Peptides with continuare action

 

(57) Abstract:

The invention relates to medicine and can be used to treat hypoglycemia, in particular of hypoglycemic events, caused by increased blood levels of insulin endogenous or exogenous origin. For the correction of hypoglycemic events offered synthesized peptides of human insulin containing amino acid residues included in the binding site of the insulin with the cellular receptor, selected from the group of C - terminal peptide of a-chain of insulin (A12-A21), C - terminal peptides of the b-chain of human insulin containing amino acid residues 14-23, dimer, containing C-terminal peptide of a-chain (A18-A21) and C-terminal peptide of the b-chain (B17-B30) connected by a disulfide bridge, as well as mixtures thereof. The proposed tool has a more prolonged effect. table 2.

The invention relates to medicine and can be used to treat hypoglycemia, in particular of hypoglycemic events, caused by increased blood levels of insulin endogenous or exogenous origin.

In addition, the invention can be used in experimental medicine and biology experiments which cases, where it may be necessary continually effect.

Currently, worldwide there is an increase in emotional tension, stress. Among the stress caused diseases are important hypoglycemic events, which are called by lowering the level of glucose in the blood (less than 50 - 70 mg %) and have a different etiology. There are:

1) hypoglycemia caused by a functional deficiency of the adrenal cortex (asthenic state) in which the rate of gluconeogenesis - formation of glucose from neugeborne products (glucogenic amino acids) is significantly reduced;

2) hypoglycemia liver type associated with impairment of liver function;

3) hypoglycemia caused by increased excretion of glucose from the body (impaired resorption of glucose in the kidney);

4) hypoglycemia, caused by increased decomposition of glucose in the tissues (tumor of the islets of Langerhans, hyperplasia and hypertrophy of the islets of Langerhans, the transient hyperinsulinism and others) (Great medical encyclopedia. - 1977. - So 6. - S. 198);

5) hypoglycemia resulting from overdose of insulin, administered with a therapeutic purpose, such as diabetes or for the treatment of mental Zabol the sport of glucose from the blood into the tissues, the inhibitory effect of insulin on gluconeogenesis and glucosazone in the liver and kidneys, followed by a slower inflow of glucose from these bodies in the bloodstream, and when hypoglycemia renal origin - accelerated excretion of glucose from the blood into the urine (Great medical encyclopedia. - 1977. - So 5. - S. 482 - 483).

The above five types of hypoglycemia can be roughly grouped into two groups:

I. Hypoglycemia caused by insufficient glucose in the blood or rapid excretion from the body (1 - 3).

II. Hypoglycemia caused by increased transport of glucose from the blood into the cell due to high blood insulin exogenous or endogenous origin (4 - 5).

There are several tools and methods for the treatment of hypoglycemic events. Correction of hypoglycemia in the acute form can be performed by intramuscular or subcutaneous injection of glucagon (Mashkovsky, M. D. - 1993. - 1 o'clock. - S. 652) or glucagon and Amylin (U.S. Pat. 5234906 USA). In alimentary hypoglycemia, as well as disease Guirec shown adrenaline (1 ml 0.1% solution), which quickly mobilize liver glycogen, providing glucose in the blood (Most of the medicines whom (ibid; Mashkovsky M. D. Medicines. - 1993. - 2 hours. - S. 219).

The above methods for correction of hypoglycemic events have a temporary effect and is not directed at relief of hormonal effect of insulin. Their use can be effective in cases 1 - 3. In the cases 4 and 5, associated with a pronounced manifestation of hormonal effect of insulin, they are not efficient enough. In these cases it would be preferable to use proposed in this application the peptides with continuare action. They can be used both independently and in combination with the above means. For example, in the correction of hypoglycemic events by introducing glucose is very difficult to find an adequate dose of glucose if the patient has a high sensitivity of tissues to insulin. Put glucose is rapidly cleared from the blood into the cells under the influence of insulin and again there comes a hypoglycemic state. The introduction of glucose in conjunction with the proposed contentware peptides would be able to solve this problem.

