WO2023020212A1 - Utilisation d'inhibiteur de drd2 dans la préparation d'un médicament pour le traitement de maladies associées à la fibrose hépatique - Google Patents

Utilisation d'inhibiteur de drd2 dans la préparation d'un médicament pour le traitement de maladies associées à la fibrose hépatique Download PDF

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WO2023020212A1
WO2023020212A1 PCT/CN2022/107831 CN2022107831W WO2023020212A1 WO 2023020212 A1 WO2023020212 A1 WO 2023020212A1 CN 2022107831 W CN2022107831 W CN 2022107831W WO 2023020212 A1 WO2023020212 A1 WO 2023020212A1
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liver
yap
drd2
fibrosis
macrophages
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WO2023020212A9 (fr
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丁楅森
曹中炜
卿杰
任亚锋
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四川大学华西第二医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biomedicine, and mainly relates to the application of DRD2 inhibitors in the preparation of medicines for treating diseases related to liver fibrosis.
  • Fibrosis can occur in multiple organs and is the final pathological outcome of many common chronic inflammatory, immune-mediated metabolic diseases and the main cause of morbidity and mortality in these diseases.
  • a variety of noxious stimuli including toxins, infectious pathogens, autoimmune responses, and mechanical stress, can induce fibrotic cellular responses. Fibrosis affects all tissues in the body and, if left unchecked, can lead to organ failure and death.
  • Fibrosis is a repair response to protect the relative integrity of tissues and organs after tissue damage.
  • myofibroblasts derived from multiple sources including resident fibroblasts, mesenchymal cells, circulating fibroblasts, and transdifferentiation of other cell types can initiate wounding by remodeling the extracellular environment Healing response to restore tissue integrity and promote parenchymal cell replacement. Normally, this pro-fibrotic program is switched off while the tissue is healing.
  • ECM extracellular matrix
  • collagen including collagen, laminin, and fibronectin
  • myofibroblast activity Up-regulation of , resulting in a chronic inflammatory environment infiltrated by macrophages (and monocytes) and immune cells.
  • this "excessive deposition" although it repairs the damage, does not have the structure and function of the parenchymal cells of the organ, and will instead cause fibrosis and dysfunction of the organ.
  • liver has the unique ability to regenerate damaged liver tissue.
  • chronic or excessive liver injury often causes dysfunctional/dysregulated repair and a pronounced scarring response, leading to excessive scarring and fibrosis.
  • Hepatic fibrosis often leads to cirrhosis and liver failure and is a pathological feature of liver diseases such as nonalcoholic steatohepatitis (NASH).
  • NPCs parenchymal hepatocytes and nonparenchymal cells
  • HSCs hepatic stellate cells
  • ECs hepatic sinusoidal endothelial cells
  • the balance between liver regeneration and fibrosis may be modulated by "context-specific" mechanisms of the liver microenvironment.
  • the invention provides the application of the DRD2 inhibitor in the preparation of medicines for treating diseases related to liver fibrosis.
  • the DRD2 inhibitor is fluphenazine (Flu) and pharmaceutically acceptable salts thereof.
  • the disease associated with liver fibrosis is caused by chronic liver injury.
  • the diseases associated with liver fibrosis include nonalcoholic steatohepatitis, autoimmune hepatitis, congenital liver fibrosis, nonalcoholic fatty liver disease, cholestatic liver disease, alcoholic hepatitis, viral One or more of hepatitis.
  • the symptoms of the disease associated with liver fibrosis include increased levels of Yes-associated protein (YAP) in liver macrophages.
  • YAP Yes-associated protein
  • the symptoms of a disease associated with liver fibrosis include increased levels of one or more of the following molecules: ⁇ -smooth muscle actin ( ⁇ -SMA), connective tissue growth factor (CTGF), Vascular cell adhesion factor 1 (VCAM-1), collagen I, Yes-associated protein (YAP), phosphorylated YAP, serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST) and liver Hydroxyproline.
  • ⁇ -SMA ⁇ -smooth muscle actin
  • CTGF connective tissue growth factor
  • VCAM-1 Vascular cell adhesion factor 1
  • collagen I Yes-associated protein
  • YAP Yes-associated protein
  • ALT serum alanine aminotransferase
  • AST serum aspartate aminotransferase
  • liver Hydroxyproline liver Hydroxyproline
  • said medicament is used to selectively target said Yes-associated protein (YAP) pathway in said liver macrophages.
