A compound for treating liver cirrhosis portal hypertension and application thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a compound for treating liver cirrhosis portal hypertension and application thereof. The compound can be used for preparing a medicine for treating liver cirrhosis portal hypertension. The medicine can achieve the treatment purpose of improving LSEC dysfunction and reducing liver cirrhosis portal pressure without affecting the circulating pressure.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a compound for treating portal hypertension in liver cirrhosis and its applications. Background Technology

[0002] Portal hypertension (PH) in cirrhosis refers to a pathological increase in portal vein pressure caused by increased hepatic vascular resistance and congestion in the portal venous system due to chronic end-stage liver disease. PH is a driving factor for a series of complications of cirrhosis, such as ascites, gastrointestinal bleeding, hepatorenal syndrome, and hepatic encephalopathy; it is the pathological basis for these complications. Cirrhotic patients with complications of portal hypertension often have a poor prognosis, require repeated hospitalizations, and are described as having unstable decompensated cirrhosis.

[0003] Dysfunction of liver sinusoidal cells (LSECs) is a key factor leading to and exacerbating portal hypertension; however, the pathogenesis of LSEC dysfunction remains unclear. In-depth exploration of the pathogenesis of portal hypertension in cirrhosis is of great significance for the treatment and prognosis improvement of patients with cirrhosis. Summary of the Invention

[0004] This invention addresses the aforementioned technical problems by providing a drug for reducing ROS production in hepatic sinusoidal endothelial cells and / or increasing eNOS activity in hepatic sinusoidal endothelial cells and / or treating portal hypertension in cirrhosis, as well as a method for preparing the drug.

[0005] This invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a medicament for reducing the production of ROS in hepatic sinusoidal endothelial cells and / or increasing the activity of eNOS in hepatic sinusoidal endothelial cells and / or treating portal hypertension in cirrhosis, characterized in that the medicament comprises a complement inhibitor and a pharmaceutically acceptable carrier or excipient.

[0007] Optionally, the aforementioned complement inhibitors can specifically block complement activation via the classical pathway, and / or complement activation via the lectin pathway, and / or specifically block complement activation via the alternative pathway, and / or nonspecifically block complement activation via the terminal pathway.

[0008] On the other hand, another object of the present invention is to provide the use of complement inhibitors in the preparation of drugs that reduce ROS production in hepatic sinusoidal endothelial cells.

[0009] Optionally, the aforementioned drug contains complement inhibitors and pharmaceutically acceptable carriers or excipients.

[0010] Optionally, the aforementioned complement inhibitors can specifically block complement activation via the classical pathway, and / or complement activation via the lectin pathway, and / or specifically block complement activation via the alternative pathway, and / or nonspecifically block complement activation via the terminal pathway.

[0011] On the other hand, another object of the present invention is to provide the use of complement inhibitors in the preparation of drugs that enhance the eNOS activity of hepatic sinusoidal endothelial cells.

[0012] Optionally, the aforementioned drug contains complement inhibitors and pharmaceutically acceptable carriers or excipients.

[0013] Optionally, the aforementioned complement inhibitors can specifically block complement activation via the classical pathway, and / or complement activation via the lectin pathway, and / or specifically block complement activation via the alternative pathway, and / or nonspecifically block complement activation via the terminal pathway.

[0014] On the other hand, another object of the present invention is to provide the use of complement inhibitors in the preparation of medicaments for treating portal hypertension in cirrhosis.

[0015] Optionally, the aforementioned drug contains complement inhibitors and pharmaceutically acceptable carriers or excipients.

[0016] Optionally, the aforementioned complement inhibitors can specifically block complement activation via the classical pathway, and / or complement activation via the lectin pathway, and / or specifically block complement activation via the alternative pathway, and / or nonspecifically block complement activation via the terminal pathway.

