Composition containing sumo inhibitor and application

Abstract

A composition containing SUMO inhibitor, and belongs to the technical field of medicine includes FXR agonist and SUMO inhibitor. In activated hepatic stellate cells, the FXR agonist does not have an effect in inhibiting the activation of hepatic stellate cells. After the FXR agonist and the SUMO inhibitor are compounded according to the present invention, the activation of hepatic stellate cells can also be inhibited for those cells under activated state. Of note, the hepatic stellate cells of an individual with hepatic fibrosis symptoms have been in an activated state, therefore a good anti-fibrotic effect cannot be achieved by using the FXR agonist alone.

Claims

1. A composition for treating hepatic fibrosis comprising a FXR agonist and a SUMO inhibitor.

2. The composition according to claim 1, wherein the FXR agonist is one or more selected from the group consisting of obeticholic acid, GW4064 and WAY-362450.

3. The composition according to claim 1, wherein the SUMO inhibitor is spectinomycin or/and ginkgolic acid.

4. A method for treating hepatic fibrosis comprising a step of administrating to a subject in need a therapeutically effective amount of the composition of claim 1.

5. The method according to claim 4, wherein activation of hepatic stellate cells is inhibited in the subject.

6. The method according to claim 4, wherein deposition of collagen fibers is reduced in the subject.

7. A pharmaceutically acceptable medicine comprising the composition of claim 1 as active ingredient, and a pharmaceutically acceptable carrier.

8. The pharmaceutically acceptable medicine according to claim 7, wherein the pharmaceutically acceptable medicine is made in a form selected from a group consisting of a tablet, a capsule, a granule, a pill, powder and an injection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A is mRNA level analysis of Fxr in quiescent (vehicle) and activated (TGF-β) HSCs treated with OCA.

(2) FIG. 1B is mRNA level analysis of Shp in quiescent (vehicle) and activated (TGF-β) HSCs treated with OCA.

(3) FIG. 1C is mRNA level analysis of Fxr in quiescent (vehicle) and activated (TGF-β) HSCs treated with GW4064.

(4) FIG. 1D is mRNA level analysis of Shp in quiescent (vehicle) and activated (TGF-β) HSCs treated with GW4064.

(5) FIG. 1E is mRNA level analysis of Fxr in quiescent (vehicle) and activated (TGF-β) HSCs treated with WAY-362450.

(6) FIG. 1F is mRNA level analysis of Shp in quiescent (vehicle) and activated (TGF-β) HSCs treated with WAY-362450. *P<0.05. **P<0.01. *** P<0.001.

(7) FIG. 2 is SUMOylation level analysis of FXR proteins after activation of HSCs. ***P<0.001.

(8) FIG. 3 is SUMOylation level analysis of FXR proteins in activated HSCs treated with

(9) SUMO inhibitors SP and GA. ***P<0.001. ##P<0.01. ###P<0.001.

(10) FIG. 4A is mRNA level analysis of Fxr in activated HSCs treated with a combination of OCA and SP.

(11) FIG. 4B is mRNA level analysis of Shp in activated HSCs treated with a combination of

(12) OCA and SP.

(13) FIG. 4C is mRNA level analysis of Fxr in activated HSCs treated with a combination of GW4064 and SP.

(14) FIG. 4D is mRNA level analysis of Shp in activated HSCs treated with a combination of GW4064 and SP.

(15) FIG. 4E is mRNA level analysis of Fxr in activated HSCs treated with a combination of WAY-362450 and SP.

(16) FIG. 4F is mRNA level analysis of Shp in activated HSCs treated with a combination of WAY-362450 and SP.

(17) FIG. 4G is mRNA level analysis of Fxr in activated HSCs treated with a combination of OCA and GA.

(18) FIG. 4H is mRNA level analysis of Shp in activated HSCs treated with a combination of OCA and GA. ns P>0.05. *P<0.05. **P<0.01.

(19) FIG. 5A is mRNA expression level analysis of Acta-2 treated with a combination of OCA and SP.

(20) FIG. 5B is mRNA expression level analysis of Acta-2 treated with a combination of GW4064 and SP.

(21) FIG. 5C is mRNA expression level analysis of Acta-2 treated with a combination of WAY-362450 and SP.

(22) FIG. 5D is mRNA expression level analysis of Acta-2 treated with a combination of OCA and GA. ns P>0.05. *P<0.05.

