CN1794995A - Use of tyrosine kinase inhibitor to treat diabetes - Google Patents

Abstract

The invention relates to the use of a c-Abl-. PDGF-R-, or c-kit- tyrosine kinase inhibitor, e.g. 4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino]phenyl]-benzamide, Bis (1H-2-indolyl)-1-methanones, AG1295, CT52923, RP-1776; GFB-111; pyrrolo[3,4-c]-beta-carboline-diones, SU 102, AG1296, RPR101511A, CDP 860, Zvegf3, CP673451, PD 170262, KI 6783, KN 1022, AG 13736, CHIR 258, MLN 518, SU 11248, Leflunomide, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of diabetes, e.g. type I diabetes, type II diabetes.

Description

The purposes of tyrosine kinase inhibitor in the treatment diabetes

Technical field

The present invention relates to 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl]-Benzoylamide (hereinafter being referred to as " Compound I ") or its pharmaceutically acceptable salt be used for the treatment of purposes in the pharmaceutical composition of diabetes such as type i diabetes or type ii diabetes in preparation, relate to Compound I or its pharmaceutically acceptable salt purposes in treatment diabetes such as type i diabetes or type ii diabetes, relate to the method for the treatment of the homoiothermic animal of suffering from diabetes such as type i diabetes or type ii diabetes, described homoiothermic animal comprises mammal, especially people, described method realizes by Compound I from the described disease effective dose of antagonism to the described animal of this class treatment of needs or its pharmaceutically acceptable salt of using.

Description of drawings

Fig. 1. in bTC-6 cell and isolated rat islets of langerhans, the NO of cytokine induction produces and not influenced by 10 μ M Compound I such as salt I.The result is the meansigma methods ± SEM of three independent observations.

Fig. 2. Compound I such as salt I are partly at nitric oxide protection human pancreatic island cell.The result is the meansigma methods ± SEM from three independent donors.

Fig. 3. the apoptosis speed of the bTC-6 cell of handling with mixed and disorderly siRNA or c-Abl-specific siRNA.Cytokine is handled (IL-1 β+IFN-γ+TNF-α) and was started from cell analysis preceding 24 hours.Apoptosis is undertaken quantitatively by flow cytometry.The result is the meansigma methods ± SEM of 3-4 observation.

Background of invention

American more than 1,000,000 suffers from type i diabetes, also is referred to as insulin dependent diabetes mellitus (IDDM) (being abbreviated as IDDM) or juvenile onset diabetes.In type i diabetes, people's pancreas produces and seldom or not produces insulin-a kind of necessary hormone that earns a bare living.Although reason is also not exclusively known, type i diabetes is multifactorial autoimmune disease, and it is to be caused by the specificity of the β cell of insulin-producing in the pancreas and carrying out property destruction.Spend one of the highest chronic disease childhood period that it being, and be the disease that people can't remove forever.Though insulin can make the people be survived, it can not cure diabetes, can not prevent its final and destructive result: renal failure, blind, nerve injury, amputation, heart attack and apoplexy.In order to survive, the people who suffers from type i diabetes must accept repeatedly injection of insulin every day or by pump infusion of insulin constantly, and must sting every day and refer to take a blood sample six times or more times measures their blood glucose.When making great efforts balance injection of insulin and its food ration, the people who suffers from type i diabetes also must prepare always potential hypoglycemia be hypoglycemia and blood glucose too high be that hyperglycemia is reacted, but these reaction life-threatenings.Although strict note keeping fit meals, workout scheme are also always injected an amount of insulin, but many other factorses can influence people's glycemic control unfriendly, this comprise stress, hormone variation, growth cycle, body movement, Drug therapy, illness/infection and fatigue.Even use insulin, type i diabetes also cause the rapid decline of quality of life usually, and make average life shorten 15 years.Annual about 30,000 Americans are diagnosed as suffers from type i diabetes, and wherein surpassing 13,000 people is the child.Just every day 35 children.

Type ii diabetes is also referred to as non-insulin-dependent diabetes mellitus (being abbreviated as NIDDM) or maturity-onset diabetes, and is relevant with the relative shortage of obesity, insulin resistance and insulin usually.Although the diabetes of this form are non-insulin requirements as a rule, exist surprising similarity between it and the type i diabetes.For example, now people are consistent thinks: also exist the absolute shortage of the β cell of insulin-producing in type ii diabetes, this beta cell lacks and is likely because the rate of death increase of beta cell causes.Therefore, generation may can be used as the treatment of I type and type ii diabetes at the pharmacological treatment of the protective effect of beta cell death.

Summary of the invention

Surprisingly, find c-Abl-, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor or its pharmaceutically acceptable salt for example Compound I or its pharmaceutically acceptable salt such as salt I be particularly useful for treating diabetes, as type i diabetes or type ii diabetes.Unexpectedly, find c-Abl-, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor or its pharmaceutically acceptable salt for example Compound I or its pharmaceutically acceptable salt such as salt I can be used for curing or prevent diabetes, as I type or type ii diabetes.

Compound I is 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl with following formula]-Benzoylamide:

Compound I free alkali, its acceptable salt and preparation thereof are open in European granted patent 0564409.The Compound I free alkali is equivalent to its active part.

Single added methanesulfonic acid salify of Compound I (hereinafter being referred to as " salt I ") and preferred crystal form thereof such as beta-crystalline form have description on January 28th, 1999 among the disclosed PCT patent application WO99/03854.

The present invention relates to c-Abl-, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor or its pharmaceutically acceptable salt purposes as anti-diabetic such as I type or type ii diabetes medicine.Most preferably, the present invention relates to Compound I or its pharmaceutically acceptable salt such as salt I purposes as anti-diabetic such as I type or type ii diabetes medicine.

