CN104001177B

CN104001177B - A kind of compound medicament composition for the treatment of type ii diabetes or metabolism syndrome

Info

Publication number: CN104001177B
Application number: CN201410271356.9A
Authority: CN
Inventors: 傅继华; 李涛; 郭可可; 曲伟; 李鑫; 安闪闪; 马少欣
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2014-06-17
Filing date: 2014-06-17
Publication date: 2016-01-20
Anticipated expiration: 2034-06-17
Also published as: CN104001177A

Abstract

The present invention relates to field of medicaments, be specifically related to a kind of compound medicament composition for the treatment of type ii diabetes or metabolism syndrome, more specifically, disclose the compound medicament composition of a kind of 5-hydroxy tryptamine 2 receptor antagonist Sarpogrelate and the combination of 5-hydroxy tryptamine synthetic inhibitor, compound medicament composition of the present invention can be used for improving insulin resistant thus treatment type ii diabetes and metabolism syndrome.

Description

A kind of compound medicament composition for the treatment of type ii diabetes or metabolism syndrome

Technical field

The present invention relates to field of medicaments, be specifically related to the compound medicament composition of 5-hydroxy tryptamine 2 receptor antagonist Sarpogrelate and the combination of 5-hydroxy tryptamine synthetic inhibitor, compound medicament composition of the present invention can be used for improving insulin resistant thus treatment type ii diabetes and metabolism syndrome.

Background technology

Insulin resistant (Insulinresistance, IR) " a variety of causes makes insulin promote cellular uptake and utilize the efficiency of glucose to decline; the supersecretion insulin of body compensatory produces hyperinsulinemia, to maintain the stable of blood glucose " this phenomenon is referred to.A people with homergy, insulin be on the feed after by the islet β cell in pancreas, insulin induction tissue in its transmission of signal donor, make surface of cell membrane produce glucose transporter (Glucosetransporter, GLUT) absorb glucose thus reduce blood sugar concentration.The main of IR is organized as liver, fatty tissue and skeletal muscle.When IR occurs cell, the insulin of normal level cannot induce these cells to produce enough GLUT, thus completes normal glucose absorption function.In order to compensate this, islets of langerhans need discharge more insulin, absorbs glucose to make enough cell GLUT be excited.Therefore the main cause of IR be " cell reduces the sensitivity of insulin, in insulin-induced cell on GLUT transporte to cells film thus increase glucose absorption ability decline ".The essence of its pathological changes is intracellular, and certain reason makes cell GLUT ability to express decline, and GLUT gene or protein expression decline, thus reduce the ability that cell absorbs glucose under insulin stimulating, thus occur IR.

Many reasons can cause IR, as insulin resistance hormone increase in obesity, long term hyperglycemia, high free fatty acid hyperlipemia, high glucocorticoid mass formed by blood stasis, gestation and body etc.But generally acknowledge, obesity causes the topmost reason of IR, especially to neutral fat; And high glucocorticoid mass formed by blood stasis, high free fatty acid hyperlipemia may be the immediate causes that obesity causes IR.The disease directly related with IR mainly contains two kinds: type 2 diabetes mellitus (Diabetesmellitustype2, DM-II) and metabolism syndrome metabolicsyndrome, MS).DM-II accounts for diabetics more than 90%, and the main manifestations of patient is exactly fat and IR, and body insulin lacks relatively, only has that sb.'s illness took a turn for the worse, patient DM-II in later stage is just because islets of langerhans is destroyed the absolute shortage occurring insulin.MS is the pathological state of multiple Metabolite abnormal aggregation, a complex set of metabolism disorder disease group, be the risk factor causing diabetes, cardiovascular and cerebrovascular disease (atherosclerosis, apoplexy, coronary heart disease), think that its collection bunch core occurred is IR at present.Clinically, the main standard of MS is diagnosed to have: abdominal obesity (namely to neutral fat), IR, hyperlipemia (hypertriglyceridemia, high low density lipoprotein and low hdl mass formed by blood stasis).Therefore, improve the sensitivity of body to insulin, reduce the first-selection that IR becomes treatment DM-II, MS.Biguanides (as metformin) and thiazolidinediones (as rosiglitazone and pioglitazone) are clinical conventional increase insulin sensitivities, improve the medicine of IR.

The most popular method that IR detects:

I .HOMA-IR index HOMA-IR index full name is " steady-state model insulin resistance index ", and this weighs the most frequently used method of body IR, and method is simple, conclusion is reliable.Measure fasting glucose and fasting insulin concentration, calculating HOMA-IR index (HOMA-IR=fasting glucose × fasting insulin ÷ 22.5), during experiment, determining IR by comparing with Normal group;

II. if glucose in urine quantitative determination blood sugar concentration enough high (exceeding renal glucose threshold), can detect glucose (this is the cause of diabetes title) in urine.In the urine that in unit interval, human or animal discharges glucose content number, the order of severity of IR and diabetes can be reflected, glucose in urine amount is higher show IR and diabetes more serious.

