CN109806386A - The pharmaceutical composition and purposes of FXR agonist and GLP-1 analog - Google Patents

Abstract

The present invention relates to the pharmaceutical compositions and purposes of FXR agonist and GLP-1 analog.Combine the purposes in the drug of preparation treatment and/or prevention non-alcohol fatty liver with GLP-1 analog specifically, the present invention relates to a kind of FXR agonists.

Description

The pharmaceutical composition and purposes of FXR agonist and GLP-1 analog

Technical field

The invention belongs to a kind of FXR agonist and GLP-1 analog drug combination and its preparation treatment and/ Or the purposes in the drug of prevention non-alcohol fatty liver.

Background technique

Farnesoid X receptor (farnesoid X receptor, FXR) is distributed mainly on liver, by inhibiting sterol to adjust Element conjugated protein -1c (SREBP-1c) inhibits the de novo formation (de novo lipogenesis, DNL) of fatty acid, reduces The fat content of liver.Since cytochromes 7A1 (CYP7A1) is the rate-limiting enzyme of cholic acid biosynthesis, and FXR agonist passes through FXR is activated, inhibits the gene expression of CYP7A1 indirectly, inhibits the bile acid biosynthesis of liver, alleviating bile, hyper acid to liver cell Toxicity.Currently, FXR agonist granted treatment primary biliary cholangitis (PBC).WO02072598 discloses one kind FXR agonist --- shellfish cholic acid (obeticholic acid, OCA) difficult to understand, is a kind of chenodeoxycholic acid derivative, and chemistry is entitled 3 α ,-24 acid of 7-6 α of alpha-dihydroxy-- 5 β of ethyl-cholane, structure are as follows.Shellfish cholic acid difficult to understand can inhibit cholic acid to synthesize, and be used for Treat biliary atresia, cholestatic liver disease, chronic liver disease, primary gallbladder cirrhosis, alcoholic fatty liver, non-alcoholic rouge The hepatopathys such as fat hepatopathy.

Glucagon-like-peptide-1 (glucagon-like peptide-1, GLP-1) is a kind of polypeptide of 30 amino acid Hormone, it can promote islet β cell insulin in a manner of concentration of glucose dependence, and reduce alpha Cell of islet secretion pancreas Glucagons (glucagon), to reduce blood glucose and weight-reducing.CN101987868B discloses a kind of GLP-1 analog, knot Structure is as follows.The GLP-1 analog has higher activity and longer Half-life in vivo compared with endogenous GLP-1, can be used for Treat the diseases such as insulin-dependent diabetes mellitus, adult-onset diabetes, obesity.

Nonalcoholic fatty liver disease (nonalcoholic steatohepatitis, NASH) is nonalcoholic fatty liver A kind of extreme development form, be defined as the appearance of the steatosis phenomenon with inflammation and hepatocellular injury.NASH can be led Cause the generation of advanced liver fibrosis, cirrhosis, hepatic failure and liver neoplasm.In past 20 years, NAFLD morbidity number has been turned over Times, become liver diseases most commonly seen in western countries.The Major Risk Factors for leading to NASH include fat, 2 types sugar Urinate sick (T2DM) and dyslipidemia and metabolic syndrome.In the U.S., non-alcoholic fatty liver disease (Nonalcoholic fatty Liver disease, NAFLD) disease incidence account for about the 10-46% of total population, wherein the patient of about 10-30% can develop into NASH.Hepatic pathology diagnosis is the sole criterion for judging NASH.NAFLD mobility scores (NAS), and wherein steatosis 3 divides, is scorching Disease 3 is divided, balloon sample becomes 2 points.When total score >=5 point, and at least 1 point of each classification, it is judged as patient NASH.Currently, NASH is related Cirrhosis be U.S.'s liver transfer operation the third-largest common cause, it is contemplated that main cause will be become in the year two thousand twenty.