Used c-Abl-, PDGF-R-, c-kit-or the ARG-tyrosine kinase inhibitor of the present invention is selected from the group that comprises following chemical compound: 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl]-Benzoylamide, it is called as Compound I in this article, be the inhibitor of a kind of PDGF-receptor isotype, Bcr-Abl and c-Kit, it is with efficient and oral availability and be celebrated; Two (1H-2-indyl)-1-ketone classes, promptly another kind of tyrosine kinase inhibitor, it has been characterized as being PDGF-R TK inhibitor, and as Mahboobi S etc., J.Med.Chem.2002 described in the 45:1002-1018, is incorporated herein by reference hereby; CAS is numbered the pdgf receptor kinase blocker AG1295 of 71897-07-9; AG1295/96, as Kovalenko M etc., Cancer Res.199454:6106-6114 and Ludewig D etc., Cell Tissue Res.2000 described in the 299:97-103, is incorporated herein by reference hereby; CT52923 (4-(6,7-dimethoxy-4 '-quinazolyl)-N-(3, the 4-methylenedioxy benzyl)-1-piperazine thioformamide); RP-1776; GFB-111; Pyrrolo-[3,4-c]-B-carboline-two ketone; SU 102 (by the SUGEN exploitation); CAS is numbered the AG1296 of 146535-11-7; RPR101511A by Aventis Pharma exploitation; CDP 860 and Zvegf3 by the ZymoGenetics exploitation; CP 673451 and PD 170262 from Pfizer; CAS is numbered the KI 6783 of 190726-45-5, and it is a kind of PDGF-R inhibitor by Japanese Kirin Brewery exploitation; KN 1022 by Japanese Kyowa Hakko and U.S. Millenium Pharmaceuticals exploitation; AG 13736 by the Pfizer exploitation; CHIR 258 by Chiron Corporation exploitation; From the MLN 518 of Millenium Pharmaceuticals with from the SU 11248 of SUGEN-Pfizer, leflunomide; Perhaps their pharmaceutically acceptable salts.

CT52923 has had description: Matsuno K etc. in following document, " the synthetic and structure activity relationship of pdgf receptor phosphorylation inhibitor-1 ", the 18th pharmaceutical chemistry seminar (18th Symposiumon Medicinal Chemistry), 25-27 day in November, 1998; The capital of a country, Japan, Japanese pharmacy association, pharmaceutical chemistry portion, Tokyo, Japan: summary 2-P-05.

RP-1776 is a kind of cyclic peptide, separates obtaining from the culture fluid of streptomyces strain (Streptomyces sp) KY11784.It is for example at Toki S, Agatsuma T etc., J.Antibiot. (Tokyo) May calendar year 2001; 54 (5): description is arranged among the 405-14.

GFB-111 is for example at Blaskovich MA etc., Nat.Biotechnol.2000 October; 18 (10): 1065-70 and Delarue F. etc., the 91st annual meeting (91 of american association of cancer research ^ThAnnual meeting of the American Association for Cancer research) 41:458 has description in 2000.

Pyrrolo-[3,4-c]-B-carboline-two ketone is for example at Teller S, Eur.J.Med.Chem.2000 April; 35 (4): description is arranged among the 413-27.

CDP 860 be derived from human anti--the PEGization antibody fragment of platelet derived growth factor beta receptor antibody.

CP 673451 targeting are in pdgf receptor.

PD 170262 or 2-[4-(2-diethylamino ethoxy) phenyl amino]-8-methyl-6-(3-thienyl) pyrido [2,3-d] pyrimidines-7 (8H)-ketone is potent tyrosine kinase inhibitor, and the platelet derived growth factor tyrosine kinase is had selectivity.Synthetic and the tyrosine-kinase enzyme inhibition activity of a series of 2-amino-8H-pyrido [2,3-d] miazines is for example at Klutchko S. etc., the meeting (213 of the 213rd country of american chemical association ^ThAmerican Chemical Society National meeting): summary MEDI 201 (placard), 1997, description is arranged in the U.S..

KI 6783 or 4-(3,4-dimethoxy phenoxy group)-6, the 7-dimethoxy-quinoline is for example at Kubo K. etc., Bioorganic and Medicinal Chemistry Letters 7:2935-2940,1997 and YagiM. etc., Exp.Cell Research 234:285-92 has description in 1997.

The KN1022 or 6 that suppresses the PDGFR phosphorylation, 7-dimethoxy-4 '-[4-(4-nitrobenzophenone) amino carbonyl piperazine-1-yl]-quinazoline are for example in the meeting of the 217th country of american chemical association: summary MEDI 061 part 1, and 1999, description is arranged in the Japan.

AG 013736 or N-methyl-2-[3-[2-(2-pyridine radicals) vinyl]-1H-indazole-6-base sulfonyl]-Benzoylamide is for example at Heller etc.; AG 013736, be the pharmacological activity of a kind of micromolecular inhibitor of VEGF/PDGFR tyrosine kinase; the 93rd annual meeting of american association of cancer research; 43:1082; 2002, open in the U.S..

CHIR 258 is orally active amino-benzimidazole quinoline growth factor kinase inhibitor, and it shows at receptor tyrosine kinase, for example from the inhibition activity profile of the receptor tyrosine kinase of PDGFR family.CHIR 258 is for example at Steigerwalt R etc. and Lee SH etc., the 94th annual meeting of american association of cancer research, and respectively at 753 (adding placard) summary 3783 and 934 (adding placard) summary R4702,2003, open in the U.S..

SU11248 or 5-[3-fluoro-2-oxo-1, the 2-indoline-(3Z)-and ylidenylmethyl]-2,4-dimethyl-1H-pyrroles-3-formic acid (2-diethylamino ethyl) amine is the inhibitors of kinases of many targets, and for example PDGFR is had selectivity.SU11248 is for example at Xin L. etc., the 93rd annual meeting of american association of cancer research, and 43:1081 (adding placard), 2002, open in the U.S..

MLN 518 is formula 4-[4-(N-right-isopropyl phenyl carbamoyl)-1-piperazinyl]-the quinazoline Piperazino derivs of 6-methoxyl group-7-(piperidino propyl group oxygen)-quinazoline; it suppresses for example PDGF R phosphorylation in conjunction with test; it is for example at Stone RM etc.; Blood 102:65-66; 2003, Kelly LM etc.; Cancer Cell 1:421-23 has description in 2002.

Leflunomide (SU 101) or 4-Isoxazolecarboxamidederivatives, 5-methyl-N-[4-(trifluoromethyl) phenyl] be tyrosine kinase inhibitor.

SU11654 suppresses the tyrosine kinase activity of c-kit.

Structure by the activating agent of code, common name or trade (brand) name sign can be obtained by the standard compilation " The Merck Index " of current edition or by data base such as Patent International (as IMS WorldPublication).Hereby its corresponding contents is incorporated herein by reference.

The invention still further relates to 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl]-Benzoylamide, two (1H-2-indyl)-1-ketone classes, AG1295, CT52923, RP-1776, GFB-111, pyrrolo-[3,4-c]-B-carboline-two ketone, SU 102, AG1296, RPR101511A, CDP 860, Zvegf3, CP 673451, PD 170262, KI6783, KN 1022, AG 13736, CHIR 258, MLN 518, SU 11248, leflunomide or its pharmaceutically acceptable salt purposes in the medicine of preparation treatment diabetes such as type i diabetes or type ii diabetes is preferably used Compound I or its pharmaceutically acceptable salt.