IR is actually the one performance of cellular metabolism disorder.During IR, carbohydrate metabolism, the lipid metabolism of body cell all get muddled, especially at adipose cell, hepatocyte and Skeletal Muscle Cell, the disorder of this energy metabolism is obvious especially, adipose cell is caused to secrete some hormonelike factor disorders (as leptin, phylaxin secretion increase, adiponectin secretion reduces), hepatocyte lipidosis, inflammatory factor produce to be increased, and Skeletal Muscle Cell is to the utilization ratio reduction etc. of glucose.But cause the essential reason of IR to be unclear at present, although find glucocorticoid, free fatty, some inflammatory factors (as tumor necrosis factor α) directly can cause cell to produce IR, suppress the cell inner expression of GLUT, they by what approach, how to change intracellular metabolic pathway and signal path thus cause IR? there is a kind of common action character? do not understand fully at present.Our research finds, glucocorticoid, free fatty cause there is common action character during IR: cause hepatocyte, adipose cell and the synthesis of Skeletal Muscle Cell 5-hydroxy tryptamine or expression of receptor to change; The IR that can block these factors and cause is changed by the 5-HT system of reverse both.

5-hydroxy tryptamine (5-hydroxytryptamine, 5-HT) be also serotonin, is a kind of small-molecule substance being present in periphery and maincenter, and the physiological function of 5-HT is complicated, does not understand fully completely so far.

The synthesis of I .5-HT in two steps, the first step: tryptophan is at tryptophan hydroxylase (tryptophanhydroxylase, TPH) (TPH can be divided into two hypotype: TPH1 and TPH2 to change 5-hydroxyryptophan under catalysis into, TPH1 is present in periphery, and TPH2 is present in maincenter); Second step: 5-hydroxyryptophan changes 5-HT under the catalysis of AADC (aromaticaminoaciddecarboxylase, AADC).Therefore, can realize respectively by suppressing TPH or AADC the suppression that 5-HT is synthesized.

Fenclonine (parachlorophenylalanine or p-chlorophenylalanine, pCPA), another name: DL-4-chlorophenylalanine, DL-fenclonine etc., molecular formula: C ₉h ₁₀clNO ₂, be TPH inhibitor, report without clinical practice.

Carbidopa (Cabidoba, CDP), molecular formula: C ₁₀h ₁₄n ₂o ₄.

Benserazide (Benserazide, BSA), another name: trihydroxy seryl hydrazine, Benseraside, Benseraside, benserazide, molecular formula: C ₁₀h ₁₅n ₃o ₅.

CDP and BSA is AADC inhibitor, clinical in parkinsonian auxiliary treatment, their combined effect feature is periphery decarboxylase inhibitor, not easily enter maincenter, periphery levodopa is only suppressed to be converted into dopamine, levodopa content in circulation is increased, 5-hydroxyryptophan also can be suppressed to be converted into 5-HT, make the cell of synthesis 5-HT produce 5-HT and reduce.CDP and BSA is also not used for improving IR, thus the research report for the treatment of DM-II or MS.

II .5-HT receptor 5-HT receptor (5-HTreceptor, 5-HTR) is present on cell membrane, belongs to membrane receptor, and 5-HT receptor subtype is complicated, has had now found that 7 large receptoroids exist, i.e. 5-HT _1-7r, 5-HT _{isosorbide-5-Nitrae, 5}r is mainly distributed in maincenter, 5-HT _2,3,6,7r is mainly distributed in periphery.This 7 receptoroid is broken into further again several hypotypes.5-HT ₂r can be divided into 5-HT _2Ar, 5-HT _2Br and 5-HT _2Cr.That periphery and maincenter all have distribution is 5-HT _{2A, 2B}r, 5-HT _2Cr is mainly present in central nervous system, and liver, skeletal muscle, visceral adipose tissue are distributed with 5-HT _{2A, 2B}r.There is selective exclusion 5-HT ₂the medicine of R is few, and known Sarpogrelate belongs to selectivity 5-HT ₂r antagonist.

Sarpogrelate (Sarpogrelate, SAR) is a species specificity 5-HT ₂r blocker, molecule: C ₂₄h ₃₁nO ₆.In clinical practice, SAR can suppress the platelet aggregation that strengthened by 5-HT and suppress vasoconstrictor effects etc., clinical in improving all symptoms of ischemic such as ulcer, pain and creeping chill caused by chronic arteria occlusion disease.There is although clinical SAR to treat the report of diabetic complication arterial obliterans of lower extremity, there is no the research report that can improve IR.

Summary of the invention

The invention discloses the compound medicament composition of a kind of 5-hydroxy tryptamine 2 receptor antagonist Sarpogrelate and the combination of 5-hydroxy tryptamine synthetic inhibitor, said composition has to work in coordination with to be improved insulin resistant thus can be used for treatment type ii diabetes and metabolism syndrome.