In case 4, with the development of hypoglycemia on the background of the tumor process introduction one glucose is impractical because it stimulates tumor growth. the peptides, which is the subject of this application. Kontribusinya peptides in this case will have a cytotoxic effect.

A patent search was conducted by headings MKI5A 61 K 37/26 and From 07 To 7/40 - 7/42, and sections of the IPC6A 61 K 38/28 and From 07 To 14/62.

Analysis of patent documents identified in these sections showed that the resulting patent documents relate to the solution of the following tasks:

production and purification of insulin.

- obtaining analogs and derivatives of insulin;

- development of forms and methods of administering insulin in the body;

- insulin therapy (the treatment of diabetes, mental illness, etc.).

There are several patent documents, which describes the peptides are fragments of insulin (and.with. 1149603 the USSR, the application 89825-91 Australia, patent 915933 Finland). However, in all these cases, the task of creating peptides with hypoglycemic, i.e. insulin-like activity.

Also found the patent documents, where it is

"Oh hyperglycemic drugs (patent 5234906 USA)

- the method and product for treatment of diabetes and hypoglycemia (patent 5321008 USA).

In the first case is used with glucagon and the years of peptides - fragments of insulin, which would be proposed for the correction of hypoglycemic events.

The invention

The essence of the invention lies in the fact that for the correction of hypoglycemic events offered synthesized peptides of human insulin containing part of amino acid residues comprising the binding site of the insulin with the cellular receptor, and in the absence of insulin-like actions of the synthesized peptides manifest themselves as active competitors insulin for cell receptor, resulting in preventing the transport of glucose from blood into cells and inhibit the development of hypoglycemia. This mechanism is not used in any of the existing means of correction hypoglycemic state.

The structure of synthetic peptides

1. C-terminal peptide "10" a-chain of insulin:

S--L--Y--Q--L--E--N--Y--C -- N

12 13 14 15 16 17 18 19 20 21

2. C-terminal peptide "14" b-chain of insulin:

L--V--C--G--E--R--G--F--F--Y--T--P--K-T

17 18 19 20 21 22 23 24 25 26 27 28 29 30

3. C-terminal peptide "16" b-chain of insulin:

L--Y--L--V--C--G--E--R--G--F--F--Y--T--P--K-T

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

4. C-terminal peptide "23" b-chain of insulin:

G--S--H--L--V--E--A--L--Y--L--V--C--G--E--R--G--F--F--Y-

8 9 10 11 12 13 14 15 16 17 18 C-terminal peptide of a-chain (18 - AND 21)

< / BR>
where S - series, L - leucine, Y is tyrosine, Q - glutamine, E is glutamic acid, N - asparagine, cysteine, V - valine, R-arginine, F - phenylalanine, T - threonine, d - Proline, lysine, G is glycine, H is histidine, And alanine.

Insulin is a small globular protein with a known primary and tertiary structure consisting of two peptide chains. The a-chain of insulin contains 21 amino acid residue, b-chain of 30 amino acid residues. The binding site of the insulin receptor, according to literature data, consists of two contiguous in space of the surface of the insulin molecule. The first section includes the remains of the b-chain: Val-12, Tyr-16, Gly-23, Phe-24, Phe-25 and Tight-26. The second section includes the amino acid a-chain: Gly-1, Glu-4, Gln-5 and Asn-21. It is shown that the mutant insulin replacement Tight-B16 on His saves only 43% of the activity of native human insulin, and replacement of residue Phe-B24 on serine leads to a complete loss of binding effect.

This shows that the binding of insulin to its receptor is a complex and delicate process. Therefore, it was not clear whether to sit on the receptor and compete with insulin synthesized peptides containing part of amino acid residues comprising the binding site of the receptor, i.e. boudal, that offer really peptides inhibit the transport of glucose from the blood into the cells, acting as kontribusinya peptides.