The invention still further relates to c-Abl-, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor or its pharmaceutically acceptable salt purposes in the medicine of preparation healing diabetes such as type i diabetes or type ii diabetes, preferably, described c-Abl-, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor are selected from the group that comprises following chemical compound: 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl]-Benzoylamide, two (1H-2-indyl)-1-ketone classes, AG1295, CT52923, RP-1776, GFB-111, pyrrolo-[3,4-c]-B-carboline-two ketone, SU 102, AG1296, RPR101511A, CDP 860, Zvegf3, CP 673451, PD 170262, KI 6783, KN 1022, AG 13736, CHIR 258, MLN 518, SU 11248, leflunomide or their pharmaceutically acceptable salts, preferred 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl]-Benzoylamide.

In this manual, term " treatment " had both comprised precaution or preventative treatment, comprised the treatment of healing property or disease inhibition again, comprise the patient that is among the risk of diabetes and patient's treatment.This term also comprises the treatment that is used to postpone progression of disease.

The applicant with " suppress and/or reverting diabetes " mean in the patient, no longer exist the diabetes state of an illness or described disease not as treatment before or serious when not having treatment.

Term used herein " healing " means treatment and causes diabetes or the outbreak of ongoing diabetes to alleviate.

Term " precaution " or " prevention " mean the outbreak or the recurrence of prevent diabetes.

The disease progression that term used herein " postpone progress " means when not using reactive compound is compared, and uses reactive compound and wards off disease and further develop or the advancing of disease of slowing down to being in prediabetes or early stage patient.

Pharmaceutical composition of the present invention can be prepared by known mode itself, and be to be suitable for through intestinal such as oral or rectum and to be applied to those compositionss of the homoiothermic animal that comprises the people through parenteral, it comprises at least a independent of treatment effective dose or with one or more pharmaceutically acceptable carriers, especially be suitable for through intestinal or through the pharmacologically active principles of the carrier combinations of parenteral application.The preferred route of administration of dosage form of the present invention is oral.

Therefore, the invention still further relates to the method that treatment suffers from the homoiothermic animal of diabetes such as type i diabetes or type ii diabetes, this method comprises Compound I or its pharmaceutically acceptable salt to the described animal administering therapeutic effective dose of this class treatment of needs.

The present invention relates to the acid-addition salts of the human individual's administered compound I that suffers from diabetes such as type i diabetes or type ii diabetes, preferred type i diabetes and preferred salt I, be 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl]-method of single mesylate of Benzoylamide.

Those skilled in the relevant art can select relevant test model to confirm the beneficial effect for diabetes such as type i diabetes or type ii diabetes that this paper is mentioned fully.The pharmacological activity of this chemical compound can be for example by hereinafter described embodiment, by in vitro tests and in vivo test or in suitable clinical study, prove.Suitable clinical study is open sign, derandominzation, the dose escalation study of for example carrying out in the patient who suffers from diabetes such as type i diabetes or type ii diabetes.In these researchs, for example estimate disease and determine curative effect based on the contrast that realizes with placebo by per 4 weeks.

The effective dose of Compound I can change according to the particular compound that is adopted or pharmaceutical composition, method of application, the diabetes type of being treated such as I type or II type or its order of severity.Dosage regimen is selected according to multiple other factors, comprises patients " renal function and liver function.The doctor of ordinary skill, clinician or veterinary can easily determine and open prevention, antagonism or stop the chemical compound of the required effective dose of disease progression.

Different according to age, individual state, method of application with related clinical setting, to the homoiothermic animal administered compound I of the about 70kg of body weight or the effective dose of its pharmaceutically acceptable salt, for example be equivalent to 100 to 1000mg, especially 800mg is as the daily dose of the free alkali of active part.Preferably, homoiothermic animal is behaved.For to the hyporeactive patient of daily dose, can consider to increase dosage safely, and as long as the patient by being benefited in the treatment and not having restrictive toxicity, just can treat the patient.

The invention still further relates to the human individual's administered compound I that suffers from diabetes such as type i diabetes or type ii diabetes or the method for its pharmaceutically acceptable salt, this method comprises that using the Compound I of pharmacy effective dose or its pharmaceutically acceptable salt once a day to the human individual reaches and surpass 3 months time.The invention particularly relates to such method, use to the adult wherein that daily dose is 400 to 800mg, the Compound I of preferred 800mg.

The present invention also provides a kind of medicated bag, it comprises c-Abl-, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor or its pharmaceutically acceptable salt, as Compound I or its pharmaceutically acceptable salt such as salt I, and the printed instructions about using to the patient who suffers from diabetes such as type i diabetes, type ii diabetes.

Do embodiment 1: Compound I such as salt I provide protection at beta cell death and diabetes?

In following embodiment, β-TC6 cell is meant the beta-TC6 cell.

Insulin-dependent (I type) diabetes (IDDM) are multifactorial autoimmune diseasees, and it is to be caused by the specificity of the β cell of insulin-producing and carrying out property destruction.Think that the dysfunction and the damage of beta cell are to soak into cell (macrophage, CD4 because of itself and islets of langerhans ⁺Or CD8 ⁺(NK) direct contact T-cell) and/or be exposed to the cytotoxicity medium that these cells produce is as proinflammatory cytokine (IL-1, TNF-α, IFN-γ), free radical, Fas part, TRAIL and perforin.At the autoimmunity of beta cell can by environmental factors such as beta cell toxin, nutritional labeling, stress, initiation such as metabolism over loading, virus.In type i diabetes, possible beta cell participates in the destruction of himself on one's own initiative by outside dead signal is converted into inner cell apoptotic event.The apoptosis and the necrosis of beta cell induced in the combination of proinflammatory cytokine, particularly IL-1 and IFN-γ.Therefore, can imagine that these cytokines not only regulate islets of langerhans and soak into activity of immune cells, and in the pathogenesis of type i diabetes, beta cell be applied direct illeffects.As if stimulate beta cell to cause a plurality of signal conduction incidents with IL-1, comprise the activation of protein kinase (PKC, p38, JNK, ERK, MSK1), lipase (PLC, PLD, sphingomyelinase), cyclo-oxygenase and transcription factor (NF-κ B, ATF-2, c-jun, Elk-1, CREB, cEBP-β, IRF-1, STAT-1).Albumen that is inducible nitric oxide synthase (iNOS) after these incidents and stress be relevant such as hsp70, Heme oxygenase, Mn-SOD, ICE etc. induce.Unfortunately, which change that it be unclear that in the gene expression is essential for the death of beta cell in the type i diabetes.Apoptosis mainly is that response ultraviolet light or DNA repair inhibition and generation in beta cell system.Take place that the p53 tumor suppressor protein is induced before this, the generation of reactive oxygen element, PARP inhibition, S-and G ₂-cell cycle is prevented and mitochondrial membrane potential descends.Cytokine mainly promotes necrosis, only promotes apoptosis on less degree, and its activation is repaired inhibition or the identical signal conduction step of ultraviolet light with DNA in fact.According to these experiment in vitro, obviously the dead mode of beta cell can change with the difference of dead signal, and similarly the signal conducting path can be used to realize multi-form beta cell death.

