the pH of the solution was 2.3, which ensured the complete dissolution of the protein. Add the 6.5 ál aliquot of 10% NaOH to bring the pH to 7.00. To the solution was added 71 μl of 0.28 M dibasic phosphate, pH 7.0, which increases the pH of the solution to 7.26. To the solution was added 620-ál aliquot of water for injection. Additionally, add 10% HCl (0.2 µl) and NaOH (0.6 μl), and the final volume of the solution was adjusted to 1.42 ml water for injection, receiving the final pH 7,42. The solution is filtered through the 0.2-µm SuporRAcrodiscR13, Gelman Scinces) filter before use.

Proteinemia mixed solution obtained by dissolution of Protamine sulfate in a solution containing 1.6 mg/ml m-cresol, 0.65 mg/ml phenol, 16 mg/ml glycerol and 14 mm dibasic phosphate. The final pH of the solution was adjusted to 7.4, and the final concentration of Protamine is 0.60 mg/100E in the calculation of the free base. The solution ACIA is achieved by mixing the solution of Aps (B28)-human insulin in a ratio of 1:1 with a solution of Protamine, as described in example 12, at controlled temperatures 13, 15, 17 and 23oC. the Results are presented in the table. End conditions of the mixture - 3,74 mg/ml Aps (B28)-human insulin, 0,0359 mg/ml (0,9%) zinc ions, 1.6 mg/ml m-cresol, 0.65 mg/ml phenol, 16 mg/ml glycerol, 14 mm dibasic phosphate and 0.30 mg/100E Protamine at a pH of 7.4. Evaluate four different crystallization temperature. 1-ml aliquot of ApsB28-human insulin, stable, 15oC, mixed with 1 ml proteinovogo solution, brought to the same temperature. After gentle shaking during rotation, the composition is left stationary during the 15oC. Another sample is obtained by stabilizing solution of 100 μl Asp(B28)-human insulin in the 13oC and then mixing with 100 μl proteinovogo solution, brought to the same temperature, the final mixture was incubated at 13oC. the Third sample receive a similar way, except that two 100 - ál aliquots stabilize, mixed and then incubated at 17oC. the Final solution is obtained by mixing stable at ambient temperature solutions 80-µl aliquot Asp (B28)-human insulin and Protamine and incubation at a temperature of time intervals, listed in the table.

The crystals obtained in accordance with the above examples, are illustrated in Fig. 2 and 3.

1. The complex analogue of insulin and Protamine, which includes human insulin, in which Pro at position B28 is substituted by Asp, Lys, Leu, Val or Ala, and Lys in position V represents Lys or Pro; human insulin, in which amino acids B28-B30 deleterows; or human insulin, in which the amino acid B27 demeterova, and Protamine, zinc, and phenol derivative, provided that when insulin is a AspB28- human insulin, the concentration of Protamine is less than 10% by weight.

2. The complex analogue of insulin and Protamine under item 1, which contains LysB28ProW- human insulin, Protamine, zinc, and phenol derivative.

3. Complex p. 2, which contains 0.2 - 0,32 mg Protamine per 100 units of insulin analogue, 0,35 - 0,9% zinc by weight, and a phenolic derivative.

4. Complex under item 1, which contains AspB28- human insulin, 0.2 to 0.35 mg Protamine/100 Units insulin analogue, 0,35 - 0,9% zinc by weight, and a phenolic derivative.

5. The complex according to any one of paragraphs.1, 2, 3 or 4, in which the complex has insulina and Protamine, which includes complex on PP.1, 2, 3, 4 or 5.

7. The composition according to p. 6, which includes 0.2 to 1.5 mg of Protamine per 100 units of insulin analogue, 0,35 - 0,9% zinc by weight and a phenolic derivative.

8. Pharmaceutical composition for parenteral administration, which includes LysB28ProW- human insulin, 0,27 - 0,32 mg Protamine per 100 units of insulin analogue, and 0.35 - 0.9% zinc by weight and a phenolic derivative.

9. Pharmaceutical composition for parenteral administration, which includes LysB28ProW- human insulin, 0.3 mg of Protamine per 100 units of insulin analog, 0.7% of zinc by weight, 1.7 mg/ml m-cresol, 0.7 mg/ml phenol, 16 mg/ml glycerol and of 3.78 mg/ml Doonbeg phosphate.

10. Pharmaceutical composition for parenteral administration, which includes AspB28- human insulin, a 0.27 - 0.35 mg of Protamine per 100 units of insulin analogue, 0,35 - 0,9% zinc by weight and a phenolic derivative.

11. Pharmaceutical composition for parenteral administration according to any one of paragraphs.6 to 10, which further comprises a soluble insulin analogue.