[0017] On the other hand, the present invention provides a method for preparing a drug that reduces ROS production in hepatic sinusoidal endothelial cells, characterized in that the method includes preparing the drug by using a complement inhibitor as an active ingredient.

[0018] Optionally, the aforementioned drug contains complement inhibitors and pharmaceutically acceptable carriers or excipients.

[0019] Optionally, the aforementioned complement inhibitors can specifically block complement activation via the classical pathway, and / or complement activation via the lectin pathway, and / or specifically block complement activation via the alternative pathway, and / or nonspecifically block complement activation via the terminal pathway.

[0020] On the other hand, the present invention provides a method for preparing a drug that enhances the eNOS activity of hepatic sinusoidal endothelial cells, characterized in that the method includes preparing the drug by using a complement inhibitor as an active ingredient.

[0021] Optionally, the aforementioned drug contains complement inhibitors and pharmaceutically acceptable carriers or excipients.

[0022] Optionally, the aforementioned complement inhibitors can specifically block complement activation via the classical pathway, and / or complement activation via the lectin pathway, and / or specifically block complement activation via the alternative pathway, and / or nonspecifically block complement activation via the terminal pathway.

[0023] On the other hand, the present invention provides a method for preparing a drug for treating portal hypertension in cirrhosis, characterized in that the method includes preparing the drug by using a complement inhibitor as an active ingredient.

[0024] Optionally, the aforementioned drug contains complement inhibitors and pharmaceutically acceptable carriers or excipients.

[0025] Optionally, the aforementioned complement inhibitors can specifically block complement activation via the classical pathway, and / or complement activation via the lectin pathway, and / or specifically block complement activation via the alternative pathway, and / or nonspecifically block complement activation via the terminal pathway.

[0026] Optionally, the drugs in any of the aforementioned regimens may also include other compounds or drugs used to treat portal hypertension in cirrhosis, such as non-selective beta-blockers, vasodilators, somatostatin and its analogues, antibiotics, endothelin receptor antagonists, diuretics, terlipressin, etc.

[0027] Optionally, the excipients in any of the foregoing formulations may be lactose, microcrystalline cellulose, sodium carboxymethyl starch, magnesium stearate, etc., for forming tablets or capsules. Alternatively, they may be mannitol, disodium hydrogen phosphate, sodium chloride, for preparing injection solutions or lyophilized powders.

[0028] Preferably, in any of the aforementioned schemes, the complement inhibitor is a complement inhibitor that can specifically block complement activation via the classical pathway.

[0029] Preferably, in any of the aforementioned schemes, the complement inhibitor is complement component 1 inhibitor (C1-INH).

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] 1. Previous studies on the key etiology of portal hypertension in cirrhosis, "cellular dysfunction of LSECs," have mostly focused on hemodynamics. This study is the first to discover that complement activation in the portal vein promotes oxidative stress in LSECs by inducing mitochondrial dysfunction, leading to LSEC dysfunction and thus participating in the development of portal hypertension. This study explores and elucidates the pathogenesis of portal hypertension in cirrhosis from a pathophysiological perspective. This invention is the first to propose treating portal hypertension in cirrhosis by inhibiting complement activation in the portal vein.

[0032] 2. Furthermore, this invention is the first to discover that complement inhibitors can improve LSEC dysfunction and reduce portal pressure in cirrhosis by inhibiting complement activation in the portal vein without affecting circulatory pressure. Attached Figure Description

[0033] The products, methods, and their beneficial effects of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] Figure 1 This is the transcriptomics of human hepatic sinusoidal endothelial cells incubated with portal vein serum (PVS) / complement heat-inactivated serum (HIS) from patients with cirrhosis. Figure A is a schematic diagram of the mitochondrial oxidative phosphorylation (OXPHOS) pathway, showing the expression of all components in the transcriptome. Figure B is a heatmap of all components of the oxidative phosphorylation (hsa00190) pathway in PVS from decompensated patients and peripheral serum / HIS from healthy controls. Figure C is a GSEA analysis (gene set enrichment analysis) of hsa00190 pathway expression in the transcriptome.