  • YAP Yes-associated protein
  • the medicament is for upregulating the type I interferon response.
  • the medicament is used to alleviate one or more of the following symptoms: liver fibrosis, liver damage, and intrahepatic lipid deposition.
  • the medicament is for promoting hepatocyte proliferation and/or promoting liver regeneration.
  • said medicament is used to reverse said liver fibrosis.
  • DRD2 inhibitor refers to a substance that reduces or eliminates the activity of dopamine D2 receptors.
  • DRD2 inhibitors may be compounds such as phenothiazines, clozapines.
  • the DRD2 inhibitor can be fluphenazine (Fluphenazine, a phenothiazine piperazine derivative) and pharmaceutically acceptable salts thereof, such as fluphenazine hydrochloride, fluphenazine heptanoate, Fluphenazine decanoate, etc.
  • the DRD2 inhibitor can be a polypeptide or protein, such as an antibody targeting dopamine D2 receptor.
  • the DRD2 inhibitor can also be a gene editing tool capable of reducing or eliminating the expression of dopamine D2 receptor.
  • DRD2 inhibitor and “DRD2 antagonist”, “DRD2 inhibition” and “DRD2 antagonism” have the same meaning.
  • chronic liver injury refers to damage to the liver caused by infection, exposure to drugs or toxic compounds, alcohol, impurities in food, abnormal accumulation of normal substances in the blood, autoimmune processes, genetic defects, or other factors chronic damage. Chronic liver damage may lead to liver fibrosis and cirrhosis.
  • liver fibrosis refers to a class of diseases characterized by chronic liver damage and fibrosis.
  • exemplary “diseases associated with liver fibrosis” include autoimmune hepatitis, congenital liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), cholestatic liver disease, alcoholic Hepatitis, viral hepatitis.
  • NASH nonalcoholic steatohepatitis
  • NAFLD nonalcoholic fatty liver disease
  • cholestatic liver disease alcoholic Hepatitis
  • viral hepatitis viral hepatitis.
  • reduce generally mean to reduce by a statistically significant amount, for example, by between about 10% and Any amount between approximately 100%.
  • increase”, “enhance”, “enhance”, “activate”, “upregulate” and “induce” generally mean increasing by a statistically significant amount, e.g., increasing relative to a reference level by about Any amount between 10% and about 100% or an increase of about 2-fold or more relative to a reference level.
  • liver regeneration is a repair process in which the remaining hepatocytes in the liver grow out of a morphologically and/or functionally the same structure as before the damage or resection by proliferating after the liver is damaged or partially resected.
  • enhancing refers to the further improvement of the described object at the existing level, and the existing level includes one or more of quantitative level, expression level, functional level, ability level.
  • reverse fibrosis refers to transforming an organ in a fibrotic state to a regenerative direction, including reducing the level of fibrosis, reducing the severity of symptoms caused by fibrosis, enhancing regenerative ability, and the like.