The described preferred fenclonine of 5-hydroxy tryptamine synthetic inhibitor, carbidopa or benserazide.

Preferred 15:1 ~ the 1:15 of weight ratio of Sarpogrelate and 5-hydroxy tryptamine synthetic inhibitor.

The weight ratio of Sarpogrelate and fenclonine is more preferably 12:1 ~ 1:2.

The weight ratio of Sarpogrelate and carbidopa is more preferably 9:1 ~ 1:2.

The weight ratio of Sarpogrelate and benserazide is more preferably 8:1 ~ 1:3.

We find under study for action:

I. all there is 5-HT synthesis system (TPH1, AADC) in hepatocyte and adipose cell.Glucocorticoid medicine--dexamethasone (dexamethasone, or fatty acid (oleic acid DEX), oleicacid, OA) stimulating when causing hepatocyte to produce IR (GLUT expression of receptor is lowered), also having that 5-HT synthesis system is raised simultaneously, 5-HT content increases in cell; Cause rat to occur in the experiment of IR at DEX, observe simultaneously liver and interior fat TPH1, AADC express is raised, 5-HT content raises, i.e. 5-HT synthesis increase in two kinds of tissues;

II. all there is 5-HT in hepatocyte, adipose cell and Skeletal Muscle Cell _{2A, 2B}r expresses, and Cultured Hepatocytes in vitro DEX or OA stimulates, or when IR animal model is set up in zoopery DEX stimulation, organizes 5-HT for three kinds _{2A, 2B}expression of receptor is all obviously raised, and synchronously occurs IR symptom.

III. during cell culture experiments in vitro, stimulate with DEX or OA and set up hepatocyte IR model, 5-HT synthetic inhibitor-pCPA, CDP, BSA, 5-HT ₂the GLUT gene expression that R blocker--SAR all can obviously suppress DEX or OA to cause is lowered, and SAR and pCPA or CDP or BSA coupling, then make GLUT be raised further, demonstrate cooperative effect.

IV. during animal experiment study, stimulate with DEX and set up IR animal model, form different compound recipes with SAR+pCPA, SAR+CDP, SAR+BSA by the weight proportion that two kinds of medicines are different and model is treated.Result shows, IR (hyperglycemia, high blood insulin concentration, the rising of HOMA-IR index that DEX causes, obvious glucose in urine content) obviously reversed, show as: blood glucose, blood insulin concentration reduce, HOMA-IR index reduces, and in urine, glucose content reduces.Further, the certain weight proportion of medicine pressed by SAR+pCPA, SAR+CDP, SAR+BSA tri-kinds of compound recipes, and demonstrate obvious synergism to treatment IR, drug combination can significantly improve curative effect of medication.Wherein, the drug weight ratio of SAR+pCPA compound recipe generation synergism curative effect is: during 12:1 ~ 1:2, effect is better; The drug weight ratio that SAR+CDP compound recipe produces synergism curative effect is: during 9:1 ~ 1:2, effect is better; The drug weight ratio that SAR+BSA compound recipe produces synergism curative effect is: during 8:1 ~ 1:3, effect is better.Further, selected SAR+pCPA compound recipe is that the compound effect of 4:1 is best with weight proportion; Selected SAR+CDP compound recipe is that the compound effect of 3:1 is best with weight proportion; Selected SAR+BSA compound recipe is that the compound effect of 2:1 is best with weight proportion.

In sum, with Sarpogrelate and 5-hydroxy tryptamine synthetic inhibitor as fenclonine or carbidopa or benserazide form compound recipe in certain weight ratio, there is obvious synergism to improving insulin resistant, can be used for the treatment of type 2 diabetes mellitus because insulin resistant causes or metabolism syndrome.

Compound medicament composition of the present invention is prepared into the dosage form of applicable Clinical practice by adding pharmaceutically acceptable carrier, as tablet, capsule, liquid preparation etc.Described pharmaceutically acceptable carrier is as disintegrating agent, diluent, binding agent, lubricant etc.

Below by embodiment, compound medicament composition of the present invention is done to the elaboration of further pharmacodynamic experiment result.

Accompanying drawing explanation

Fig. 1 is that DEX or OA is to Cultured Hepatocytes in vitro TPH1, AADC, 5-HT _2Ar, 5-HT _2Bthe impact (A) of R gene expression and DEX or OA stimulate impact (B) on Cultured Hepatocytes in vitro 5-HT content ( n=3)

Fig. 2 is the Cultured Hepatocytes in vitro GLUT2 down regulation of gene expression model caused at DEX, SAR, pCPA, CDP, BSA treat this downward of reversible, SAR and pCPA, CDP, BSA drug combination then strengthen this unkehr effect (A) further, at the hepatocyte GLUT2 down regulation of gene expression model that OA causes, this effect also exist (B) ( n=3)