[0035] Figure 2 This is a heatmap showing the downregulation of transcriptional levels of the MT-ND series genes in the PVS-treated group.

[0036] Figure 3 These are the results of ROS detection in cells; Figure A shows the fluorescence signal of ROS detected by the superoxide fluorescent probe; Figure B shows the statistics of relative ROS content, where * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0037] Figure 4 This represents the relative ROS content after complement activation products stimulate LSECs. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0038] Figure 5 The figures show the expression of ND1, 2, 4, 5, and 6 at the transcriptional and protein levels after LSECs were stimulated by complement activation products. Figures A through E show the transcriptional levels of ND1, 2, 4, 5, and 6, respectively. Figure F shows the protein levels of ND1, 2, 4, 5, and 6. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0039] Figure 6 The results show the protein detection results of eNOS and PeNOS in LSECs caused by complement activation products; Figure A shows the C3a treatment; Figure B shows the C5a treatment; and Figure C shows the sC5b9 treatment.

[0040] Figure 7 It represents the proportion of complement activation caused by different pathways in patients with decompensated cirrhosis.

[0041] Figure 8 Figure A shows the relative content of ROS after treatment with complement inhibitors, and the protein expression of eNOS and peNOS (Figure B).

[0042] Figure 9 This is a schematic diagram of establishing a portal hypertension model of liver cirrhosis using SD rats.

[0043] Figure 10 These are liver photographs (Figure A) of a rat model of portal hypertension with cirrhosis at different stages of development, showing nodular fibrosis of hepatocytes (Figure B), and changes in body weight (Figure C), portal vein pressure (Figure D), alanine aminotransferase (ALT) (Figure E), and aspartate aminotransferase (AST) (Figure F).

[0044] Figure 11 These figures show the changes in sC5b-9 levels in the portal vein at different stages of a rat model of portal hypertension with liver cirrhosis; Figure A shows changes in intrahepatic MAC deposition; and Figure B shows changes in sC5b-9 levels.

[0045] Figure 12 This is a schematic diagram of using C1-INH to treat rats with cirrhotic portal hypertension.

[0046] Figure 13 These are changes in serum sC5b-9 levels, portal vein pressure, arterial pressure, and heart rate after C1-INH treatment.

[0047] Figure 14 This is a correlation analysis between portal vein sC5b-9 and portal vein pressure.

[0048] Figure 15 The values ​​represent the ROS fluorescence intensity of rat LSECs before and after C1-INH treatment.

[0049] Figure 16 This is a scanning electron microscope showing the fenestrations on the surface of hepatic sinusoidal endothelial cells. The complement activation products were added to heat-inactivated peripheral serum from healthy individuals and then treated with LSECs for observation.

[0050] Figure 17 This is a scanning electron microscope showing the fenestrations on the surface of hepatic sinusoidal endothelial cells (LSECs) after treatment with complement inhibitors. Detailed Implementation

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0053] Example 1: Paired transcriptome analysis of portal vein serum (PVS) / complement heat-inactivated serum (HIS) from the same patient

[0054] Portal vein serum was collected from 10 patients in the decompensated stage of portal hypertension cirrhosis. The transcriptome of human hepatic sinusoidal endothelial cells (LSECs) incubated with the 10 pairs of patient serum was analyzed by pairing the patient's portal vein serum (PVS) with complement heat-inactivated serum (HIS).

[0055] Patient selection criteria: (a) Inclusion criteria (meeting all criteria): confirmed cirrhosis by tissue biopsy or at least two imaging examinations; planned TIPS surgery due to various decompensated events (refractory ascites, esophageal variceal bleeding, etc.); age ≥18 years and ≤85 years; subjects voluntarily participate in the study and have signed informed consent.