Although medical demand is urgent, FDA so far not yet ratifies any drug and treats for NASH.Although early stage Clinical research discovery FXR agonist or GLP-1 analog are applied alone to nonalcoholic fatty liver disease, especially contain to liver fat Amount has certain curative effect, but still cannot reach ideal therapeutic effect, also, FXR agonist has raising low-density lipoprotein gallbladder The adverse reaction of sterol (LDL-c) and serious itch, therefore drug effect is increased by drug combination, reducing adverse reaction has very Important meaning.

Summary of the invention

The present invention provides a kind of FXR agonist and combines with GLP-1 analog in preparation treatment and/or prevention non-alcoholic rouge Purposes in the drug of fat hepatopathy, the drug combination have synergistic effect.

It is selected from and is administered simultaneously or consecutive administration in administering drug combinations mode of the present invention.

In a scheme of the invention, the FXR agonist is selected from shellfish cholic acid difficult to understand or its officinal salt.Austria's shellfish gallbladder Sour officinal salt can be obtained by shellfish cholic acid difficult to understand and alkaline matter at salt, these alkaline matters include ammonium, alkali metal or alkaline earth gold Hydroxide and carbonate, the bicarbonate of category, typically there is sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, carbon Sour potassium etc..

In a scheme of the invention, the GLP-1 analog is selected from structure compound as follows or it can medicine Use salt.

GLP-1 analog of the present invention belongs to amphoteric compound, and one of ordinary skill in the art can by well-known technique Salt is reacted into using acidic or basic compound, the acid of the formation acid-addition salts generallyd use are as follows: hydrochloric acid, hydrobromic acid, hydrogen Acid iodide, sulfuric acid, phosphoric acid, p-methyl benzenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, Acetic acid；Salt includes sulfate, pyrosulfate, trifluoroacetate, sulphite, bisulfites, phosphate, hydrophosphate, phosphorus Acid dihydride salt, metaphosphate, pyrophosphate, hydrochloride, bromide, iodide, acetate, propionate, caprylate, acrylic acid Salt, formates, isobutyrate, caproate, enanthate, propiolate, oxalates, malonate, succinate, suberate, Fumarate, maleate, butine-Isosorbide-5-Nitrae-diacid salt, hexin -1,6- diacid salt, benzoate, chloro benzoate, methylbenzene Formates, dinitro-benzoate, hydroxy benzoate, methoxy benzoic acid salt, phenylacetate, phenpropionate, benzenebutanoic acid salt, Citrate, lactate, gamma hydroxybutyrate, glycol hydrochlorate, tartrate, mesylate, propane sulfonic acid salt, how -1- sulfonic acid Salt, how -2- sulfonate, mandelate etc..Alkaline matter can also with GLP-1 analog at salt, these alkaline matters include ammonium, Hydroxide and carbonate, the bicarbonate of alkali or alkaline earth metal, typically there is sodium hydroxide, potassium hydroxide, hydrogen-oxygen Change ammonium, sodium carbonate, potassium carbonate etc..

In a scheme of the invention, the non-alcohol fatty liver be selected from non-alcoholic simple fatty liver, Nonalcoholic fatty liver disease and its related liver cirrhosis or hepatocellular carcinoma.

Purposes of the present invention, wherein the dosage of the FXR agonist is 0.1 to 100mg, preferably 1mg, 2mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 40mg, 50mg, 70mg or 90mg.

Purposes of the present invention, wherein the dosage of the GLP-1 analog is 0.1 to 50mg, preferably 0.5mg, 1mg、1.5mg、2mg、2.25mg、2.5mg、2.75mg、3mg、3.25mg、3.5mg、3.75mg、4mg、4.5mg、5mg、10mg、 15mg, 20mg, 25mg, 30mg or 40mg.

In a scheme of the invention, the mass ratio of the FXR agonist and the dosage of GLP-1 analog is 100:1 to 1:10, preferably 10:1 to 1:1, most preferably 7:1,6.7:1,5:1,4:1,3:1,2.7:1,2.5:1,2:1,1.7:1, 1.5:1 or 1.3:1.

In a scheme of the invention, the FXR agonist recommendation is administered once for one day or one day administered twice, preferably It is administered once within one day.

In a scheme of the invention, the GLP-1 analog recommendation is administered once for one day or one day administered twice, excellent Select be administered once within one day.