(23) FIG. 6A is mRNA expression level analysis of Fxr.

(24) FIG. 6B is mRNA expression level analysis of Shp. *P<0.05.

(25) FIG. 7A is mRNA expression level analysis of Acta-2. **P<0.01.

(26) FIG. 7B is liver Sirius red staining.

(27) Note: in all figures of the present invention, P value below 0.05 indicates a statistical difference.

DETAILED DESCRIPTION OF THE INVENTION

(28) The present invention is further illustrated below in conjunction with embodiments and drawings.

(29) The present invention provides a composition containing SUMO inhibitor. The composition includes FXR agonist and SUMO inhibitor. The present invention does not have specific limitation to a mass ratio of the FXR agonist to the SUMO inhibitor, and the mass ratio may be any value.

(30) According to the present invention, the FXR agonist preferably includes, but is not only limited to, one or more of obeticholic acid, GW4064 and WAY-362450. The more specifically refers to two or more. According to the present invention, when there are two or more FXR agonists, all components are mixed according to any mass. The FXR agonists are not limited to the above three agonists according to the present invention, their sources are not specifically limited, and the FXR agonists preferably include various FXR agonists of a natural source, a semisynthetic source or a chemosynthetic source.

(31) According to the present invention, the SUMO inhibitor preferably includes, but is not only limited to, spectinomycin and/or ginkgolic acid. According to the present invention, when the SUMO inhibitor preferably includes the spectinomycin and the ginkgolic acid, the spectinomycin and the ginkgolic acid are mixed according to any mass ratio. The SUMO inhibitors are not limited to the above two SUMO inhibitors, their sources are not specifically limited, and the SUMO inhibitors preferably include various SUMO inhibitors of a natural source, a semisynthetic source or a chemosynthetic source.

(32) According to the present invention, hepatic stellate cells in activated state lose responsiveness to the FXR agonist, and the FXR agonist does not have an effect of inhibiting activation of the hepatic stellate cells. The SUMO inhibitor can significantly improve the responsiveness of the activated hepatic stellate cells to the FXR agonist, and can significantly enhance the inhibition effect of the FXR agonist on the activation of the hepatic stellate cells. The hepatic stellate cells of an individual with hepatic fibrosis symptoms have been in an activated state, and thus an expected anti-fibrotic effect cannot be achieved by using the FXR agonist alone. The composition of the FXR agonist and the SUMO inhibitor provided by the present invention can significantly inhibit the activation of the hepatic stellate cells and reduce deposition of collagen fibers to further combat liver fibrosis.

(33) The present invention further provides application of the composition according to the above technical solution to preparation of anti-hepatic fibrosis medicine.

(34) The present invention further provides application of the composition according to the above technical solution to preparation of anti-hepatic fibrosis symptom medicine.

(35) The present invention further provides application of the composition according to the above technical solution to preparation of medicine for inhibiting activation of hepatic stellate cells.

(36) The present invention further provides application of the composition according to the above technical solution to preparation of medicine for reducing deposition of collagen fibers.

(37) According to the present invention, dose forms of the medicine independently include a tablet, a capsule, a granule, a pill, powder or an injection. Preparation methods of the above dose forms are not specifically limited according to the present invention. Conventional preparation methods for preparing corresponding dose forms and corresponding used auxiliary materials may be used for preparation.

(38) According to the present invention, administration methods of the composition preferably include oral administration, intravenous injection, intravenous drip, intramuscular injection, and a combination of various administration modes.

(39) The composition containing the SUMO inhibitor and application provided by the present invention are illustrated in detail below in conjunction with the embodiments, but they are not to be interpreted as limitation to the protection scope of the present invention.

Embodiment 1

(40) Reason Analysis on Responsiveness Reduction of HSCs to FXR Agonist After Activation

(41) 1. Experiment Materials

(42) HSC-T6 cell line used in the present invention was purchased from Central South University Cell Bank.

(43) OCA, GW4064 and WAY-362450 used in the present invention were purchased from MCE Company. TGF-β recombinant proteins were purchased from R&D Systems Company. Reverse transcription kits were purchased from Applied Biosystems Company. Trizol RNAiso plus was purchased from TAKARA Company. Used primers were synthesized by Life Invitrogen Company. SUMOylation ELISA detection kits were purchased from Epigentek Company. Other reagents were all commercially available.