[0056] (b) Exclusion criteria (any one of the following): Patients with CTP grade C > 14; patients found to have hepatocellular carcinoma or extrahepatic tumors, severe organ dysfunction or other diseases affecting expected survival during preoperative evaluation; patients undergoing TIPS recanalization due to shunt failure; women who are planning to become pregnant or are already pregnant or breastfeeding; patients who refuse to sign informed consent forms.

[0057] Table 1. Information on enrolled patients

[0058]

[0059] CHB: Chronic viral hepatitis; ALD: Alcoholic liver disease; NASH: Nonalcoholic steatohepatitis.

[0060] Using complement-heat-inactivated serum (HIS) as the control group, differentially expressed genes were screened at a fold change of 1.5, resulting in 7247 genes showing differential transcriptional levels. The KEGG pathway enriched multiple disease signaling pathways involved in the pathogenic mechanism of mitochondrial dysfunction. Further analysis of mitochondrial oxidative phosphorylation (OXPHOS) gene expression revealed overall upregulation of oxidative respiratory chain genes in the PVS-treated group, suggesting possible abnormal activation and energy metabolism in LSECs of the PVS-treated group. Figure 1Additionally, complement activation treatment (portal vein serum) <pvs>The MT-ND series genes encoded by mitochondrial DNA in the group were downregulated. Figure 2 This suggests that complement activation may lead to oxidative stress and dysfunction of hepatic sinusoidal endothelial cells by causing downregulation of ND gene expression in the mitochondrial respiratory chain and mitochondrial dysfunction.

[0061] Example 2: Detection of mitochondrial ROS

[0062] To verify whether complement-activated patient portal serum could increase reactive oxygen species (ROS) production in LSECs, a superoxide fluorescent probe was used to detect ROS generation. It was found that after stimulation of LSECs with complement-activated patient serum, intracellular ROS significantly increased compared to complement-heat-inactivated serum (HIS). Figure 3 (A and 3B). This validates the transcriptomic findings that complement-activated portal serum can induce increased ROS production in LSECs, leading to cellular oxidative stress.

[0063] Example 3: Portal vein serum complement activation induces oxidative stress in hepatic sinusoidal endothelial cells, leading to endothelial dysfunction.

[0064] 1. Our previous findings revealed abnormal increases in C3a, C5a, and sC5b-9 in the portal blood of patients with cirrhosis, with these levels rising with disease progression and increased portal pressure. Furthermore, we observed specific deposition of the complement activation product C5b-9 in and around the portal vein. This suggests that cellular damage may play a key role in the production of complement activation products C3a, C5a, and sC5b-9. Therefore, we further investigated the effects of C3a, C5a, and sC5b-9 on cellular respiratory stimulation and ROS production.

[0065] LSECs cells were stimulated for 4 hours with different concentration gradients of anaphylatoxins C3a, C5a, and sC5b-9. The results showed that C3a, C5a, and sC5b-9 all increased intracellular ROS production. Figure 4 This suggests that three key complement-activating components in patient serum can reduce NO utilization by causing increased ROS in hepatic sinusoidal endothelial cells, leading to cellular oxidative stress.

[0066] 2. To investigate whether the increased ROS production in LSECs cells induced by complement activation products is caused by a decrease in ND expression, LSECs were stimulated in vitro based on the highest concentrations of complement activation products C3a, C5a, and sC5b-9 detected in patient portal blood. The results showed that the transcriptional levels of ND1, ND2, ND4, ND5, and ND6 in the cells were significantly downregulated compared to the control group (HNHS group). Figure 5 Consistent results were also observed in the protein levels of ND1, ND2, ND4, and ND6 in cells (AE). Figure 5 F).