In a preferred embodiment of the invention, the FXR agonist is to be administered once for one day with GLP-1 analog.

The pharmaceutical composition for further relating to a kind of FXR agonist and GLP-1 analog of the invention, comprising optional one kind or A variety of pharmaceutical carriers, excipient and/or diluent.Pharmaceutically acceptable any dosage form can be made in described pharmaceutical composition. For example, the pharmaceutical preparation of FXR agonist and GLP-1 analog can be formulated as tablet, capsule, pill, granule, solution Agent, suspension, syrup, injection (including injection, injection sterile powder and concentrated solution for injection), suppository, inhalant Or spray.

In addition, described pharmaceutical composition of the invention can also be with any suitable administration mode, such as take orally, stomach Outside, the modes such as rectum, transpulmonary or local administration are applied to the patient or subject for needing this treatment.When for being administered orally When, described pharmaceutical composition can be made into oral preparation, such as oral solid formulation, such as tablet, capsule, pill, granule； Or oral liquid, such as oral solution, oral suspensions, syrup.When oral preparation is made, the pharmaceutical preparation It also may include suitable filler, adhesive, disintegrating agent, lubricant etc..

" joint " of the present invention is a kind of administration mode, refers to and gives at least one dosage in the certain time time limit FXR agonist and GLP-1 analog.The time limit can be in a dosage period, in preferably 4 weeks, in 3 weeks, and 2 weeks It is interior, in 1 week or within 24 hours.FXR agonist and GLP-1 analog can simultaneously or sequentially be given.This time limit includes this The treatment of sample exempts from FXR agonist and GLP-1 analog wherein giving by identical administration route or different way of administration.This hair The bright united administration mode, which is selected from, to be administered simultaneously, independently prepare and is total to administration or independently prepare and be administered in succession.

The present invention also provides a kind of methods for treating non-alcohol fatty liver, including give to cancer patient aforementioned FXR agonist and GLP-1 analog.

Detailed description of the invention

Fig. 1 hepatic tissue NAS scoring

The scoring of Fig. 2 hepatic cell fattydegeneration

TC detected value in Fig. 3 liver

TG detected value in Fig. 4 liver

Fig. 5 animal peripheral blood ALT detected value

Fig. 6 animal peripheral blood AST detected value

Fig. 7 animal peripheral blood TC detected value

Fig. 8 animal peripheral blood TG detected value

Fig. 9 animal peripheral blood HDL detected value

Figure 10 animal peripheral blood LDL detected value

Specific embodiment

Exemplary tests scheme of the composition of the invention presented below in non-alcohol fatty liver medical usage, To show the favorable activity or advantageous effects of the present composition.It is understood that following testing programs are only pair The example of the content of present invention, rather than limiting the scope of the invention.Those skilled in the art are under the introduction of this specification, energy It is enough that modifications or changes appropriate is carried out to technical solution of the present invention, without departing from the spirit and scope of the invention.

Embodiment 1: clinical trial of the present composition in the HFD+CHOL C57bl/6 male mice NASH model induced

Experimental material

Test sample:

Shellfish cholic acid (OCA) difficult to understand can prepare according to method disclosed in WO02072598；(structure is such as GLP-1 analog Shown in lower) it can be prepared according to method disclosed in patent CN101987868B；GLP-1 is similar by GLP-1 similar to composition granule Object, SNAC and conventional pharmaceutical adjuvants composition, the particle can be prepared by conventional method of granulating, for example, by GLP-1 analog, SNAC and other auxiliary materials mixing after, by dry granulation, whole grain, total mix and etc. preparation.It is similar without GLP-1 in blank granules Object, other pharmaceutic adjuvants and preparation process are with GLP-1 similar to composition granule.

Experimental animal:

SPF grades of mouse of C57BL/6 male, 26-30 grams of weight, be purchased from Shanghai Ling Chang Biotechnology Co., Ltd, raising in Keyes's Chinese mugwort biotechnology (Suzhou) Co., Ltd (KCI) animal center, animal certificate number: 2013001816031, animal uses Licensing: SYXK (Soviet Union) 2017-0041.