Fig. 3 is when DEX causes hepatocyte 5-HT content to raise, this effect can be obviously suppressed with pCPA, CDP, BSA treatment, and SAR does not have inhibitory action, and SAR and pCPA, CDP, BSA administering drug combinations do not have synergism (A) yet, when OA causes hepatocyte 5-HT content to raise, also exist this effect (B) ( n=3)

Fig. 4 is that rat obviously raises (skeletal muscle have no TPH1, AADC express) (A) to liver, fatty tissue TPH1, AADC protein expression after DEX15 days continuously and rat obviously raises to liver, fatty tissue 5-HT content after DEX15 days continuously, when treating with pCPA, CDP, BSA then 5-HT content obviously reduce (B) ( n=6)

Fig. 5 is that rat is continuously to liver, fatty tissue, skeletal muscle 5-HT after DEX15 days _2Ar, 5-HT _2Br gene expression is obviously raised

Fig. 6 is that rat is obviously lowered to liver GLUT2, fatty tissue and skeletal muscle GLUT4 gene expression after DEX15 days continuously, obviously raises GLUT2, GLUT4 gene expression with SAR, pCPA, CDP, BSA treatment

Detailed description of the invention

Embodiment 1

Hepatocyte Cell culture invitro is studied

" hepatocyte 5-HT synthesis system, 5-HT _{2A, 2B}receptor Gene Expression, GLUT gene expression, the change when DEX or OA stimulates; And suppress 5-HT synthesis or block 5-HT _{2A, 2B}receptor (5-HT _{2A, 2B}r) to the improvement result of the IR that DEX or OA causes time " result of study.

1.1 experimental technique

(1) cell culture

Rat BRL-3A liver cell line In vitro culture, test passage 10-20 for time complete.Cell culture 37 DEG C, containing 5%CO ₂incubator in, with containing the PRMI-1640 culture medium culturing of 10% hyclone, and combine antibacterial with penicillin/lotus mycin.Cell implantation concentrations 5 × 10 ⁵individual/bottle, cultivated after 24 hours and can carry out administration when cell proliferation reaches 70-80% bottle floor space.Use dexamethasone (DEX) or oleic acid (OA) to stimulate respectively and set up IR cell model.DEX stimulates IR model administration grouping: matched group-normal incubation medium is cultivated, 30 μMs/LDEX is added in DEX stimulation model group-culture medium, pCPA group--add 30 μMs/LDEX and 25 μM/LpCPA in culture medium, 30 μMs/LDEX and 25 μM/LCDP is added in CDP group-culture medium, 30 μMs/LDEX and 25 μM/LBSA is added in BSA group-culture medium, SAR treatment group--add 30 μMs/LDEX and 25 μM/LSAR in culture medium, SAR and pCPA administering drug combinations group--add 30 μMs/LDEX and 25 μM/LSAR+25 μM/LpCPA in culture medium, SAR and CDP administering drug combinations group--add 30 μMs/LDEX and 25 μM/LSAR+25 μM/LCDP in culture medium, SAR and BSA administering drug combinations group--add 30 μMs/LDEX and 25 μM/LSAR+25 μM/LBSA in culture medium, OA stimulates IR model administration grouping: matched group-normal incubation medium is cultivated, 200 μMs/LOA is added in OA stimulation model group-culture medium, pCPA group--add 200 μMs/LOA and 25 μM/LpCPA in culture medium, 200 μMs/LOA and 25 μM/LCDP is added in CDP group-culture medium, 200 μMs/LOA and 25 μM/LBSA is added in BSA group-culture medium, SAR treatment group--add 200 μMs/LOA and 25 μM/LSAR in culture medium, SAR and pCPA administering drug combinations group--add 200 μMs/LOA and 25 μM/LSAR+25 μM/LpCPA in culture medium, SAR and CDP administering drug combinations group--add 200 μMs/LOA and 25 μM/LSAR+25 μM/LCDP in culture medium, SAR and BSA administering drug combinations group--add 200 μMs/LOA and 25 μM/LSAR+25 μM/LBSA in culture medium.All experimental grouies use lysate cell lysis after cultivating 48 hours, and gained solution can carry out further experiment, for intracellular matter content detection and PCR test.

(2) dosage

Dexamethasone (DEX)--30 μMs/L

Fenclonine (pCPA)--25 μMs/L

Carbidopa (CDP)--25 μMs/L

Benserazide (BSA)--25 μMs/L

Sarpogrelate (SAR)--25 μMs/L

Oleic acid (OA)--200 μMs/L

1.2 experimental index detection methods

(1) cell 5-HT content detection

Euzymelinked immunosorbent assay (ELISA) (ELISA) detects 5-HT content.