[0067] 3. The preceding results suggest that complement activation products can increase cellular ROS production by downregulating ND protein expression in LSECs cells. ROS, in turn, can reduce eNOS activity by inhibiting phosphorylation of endothelial NO synthase (eNOS), leading to endothelial cell dysfunction. After treating LSECs with different doses of complement activation products, we found that with increasing doses of C3a, C5a, and sC5b-9, eNOS phosphorylation was inhibited (peNOS expression downregulated), and eNOS activity decreased (…). Figure 6 A-6C)

[0068] 4. In the portal blood of 10 patients with decompensated cirrhosis and portal hypertension, detected using a complement assay kit (Eagle Bioscience series), complement activation caused by the classical pathway, alternative pathway, and lectin pathway accounted for 41%, 39%, and 20%, respectively, while complement activation caused by the classical pathway and lectin pathway accounted for 61% in total. Figure 7 ).

[0069] 5. We designed a rescue experiment to verify whether complement inhibitors could improve oxidative stress and dysfunction in LSECs caused by complement activation. LSECs were treated with portal vein serum from patients with complement activation to block different complement activation pathways, and the final ROS generation and eNOS activity were examined.

[0070] Specific inhibition strategies: Complement factor C1 inhibitors (SerpinG1, C1-INH) specifically block complement activation via the classical and lectin pathways; complement factor D inhibitors (CFD-INH) specifically block alternative pathway activation.

[0071] Complete inhibition strategy: Complement factor C3 inhibitors (C3-INH) are used to non-specifically block complement activation at the terminal pathway.

[0072] The results show ( Figure 8 The use of complement inhibitors can significantly reduce ROS production, with the C1-INH group showing a more pronounced effect. Complement inhibitors can increase peNOS expression, enhance eNOS activity, and improve endothelial dysfunction in LSECs. However, the improvement in hepatic sinusoidal endothelial function after C3-INH treatment was not as significant as that after C1-INH, it may be because physiological (low-level) complement activation can play a beneficial role, such as promoting the regeneration and proliferation of damaged endothelial cells. Combined with the discovery of complement activation pathways in patients' portal blood—the classical pathway and the lectin pathway causing complement activation exceed 60%—these may explain the better therapeutic effect of C1-INH. C1-INH may improve portal hypertension in cirrhosis by inhibiting complement activation via the classical and lectin pathways in the portal vein.

[0073] Example 4: C1-INH can reduce portal vein pressure in patients with portal hypertension due to cirrhosis.

[0074] 1. We used SD rats to establish a portal hypertension model of liver cirrhosis. Each rat was repeatedly injected intraperitoneally with carbon tetrachloride, and after 8 weeks of induction, the rats reached the level of portal hypertension. Figure 9 ).

[0075] The results show ( Figure 10 After 4 weeks of repeated CCL4 injections, Masson observed a small number of irregularly proliferating hepatocytes in the liver, along with a small amount of nodular fibrosis and a small amount of fibrous tissue formation. At this time, the rats in the model group began to experience weight loss, increased liver enzymes, and elevated portal pressure. After 6 weeks of repeated CCL4 injections, Masson observed that the fibrosis was more severe, and pseudolobules began to appear in the liver, indicating significant damage to the liver tissue structure. At this time, the weight of the rats in the model group was still lower than that of the control group, and liver enzymes and portal pressure further increased. After 8 weeks of CCL4 injections, diffuse yellowish-brown nodules were visible on the surface and cut surface of the liver. Widely distributed pseudolobules were visible in the liver tissue, surrounded by coarsely proliferating fibrous septa. In addition to the severe destruction of the normal liver structure, the hepatocytes within the pseudolobules were disordered, and a large number of inflammatory cells were infiltrated. At this time, the portal pressure of the rats in the model group exceeded 10 mmHg, and ascites appeared.