Modeling feed:

HFD+HCOL (60%+ cholesterol 1.25% high in fat), is purchased from Nantong Te Luofei feed technology Co., Ltd, and card is raised by Soviet Union (2014)06092。

Test-compound and positive reference substance preparation program:

Group -1: the GLP-1 analog blank granules of the solvent+pH6.8 phosphate buffer configuration of animal OCA are given.

Group -2: the GLP-1 analog blank granules of the solvent+pH6.8 phosphate buffer configuration of animal OCA are given.

Group -3: using 1% hydroxypropyl methyl cellulose (HPMC) as solvent, and the OCA that weighed amount is added is ground in 1%HPMC Mill uniformly, adds 1%HPMC to aequum, and ultrasound can be administered after 15 minutes, is stored in 4 DEG C, configuration in 4 days is primary.

Group -4: it is added in prepared pH6.8 phosphate buffer in advance, magnetic stirrer at least 15 minutes, matches It is uniform with magnetic stirrer after to required volume.Then it is settled to required volume.Prepared medical fluid is stored in 4 DEG C of conditions Under, until administration terminates, drug need to be protected from light preparation, and be stored in brown volumetric flask.Test sample is prepared once daily.

Group -5: using 1%HPMC as solvent, and the OCA that weighed amount is added is ground uniformly in 1%HPMC, adds 1% HPMC can be administered after 15 minutes to aequum, ultrasound, be stored in 4 DEG C, configuration in 4 days is primary.

Group -6: using 0.1%HPMC as solvent, and the OCA that weighed amount is added is ground uniformly in 1%HPMC, adds 1% HPMC can be administered after 15 minutes to aequum, ultrasound, be stored in 4 DEG C, configuration in 4 days is primary.Prepared pH6.8 phosphorus in advance is added In phthalate buffer, magnetic stirrer at least 15 minutes, it is assigned to uniform with magnetic stirrer after required volume.Then It is settled to required volume.Prepared medical fluid is stored under the conditions of 4 DEG C, until administration terminates, drug need to be protected from light preparation, and store In brown volumetric flask.Test sample is prepared once daily.

Laboratory apparatus:

Full-automatic blood biochemistry analyzer: Hitachi, Amada Co., Ltd. new and high technology, model: 7060

Scale: JJ500, G&G, Jiangsu, China

Intragastric administration on mice device: #8 gastric perfusion needle

Tissue processor: HistoCore Pearl, Leica

Embedding machine: HistoCore Arcadia, Leica

Slicer: RM2235, Leica

Automatic staining machine: LEICA Autostainer ST5020

Slice scanner: NanoZoomer Digital Pathology (S210) (shore pine, Japan)

Liquid-transfering gun and pipette tips: RAININ (2 μ l, 10 μ l, 20 μ l, 100 μ l, 200 μ l, 1000 μ l)

Liquid-transfering gun: RAININ

Ultra low temperature freezer: SANYO UITRA LOW；MDF-382E

Experimental method

Model foundation:

Implement the involved every operation of this experiment in the case where KCI zoopery operates SOP guideline.Animal is in barrier Interior adaptation enters experiment after a week.It is grouped at random on the basis of the weight of animals according to serpentine curve, starts to feed related experiment use Animal feed (intact animal standard feed feeding, animal pattern with to want animal it is high in fat+high cholesterol diet feed), continuous 10 Week.Daily measurement animal feed intake, the weight of animals of measurement in every two days continue 10 weeks.

Experimental group and administration:

6 groups of experiment point, intact animal group (group -1, n=3), model group (group -2, n=8), OCA-30mpk group (group -3, n =8), GLP-1 is similar to composition granule 30mpk group (group -4, n=8), and OCA-20mpk group (group -5, n=8), GLP-1 is similar to composition granule 30mpk+OCA-20mpk group (group -6, n=8).Start to be administered orally within modeling the 5th week.Model group gives compound solvent.Administration Approach is to be administered orally, administered volume 10mL/kg, once a day, continuous 42 days (table 1).

