CN104327163A

CN104327163A - Insulin amyloid polypeptide inhibitor, preparation method and application thereof

Info

Publication number: CN104327163A
Application number: CN201410509099.8A
Authority: CN
Inventors: 罗瑞雪
Original assignee: Suzhou Puluoda Biological Science and Technology Co Ltd
Priority date: 2014-09-28
Filing date: 2014-09-28
Publication date: 2015-02-04
Anticipated expiration: 2034-09-28
Also published as: CN104327163B

Abstract

Insulin amyloid polypeptide inhibitor, preparation method and application thereof. The invention relates to the field of drugs and particularly relates to an inhibitor 1 compound which can treat diabetes. A sequence of the inhibitor 2 is VLSVAALNHLDKATPIESH. A preparation method of the inhibitor 2 is carried out through Fmoc-protected solid-phase synthetic technology, with Rink amide MBHA resin and Fmoc-Gly-Wang resin as supporters, with 6-chloro-1-hydroxylbenzotriazole and N,N-diisopropyl carbodiimide as condensing agents, and with trifluoroacetic acid as a cutting reagent. The invention also provides an application of the inhibitor 2 for preventing or treating diabetes and complications thereof, wherein the complications comprise diabetic nephropathy, diabetic hypertension, diabetic eye diseases and diabetic neurogenic lesion. The inhibitor 2 can be employed for preventing or treating the diabetes and the complications thereof through various dosing methods, such as subcutaneous injection or intramuscular injection, intravenous injection or intravenous drip, and oral medication, such as pills, capsules, nasal spray agents and the like. The islet amyloid polypeptide inhibitor 2 can targetedly inhibit generation of islet amyloid polypeptide, thereby preventing or treating diabetes.

Description

AC2993 inhibitor and preparation method thereof, application

Technical field:

The present invention relates to pharmaceutical field, be specifically related to the polypeptide compound being used for the treatment of diabetes.

Background technology

The metabolic disease of diabetes to be one group with hyperglycemia be feature is a kind of syndromes of glucose, protein and the lipid metabolism disorders caused by the cellular metabolism effect defect of insufficient insulin or Regular Insulin.In recent years, the number of diabetic subject increases gradually, is divided into type i diabetes and type ii diabetes.Wherein the number of type ii diabetes patient accounts for the 85-90% of diabetes total number of persons, and the health and lives of patient in serious threat.If diabetes can not get good control, a lot of complication may be caused, such as: diabetic nephropathy, retinopathy, hypertension etc.

Recent research shows to send out in process in the disease of diabetes B, and ever-increasing beta cell apoptosis causes the major cause that beta cell fails and hyperglycemia occurs.At present, Most patients helps blood sugar in control volume by orally-taken blood sugar reducing medicine, but but cannot improve the activity of beta cell, and promote the secretion of Regular Insulin, a lot of patient finally still wants use of exogenous insulin.

Contact is there is between the sex change of islet amyloid sample and diabetes.The fiber yarn formation of islet amyloid polypeptide can cause beta cell dead by apoptosis, and amyloid deposition directly may can kill cell.Suppress islet amyloid polypeptide to be formed, the cytotoxicity of islet amyloid polypeptide to beta cell can be reduced, promote the secretion of Regular Insulin.Therefore, islet amyloid polypeptide suppresses the target spot thinking treatment diabetes.However, the islet amyloid polypeptide inhibitor of unripe exploitation comes out, for prevention or treatment diabetes.

Summary of the invention:

The present invention seeks to the feature for existing hypoglycemic medicine, design a kind of inhibitor 2, effectively can treat diabetes.

Technical scheme

A kind of islet amyloid polypeptide inhibitor 2, its sequence is VLSVAALNHLDKATPIESH.The preparation method of described inhibitor 2; it is characterized in that: the solid phase synthesis technique preparation adopting Fmoc protection; adopt the solid phase synthesis technique preparation of Fmoc protection; Rink acyl ammonia mbha resin and Fmoc-Gly-Wang resin are as carrier; the chloro-1-hydroxy benzo triazole of 6-and N; N-DIC is condensing agent, and trifluoroacetic acid is cutting reagent.The application of described inhibitor 2 in prevention or treatment diabetes and diabetic complication.Described diabetic complication is diabetic nephropathy, diabetic hypertension, diabetes eye illness, diabetic neuropathic pathology.Inhibitor 2, by the prevention of multiple administering mode or treatment diabetes and diabetic complication, comprises subcutaneous or intramuscular injection, intravenous injection or intravenous drip, oral administration as pill, capsule etc., nasal spray etc.

Beneficial outcomes:

Islet amyloid polypeptide inhibitor 2 in the present invention, can be formed by targeted inhibition islet amyloid polypeptide, reaches the effect of prevention or treatment diabetes.Be more preferably the purposes in treatment diabetes and/or diabetes complicated disease drug.Described diabetes comprise type i diabetes and type ii diabetes; Described diabetic complication includes but not limited to: diabetic nephropathy, diabetic hypertension, diabetes eye illness, diabetic neuropathic pathology etc.