C-Abl is the protein tyrosine kinase of wide expression, and it has the molecular weight of about 145kDa.Under physiological condition, proved that c-Abl participates in regulating cytoskeleton function such as migration and cellularity and cell cycle progression.Yet, when cellular exposure in multi-form stress the time, the c-Abl highly activation that becomes, this causes cell cycle to be prevented and apoptosis.

In sum, the broad research of non-beta cell proved c-Abl to dissimilar stress response in promote apoptosis.The deduction effect of c-Abl in insulin-producing cells do not illustrated as yet.Whether therefore, it be unclear that this albumen plays a role in the selection between beta cell death and apoptosis and necrosis.

Meaning for type i diabetes.Although as if the pathogenesis of type i diabetes very complicated, might cause the interior path of born of the same parents of beta cell death on a certain specified point, to be converged.Imagination ground, this can only adopt a kind of approach to block a plurality of dead signals, realize the probability of beta cell survival thus for we provide.

Tyrosine kinase c-Ab 1 may be a kind of important mediators of beta cell death.C-Abl has expression in bTC-6 cell and isolated rat islets of langerhans, and no matter be to use the pharmacologically active agents Compound I still by knock out with the RNAi technology C-Abl express the C-Abl realized active suppress all to produce at proinflammatory cytokine or nitric oxide donors the protective effect of inductive beta cell death.C-Abl plays a role by promoting nitric oxide to produce, and can not resist the inductive nitric oxide generation of cytokine institute because suppress C-Abl.

Based on these data, the c-Abl conduct stress play a role with the sensor of outside dead signal, and the activation of c-Abl can cause JNK and p38MAP tyrosine phosphorylation and activation, NF-κ B and PI3K inactivation, mitochondrion to discharge short antiapoptotic factors and finally cause beta cell death.Compound I can be blocked different dead signals, realizes the survival of beta cell thus and at the protective effect of diabetes.

The result:In order to determine Compound I such as whether salt I intervenes or whether c-Abl participates in causing the signal transduction cascade of beta cell death, make the isolated rat islets of langerhans be exposed to the nitric oxide donors DETA/NO (0.5mM) of slow release or the combination that is exposed to IL-1 β (25U/ml)+IFN-γ (1000U/ml)+TNF-α (1000U/ml) reaches 24 hours.In both cases, between whole incubation period, all having or do not having under the condition of 10 μ M Compound I such as salt I and cultivate islets of langerhans.After incubation period, islets of langerhans lived with iodate third ingot (propidium iodide)+Bb (bisbenzimide) dye and under fluorescence microscope, take pictures.The percentage ratio of total cell number is counted and be expressed as to (the white shrinkage or the broken nuclear) of apoptosis and downright bad (red or pink broken nuclear) cell.Itself does not influence the viability of islet cells salt I such as Compound I.DETA/NO induces 10% islet cells necrosis, and combination of cytokines is induced about 40% necrosis.It is worth noting that NO donor combined significantly thing I of inductive cell death of institute such as salt I resist (table 1).Equally, the islet cells necrosis of cytokine induction is partly reduced by the c-Abl inhibitor.In all groups, the frequency of apoptotic cell was lower than for 10% (result does not show).

Table 1. Compound I such as salt I at NO-and cytokine the protective effect of inductive islet cells death.The result is the meansigma methods ± SEM of three independent observations.

Handle	No Compound I	10 μ M Compound I are arranged
			Contrast	1,4±0,3	3,2±0,9
DETA/NO	9,3±1,2	4,9±1,2 *
			IL-1+IFN-γ+TNF-α	41,6±5,5	32,8±3,2 *

Measured and come leisure to have or do not have the nitrite level of hatching 24 hours cell under the condition of 1 or 10 μ M Compound I such as salt I with above given combination of cytokines.The nitric oxide that Compound I such as salt I do not suppress cytokine induction produces (Fig. 1).On the contrary, the nitric oxide output under salt I exists is higher, may be owing to the cell viability that has contacted salt I is higher, compares with independent cytokine, and the cell that has contacted cytokine+Compound I discharges many 35% nitrite.

Tested Compound I and whether provide protection at causing the Rezulin streptozotocin external.Under the 0.4mM streptozotocin, described protective effect highly significant, and under the 0.75mM streptozotocin, Compound I only provides weak protective effect (table 2).Under the 0.6mM streptozotocin, the protection effect of Compound I is medium.

Table 2. Compound I such as salt I at streptozotocin the protective effect of inductive islet cells death

Handle	No Compound I	10 μ M Compound I are arranged
			Contrast	0,9±0,2	0,5±0,2
0.4mM streptozotocin	86,3±0,3	7,3±2,4 ***
			0.6mM streptozotocin	85,3±5,5	41,3±6,2 **
0.75mM streptozotocin	91±3,5	75,3±5,2 *

24 hours interpolation Compound I (10 μ M) before streptozotocin.Collect islets of langerhans and after adding streptozotocin, took pictures in 6 hours.The percentage ratio of total cell number is counted and be expressed as to (red or pink broken nuclear) cell of necrosis.The result is a non-viable non-apoptotic cell percentage ratio, is expressed as the meansigma methods ± SEM of three independent observations. ^{* *}, ^*With ^*The paired t-check of student is used in expression p＜0.001,0.01 and 0.05.

In order to study whether also regulating cell death in human pancreatic island cell of Compound I such as salt I, under the condition that has or do not have Compound I such as salt I (10 μ M), DETA/NO (2mM) and brefeldin A (10 μ M), hatch people's islets of langerhans.As viewed with rat Langerhans islet, it equally partly provides protection (Fig. 2) at the toxic level behaviour islets of langerhans of NO.Therefore, salt I partly resists the inductive islet cells death of brefeldin B (ER stress).