Table 1, experimental group and drug administration information

Experimental endpoints:

24 hours beginning animal fasting 6 hours after the last administration, according to KCI animal euthanasia standard SOP by all animals Euthanasia is executed, blood sample is acquired, prepares serum, freezen protective.Animal collects liver through phosphate buffer total body perfusion, claims Weight, acquisition liver substantially photo.Part hepatic tissue liquid nitrogen flash freezer, minus 80 DEG C of preservations, remaining hepatic tissue are fixed on 10% neutral good fortune Subsequent histopathology coherent detection is carried out in your Malin's solution.

Blood biochemistry detection

Glutamic-pyruvic transaminase (ALT) in serum, glutamic-oxalacetic transaminease (AST), highly dense is detected after fasting using full automatic biochemical apparatus Spend the blood biochemistry index such as lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG), cholesterol (TC).Detect unit Triglycerides (TG), cholesterol (TC) in weight liver organization.

Triglycerides and cholesterol detection in liver

Suitable frost hepatic tissue is weighed, with liver weight (g): solvent is added in solvent (mL)=1:9 ratio, is homogenized (ice bath)； 2500rpm low-temperature centrifugation 10min collects supernatant, draws a certain amount of supernatant, being diluted to 50 times, (that is: liver tissue homogenate's liquid is dense 2%), with 7060 biochemical instruments of Hitachi degree detects TC and TG content.

Histopathology

Liver organization dehydration, paraffin mass production, paraffin section, 3 μm of piece thickness are carried out according to KCI pathological criteria SOP.It follows KCI pathological criteria dyes SOP and carries out HE dyeing, sirius red dyeing, and passes through NanoZoomer Digital Pathology (S210) (shore pine, Japan) slice scanner carries out the scanning of sirius red stained slice full wafer.Two diseases are passed through using double-blind study Neo-confucian carries out hepatic injury pathological analysis and scoring (table 2) to HE stained slice；Sirius red stains are sliced and carry out liver fiber Change areal calculation, liver fibrosis %=fibrosis area/total reference area × 100.

Table 2, hepatic injury pathological score standard

Data analysis

Using 5.0 software of graphpad prism, mean ± sd and mean ± sem is calculated, significance test of difference uses T-test and One-way ANOVA is examined；There is between two groups statistical significance when P < 0.05.

Experimental result

Animal basic physiological is observed during administration

Animal clinical observation Non Apparent Abnormality variation during experiment.

The weight of animals changes during test

Growth trend is presented by high cholesterol diet high in fat raising weight in all animals during experiment, after giving tested material Body weight increase tends towards stability, and weight mean value is without obvious statistical difference between each experimental group.

Liver weight and liver body are than variation

Each group hepatic tissue surface is smooth, and model group has growth trend compared with Normal group liver again, meanwhile, with model group phase Than again each tested material processing group liver has decreasing trend, but not statistically significant.The variation of liver weight/weight ratio changes with liver weight Unanimously, no difference of science of statistics between each group.

Histopathology

HE dyeing liver histological observes the different degrees of liver cell diffusivity liver cell of visible high cholesterol induction high in fat Steatosis is dispersed in multifocal cell infiltration, is distributed in the different zones of hepatic tissue.The visible OCA of NAS overall score and tested material Each group has downward trend compared with model group, and GLP-1 is shown compared with model group similar to composition granule and OCA drug combination group Work property reduces (P < 0.01), has statistical significance (Fig. 1, ##p < 0.001vs. Normal group；* p < 0.01vs. model Group).The visible hepatic cell fattydegeneration of OCA treatment group mitigates, and cell infiltration is reduced.Visible difference after the treatment of test-compound group Hepatic cell fattydegeneration, necrosis and the reduction of cell infiltration of degree.GLP-1 is similar to composition granule and GLP-1 analog Particle with the drug combination group of OCA compared with model group, hepatic cell fattydegeneration conspicuousness reduce (P < 0.05) (Fig. 2, #p < 0.05vs. Normal group；* p < 0.001vs. model group；T-test:$ p < 0.05vs. model group).