(44) 2. Experiment Methods

(45) 2.1 HSC-T6 cell line culture and modeling

(46) HSC-T6 cells, seeded and cultured in a proper density, were treated with TGF-β(10 mg/ml) or OCA (5 μM) or GW40064 (5 μM) or WAY-362450 (5 μM) for 12 h before collection.

(47) 2.2 Real-time quantitative PCR

(48) 2.2.1 Total RNA extraction of cell samples

(49) 1) After the cells were cleaned by PBS, 800 μl of a Trizol reagent was added. After repeated blowing by a pipette, the cells were transferred into an EP tube to be subjected to still standing for 5 min at the room temperature.

(50) 2) 160 μl of chloroform was added, and violent oscillation was performed for 15 sec. After still standing for 5 min at the room temperature, 12000 g centrifugation was performed for 15 min. The sample was divided into three layers including a bottom layer being a yellow organic phase, an upper layer being a colorless water phase, and a middle layer.

(51) 3) 300 μl of the upper layer water phase was carefully transferred into a new tube. 300 μl of isopropanol was added. After uniform mixing through reversing, still standing was performed for 10 min at the room temperature. Then, 12000 g centrifugation was performed for 10 min. Supernatant was abandoned.

(52) 4) RNA precipitates were washed by 1.0 ml of precooled 75% ethanol. Then, 12000 g centrifugation was performed for 5 min. Supernatant was abandoned to obtain total RNA. Redissolution was performed by 10 μl of DEPC water. After quantification, dilution was performed to 0.5 μg/μl.

(53) 2.2.2 Reverse transcription

(54) RNA solutions and kit components were prepared into a system with a total volume of 20 μl according to a system proportion required in the specification, and a program temperature was set for reverse transcription. Specific proportion requirements are shown in Table 1:

(55) TABLE-US-00001 TABLE 1 Proportion for reverse transcription Reagent name Dose 10*RT Buffer 2.0 μl 25*dNTP Mix(100 mM) 0.8 μl 10*RT Random Primers 2.0 μl MultiScribeReverseTransscriptase 1.0 μl Rnase free dH.sub.2O 4.2 μl Total 10 μl Total RNA 10 μl

(56) Use conditions of reverse transcription are as follows:

(57) Stage 1: reverse transcription at 37° C. for 15 min.

(58) Stage 2: denaturation at 85° C. for 5 s.

(59) 2.2.3 PCR

(60) A PCR system is shown in Table 2:

(61) TABLE-US-00002 TABLE 2 PCR system Reagent name Dose SYBR Green 7.5 μl PCR Forward Primer (10 μM) 1.0 μl PCR Reverse Primer (10 μM) 1.0 μl cDNA 1.0 μl Rnase free dH.sub.2O 4.5 μl Total 15.0 μl

(62) Use conditions of PCR are as follows:

(63) Stage 1: initial denaturation at 95° C. for 1 min.

(64) Stage 2: PCR reaction at 95° C. for 15 sec; for example, at 60° C. for 30 sec for 40 Cycles; and at 72° C. for 30 sec.

(65) Stage 3: dissociation curve analysis at 65 to 95° C., 0.5° C./5s.

(66) Primer sequences of genes to be detected are shown in Table 3:

(67) TABLE-US-00003 TABLE 3 Primer sequence Genename Forward primer Reverse primer Rat Fxr SEQ ID No. 1 TGGACTCATACAG SEQ ID No. 2 GTCTGAAACCCTGG CAAACAGAGA AAGTCTTTT Rat Shp SEQ ID No. 3 CCTGGAGCAGCCC SEQ ID No. 4 AACACTGTATGCAA TCGT ACCGAGGA Rat SEQ ID No. 5 GCTCCATCCTGGC SEQ ID No. 6 TAGAAGCATTTGCG Acta2 TTCTCTA GTGGAC Rat SEQ ID No. 7 AACGGCACAGTC SEQ ID No. 8 ACGCCAGTAGACTC Gapdh AAGGCTGA CACGACAT Mouse SEQ ID No. 9 GCACGCTGATCAG SEQ ID No. 10 CAGGAGGGTCTGTT Fxr ACAGCTA GGTCTG Mouse SEQ ID No. 11 GTACCTGAAGGG SEQ ID No. 12 GTGAAGTCTTGGAG Shp CACGATCC CCCTGGT Mouse SEQ ID No. 13 GCACCCAGCATG SEQ ID No. 14 TCTGCTGGAAGGTA Acta2 AAGATCAAG GACAGCGAAG Mouse SEQ ID No. 15 TTGATGGCAACAA SEQ ID No. 16 CGTCCCGTAGACAA Gapdh TCTCCAC AATGGT

