A complex containing an analogue of human insulin, and pharmaceutical composition based on it

 

(57) Abstract:

Describes the review of the new stable complex containing six molecules of 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 substituted by Pro; or des (B28-B30) human insulin; or des (B27) human insulin; two zinc ions and at least three molecules of phenol derivative selected from the group comprising m-cresol, phenol, or a mixture of m-cresol and phenol. He is Monomeric analogue of human insulin and is a review of the complex induced by zinc-phenol, which is rapidly absorbed, has a good stability against chemical degradation. Also described pharmaceutical composition based on it. 2 S. and 11 C.p. f-crystals, 3 ill.

The invention relates to Monomeric analogues of human insulin. More specifically, the invention concerns a review of the complex containing the insulin analogue, zinc and phenol derivative.

Since the introduction of insulin in 20-ies of XX century was made constant attempts to improve the treatment of diabetes. Great progress has been made in obtaining sufficient motrya these achievements, treatment with subcutaneous injection does not provide the patient's need for the regulation and control of glycemia. Frequent deviations from normal levels of blood glucose throughout the life of the patient lead to Hyper - or hypoglycemia and long-term complications, including retinopathy, neuropathy, nephropathy, and micro - and macroangioapthy.

To avoid extreme levels of blood glucose diabetics often practiced therapy reusable injection of insulin when insulin is assigned with each meal. However, this therapy has not yet improved. The fast-acting insulin, commercially available, reaches its maximum action too late after injection and its effect lasts too long for optimal glucose control. Recently made significant contributions to create insulin preparations and preparations of insulin analogue, which alter the kinetics of subcutaneous absorption.

Since all commercial pharmaceutical preparations contain insulin insulin in self-assembling condition and, as a rule, in the form of zinc-hexamer believe that limiting the rate of absorption of insulin from the subcutaneous depot injection into the blood stream bound dissocia the ECJ absorption, were developed Monomeric insulin analogs. These Monomeric analogues have relatively more rapid onset of activity than insulin, while retaining the biological activity of natural human insulin. They provide quick absorption and lead time of injection and the peak action of insulin in closer temporal compatibility level of glucose in the blood, associated with a reaction to the meal. Getting different Monomeric analogues disclosed in Chance and others, EPO publication N 383472, and Brange and others, EPO publication N 214826.

Unfortunately, modification of insulin, which make these Monomeric analogues, also give a high degree of polymer formation in parenteral drugs. Because the validity of insulin is impaired in the formation of a 1% polymer (USP, 1990). to minimize this degradation is very important in reducing unwanted side effects. Therefore, the desired Monomeric analogues that would have been self-assembling condition and formed stable information, while preserving the properties of rapid absorption.

Adding ions of some metals, mainly zinc, increase chemical stability, causing insulin associationassociation insulin and induce an allosteric conformational change, when eight N-terminal amino acids of the B-chain is converted from a stretched conformation in alpha-helix (Derewenda and others, Nature, 338: 594-596 (1989)). This state conformation with the phenolic communication known as Zn (II)-R condition.

In contrast to these established points of view that insulin is easily aggregated in the presence of zinc and forms a clear stable structure of Zn-hexamer, early studies of Monomeric insulin analogues revealed that any aggregation between zinc and insulin analogue is different from the aggregation observed with insulin (B. H. Frank. The text and slides of the lecture at the Conference on insulin "self-Association and study of the conformation of human proinsulin and insulin analogs", University of York (August 29 - September 1, 1989)). In addition, highly stable Zn-review of the complex formed with the insulin was not observed with Monomeric analogues. Id. Brems and other Protein Engineering, 5:6, 527-533 (1992), reveals that Monomeric LysB28ProB29-hI less prone to dimerization and self in the form of higher molecular weight than human insulin. Brems, etc. continues to conclude that AspB28ProB29-hI, AlaB28ProB29hI and LysB28ProB29-hI show little or no SUP> insulin induced demonstrate Zn Association, but less than that of Zn-insulin. In a subsequent unpublished experimental observations of the present inventors assume that the Association with zinc is observed; however, this Association between analogue and zinc other than insulin. The Association, which is observed with these analogues is due to the many forms of high molecular weight and non-dominant, well-defined hexamers Zn-insulin. Therefore, it is clear that Monomeric insulin analogs do not form a Zn(II)-T6conformation is similar to insulin.