The present invention corresponds, thus, the criterion of "inventive step".

Information confirming the possibility of carrying out the invention

Peptides were synthesized by solid-phase method. In the work of the applied L-amino acids with relocalising 2-(4-nitrophenyloctyl)ethoxycarbonyl (NSC) Nand-protecting group. As a side protection functions of amino acids were used: for Glu, Tight, Thr - tert-butyl ester; Arg - mesitylenesulfonyl group; for Cys - trailing; Lys - tert-butyloxycarbonyl; Asn - santinello protective group. The Assembly of the peptide chain was carried out in a manual synthesizer on the Wang resin (0.8 mmol/g of Oh-groups), using as a condensing agent THIEF - reagent Castro synthetic Protocol described previously (Samukov V. V. et al.//Tetrahedron Lett. 1994. V. 35. P. 7821-7824). The release and cleavage of the peptide from the polymer was carried out according to the method of Tamm (Tam J. P.//Org. Chem. 1985. V. 50. R. 5291-5298).

Purification of the peptides was carried out prepreparation reverse-phase HPLC using a chromatograph Pharmacia-LKB 2249 (Svezia%) in 0.1% triperoxonane acid over 60 min at a flow rate of 4 ml/min The volume fractions of 4 ml Fractions containing peptides with a purity above 90% according to analytical HPLC, were pooled, the solvent was removed under reduced pressure on a rotary evaporator at a bath temperature of 30oC. the Residue was dissolved in glacial acetic acid, filtered through a glass filter, liofilizirovanny. On prepreparation column was applied to 200 mg of the crude peptide. Analytical HPLC was performed on a column (4.6 x 150 mm) with sorbent Nucleosil 100 RP-18 (5 μm). Detection was carried out spectrophotometrically by absorption at 226 nm. The amino acid composition of the peptides was determined after hydrolysis in 6 BC HCL (110oC, 24 h; in sealed ampoules) on the amino acid analyzer Biotronik LC 5001(Germany).

Obtaining dimer. 10 mg of lyophilized peptide NYCN with a free sulfhydryl group was dissolved in 5 ml of degassed 50% acetic acid was added 15 mg of 2,2'-veridicality (Merck) and 3 h and stirred the reaction mixture without air. The reaction course was monitored by HPLC. Then the reaction mixture was applied on the column and NYC(Ps)N allocated prepreparation reversed-phase HPLC in the chromatographic conditions described above. The yield of dried product is 90%.

10 mg pept is, what about the data of HPLC, a new peak appeared. After complete disappearance of the original tetradecapeptide (5 h) octadecane with a disulfide bridge was isolated from the reaction mixture by HPLC. The fractions containing the target peptide with a purity of above 95% were pooled, solvent was removed under reduced pressure, the residue was dissolved in degassed water, filtered and liofilizirovanny. The yield of dried material per tetradecapeptide 60%.

All the resulting synthesis of the peptides had the correct amino acid composition.

The synthesized peptides were injected into mice (hybrid CBA x C Black) intraperitoneally in equimolar amounts. Mice were scored by decapitation after 30, 60, 90 and 120 min after drug injection. The sugar content in the blood was determined glucoseoxidase method using a set of reagents "Novolak" production "Vector-best" (Novosibirsk).

The results obtained are presented in table.1 and 2. From the presented data shows that all we synthesized peptides had no hormonal (proinsulin) effect (table.1), but all had continuare action, delaying the absorption of glucose from the blood under the influence of the hormone (PL.2).

Tool, obladayuschego peptide a-chain of insulin, comprising 10 amino acid residues (A12 - A21), C - terminal peptides of the b-chain of insulin, comprising amino acid residues 14-23, dimer containing joined by a disulfide bridge With the leaf tetrapeptide a-chain (A18 - A21) and C-terminal tetradecapeptide b-chain of insulin (B17 - B30), or mixtures thereof.

 

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