(68) 2.3 SUMOylation ELISA detection kit

(69) After cell nuclear proteins were extracted according to specification requirements of an ELISA detection kit, primary antibodies and nuclear proteins of a cell sample were incubated for 60 min at the room temperature in a plate hole. SUMO specific detection antibodies were then supplemented and added according to the specification requirements. A color sensitizer was further added for color development. Then, absorbance at 655 nm was fast read. Relative quantitative analysis was performed.

(70) 3. Experiment Results

(71) 3.1 Reduced responsiveness of activated HSCs to the FXR agonist

(72) In cultured HSCs in vitro, activation of the HSCs was promoted by incubation with TGF-β1. According to results of real-time RT-PCR (FIGS. 1A-F), the capability of the FXR agonist in regulating FXR downstream target genes is reduced in HSC-T6 cell in activated state caused by TGF-β1 incubation. In quiescent cells, FXR agonists OCA, GW4064 and WAY-362450 could significantly up-regulate the mRNA level of Shp, an FXR target gene (FIG. 1B, FIG. 1D, and FIG. 1F). In the activated state, none of the FXR agonists OCA, GW4064 and WAY-362450 could significantly up-regulate the mRNA level Shp (FIG. 1A, FIG. 1B, and FIG. 1F).

(73) 3.2 Improved SUMOylation level of FXR proteins in activated HSCs

(74) According to ELISA kit detection aiming at the SUMOylation level (FIG. 2), the SUMOylation level of FXR proteins in the activated HSC-T6 cells caused by TGF-β1 incubation was significantly elevated.

Embodiment 2

(75) Effect of SUMO Inhibitor on FXR Pathway in In-vitro HSCs

(76) 1. Experiment Materials

(77) SUMO inhibitors of spectinomycin (SP) and ginkgolic acid (GA) were purchased from MCE Company.

(78) Other experiment materials were the same as those in Embodiment 1.

(79) 2. Experiment Methods

(80) 2.1 HSC-T6 cell line culture and modeling

(81) Specific methods were the same as those in section 2.1 in Embodiment 1.

(82) 2.2 RT-PCR

(83) Specific methods were the same as those in section 2.2 in Embodiment 1.

(84) 2.3 SUMOylation ELISA detection kit

(85) Specific methods were the same as those in section 2.3 in Embodiment 1.

(86) 3. Experiment Results

(87) 3.1 SUMO inhibitors inhibited SUMOylation of FXR proteins in activated HSCs

(88) Through detection by SUMOylation ELISA kits, it was found that SUMOylation inhibitors SP and GA could significantly reduce the SUMOylation level of FXR proteins in activated HSCs (FIG. 3).

(89) 3.2 SUMO inhibitors improved the responsiveness of activated HSCs to FXR agonists

(90) In Embodiment 1, it was found that in activated HSCs, FXR agonists OCA, GW4064 and WAY-362450 lost their regulation effects on the FXR signaling pathway, manifested by loss of an induction effect on Shp. In the study of this section, the regulation effect of the FXR agonist on the FXR signaling pathway in the presence or absence of SUMO inhibitors was investigated. According to the results of RT-PCR (FIGS. 4A-H), FXR agonists OCA, GW4064 and WAY-36250 significantly up-regulated the mRNA expression of Shp in the presence of SUMO inhibitor SP in activated HSCs (FIG. 4B, FIG. 4D, and FIG. 4F). Similar phenomenon was observed in the presence of another SUMO inhibitor GA (FIG. 4H).

(91) 3.3 SUMO inhibitors enhanced the inhibitory effect of FXR agonists on activation of HSCs

(92) Above results show that SUMO inhibitors can inhibit the SUMOylation of FXR, and further enhance the regulation effects of the FXR agonists on the FXR signaling in activated HSCs. Then, the effect of SUMO inhibitors on HSC activation resistance of FXR agonist was investigated. Identically, according to the results of RT-PCR (FIG. 5), after administration of SP to the activated HSCs, administration of FXR agonist OCA, GW4064 and WAY36250 significantly reduced the expression of a fibrotic-related gene Acta2 in hepatic stellate cells; similarly, after administration of the GA to the activated HSCs, OCA could significantly inhibit expression of the Acta2. Above results show that the SUMO inhibitors SP and GA can reduce the SUMOylation level of the FXR proteins, and enhance the inhibitory effect of FXR agonists on the expression of fibrosis-related genes in activated HSCs, thereby improving the anti-fibrotic efficacy.