Against the background of these publications present invention offers Monomeric insulin analogs in the form of a clearly defined, stable zinc-phenolic review of the complex. This review of the complex is completely different from the complexes with insulin under identical conditions. Insulin complexes with zinc and phenol have Zn(II)-R6conformation. Review of the complex of the present invention is not identical to this conformation. And that was very surprising, review of complex insulin analogue has a much greater propensity for dissociation than insulin. This tendency to dissociates broadcast is e fast stable monomers insulin and says, what is the obvious way to create a fast-acting insulin is preventing dimer formation or hexamer. Similarly, Brange and others in the Diabetes Care 13: 923-954 (1990) reveal that if insulin is administered as hexamer, besides the fact that it is more slowly undergoes free diffusion, hexamer should be more spatial difficult than the monomer, the movement in the subcutaneous layer and/or in its passage through the capillary membrane. In addition, when the hypodermic injection of Zn(II)-R6conformation is not dissociated directly, but must transfairusa through Zn(II)-T6conformation. These conformational changes and, consequently, the dissociation delay the beginning of the activity. Therefore, the expert in this area at that time understood that attempts to chemically stabilize the Monomeric insulin analogue zinc with the formation of a clear review of the complex will not be successful or, if successful, the action will not be quick.

This drug is a review of the complex induced by zinc-phenol, which is rapidly absorbed. The absorption rate review of the complex at least two times faster than the speed of insulin. And this review of the complex obscuritatem of the present invention, it converts the Monomeric insulin analog in a clear stable centenary review of the complex. It is noteworthy that during the formation of this review of the complex retains the fast-acting properties associated with Monomeric insulin analogue. Accordingly, the present invention provides parenteral drugs review of the complex analogue of insulin, which are stable and act quickly.

This invention offers a complex analogue of human insulin, which includes: six molecules of similar to human insulin, two zinc ions and at least three molecules of phenol derivative selected from the group consisting of: m-cresol, phenol, or a mixture of m-cresol and phenol; analog complex is hexameron. Further, the invention provides preparations of parenteral products, including review of the complex.

Fig. 1 is a graph of the profile actions LysB28ProB29hI and human insulin. The graph shows the average rate of saturation of glucose. The drawing demonstrates the advantages of the present invention.

Fig. 2 is a graph showing the stability LysB28ProB29human is then compared with the Monomeric LysB28ProB29human insulin and insulin. The drawing demonstrates the advantages of the present invention.

Fig. 3 is a graph showing the dissociation LysB28ProB29-human insulin in a review of the complex. The graph shows the in vitro dissociation molded in insulin preparations (a); LysB28ProB29-hI drug review complex (); nevorovannoe in insulin product (); and Monomeric LysB28ProB29-hI (*) under static light scattering at 488 nm at an angle of 90o. The samples contained 0.5 mol Zn per mole of protein. 1.25 mg/ml m-cresol and 1.09 mg/ml phenol, 7 mm of sodium phosphate and 16 mg/ml glycerol. Monomeric samples and samples neformalbnye in the preparation did not contain additional fillers. The drawing shows the advantages of the present invention.

As mentioned above, the invention provides Monomeric complex analogue of human insulin in the form of hexamer. The term "Monomeric insulin analog" or "analog of human insulin" here refers to human insulin, in which Pro at position B28 is substituted by Asp, Lys, Leu, Val or Ala; Lys at position B29 is a lysine or replaced by Proline;

des (B28-B30); or

des (B27).

is turned off here by reference. Monomeric insulin analogs are less prone to dimerization or self than insulin.