12. Pharmaceutical composition for parenteral administration comprising a mixture of R is th, and crystals of insulin analog-Protamine, in which the weight ratio of the two components is from 1 : 99 to 99 : 1 analogue insulin crystals of insulin analog-Protamine, with the specified analogue insulin is a human insulin in which Pro at position B28 is substituted by Asp, Lys, Leu, Val or Ala, and Lys in position B is Lys or Pro human insulin, in which amino acids B28 - B30 deleterows; or human insulin, in which the amino acid B27 demeterova, provided when insulin is a AspB28- human insulin, the concentration of Protamine is less than 10% by weight.

13. Pharmaceutical composition for parenteral administration comprising a mixture of a solution of soluble insulin analogue containing analogue of human insulin, zinc and phenol derivative, and the crystals of insulin analog-Protamine, in which the weight ratio of the two components is from 1 : 99 to 99 : 1 analogue insulin crystals of insulin analog-Protamine, and wherein said insulin analogue is a LysB28ProW- human insulin.

14. Pharmaceutical composition for parenteral administration under item 12 or 13, in which the weights of Teroldego introduction by p. 14, in which the weight ratio of the two components is 50 : 50.

15. Pharmaceutical composition for parenteral administration by p. 14, which includes LysB28ProW- human insulin and crystals LysB28ProW- human insulin-Protamine.

17. Pharmaceutical composition for parenteral administration by p. 16, in which the weight ratio of the two components is 50 : 50, 75 : 25 or 25 : 75.

18. Pharmaceutical composition for parenteral administration, containing an insulin analog-Protamine, which includes a mixture of a solution of a soluble analogue of human insulin, including analogue of human insulin, zinc and phenol derivative, characterized in that essentially all soluble analogue is a review of the complex and the complex of the insulin analog-Protamine, in which the weight ratio of the two components is from 1 : 99 to 99 : 1 analog soluble insulin to the insulin analog-Protamine, with the specified analogue insulin is a human insulin in which Pro at position B28 is substituted by Asp, Lys, Leu, Val or Ala, and Lys in position B is Lys or Pro, human insulin, in which amino acids B28 - B30 deleterowpointer introduction, containing an insulin analog-Protamine, p. 18, in which the weight ratio of the two components is from 75 : 25 to 25 : 75.

20. Pharmaceutical composition for parenteral administration containing an insulin analog-Protamine, p. 18 or 19, which includes soluble AspB28review of the complexes and complexes AspB28- Protamine.

21. Pharmaceutical composition for parenteral administration, containing an insulin analog-Protamine, p. 18 or 19, which includes soluble LysB28ProWreview of the complexes and complexes LysB28ProW- Protamine.

22. Pharmaceutical composition for parenteral administration, containing an insulin analog-Protamine, according to any one of paragraphs.18, 19, 20 or 21, which contains crystals of a complex of an insulin analog-Protamine.

23. Pharmaceutical composition for parenteral administration, containing an insulin analog-Protamine, according to any one of paragraphs.18 to 22, in which a solution of a soluble analogue of human insulin consists of 0.35 - 0,09% Zn by weight.

24. Pharmaceutical composition for parenteral administration, containing an insulin analog-Protamine, p. 23, in which the analogue of human insulin is a AspB28B28ProW- human insulin.

26. The method of producing complex in any of paragraphs.1 - 5, including a connection of similar Monomeric insulin, Protamine, zinc, and phenol derivative in an aqueous solvent and curing the mixture to form a complex.

27. The method of obtaining crystals LysB28ProW- human insulin-Protamine, which includes the connection of an aqueous solution LysB28ProW- human insulin in a review of the associative condition and solution of Protamine at a temperature of 8 - 22oC, with specified an aqueous solution containing 0.35 to 0.9% zinc by weight, LysB28ProW- human insulin and phenolic derivative at pH of 7.1 and 7.6, and this solution contains Protamine Protamine at pH of 7.1 and 7.6, so that the final concentration of Protamine was 0,27 - 0,32 mg Protamine/100 Units of insulin analogue.

28. The method according to p. 27, in which the temperature is 15oC, the concentration of zinc is 0.7 to 0.9%, and the concentration of Protamine is 0.3 mg/100 Units of insulin analogue.

29. The method of obtaining crystals AspB28- human insulin-pretative state and solution of Protamine at a temperature of 13 - 17oC, with specified an aqueous solution containing 0.35 to 0.9% zinc by weight, AspB28- human insulin and phenolic derivative at pH of 7.1 and 7.6, this solution contains Protamine Protamine at pH of 7.1 and 7.6, so that the final concentration of Protamine was 0,27 - 0,32 mg Protamine/100 Units of insulin analogue.

30. A method of obtaining a pharmaceutical composition for parenteral administration by any and PP.6 - 17, which includes a suspension of crystals of insulin analog-Protamine in a pharmaceutically acceptable diluent.

31. Pharmaceutical composition for parenteral administration according to any one of paragraphs.6 - 25 for use in the treatment of diabetes.

 

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 molecular biology, in particular the production of insulin from predecessor
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.

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