(2) TPH1, AADC, 5-HT _{2A, 2B}receptor, GLUT2 gene expression detect

Hepatocyte correlation factor gene expression: reverse transcription PCR detects TPH1, AADC, 5-HT2A, 2B receptor (5-HT _2Ar, 5-HT _2Br), GLUT2 gene (mRNA) is expressed.Wherein, hepatocyte GLUT is GLUT2.Primer:

THP1 forward primer 5'ACTGCGACATCAACCGAGAA3'

Downstream primer 5'GGCTAACCCTGACAGGAAAT3'

AADC forward primer 5 ' AGAACTGACCTAACCGAAGC3 '

Downstream primer 5 ' CAGACCAACCCGAGGATGAC3 '

5-HT _2Ar forward primer 5'GGATTTACCTGGATGTGC3'

Downstream primer 5'TGGATGGACCGTTGGAAG3'

5-HT _2Br forward primer 5'CAGCAGCAGAGGAAATGA3'

Downstream primer 5'ATCCAGGGAAATGGCACA3'

GLUT2 forward primer 5'GGTGTTGCTGGATAAGTTCA3'

Downstream primer 5'AGGATTCGAGTTAAGAGGGA3'

Internal reference GAPDH forward primer 5'TATCGGACGCCTGGTTAC3'

Downstream primer 5'TGCTGACAATCTTGAGGGA3';

1.3 experimental result

Between all data statistics detection groups, Student-t detects, and p<0.05 indicates obvious significant difference, and p<0.01 indicates clearly significant difference.Data means standard deviation in following all figure represent, N=3; * p<0.05, * * p<0.01 compares with matched group; $ p<0.05, $ $ p<0.01 and DEX or OA stimulates model group to compare; #p<0.05, ##p<0.01 drug combination compares with the alone medicine of SAR; & p<0.05, & & p<0.01 drug combination compares with the alone medicine of pCPC or CDP or BSA.

(1) DEX stimulation or OA stimulation cause there is obvious 5-HT during hepatocyte IR and synthesize increase and 5-HT2A, 2BR up-regulated

The results are shown in Figure 1.Visible, there is 5-HT synzyme TPH1, AADC gene expression in normal liver cell, and 5-HT content detected, also 5-HT detected _{2A, 2B}r gene expression; DEX stimulates or after OA stimulation, hepatocyte 5-HT synthesizes the increase of increase-5-HT content, TPH1, AADC, 5-HT _{2A, 2B}r gene expression is raised.Prompting hepatocyte is when DEX or OA stimulates, and 5-HT synthesis system is activated, 5-HT _{2A, 2B}r is raised.

(2) SAR and pCPA, CDP, BSA coupling have synergism to improving the hepatocyte IR that DEX or OA cause

Individually dosed DEX or OA that all can obviously reverse of SAR, pCPA, CDP, BSA stimulates the hepatocyte GLUT2 down regulation of gene expression caused; The curative effect of SAR and pCPA, CDP, BSA drug combination is more obvious, and demonstrate obvious synergistic function, the gene expression relative quantity of GLUT2 is close to matched group.The results are shown in Figure 2.

Raise the hepatocyte 5-HT content that DEX or OA causes, pCPA, CDP, BSA all have inhibitory action, and 5-HT content in hepatocyte is declined.SAR does not have this effect, and SAR and pCPA, CDP, BSA administering drug combinations do not have synergism yet.The results are shown in Figure 3.

Result shows, hepatocyte 5-HT synthesizes to be increased and 5-HT _{2A, 2B}r up-regulated is the common feature of DEX or OA when causing hepatocyte IR, may be the common cause causing IR; Synthesized by the 5-HT of T suppression cell, or by blocking 5-HT _{2A, 2B}receptor, can improve the hepatocyte IR that DEX or OA causes independently; When suppressing 5-HT to synthesize and blocking-up-HT simultaneously _{2A, 2B}during receptor, then there is synergism to the improvement effect of the hepatocyte IR that DEX or OA causes.

Embodiment 2

The rat IR that animal experiment study-DEX causes and diabetes model

Relatively SAR and pCPA, CDP, BSA administering drug combinations, and individually dosed therapeutical effect DEX being caused to rat IR and diabetes model separately, thus verify SAR and pCPA, CDP, BSA administering drug combinations cooperative effect to IR and treating diabetes.

2.1 experimental technique

(1) animal process

108 male Wistar rats are divided into 18 groups (often organizing 6) at random: matched group, IR model group, IR model SAR treatment group, IR model SAR+pCPA compound treatment group (SAR:pCPA is by 4 kinds of compound recipes of Different Weight proportioning), IR model pCPA treatment group, IR model SAR+CDP compound treatment group (SAR:CDP is by 4 kinds of compound recipes of Different Weight proportioning), IR MODEL C DP treatment group, IR model SAR+BSA compound treatment group (SAR:BSA is by 4 kinds of compound recipes of Different Weight proportioning), IR Model B SA treatment group.Animal is raised in cages in common mouse cage, and maintenance room temperature 24 ± 2 DEG C, illumination control to ensure that 12h is bright, dark condition, and light water is fed, successive administration 15 days.