The specialist of this area is understood that other modifications. These modifications are widely accepted in the art and include the substitution of the histidine residue at position B10 aspartic acid; substitution of the phenylalanine residue at position BI aspartic acid; replacement of a residue of threonine in position B30-alanine; the substitution of the serine residue at position B9 aspartic acid; a deletion of amino acids only in the position B1 or in combination with a deletion at position B2; and a deletion of threonine in position B30.

All abbreviations of the amino acids used in this disclosure, adopted by the Office of the United States patent and trademark office, as set forth in section 37 of the Code of Federal regulations, 1.822 (b) (2). The preferred Monomeric insulin analog is LysB28ProB29-human insulin (B28 is Lys; B29 - Pro).

The term "treatment" here describes the manipulation and care of patients with the aim of combating the disease, condition or disorder and includes the purpose of the compounds of the present invention to prevent the onset of symptoms or complications, alleviating the symptoms or complications, or say physiologically tolerated and gives the desired toychest drug in order to prevent the current through the cell membrane. For such purposes, with known concentrations of commonly used compounds such as glycerin.

The term "phenolic derivative" or "phenolic" means m-cresol, phenol, or a mixture of m-cresol and phenol. It is desirable that it was a m-cresol.

The term "physiologically tolerable buffer known. Physiologically tolerable buffer, preferably a phosphate buffer such as sodium phosphate. Other physiologically tolerated buffers include TRIS, sodium acetate or sodium citrate. The choice and concentration of the buffer is known in the technical field.

The insulin analogs of the present invention complexesa with ions of zinc and derivatives of phenol to form stable review of the conformation. And zinc, and a derivative of phenol are important components in getting a complex that is stable and able to quickly dissociates and start acting. Review of the complex consists of two zinc ions on hexamer analogue of human insulin and at least three molecules derived phenol selected from the group consisting of m-cresol, phenol, or a mixture of m-cresol and phenol.

Soluble Monomeric analogue preobraze and a pH of about 7.5 and adding zinc. Zinc is mainly added as a salt. Examples of zinc salts include zinc acetate, zinc bromide, chloride, fluoride, iodide and sulphate of zinc. Specialist known for many other zinc salts, which can also be used in the method of the present invention. Preferably used zinc acetate or zinc chloride, as these salts do not add new chemical ions to commercially acceptable methods.

Dissolution of similar may contribute to what is commonly referred to as acid dissolution, i.e., the pH is reduced to about 3.0 to 3.5 physiologically acceptable acid, HCl better, which helps dissolving the Monomeric analogue. Other physiologically acceptable acids include acetic acid, citric acid and phosphoric acid. Then the pH is brought physiologically acceptable base, for example sodium hydroxide, up to about 7.4 and 7.5. Other physiologically acceptable bases include potassium hydroxide and ammonium hydroxide.

Review of the complex can be entered in a stable fast-acting parenteral drugs. The concentration of the insulin analogue in the product is about 0.5 mg/ml 20 mg/ml; about 1.2 mg/ml of 17.5 mg/ml; even better about 3.5 mg/ml total concentration of zinc ion zinc associated with each hexameron. In the molded product review of the complex binds to seven phenols. Usually when molding the drug six phenols contact hexameron. Accordingly, it is desirable to add to the drug excess phenol. Phenol acts as a preservative. Therefore, the preferred concentration is about 23 mm - 35 mm, better than 29 mm. Better if phenol is m-cresol.

The drug can be added as an isotonic agent glycerin. The concentration of the isotonic agent is the range known in the field of technology for preparations of insulin, preferably about 16 mg/ml. pH of the drug botherered physiologically tolerated buffer, preferably a phosphate buffer such as sodium phosphate.