Whether regulate and control streptozotocin inductive diabetes of institute and/or Compound I such as salt I and whether also provide protective effect in order to study in vivo c-Abl, use following operation: from Taconic M ﹠amp at the inductive diabetes of streptozotocin; B, Sollentuna, Sweden buys the male NMRI mice of heavily about 25g.In whole research, animal can freely absorb tap water and granular food.Change bedding and padding weekly.Before experiment, measure body weight and blood glucose with Pen pick off (MediSense, Waltham, MA, the U.S.).(the-1,0,1 day) was dissolved in 0 with the 200mg/kg body weight to animal tube feed 200 microlitres in continuous three days, the Compound I of 9%NaCl such as salt I.At the 0th day, with the streptozotocin (Sigma-Aldrich Co, St.Louis, MO, the U.S.) of mouse tail vein injection 120 or 160mg/kg body weight.Before the injection streptozotocin is dissolved among 0,9% NaCl.Surveyed body weight at the 0th, 1,2,3,5,7,9 day and the blood sample of collecting from afterbody is surveyed blood glucose.Put to death animal at the 9th day by disconnected neck.All zooperies have all obtained the approval of local animal ethics committee (Tierp, Sweden).

The Compound I treatment fully provides protection (table 4) at the streptozotocin injection of 120mg/kg.In addition, Compound I such as salt I partly provide protection (table 3) at higher streptozotocin dosage (160mg/kg).

Table 3. Compound I such as salt I to the 160mg/kg streptozotocin the effect of inductive mice diabetes

My god	Saline	Compound I	STZ	The STZ+ Compound I
					-2	8,6±0,6	7,5±0,7	8,2±0,9	7,9±0,4
0	7,4±0,3	7,5±0,6	8,5±0,5	6,8±0,4 *
					1	7,6±0,3	8,5±0,8	10,3±0,7	8,0±0,3 **
2	8,5±0,3	9,0±0,5	20,2±1,6	11,7±0,7 ***
					3	8,5±0,4	9,1±0,4	20,7±0,5	13,5±1,3 ***
5	7,6±0,2	8,5±0,6	21±1,3	14,4±1,4 **
					7	7,7±0,3	8,5±0,5	25,6±0,9	18,1±2,1 **
9	8,7±0,2	9,0±0,4	27,2±0,4	20,0±2,4 **

At the-1,0 and 1 day the NMRI mice is filled out hello 200mg/kg Compound I by gavage once a day.At the 0th day, with injection 160mg/kg streptozotocin in the mouse vein, and each given in the drawings day detection blood glucose. ^*, ^*With ^{* *}Expression is used student t-check with respect to STZ p＜0.05,0.01 and 0.001.Observation frequency is 5 (saline and Compound I) and 10 (STZ and STZ+ Compound I).

Table 4. Compound I to the 120mg/kg streptozotocin the effect of inductive mice diabetes

My god	STZ	The STZ+ Compound I
			0	8,9±0,4	7,8±0,3
1	9,9±0,2	7,4±0,3 ***
			2	9,5±0,5	7,4±0,4 *
3	10,0±0,8	8,2±0,9
			5	12,7±1,5	9,0±0,9 *
7	12,6±1,2	8,8±0,6 *

At the-1,0 and 1 day the NMRI mice is filled out hello 200mg/kg Compound I by gavage once a day.At the 0th day, with injection 120mg/kg streptozotocin in the mouse vein, and each given in the drawings day detection blood glucose. ^*With ^{* *}Represent when comparing (student t-check) p＜0.05 and 0.001 respectively with corresponding STZ group.Observation frequency is 10.

Research design and method

1. identify that the path that suppresses by Compound I promotes the other dead signal of people's beta cell death And stressors.With 10uM salt I such as Compound I handler islet cells, and measure the islet cells death that responds following cytotoxic substance: hydrogen peroxide (150 μ M; Oxidative stress), staurosporin (200nM, PKC-suppresses), FCCP (5 μ M; Uncoupling and mitochondrial membrane permeability change), brefeldin A (10 μ M, ER stress), thapsigargin (thapsigargin) (200nM, ER stress with the Ca that increases ²⁺) and amycin (2 μ M, DNA damage).Dye succeeded by the fluorescence microscopy microscopy by iodate third ingot and Bb work and to come observation of cell death.Use 10 islets of langerhans of two groups.This operation make can to the apoptosis of complete islets of langerhans and downright bad both carry out quantitatively.Described dyeing technique alive and XTT detection method (the MTT detection method of reduced form) combination, this provides the Screening test method of simple and rapid islets of langerhans viability for us.

2. whether the research Compound I influences the development of diabetes in the type i diabetes animal model.As mentioned above, injection provides protection at the single dose streptozotocin for Compound I such as salt I.In order to extend this observed result, estimated the diabetes developing effect of c-Abl at the c57KSJ/ black mouse model of multiple dose streptozotocin processing.The Compound I treatment (the 200mg/kg Compound I in 200 μ l 0.9%NaCl) of every day starts from preceding 1 day of streptozotocin processing first (every day, low dosage 40mg/kg injection was 5 days), and continues 10 days or two weeks, this moment occurs obvious diabetes.By every day the measuring blood value estimate described treatment.After 10 days or two weeks, put to death mice, take out pancreas and fixing to carry out immunohistochemistry and the morphometry credit is analysed.To islets of langerhans inflammation and the score of beta cell amount.

Studied the importance of c-Abl in the palindromia of non-obese diabetes mice (being abbreviated as the NOD mice).Use above given similar scheme.But, in this case, give the Compound I treatment to the female NOD mice of diabetes.By gavage behind the administered compound I 1 day first, will be from young NOD mice isolating 300 islet transplantations under the scrotum.Measuring blood value and put to death mice after 7 days.Point at this moment, the immune cell destruction that most beta cells of transplanting have been activated, Compound I all is detectable to any deduction effect of beta cell survival.Reclaim graft and fixing to analyze.

The 3rd, studied the effect of Compound I to born diabetic duration in the NOD mice.Make the miniature osmotic pumps of the subcutaneous Alzet of acceptance of female NOD mice in age in 4-5 week (this moment not or have minimum insulitis), this osmotic pumps per hour discharges the dense Compound I of 0.25 μ l or independent excipient reached for 4 weeks.Mices are put to death in 4 week backs, take out pancreas and fixing to carry out immunohistochemistry and the morphometry credit is analysed.Degree and the score of beta cell amount to insulitis.

The pharmacology who considers c-Abl suppresses the problematic probability of possibility, has attempted the gene approach.For this purpose, from c57/KSJ black and NOD mice, separate islets of langerhans, disperse islet cells by trypsin treatment.Instruct the recombinant adenoviral vector transduction islet cells that c-Abl specific siRNA molecule transcribes with 5MOI.In contrast, use the adenovirus vector of the mixed and disorderly siRNA sequence of coding.Cell was reassembled 5 days external, be transplanted to afterwards under the scrotum of homology mice.Mice is treated and given as mentioned analyzing like that.