[0076] Figure 11 The results showed that the level of sC5b-9 in the portal vein of rats in all groups increased with the modeling time. At CCL 4-4 weeks, although some rats showed increased sMAC (soluble membrane attack complex) in the portal vein, there was no overall difference compared to the control group. Portal vein deposition of MAC (membrane attack complex) was observed in the liver of rats, at this time MAC was only deposited within the portal vein. At CCL 4-6 weeks, the level of sC5b-9 in the portal vein of rats was higher than that in the control group; at this time, more obvious portal vein deposition of C5b-9 was observed in the liver of rats. By week 8, the level of sC5b-9 in the portal vein further increased to 690.83±29.6 ng / ml, and a large amount and extensive sC5b-9 deposition was observed in the liver tissue of rats in the modeling group. At this time, sC5b-9 was not only limited to the portal vein, but also appeared in the lymphatic vessels, fibrous tissue, and even hepatocytes surrounding the portal vein.

[0077] 2. Based on the above research, 8 weeks was ultimately selected as the CCL4 modeling endpoint. Eight weeks after CCL4 modeling, intraperitoneal injections were stopped, and the 18 successfully modeled SD rats were randomly divided into two groups: the CCL4 discontinuation group (C1-INH-Stopped) (n=6) and the intervention group (n=12). The intervention group received C1-INH injections three times a week via the tail vein for two weeks. They were further divided into two different treatment dose groups: a low-dose group of 100U C1-INH (C1-INH-Low) (n=6) and a high-dose group of 400U C1-INH (C1-INH-Hight) (n=6). Figure 12 ).

[0078] The results show ( Figure 13 In the C1-INH-Low group, the portal vein sC5b-9 level in rats decreased to 539.38±58.15 ng / ml (vs 616.61±48.52), and the portal vein pressure (PP) decreased to 8.31±0.58 mmHg (vs 10.49±0.35). In the C1-INH-High group, the portal vein sC5b-9 level in rats decreased to 470.60±33.67 ng / ml, and the portal vein pressure (PP) decreased to 8.00±1.19 mmHg. There were no significant differences in arterial pressure and heart rate between the intervention group and the control group after drug withdrawal.

[0079] Correlation analysis was performed on portal vein sC5b-9 and PP in all rats (n=36) in the natural disease model group and the treatment group to explore the relationship between these two indicators. The results showed a close correlation between portal vein sC5b-9 and PP (r=0.421, P<0.0001). Figure 14 ).

[0080] To verify in vivo the effect of complement inhibitors on improving oxidative stress in rat hepatic sinusoidal endothelial cells (LSECs), ROS levels in rat liver tissue were measured. The results showed that after two weeks of C1-INH treatment, the ROS fluorescence intensity of rat LSECs was significantly reduced, indicating a decrease in ROS production. Figure 15 )

[0081] Example 4: Fenholes on the surface of hepatic sinusoidal endothelial cells

[0082] Complement activation products C3a, C5a, and sMAC were added to heat-inactivated peripheral serum from healthy individuals and used to treat LSECs. Scanning electron microscopy was used to observe the fenestration. It was found that both C5a and MAC induced defenestration in LSECs—reducing the number and size of fenestrations on the cell surface, which is a major marker of capillary formation in LSECs. Figure 16 ).

[0083] Three different patients with PVS were treated with the classical pathway inhibitor C1inh (with and without), and the fenestration was observed by scanning electron microscopy. The results showed that the defenestration of LSEC was significantly reversed. Figure 17 ).

[0084] In summary, this invention is the first to discover that complement inhibitors can improve LSEC dysfunction and reduce portal pressure in cirrhosis by inhibiting complement activation in the portal vein, without affecting circulatory pressure.

[0085] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.< / pvs>

Claims

1. Application of complement inhibitor C1-INH in the preparation of drugs for treating portal hypertension in cirrhosis.

2. A method for preparing a drug for treating portal hypertension in liver cirrhosis, characterized in that, The method includes preparing the drug by using the complement inhibitor C1-INH as the active ingredient.

3. The application as described in claim 1 or the method as described in claim 2, characterized in that, The drug also contains pharmaceutically acceptable carriers or excipients.

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