Before the invention of the publication stated that the rapid absorption it is necessary to avoid aggregation. So unexpected was the fact that the review of the similar in the drug provides the beginning of an action. Unlike insulin, the drug review of complex insulin analogue has no negative effect on the time required to reach peak concentration of insulin analogue in serum. In Fig. 1 shows the average velocity saturation glucosmine LysB28ProB29- hI; and regular human insulin. The product review of the complex retains the quick action Monomeric LysB28ProB29- hI. The absorption rate is significantly higher than that of regular human insulin. Thus, the results in Fig. 1 shows: the first review of the LysB28ProB29hI and Monomeric LysB28ProB29hI have a similar rate of absorption; second, and review of, and Monomeric LysB28ProB29hI have a higher rate of absorption than insulin.

The drug, including the insulin analogue as hexamer stable. In comparative studies of Monomeric LysB28ProB29- hI shows the greatest degree of decomposition with an increase of 1.63% per week of polymer formation during the 6-week period. Human insulin is not formed in a drug undergoes the formation of the polymer to a lesser extent - 0,61% per week. However, in the preparation of the formation of high-molecular polymer is reduced to 0,095% for insulin per week. LysB28ProB29- hI in the preparation of a review of the complex shows a reduced formation of high molecular weight polymer of 0.11% per week, which sravnitel insulin of the present invention can be obtained from a variety of known technologies of synthesis of peptide, includes classical methods (dissolution), solid phase methods, semi-synthetic methods and the latest techniques of recombinant DNA. For example, Chance, etc., publication EPO N 383472, and Brange and others, the publication of the CIS countries N 214826, disclose the receipt of different Monomeric analogues.

The following examples and preparations are offered to further illustrate how insulin analogues and inventions. Scope of the invention should not be considered as consisting only of the following examples.

Preparative example 1

Obtaining mass protein

Neformalbnye in preparation samples of insulin and LysB28ProB29- hI was prepared with a concentration of 3.5 mg/ml in 7 mm sodium phosphate and 1.25 mg/ml m-cresol or without 1,09 mg/ml phenol and 16 mg/ml glycerol, depending on the conducted experiment. Samples LysB28ProB29- hI in the form of a review of the complex was prepared in an identical manner, except that there was added zinc, 19.7 mg/ml All samples passed through the acid phase to pH 3.0, when a series of compounds were added zinc. Then the pH was brought to 7.4. Protein concentration was determined before adding phenols UV absorption spectroscopy using a two-beam spectrophotometer AVIV, model 14 DS. the emer 1

Chemical stability

Decomposition was initiated by incubation of insulin and Monomeric and review of the LysB28ProB29- hI, molded and Nestorovich in drugs in the 30oC. Molded insulin and hexamer LysB28ProB29- hI, contained: 3.5 mg/ml protein, 16 mg/ml glycerol, 7 mm of dibasic heptahydrate, sodium phosphate, 1.25 mg/ml m-cresol, of 1.09 mg/ml phenol and 0,0245 mg/ml zinc oxide at pH of 7.3 to 7.4. Neformalbnye insulin and Monomeric LysB28ProB29hI contained: 3.5 mg/ml protein, 16 mg/ml glycerol, 7 mm of dibasic heptahydrate, sodium phosphate, 1.25 mg/ml m-cresol 1,09 mg/ml phenol at pH of 7.3 to 7.4. After 7 days the samples were removed from the incubation chamber and was estimated on the formation of species with high molecular weight using HPLC exceptions size. The analysis was carried out by the introduction of 20 ál of the samples in column Dupont Bond GF-250 Special (9.4 x 250 mm) using a mixture of 0.4 M ammonium bicarbonate and acetonitrile as eluent (flow rate 0.5 ml/min at ambient temperature and detection at 214 nm). Interest the formation of the polymer is determined from the ratio of the peak molecular weight to the total area of the monomer and high molecular weight peak. The results pokazanevProB29- hI, LysB28ProB29- hI in the form of a review of the complex and insulin were determined in vitro using static light scattering. As described above, was prepared three solution weight of a protein, molded and nevorovannoe in drugs, except that neformalbnye in drugs solutions weight of a protein does not contain zinc, glycerol or preservatives. Using these 3.5 mg/ml mass, was preparing a series of dilutions as for insulin, and LysB28ProB29- hI, with coverage of the protein concentration of 3.5 mg/ml to 0.2 mg/ml All dilutions were made up to a final volume of 10 ml of 7 mm buffer phosphate, pH 7,4, in order to simulate subcutaneous lesion after injection. All solutions were filtered through 0.2 μm filters Gelman low binding of the protein to perform measurements on static light scattering (SLS). The protein concentration for these samples was determined using chromatography with reversed phase.