Consider inherent toxicity of adenovirus vector and immunogenicity, it may not be suitable for purpose in the body.Made up the AAV carrier of expressing same anti-c-Abl siRNA construct.When in external dispersion, about 30% pancreatic is by AAV transduction (result does not show).This is significantly less than the transduction efficiency that obtains with adenovirus vector, but enough high making can be estimated the c-Abl in the beta cell destruction in the body.

Method and apparatus

People's islets of langerhans.(glucose in RPMI1640 of 5.6mM+10%FCS) cultivate down by free-floating in the standard culture condition for people's islets of langerhans.

Flow cytometry and cell sorting.Use has the cell sorting ability and makes the green fluorescent protein that spends stable form as the sub flow cytometer (FACSCalibur of report, Becton-Dickinson) assess effectiveness easily, and the transfectional cell of sorting simultaneously is used for further experiment or transplants.Therefore, no longer must depend on the genetically modified insulinoma cell clone's of selected stably express generation (problem of clonal vaviation).Can use isolating transient transfection cell from non-transfected cells.In addition, flow cytometer also can be assessed cell viability (dyeing of iodate third ingot) and apoptosis (anti--activation caspase-3 antibody).In addition, flow cytometer also is used for the sorting of rodent beta cell, cell cycle analysis, mitochondrial membrane potential, oxygen-derived free radicals generation and immunofluorescence research (insulin, glucagon, Bcl-2).

Embodiment 2: the 4-[(4-methyl isophthalic acid-piperazine-1-ylmethyl that contains beta-crystalline form)-and N-[4-methyl-3-[[4-(3-pyrrole The pyridine base)-and the 2-pyrimidine radicals] amino] phenyl]-capsule of Benzoylamide mesylate

Contain the capsule that is equivalent to as the 119.5mg salt I of the 100mg Compound I (free alkali) of active part with following preparation of compositions:

Compositions:

Salt I Avicel PVPPXL Aerosil magnesium stearate	119.5mg 200mg 15mg 2mg 1.5mg
			338.0mg

By mixing each component and mixture being filled into this capsule of preparation in No. 1 hard gelatin capsule.

Embodiment 3: to the inductive protective effect mechanism at beta cell death and diabetes of Compound I in the body Research

Background:The molecular weight of a known compound I inhibition c-Abl-wide expression is about the protein tyrosine kinase of 145kDa.Under physiological condition, proved that c-Abl participates in regulating cytoskeleton function such as migration and cellularity and cell cycle progression.Yet, when cellular exposure in multi-form stress the time, the c-Abl highly activation that becomes, this causes cell cycle to be prevented and apoptosis.

The nearest result who obtains:C-Abl has expression in bTC-6 cell and isolated rat islets of langerhans, use the pharmacologically active agents Compound I suppress c-Abl active produce at by streptozotocin, proinflammatory cytokine or by nitric oxide donors the protective effect of inductive beta cell death.Compound I such as salt I are the selective depressants that is used for the treatment of CML clinically.Except c-Abl, known compound I suppresses ABL oncogene, c-KIT, PDGF beta receptor and c-Abl homologue ARG.Thereby the effect that is necessary to prove salt I is via the inhibition specificity mediation of c-Abl.Therefore has specific siRNA processing bTC-6 cell with mixed and disorderly siRNA or to c-Abl.Utilize Lipofectamine reagent that siRNA is introduced cell.Follow the trail of cell 1 or 3 days then, separate total RNA afterwards.By the synthetic cDNA of RNA and to utilize c-Abl (35 circulations) and b-actin be that beta-actin (20 circulations) has specific primer it is carried out pcr amplification.On agarose gel, separate the PCR product and observe by ethidium bromide staining.Handle at siRNA and to fail to observe c-Abl band (not shown) in the back 24 hours cell.Yet at 72 hours, c-Abl took out of existing.These results suggest knock out the courier at the siRNA of c-Abl via the mediation of RNAi mechanism, and this effect only is instantaneous (data not shown) in the bTC-6 cell of breeding rapidly.After determining that c-Abl mRNA level can be reduced by the RNAi technology, whether the bTC-6 cell that next we studied disappearance c-Abl mRNA increases the combination that responds IL-1 β, IFN-γ and TFN-α with cell death.Opposite with viewed situation in former generation islet cells, the bTC-6 cell is the death by apoptosis response cytokine preferentially.And the bTC-6 cell death of cytokine induction couple of days after processing is resisted (Fig. 3) forcefully by the specific siRNA of c-Abl.This shows that the proteic effect that causes siRNA to handle of may having enough to meet the need slowly of c-Abl postpones.But what is more important, Notes of Key Data SALT I inductive protective effect at NO donor and combination of cytokines suppress mediation by c-Abl.

Relation between c-Abl and the different map kinase: studied c-Abl and different map kinase p38, the JNK that may work as c-Abl downstream effect thing and relation between the ERK.For this purpose, rat Langerhans islet with 10 μ M Compound I preincubates 24 hours, is exposed to the combination 20 minutes of DETA/NO (2mM) and IL-1 β, IFN-γ and TNF-α then.With phosphoric acid specific antibody and immunoblotting islets of langerhans is carried out p38, JNK2 and ERK1/2 analysis of Phosphorylation then.The inductive p38 of DETA/NO, JNK and ERK activation are resisted in Compound I such as salt I treatment partly (25-45%), and strengthen the MAPK activation (table 5 and 6) of cytokine induction.

Table 5. Compound I such as salt I are to DETA/NO-and cytokine-inductive p38, JNK and the activatory effect of ERK

Handle	Phosphoric acid-p38	Phosphoric acid-JNK	Phosphoric acid-ERK
				Contrast	100±44	100±60	100±69
Compound I	109±39	213±93	174±94
				DETA/NO	654±213	360±47	708±167
The DETA/NO+ Compound I	497±208	207±53 **	482±162
				Cytokine	956±252	800±233	958±148
Cytokine+Compound I	1484±321	1250±210 *	1179±64

The isolated rat islets of langerhans with 10 μ M Compound I preincubates 24 hours, is exposed to DETA/NO or cytokine IL-1 β (50U/ml) and IFN-γ (1000U/ml) 20 minutes then.Measure the phosphorylation of ERK, JNK and p38 and be expressed as " total amount of/ERK, JNK and p38 " by immunoblotting.The result is the meansigma methods of 4 independent observations with the percentage expression of contrast. ^*With ^*2-ANOVA of unit and student t-check are used in expression p＜0.01 and p＜0.05 when the respective sets with Compound I compares respectively.