Total cholesterol TC, total triglycerides TG are detected in hepatic tissue

TC, TG testing result display model group are apparently higher than Normal group, and TC in each medication group liver in hepatic tissue Content is substantially less than model group, has statistical significance (p < 0.05), and each medication group of TG has decline compared with model group in liver group Trend, and when OCA-30mg/kg/d and GLP-1 is treated similar to composition granule and OCA-20mg/kg/d drug combination and model group (p < 0.05) (p < 0.01 Fig. 3, * * is reduced compared to conspicuousness；* * p < 0.001vs. model group；#p < 0.05vs. Normal group, Fig. 4).

Peripheral blood biochemical index

Animal pattern serum glutamic oxalacetic transaminase AST, alanine aminotransferase ALT, total cholesterol TC, total triglycerides TG, height Density lipoprotein HDL, low-density lipoprotein LDL value obviously write raising, animal after OCA and test sample treatment serum AST, ALT, TC, HDL, LDL value have apparent decreasing trend compared with model group.Test-compound GLP-1 analog and GLP-1 are similar to composition granule It is significantly lower than model group with drug combination the group ALT and AST of OCA, but statistical analysis is showed no display significant difference.TC, HDL and LDL value conspicuousness compared with model group reduces, (Fig. 5, * * * p < 0.001vs. normal control as shown in Fig. 5 to Figure 10 Group；Fig. 6, * p < 0.05, * * p < 0.01vs. model group；Fig. 7, ###P < 0.001vs. Normal group；* p < 0.01vs. model Group；* * p < 0.001vs. model group；Fig. 9, ###P < 0.001vs. Normal group；* p < 0.05vs. model group；***p< 0.001vs. model group；Figure 10, ###P < 0.001vs. Normal group；* p < 0.01vs. model group；* * p < 0.001vs. mould Type group).

Conclusion

This experimental result show GLP-1 analog list medicine using or with OCA drug combination compared with model group to height high in fat The mouse NASH process of cholesterol induction shows conspicuousness therapeutic effect, especially GLP-1 analog and OCA use in conjunction Conspicuousness is used better than test-compound list medicine, while being better than the curative effect of the mono- medicine of OCA.

Claims (9)

1. a kind of FXR agonist combines the medicine in preparation treatment and/or prevention non-alcohol fatty liver with GLP-1 analog Purposes in object.

2. purposes as described in claim 1, which is characterized in that the FXR agonist is selected from shellfish cholic acid difficult to understand or its officinal salt.

3. purposes as described in claim 1, which is characterized in that the GLP-1 analog is selected from structure chemical combination as follows Object or its officinal salt:

4. purposes as described in claim 1, which is characterized in that it is simple that the non-alcohol fatty liver is selected from non-alcoholic Property fatty liver, nonalcoholic fatty liver disease and its related liver cirrhosis or hepatocellular carcinoma.

5. purposes described in any one of -4 according to claim 1, which is characterized in that the joint has collaboration drug action.

6. purposes according to any one of claims 1-5, which is characterized in that the FXR agonist and GLP-1 analog Mass ratio be 100:1 to 1:10, preferably 10:1 to 1:1, most preferably 7:1,6.7:1,5:1,4:1,3:1,2.7:1,2.5: 1,2:1,1.7:1,1.5:1 or 1.3:1.

7. purposes according to any one of claims 1-5, which is characterized in that the dosage of the FXR agonist is 0.1 to 100mg, preferably 1mg, 2mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 40mg, 50mg, 70mg or 90mg.

8. purposes according to any one of claims 1-5, which is characterized in that the dosage of the GLP-1 analog Be 0.1 to 50mg, preferably 0.5mg, 1mg, 1.5mg, 2mg, 2.25mg, 2.5mg, 2.75mg, 3mg, 3.25mg, 3.5mg, 3.75mg, 4mg, 4.5mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg or 40mg.

9. containing the pharmaceutical composition of FXR agonist and GLP-1 analog of any of claims 1-8, feature It is, includes one or more pharmaceutical excipient, diluent or carrier.