Embodiment

Embodiment 1

The chemical synthesis process of polypeptide

The solid phase synthesis technique preparation that inhibitor 2 is protected with Fmoc.Building-up reactions is carried out to N end from C end according to inhibitor 2 sequence, and Rink acyl ammonia mbha resin and Fmoc-Gly-Wang resin have free amino group as on carrier (can buy in Advanced Chem Tech company).In each step connection procedure, amino-acid residue all will activate, and has the HBTU of 4 times of free amino groups on carrier in activator mixture, HOBt, DIEA and Fmoc-amino acid.With the chloro-1-hydroxy benzo triazole of 6-and N, N-DIC is condensing agent, carries out ligation, after each amino acid whose ligation, the free amino group do not connected is closed, capping 10 minutes with the mixture of pyridine/acetic acid/N-Methylimidazole (4:1:0.5).Before next amino acid connects, all the Fmoc-group on carrier to be removed, go Fmoc-group to use the dimethyl formamide containing 20% piperidines, need 15 minutes.Finally, after all amino-acid residues are linked in sequence, inhibitor 2 98% trifluoroacetic acid cuts down from carrier, and cutting at room temperature carries out 2 hours.

Apply above-mentioned electrochemical conditions can synthesize and obtain inhibitor 2, sequence is VLSVAALNHLDKATPIESH, and this sequence is brand-new sequence.Also the raw work synthesis in Shanghai can be entrusted.

Embodiment 2

The mensuration of the apoptosis in vitro of islet amyloid polypeptide inhibitor 2

This experimental study AC2993 inhibitor 2 suppresses the effect of insulinoma cell (INS-1 cell) apoptosis.After cell is with the process of 10 μm of ol/L Insulin-Like starch polypeptide, after TUNEL dyeing, find to have occurred a lot of green bright spot compared with medium controls in explanation system, to there is the cell of apoptosis.After 20 μm of ol/L AC2993 inhibitor 2 and AC2993 are hatched altogether, microscopy sees the green bright spot more less than the control group adding merely Insulin-Like starch polypeptide.To count taking pictures and after adding up, find to be about 65.0% with the apoptosis rate of 10 μm of ol/L Insulin-Like starch polypeptide process, and 20 μm of ol/L inhibitor 2 reduce to 30.2% (p<0.05) with the apoptosis rate of Insulin-Like starch polypeptide co-treatment.And apoptosis situation and the medium controls of the cell only processed with 20 μm of ol/L inhibitor 2 there is no significant difference, illustrate that inhibitor 2 significantly reduces the apoptosis of Insulin-Like starch polypeptid induction, but the inhibitor 2 of this concentration self do not have toxic action to cell.

Embodiment 3

Hypoglycemic experiment in the body of islet amyloid polypeptide inhibitor 2

Kunming mice, the high sugar of high fat is fed to body weight 18 ~ 22g, male and female half and half, and before experiment, water 24h is can't help in fasting, abdominal cavity disposable injection streptozotocin (STZ) 50mg/kg (citrate buffer of 1% dissolves preparation).After 1 week, blood sugar (BS) is surveyed in mouse tail vein blood sampling, and BS is modeling success higher than 16mmol/L.After modeling, mouse is divided into 3 groups, often organizes 10, diabetic model group (DM group): the PBS damping fluid subcutaneous injection giving equivalent; Control group (Regular Insulin): 20 μ g/kg subcutaneous administrations (being dissolved in 0.1mol/L PBS damping fluid), one day twice, continuous 9d; Sample sets (islet amyloid polypeptide inhibitor 2): same to control group; Separately get 10 normal mouses as blank group.After medication, 9d is in tail venous blood sampling Quick Measurement blood sugar.After 9d, in drug withdrawal after 1 week, fasting be can't help water and is carried out glucose tolerance test in 16 hours.By 25% glucose solution (2g/kg) gavage, after gavage, 0,30,120 minute tail venous blood sampling detects blood sugar.

Result: islet amyloid polypeptide inhibitor 2 has the effect (P<0.05, table 1) reducing diabetic mice blood sugar; Meanwhile, can the sugar tolerance (table 2) of diabetes-alleviating model mice.The effect of its inhibitor 2 is suitable with control group insulin action.

Table 1 islet amyloid polypeptide inhibitor 2 pairs of mouse hypoglycemic activities

* p<0.05, * * p<0.01 is compared with model group

Table 2 islet amyloid polypeptide inhibitor 2 pairs of glucose tolerance in mice experimental results

* p<0.05, * * p<0.01 is compared with model group

Conclusion: islet amyloid polypeptide inhibitor 2 pairs of diabetic mices have therapeutic action.

SEQUENCE LISTING

Pu Luoda bio tech ltd, <110> Suzhou

<120> AC2993 inhibitor and preparation method thereof, application

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> PRT

<213> artificial sequence

<400> 1

Val Leu Ser Val Ala Ala Leu Asn His Leu Asp Lys Ala Thr Pro Ile

1 5 10 15

Glu Ser His

Claims

1. an islet amyloid polypeptide inhibitor, is characterized in that: its sequence is VLSVAALNHLDKATPIESH.

2. the preparation method of inhibitor described in a claim 1; it is characterized in that: the solid phase synthesis technique preparation adopting Fmoc protection; Rink acyl ammonia MBHA resin and Fmoc-Gly-Wang resin are as carrier; the chloro-1-hydroxy benzo triazole of 6-and N; N-DIC is condensing agent, and trifluoroacetic acid is cutting reagent.

3. the application of inhibitor according to claim 1 in preparation prevention or treatment diabetes and diabetes complicated disease drug.

4. purposes according to claim 3, described diabetic complication is diabetic nephropathy, diabetic hypertension, diabetes eye illness, diabetic neuropathic pathology.

5. the purposes of inhibitor according to claim 4, it is characterized in that: prevent by multiple administering mode or treat diabetes and diabetic complication, comprising subcutaneous or intramuscular injection, intravenous injection or intravenous drip, oral administration as pill, capsule etc., nasal spray.