Table 6. recomputates from the result of table 5 so that the effect of Compound I is expressed as the percentage ratio of the respective sets of no any Compound I interpolation

Handle	Phosphoric acid-p38	Phosphoric acid-JNK	Phosphoric acid-ERK
				DETA/NO	100	100	100
The DETA/NO+ Compound I	76	57	67
				Cytokine	100	100	100
Cytokine+Compound I	155	156	123

These inductive nitric oxide generations of sustenticular cell factor as a result are at least in part via c-Abl path activation JNK and p38, and this causes beta cell death.On the other hand, as if active early stage rising of p38 that occurs in the response of the pair cell factor and JNK suppressed by c-Abl.Yet, in this case, might cytokine inductive p38 and active this first peak value representative of JNK cause the physiological responses that gene expression changes and propagation increases, and be not apoptosis itself.For example, shown that the cytokine activation of p38 and JNK participates in the follow-up expression of iNOS gene.Under this background, the p38 of present viewed c-Abl mediation and JNK suppress to have explained well that viewed nitric oxide generation increases in the islets of langerhans of handling with cytokine and Compound I.

Because these data, the c-Abl performance stress with the sensor effect of outside dead signal, the c-Abl activation can cause JNK and kinase whose phosphorylation of p38MAP and activation and finally cause beta cell death.

Additional experiment:

1. the expression of research c-Abl in beta cell.The expression of c-Abl mRNA can be assessed by PCR in real time.The c-Abl mRNA level of islets of langerhans and the c-Abl mRNA level of other tissue are compared.C-Abl is had specific fluorescent probe available from TIB MOLBIOLSyntheselabor (Berlin, Germany), with reference to c-Abl cDNA standard curve fluorescence signal is carried out quantitatively with the Lightcycler instrument.C-Abl mRNA molecular number is standardized as b-actin mRNA molecule.The c-Abl mRNA content of people's islets of langerhans and the c-Abl mRNA content of liver, muscle, kidney, spleen, brain and lung are compared.Abreast, to carrying out quantitatively with the expression of the c-Abl similarity SRCA RG that plays a role with the similar mode of c-Abl.Under this background, determined whether c-Abl mRNA level stress be influenced by cytokine, oxidative stress and ER.People's islets of langerhans is exposed to IL-1 β (25U/ml)+IFN-γ (1000U/ml)+TNF-α (1000U/ml), 100mM hydrogen peroxide or brefeldin A (10 μ M) 3 hours, analyzes the expression of c-Abl mRNA then by PCR in real time.

Research to beta cell stress response in c-Abl whether by phosphorylation.By with pCDNA3/c-Abl plasmid (available from Ludwig Institute, University of Uppsala) fat transfectional cell, carry out Geneticin (geneticin) resistance then and select to produce the stable bTC-6 cell line of expressing wild type c-Abl.To cross the bTC-6 cellular exposure of expressing c-Abl then in IL-1, brefeldin A and DETA/NO 20,60,360 minutes, with determine the pair cell factor, ER stress with c-Abl in the nitric oxide production response whether by phosphorylation.Then in the presence of inhibitors of phosphatases with cell homogenates, and utilize K-12 anti--c-Abl antibody (Santa Cruz) immunoprecipitation c-Abl.At PAGE with after transferring to nylon leaching film, with carrying out tyrosine phosphorylation and total tyrosine phosphatase fractional analysis at aminoacid 245 places to the c-Abl band by two phosphoric acid specificity c-Abl antibody (Tyr245 and Thr735) and the phosphotyrosine antibody 4G10 that Cell Signaling Technology obtains.C-Abl is active during by excessive phosphorylation increases as amino acid residue Tyr245 and Thr735.

3. whether the Subcellular Localization of research c-Abl stress influence by beta cell.On cover plate, cultivate the bTC-6 cell of expressing c-Abl, be exposed to cytokine, brefeldin A and nitric oxide donors 6 hours then.After fixing, sealing and saturatingization, pass through confocal microscopy microscopy analysis of cells with Mitotracker green (MolecularProbes) (it dyes green with mitochondrion) and K-12c-Abl antibody (Santa Cruz) (itself and yoke close two of rhodamine resist c-Abl is dyed redness).If c-Abl is repositioned to mitochondrion by ER, the pattern that then dyes becomes unique yellow by dispersive redness and green.

4. identify the downstream targets of c-Abl.For this purpose, produced the CbTC-6 cell that instantaneous mistake is expressed the c-Abl of wild type or constitutive activity form.With pcDNA3/c-Abl-carrier and GFP-expression vector fat transfection (Lipofectamine+Lipofectamine plus) bTC-6 cell.This produces the 20%GFP-positive cell, and it awaits being enriched to more than 75% by FACS.With the cell coated plate of sorting and cultivated 24 hours, carry out the analysis of Phosphorylation of candidate's target ERK, JNK, p38, I κ B, p53 and AKT (the downstream effect thing of PI3K) then.Analyze preceding 20 and 120 minutes, have or the condition of salt-free I such as Compound I under, with amycin (the nuclear activation of c-Abl) or DETA/NO (the endochylema activation of c-Abl) irritation cell.Utilize the phosphorylation (ser/thr) of the phosphoric acid specific antibody (CellSignal Technology) of commercially available acquisition by the target protein of immunoblotting assay deduction.Whether this can be disclosed in candidate's effector in the activatory response of c-Abl by phosphorylation and activation.Utilize traditional Western engram technology to analyze Bcl-2, Bcl-XL and Aph2 level.In some cases, the immunoprecipitation of candidate albumen is necessary sometimes, to improve the sensitivity of immunoblotting assay.For the phosphorylation of tyrosine-specific, with candidate's effector immunoprecipitation and utilize the anti-phosphotyrosine antibody of PY20 to analyze by immunoblotting.