Embodiment 3

(93) Effect of SUMO Inhibitor on FXR Pathway Under Liver Fibrosis State of Mice

(94) 1. Experiment Materials

(95) Experiment mice (C57BL/6) were purchased from Comparative Medicine Centre of Yangzhou University.

(96) CCl.sub.4 was purchased from Shanghai Lingfeng Chemical Reagent Company, and mineral oil was purchased from Sigma-Aldrich Company.

(97) Other experiment materials were the same as those in Embodiment 1.

(98) 2. Experiment Methods 2.1 Effect of a combination of SUMO inhibitor and OCA on CCl.sub.4-induced liver fibrosis

(99) After animal adaptive feeding for one week, a total of 40 mice were randomly divided into 5 groups with 8 mice in each group. A control group, a model group, an OCA single-administration group, an SP single-administration group and an OCA+SP co-administration group were included. The model group received intraperitoneal injection of CCl.sub.4 (20%, dissolved in mineral oil). In the control group, a vehicle (mineral oil) with corresponding volume was administrated twice a week for 4 weeks. Mice received intraperitoneal intragastric administration of OCA at a dosage of 1.5 mg/kg once a day from the third week for two weeks after modeling. Additionally, in the OCA+SP co-administration group, SP was subcutaneously injected at a dosage of 200 mg/kg/day from the first week. At the end of the administration periods, the mice were killed, and the livers were isolated and stored for use.

(100) 2.2 RT-PCR

(101) Specific methods were the same as those in section 2.2 in Embodiment 1.

(102) 2.3 Liver clinicopathologic analysis

(103) After being fixed in 4% paraformaldehyde, some liver tissues were sent to Wuhan Servicebio Technology Co., Ltd. (Wuhan, China) for double-blind analysis and detection. A detection item was Sirius red staining analysis.

(104) 3. Experiment Results 3.1 Enhancement of regulation effects of OCA on FXR pathway of fibrotic mice by SUMO inhibitor

(105) Through RT-PCR experiments, mRNA relative expression of an FXR downstream gene in liver tissues was investigated (FIG. 6). It was found that mRNA level of the FXR downstream gene significantly increased by OCA treatment after combined use with SP. It showed that SUMOylation inhibitors could enhance the agonistic effect of the FXR agonist OCA on the FXR pathway in fibrotic mice.

(106) 3.2 Capability of SUMO inhibitor SP to enhance anti-hepatic fibrosis efficacy of OCA

(107) According to the expression investigation of fibrosis-related genes in liver tissues in RT-PCR experiments and staining results of liver pathological analysis, it was found that the anti-fibrotic efficacy of the OCA is significantly improved in the presence of SUMO inhibitor SP. The RT-PCR results showed that the expression of the major fibrosis gene Acta2 in the liver tissues in the co-administration group (SP+OCA) was also significantly reduced compared with that of the single administration group (FIG. 7A). Additionally, Sirius red staining results showed that only few of red collagen fibers in the liver of the control group were stained while slices in the model group and OCA single-administration group contained a great number of red collagen fibers due to hepatotoxicity, but after OCA and SP co-administration was adopted, red collagen fiber hyperplasia in the liver was reduced (FIG. 7-2).

(108) It can be concluded from the above embodiments that the hepatic stellate cells in activated state lose the responsiveness to FXR agonist, thus FXR agonist does not have an effect in inhibiting the activation of hepatic stellate cells. After the FXR agonist and SUMO inhibitor are compounded according to the present invention, hepatic stellate cells in activated state restore the responsiveness to FXR agonist. More importantly, this composition proposed in this invention exert excellent inhibitory effect on the activation of hepatic stellate cells in activated state, reducing the production of collagen fibers and thus inhibit progress of liver fibrosis.

(109) The present invention only describes preferred implementations above. It should be noted that, for those ordinarily skilled in the art, various improvements and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations shall be deemed to be within the protection scope of the present invention.