In experiment, SAR+pCPA compound recipe by four kinds of compound recipes of Different Weight proportioning is:

SAR+pCPA-1—SAR:pCPA＝12:1；SAR+pCPA-2—SAR:pCPA＝4:1；

SAR+pCPA-3—SAR:pCPA＝1:1；SAR+pCPA-4—SAR:pCPA＝1:2。

In experiment, SAR+CDP compound recipe by four kinds of compound recipes of Different Weight proportioning is:

SAR+CDP-1—SAR:CDP＝9:1；SAR+CDP-2—SAR:CDP＝3:1；

SAR+CDP-3—SAR:CDP＝1:1；SAR+CDP-4—SAR:CDP＝1:2。

In experiment, SAR+BSA compound recipe by four kinds of compound recipes of Different Weight proportioning is:

SAR+BSA-1—SAR:BSA＝8:1；SAR+BSA-2—SAR:BSA＝2:1；

SAR+BSA-3—SAR:BSA＝1:1；SAR+BSA-4—SAR:BSA＝1:3。

(2) dosage

Matched group-subcutaneous injection (s.c.) normal saline 0.50ml/kg/ time, and oral administration (p.o.) 0.5% sodium carboxymethyl cellulose (CMC-Na) solution 5.0ml/kg/ time;

IR model group-s.c.DEX0.75mg/kg/ time, and p.o.CMC-Na5.0ml/kg/ time simultaneously;

IR model drug treatment group-s.c.DEX0.75mg/kg/ time, and p.o. respectively organizes medicine simultaneously, all administration group dosage homogeneous phases are all: 20.0mg/kg/ time.

Each medicine 0.5%CMC-Na suspendible, concentration is identical to be: 4.0mg/ml; DEXs.c. administration volume is 0.50ml/kg/ time, and each group medicine p.o. administration volume is 5.0ml/kg/ time; Every day is administered twice, and morning and afternoon is respectively administered once.

(3) HOMA-IR Index for Calculation

Successive administration the 15th day, each treated animal first fasting (can't help water) 12 hours, then gets blood, surveys fasting glucose, fasting insulin concentration, calculates HOMA-IR index (HOMA-IR=fasting glucose × fasting insulin ÷ 22.5).

(4) glucose in urine content detection

Successive administration the 15th day, fasting about 7 hours before last administration, then each group gives DEX and relative medicine on request respectively, rapidly animal is put into metabolic cage after administration, freely drink water, after collection administration, urine volume in 5 hours, measures concentration of glucose in urine, glucose content in urine in calculating 5 hours.

Successive administration animal fasting 12 hours after 15 days, anesthesia, gets blood, gets liver, gets visceral adipose tissue and gets huckle skeletal muscle, to detect serum and to organize related biochemical indicator.

2.2 experimental index detection methods

(1) serum and biochemical Indexs measure

ELISA method detects serum, liver, fatty tissue 5-HT level, and ELISA method detects serum insulin concentration, spectrophotometry blood sugar concentration, glucose in urine content.

(2) GLUT2, GLUT4,5-HT2A, 2B Receptor Gene Expression detects

Liver, fatty tissue and the gene expression of skeletal muscle correlation factor: reverse transcription PCR detects GLUT2, GLUT4 gene (mRNA) and expresses.Wherein, GLUT2 is present in hepatocyte, and GLUT4 is present in adipose cell and skeletal muscle.5-HT _2Ar, 5-HT _2Br, GLUT2, the same cell experiment of internal reference GAPDH primer thing, GLUT4 primer is as follows:

GLUT4 forward primer 5'TCCAGGATGAAGGAAACAGC3'

Downstream primer 5'GCGAGGCAAGGCTAGATTTT3'.

(3) TPH1, AADC protein expression detects

Liver, fatty tissue and skeletal muscle TPH1, AADC protein expression detect: Westernblot method.Adopt epicyte protein extraction agent box to extract epicyte protein, after being separated by the albumen of SDS-PAGE gel electrophoresis to different molecular weight size, electricity turns by the protein delivery of molecular weight on pvdf membrane, and transferring film terminates to carry out closed 2 hours to film afterwards.Take out pvdf membrane, use TPH1, AADC, ATP1A1 protein antibodies (primary antibodie) to hatch pvdf membrane respectively, spend the night in 4 DEG C; With corresponding Radix Cochleariae officinalis enzyme labelling two is anti-, 2 hours are hatched to pvdf membrane again.After adding chemical luminescence for liquid reaction, pvdf membrane is put into Tanon5200 imaging system and carry out exposure imaging detection.

2.3 experimental result

Between all data statistics detection groups, Student-t detects, and p<0.05 indicates obvious significant difference, and p<0.01 indicates clearly significant difference.Data means standard deviation in following all figure represent, N=6; * p<0.05, * * p<0.01 compares with matched group; $ p<0.05, $ $ p<0.01 compares with DEX model group; #p<0.05, ##p<0.01 drug combination compares with the alone medicine of SAR; & p<0.05, & & p<0.01 drug combination compares with the alone medicine of pCPC or CDP or BSA.