5. the interaction between research c-Abl and the Shb:

Carry out the interaction that this experiment is intended to understand the deduction between c-Abl and the adaptin Shb.Shb is the albumen that contains the SH2 domain, it has the motif of proline rich at the N-end, have central PTB (phosphotyrosine combination)-domain, several potential tyrosine phosphorylation sites and the terminal SH2 domain of C-, known its produces in the signal conduction complex at response tyrosine-kinase enzyme activation and plays a role.It is worth noting that the apoptosis tendency of crossing the beta cell of expressing Shb increases.Really, when not having serum or existing when cultivating islets of langerhans under the condition of cytotoxic cytokines, express the transgenic mice that is in the SHB under the control of rat insulin promoter and show the apoptosis speed of rising.Because nearest report proof Shb family member Shd is in conjunction with c-Abl and interact with it, might be in beta cell c-Abl via playing a role with the interaction of Shb.In order to study this point, will be by transient transfection to cross expression c-Abl, the cell of Shb or Shb mutant is handled with pervanadate, then with anti--Shb-antibody mediated immunity precipitation.Then immunoprecipitate is carried out the tyrosine phosphorylation of immunoblotting with the Shb of analysis c-Abl and Shb level and c-Abl co-precipitation.Preliminary discovery shows c-Abl and Shb co-precipitation, and vice versa, and c-Abl crosses to express and causes the Shb-phosphorylation to increase (data not shown).These find to support that Shb is the viewpoint of c-Abl kinase substrate.

In order further to understand the interaction between c-Abl and the Shb, carried out fusion rotein pull-down experiment.GST, GST-ShbSH2 and GST-ShbPTB/ proline rich domain fusion rotein and the COS cell homogenates that contains high-level c-Abl are interacted.Described reaction is to carry out under the condition that has or do not have pervanadate to stimulate, and has been the importance of understanding c-Abl tyrosine phosphorylation and phosphotyrosine interpolation, has been understanding SH2-domain and the interactional importance of phosphotyrosine residue.By immunoblotting the pull-down product is carried out c-Abl and phosphoric acid-c-Abl analysis.These experiments show which domain of Shb is absolutely necessary for combining with non-phosphorylating or phosphorylation c-Abl.Using c-Abl fusion rotein (c-Abl-SH2 and c-Abl-SH3) to test to estimate which domain accordingly is vital for combining with Shb.

In order to determine whether the Shb-c-Abl interaction mediated the islet cells of expressing Shb apoptotic enhancing tendency takes place in the response that different toxicity are stimulated, from the Shb transgenic mice, separate islets of langerhans, and be exposed to cytokine, DETA/NO and streptozotocin, with or carry out pretreatment without Compound I.The pair cell apoptosis quantitatively also will be compared with the data of the control mice of parallel acquisition by the data that transgenic mice obtains with downright bad carrying out.If combined thing I of Shb islets of langerhans enhanced cell apoptosis and downright bad level such as salt I normalization might the Shb-c-Abl interaction may be necessary apoptosis regulation paths then.

Equipment: RNAi. carry out this research and be intended to utilize the RNAi technology to close gene expression in the beta cell.SiRNA (siRNA) with the price of every pair of RNA oligonucleotide 300-600 dollar available from Dharmacon Research.Utilizing the siRNA of FITC labelling to observe with Lipofectamine or Lipofectamine 2000 has been incorporated into siRNA in the cell of insulin-producing (result does not show) effectively.Unfortunately, the effect of the siRNA of liposome delivery is instantaneous.In order to produce reorganization gland-relevant (AAV) carrier, used AAV Helper-Free system (Stratagene).This test kit comprises the plasmid of the AAV-carrier that is used to produce expression β-gal that preliminary transfection efficiency institute uses.The work of carrying out with viral vector has obtained the approval of the Arbetarskyddstyrelsen of Swedish government mechanism.

PCR in real time.Lightcycler instrument (Roche) is a PCR in real time circulation instrument.This instrument is the quantification of mrna molecule fast and accurately, therefore is suitable for the research of gene expression.

Provide Confocal microscopy microscopy and electron microscopic microscopyTechnology.

Claims (10)

1.c-Abl-, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor or its pharmaceutically acceptable salt or the purposes of its pharmaceutically acceptable salt in the medicine of preparation treatment diabetes.

2.c-Abl-, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor or its pharmaceutically acceptable salt or its pharmaceutically acceptable salt cure purposes in the medicine of diabetes in preparation.

3.c-Abl-, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor or its pharmaceutically acceptable salt or the purposes of its pharmaceutically acceptable salt in the medicine of preparation prevent diabetes.

4. according to each purposes in the claim 1 to 3, c-Abl-wherein, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor or its pharmaceutically acceptable salt are selected from the group that comprises following chemical compound: 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl]-Benzoylamide, two (1H-2-indyl)-1-ketone classes, AG1295, CT52923, RP-1776, GFB-111, pyrrolo-[3,4-c]-B-carboline-two ketone, SU 102, AG1296, RPR101511A, CDP 860, Zvegf3, CP 673451, PD 170262, KI 6783, KN 1022, AG 13736, CHIR 258, MLN 518, SU 11248, leflunomide or their pharmaceutically acceptable salts.

5. according to each purposes in the claim 1 to 4, wherein diabetes are type i diabetes.

6. according to each purposes in the claim 1 to 4, wherein diabetes are type ii diabetes.

7. according to each purposes in the claim 4 to 6, wherein 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl]-Benzoylamide is the form of single mesylate.

8. treatment suffers from the mammiferous method of diabetes, this method comprises that the described people to this class treatment of needs uses the c-Abl-of the described disease effective dose of antagonism, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor or its pharmaceutically acceptable salt, described tyrosine kinase inhibitor is selected from the group that comprises following chemical compound: 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl]-Benzoylamide, two (1H-2-indyl)-1-ketone classes, AG1295, CT52923, RP-1776, GFB-111, pyrrolo-[3,4-c]-B-carboline-two ketone, SU 102, AG1296, RPR101511A, CDP 860, Zvegf3, CP 673451, PD170262, KI6783, KN 1022, AG 13736, CHIR 258, MLN 518, SU 11248, leflunomide or their pharmaceutically acceptable salts.

9. medicated bag, it comprises c-Abl-, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor and about to suffering from diabetes such as type i diabetes, the printed instructions that the patient of type ii diabetes uses, described tyrosine kinase inhibitor is selected from the group that comprises following chemical compound: 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl]-Benzoylamide, two (1H-2-indyl)-1-ketone classes, AG1295, CT52923, RP-1776, GFB-111, pyrrolo-[3,4-c]-B-carboline-two ketone, SU 102, AG1296, RPR101511A, CDP 860, Zvegf3, CP 673451, PD 170262, KI 6783, KN 1022, AG 13736, CHIR258, MLN 518, SU 11248, leflunomide or their pharmaceutically acceptable salts.

10. according to the medicated bag of claim 9, wherein c-Abl-, PDGF-R-, c-kit-or ARG-tyrosine kinase inhibitor are 4-(4-methyl piperazine-1-ylmethyl)-N-[4-methyl-3-((4-pyridin-3-yl) pyrimidine-2--amino) phenyl]-Benzoylamide or its pharmaceutically acceptable salt.