For analysis of samples molded in drugs, a control sample with the solvent without protein were prepared for each series of protein samples. These control samples contained fillers in the same concentration as the corresponding series of protein samples. For the whether these matched control samples with solvent guaranteed, the data reflect only the dispersion of the solute and nothing more because of a change in solvent.

Experiments on static scattering was carried out using autocorrelator and goniometer Brookhaven Instruments 2030AT. All measurements were taken with an aperture of 1 mm under a scattering angle 90ousing ion laser argon Lexel model 3500 installed at 488 nm. The temperature was maintained at 25oC temperature bath Neslab RTE-110. The signal at the photomultiplier tube was calibrated using 0.1 ám core of toluene.

The average molecular weight was calculated from the equations described in Canter, C. R. & Schimmel, P. R., Biophysical Chemistry, W. H. Freeman and Company, new York, pp. 838 - 843 (1982). In Fig. 3 shows the results of the study of light scattering. In vitro profiles dissociation LysB28ProB29- hI in the form of a review of the complex and insulin completely different. The results of the insulin analogue show rapid dissociation, which provides faster absorption than human insulin. Even if both drugs include a review of the state Association and the drugs are equally stable to chemical degradation, hexamer LysB28ProB29- hI has balmalcolm analogue of 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 substituted by Pro; or des (B28-B30) human insulin; or des (B27) human insulin; two zinc ions and at least three molecules of phenol derivative selected from the group comprising m-cresol, phenol, or a mixture of m-cresol and phenol.

2. Complex p. 1, where the analogue of human insulin represents LysB28ProB29- human insulin.

3. Complex p. 1, where the analogue of human insulin is a AspB28- human insulin.

4. Complex under item 1, which is a sustainable hexamer clearly defined structure.

5. Complex p. 1, which exists in the form of a solution.

6. Complex under item 5, where the solution is characterized by its performance.

7. The complex PP.4, 5 or 6, where the analogue of human insulin is a AspB28- human insulin.

8. Pharmaceutical composition for parenteral injection, possesses activity that reduces the level of glucose in the blood having a high absorption rate and high stability with respect to chemical destruction, characterized in that it is 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 substituted by Pro; or des (B28-V) human insulin; or des (B27) human insulin; two zinc ions and at least three molecules of phenol derivative selected from the group comprising m-cresol, phenol, or a mixture of metacresol and phenol, in an effective amount.

9. The pharmaceutical composition under item 8, which additionally comprises an isotonicity agent and a physiologically acceptable buffer.

10. The pharmaceutical composition according to p. 9, characterized in that the analogue of human insulin is LysB28ProB29- human insulin.

11. The pharmaceutical composition according to p. 10, characterized in that it includes about 3.5 mg/ml LysB28ProB29- human insulin, about to 19.7 µg/ml of zinc, about 7 mm of sodium phosphate, about 16 mg/ml glycerin, and about 29 mm m-cresol.

12. Pharmaceutical composition for parenteral administration under item 8, characterized in that it includes a set under item 7.

13. The composition according to p. 12, characterized in that the concentration of the complex of the insulin analogue is 0.5 - 20 mg/ml, and the concentration of zinc is 10 to 50 μg/ml

 

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