(1) DEX causes in rat IR model, the change of liver, interior fat and skeletal muscle 5-HT synthesis system-TPH1, AADC protein expression

The results are shown in Figure 4.Visible, there is TPH1, AADC protein expression in normal rat liver, interior fat, but skeletal muscle has no expression; When DEX causes rat IR and diabetes, the protein expression of liver and fatty tissue two kinds of enzymes obviously raises.Correspondingly detect that in two kinds of tissues, 5-HT content obviously raises, after suppressing 5-HT synthesis with pCPA or CDP or BSA, 5-HT content obviously reduces.

(2) DEX causes in rat IR model, the change of liver, interior fat and skeletal muscle 5-HT2A, 2BR gene expression

The results are shown in Figure 5.There is 5-HT in normal rat liver, interior fat and skeletal muscle _{2A, 2B}r gene expression; When DEX causes rat IR, organize 5-HT for three kinds _{2A, 2B}r gene expression is obviously raised.

(3) suppress 5-HT synthesis or block 5-HT2A, 2BR, DEX being caused to the impact of rat IR model liver, interior fat and skeletal muscle GLUT gene expression

The results are shown in Figure 6.Cause in rat IR model at DEX, liver GLUT2, interior fat and skeletal muscle GLUT4 gene expression are obviously lowered; Obviously GLUT2, GLUT4 is raised with SAR, pCPA, CDP, BSA treatment.

(4) SAR+pCPA compound recipe is to the therapeutical effect result of study of the IR that DEX causes

The results are shown in Table 1.DEX causes in IR model, and animal fasting plasma glucose concentration, Fasting insulin concentration all obviously raise, and HOMA-IR index obviously raises, and the urine of collecting discharge in 5 hours can detect a large amount of glucose content, and serious IR and diabetic symptom appear in prompting.With the SAR of identical dosage, pCPA, SAR+pCPA tetra-kinds of compound recipes: SAR+pCPA-1, SAR+pCPA-2, SAR+pCPA-3, SAR+pCPA-4 treat the IR that DEX causes, all obviously can reduce blood sugar concentration, blood insulin concentration and HOMA-IR index, and obviously reduce the glucose in urine amount of discharging in 5 hours; Compound recipe SAR+pCPA-2, SAR+pCPA-3 treatment all shows its blood sugar lowering, falls blood insulin, reduces HOMA-IR index and reduces the effect of glucose in urine amount treats separately significantly better than SAR or pCPA, and prompting produces obvious cooperative effect; The blood sugar lowering that compound recipe SAR+pCPA-1 treats, fall blood insulin, reduce HOMA-IR index and reduce the effect of glucose in urine amount and treat significantly better than pCPA, do not have difference also to have better curative effect trend although treat to compare with SAR; What compound recipe SAR+pCPA-4 treated fall blood insulin and reduce the effect of glucose in urine amount treats significantly better than pCPA, blood sugar lowering, reduces the effect of HOMA-IR index and also demonstrates and have better curative effect trend than pCPA treatment, and its curative effect and SAR treat quite.Result is pointed out, and SAR and pCPA forms a certain proportion of compound recipe, and have obvious synergism to treatment IR, by weight ratio, the proportion of SAR:pCPA is: 12:1 ~ 1:2, best with 4:1 compound recipe (SAR+pCPA-2) curative effect in this experiment.

Table 1 rat is given after DEX15 days continuously, and with after SAR, SAR+pCPA tetra-kinds of compound recipes, pCPA treatment, fasting plasma glucose concentration, fasting insulin concentration, HOMA-IR index and glucose in urine amount testing result in 5 hours

Note: each administration group dosage is identical, is 20.0mg/kg. time, one day twice, and morning and afternoon respectively once

$ $: P<0.01, compares with matched group; *: P<0.05, * *: P<0.01, compare with model group;

#:P<0.05, ##:P<0.01, compare with SAR group; &: P<0.05, & &: P<0.01, compares with pCPA group

(5) SAR+CDP compound recipe is to the therapeutical effect result of study of the IR that DEX causes

The results are shown in Table 2.With the SAR of identical dosage, CDP, SAR+CDP tetra-kinds of compound recipes: SAR+CDP-1, SAR+CDP-2, SAR+CDP-3, SAR+CDP-4 treat the IR that DEX causes, all obviously can reduce blood sugar concentration, blood insulin concentration and HOMA-IR index, and obviously reduce the glucose in urine amount of discharging in 5 hours; Compound recipe SAR+CDP-2, SAR+CDP-3 treatment all shows its blood sugar lowering, falls blood insulin, reduces HOMA-IR index and reduces the effect of glucose in urine amount treats separately significantly better than SAR or pCPA, and prompting produces obvious cooperative effect; Falling blood insulin and reducing the effect of glucose in urine amount of compound recipe SAR+CDP-1 treatment is treated significantly better than CDP, reduce the effect of glucose in urine amount also significantly better than SAR treatment, although blood sugar lowering, reduce the effect of HOMA-IR index and treat separately to compare with SAR, CDP and do not have difference also to have better curative effect trend; The minimizing glucose in urine amount effect that compound recipe SAR+CDP-4 treats is treated significantly better than CDP, blood sugar lowering, falls blood insulin, reduces HOMA-IR index effect and SAR, CDP and treat separately quite or have better curative effect trend.Result is pointed out, and SAR and CDP forms a certain proportion of compound recipe, and have obvious synergism to treatment IR, by weight ratio, the proportion of SAR:CDP is: 9:1 ~ 1:2, best with 3:1 compound recipe (SAR+CDP-2) curative effect in this experiment.

Table 2 rat is given after DEX15 days continuously, and with after SAR, SAR+CDP tetra-kinds of compound recipes, CDP treatment, fasting plasma glucose concentration, fasting insulin concentration, HOMA-IR index and glucose in urine amount testing result in 5 hours

#:P<0.05, ##:P<0.01, compare with SAR group; &: P<0.05, & &: P<0.01, compares with CDP group

(6) SAR+BSA compound recipe is to the therapeutical effect result of study of the IR that DEX causes

The results are shown in Table 3.With the SAR of identical dosage, BSA, SAR+BSA tetra-kinds of compound recipes: SAR+BSA-1, SAR+BSA-2, SAR+BSA-3, SAR+BSA-4 treat the IR that DEX causes, all obviously can reduce blood sugar concentration, blood insulin concentration and HOMA-IR index, and obviously reduce the glucose in urine amount of discharging in 5 hours; Compound recipe SAR+BSA-2, SAR+BSA-3 treatment all shows its blood sugar lowering, falls blood insulin, reduces HOMA-IR index and reduces the effect of glucose in urine amount treats separately significantly better than SAR or BSA, and prompting produces obvious cooperative effect; The minimizing glucose in urine amount effect that compound recipe SAR+BSA-1 treats is treated separately significantly better than CDP or SAR, blood sugar lowering, fall blood insulin concentration, reduce the effect of HOMA-IR index and treat with SAR and compare therapeutic equivalence or have better curative effect trend, blood sugar lowering, blood insulin falls, the effect of reduction HOMA-IR index treats to compare with BSA better curative effect trend; The minimizing glucose in urine amount effect that compound recipe SAR+BSA-4 treats is significantly better than BSA treatment and treat to compare with SAR and have better curative effect trend, blood sugar lowering, falls blood insulin, reduces the effect of HOMA-IR index and treat suitable with SAR and treat better curative effect trend than BSA.Result is pointed out, and SAR and BSA forms a certain proportion of compound recipe, and have obvious synergism to treatment IR, by weight ratio, the proportion of SAR:BSA is: 8:1 ~ 1:3, best with 2:1 compound recipe (SAR+CDP-2) curative effect in this experiment.

Table 3 rat is given after DEX15 days continuously, and with after SAR, SAR+BSA tetra-kinds of compound recipes, BSA treatment, fasting plasma glucose concentration, fasting insulin concentration, HOMA-IR index and glucose in urine amount testing result in 5 hours

#:P<0.05, ##:P<0.01, compare with SAR group; &: P<0.05, & &: P<0.01, compares with BSA group.

Claims

1. a compound medicament composition, containing active constituents of medicine and pharmaceutically acceptable carrier, wherein active constituents of medicine is made up of Sarpogrelate and 5-hydroxy tryptamine synthetic inhibitor, and wherein the weight ratio of Sarpogrelate and 5-hydroxy tryptamine synthetic inhibitor is 15:1 ~ 1:15.

2. the compound medicament composition of claim 1, wherein 5-hydroxy tryptamine synthetic inhibitor is fenclonine, carbidopa or benserazide.

3. the compound medicament composition of claim 1, wherein the weight ratio of Sarpogrelate and fenclonine is 12:1 ~ 1:2.

4. the compound medicament composition of claim 3, wherein the weight ratio of Sarpogrelate and fenclonine is 4:1.

5. the compound medicament composition of claim 2, wherein the weight ratio of Sarpogrelate and carbidopa is 9:1 ~ 1:2.

6. the compound medicament composition of claim 5, wherein the weight ratio of Sarpogrelate and carbidopa is 3:1.

7. the compound medicament composition of claim 2, wherein the weight ratio of Sarpogrelate and benserazide is 8:1 ~ 1:3.

8. the compound medicament composition of claim 7, wherein the weight ratio of Sarpogrelate and benserazide is 2:1.

9. the compound medicament composition any one of claim 1 to 8 is for the preparation of the purposes of medicine for the treatment of type ii diabetes or metabolism syndrome.