  • Figure 1 shows that YAP levels are increased in macrophages from human cirrhotic livers and mouse fibrotic livers
  • Figure 2 shows that deletion of myeloid-specific YAP attenuates liver fibrosis by stimulating type I interferon signaling
  • Figure 3 shows scRNA-Seq analysis to determine the altered cell populations caused by myeloid-specific deletion of YAP after CCl4 -induced chronic injury
  • Figure 4 shows the determination of crosstalk between YAP-deficient macrophages and endothelial cells
  • FIG. 5 shows G protein-coupled receptor (GPCR) ligand library screening to identify dopamine receptor DRD2 antagonists targeting macrophage pro-fibrogenesis YAP;
  • Figure 6 shows that DRD2 antagonists and myeloid-specific deletion of Drd2 attenuate liver fibrosis
  • Figure 7 shows selective targeting of the pro-fibrotic DRD2-YAP axis in macrophages attenuates liver fibrosis by stimulating type I interferon signaling
  • Figure 8 shows that DRD2 antagonists reduce liver fibrosis in a minipig NASH model
  • Figure 9 shows the percentage of F4/80 + cells relative to visual hepatocytes (related to Figure 1);
  • Figure 10 shows immunofluorescence or ⁇ -SMA and Sirius red staining for YAP (green), F4/80 (red) and DAPI (blue) for the number of injections shown in Figure 1B;
  • FIG 11 shows the knockout efficiency of myeloid-specific deletion of Yap (related to Figure 2);
  • Figure 12 shows immunofluorescent staining of F4/80 (A) or Desmin (green) (B) at the indicated number of injections (with IFN- ⁇ (red) and DAPI (blue), related to Figure 2I);
  • Figure 13 shows that IFN- ⁇ treatment protects liver function after CCl4- induced injury (related to Figure 2);
  • Figure 14 shows immunofluorescence of F4/80 (green) or CD31 (red) and DAPI (blue) showing Yap1 fl/fl Lyz2 -Cre + small Decreased number of macrophages or endothelial cells in mouse liver slices (associated with Figure 3);
  • Figure 15 shows a comparison of the frequencies of different cell populations in WT or Yap1 fl/fl Lyz2-Cre + mice (related to Figure 3);
  • Figure 16 shows a violin plot showing the expression of endothelial cell markers Cdh5 and Pecam1 in endothelial cell subsets (related to Figure 3);
  • Figure 17 shows a heatmap showing differentially expressed transcripts in endothelial cell subpopulations (related to Figure 3);
  • Figure 18 shows immunofluorescence and quantification of CTGF or VCAM1 (green), CD31 (red) and DAPI (blue) in liver sections from control or Yap1 fl/fl Lyz2-Cre + mice after 3 injections of CCl (related to Figure 4);
  • Figure 19 shows immunofluorescence showing that macrophages labeled by F4/80 were mainly aggregated with sinusoidal endothelial cells (LYVE-1) after one injection of CCl4 (scale bar, 50 ⁇ m; related to Figure 4);
  • LYVE-1 sinusoidal endothelial cells
  • Figure 20 shows immunofluorescence of DRD2, F4/80 (macrophages) and HNF- 4 ⁇ (hepatocytes) demonstrating that DRD2 is selectively induced in macrophages but not in In hepatocytes (scale bar, 100 ⁇ m; related to Fig. 5);
  • Figure 21 shows immunofluorescence of DRD2 and YAP showing that, at the indicated CCl injection times, these proteins were co-expressed in large quantities and that changes in their respective expression levels were correlated (scale bar, 100 ⁇ m; related to Figure 5);
  • Figure 22 shows body weight measured after Flu treatment in a CCl4- induced chronic liver fibrosis model (related to Figure 6);
  • Figure 23 shows the knockout efficiency of Drd2 myeloid-specific deletion (related to Figure 6);
  • Figure 24 shows qRT-PCR analysis of mRNA expression of IFIT1, OAS2, MX1, ISG15 and IFIT3 in anti-IFNAR1 treated or control mice (related to Figure 7);
  • Figure 25 shows that immunofluorescence of YAP and F4/80 demonstrates that anti-IFNAR1 treatment does not affect YAP levels (related to Figure 7);
  • Figure 26 shows that antibody blockade of IFNAR1 signaling reverses enhanced liver regeneration in myeloid-specific DRD2 or YAP deficient mice (related to Figure 7);
  • Figure 27 shows immunofluorescence of VCAM1 (green) and CD31 (red) demonstrating that antibody blockade of IFNAR1 signaling reversed the repressed VCAM1 in myeloid-specific DRD2 or YAP-deficient mice (scale bar, 100 ⁇ m; 7 related);
  • Figure 29 shows pathological examination of human control and cirrhotic sections (related to Figure 1);
  • Figure 30 shows the apparent effect of IFN- ⁇ treatment during or after liver fibrosis (repair) (related to Figure 2);
  • Figure 31 shows a summary schematic diagram of an embodiment of the invention.
  • the term "about” typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 4% of the stated value /-3%, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, even more typically +/-0.5% of the stated value.
  • FIG. 3 (A) Hepatic nonparenchymal cells (NPCs) isolated from mouse livers and subjected to scRNA-Seq after 4 CCl injections. Cell lines were inferred from the expression of marker genes and annotated: B cell, B cells; T cell, T cells; Mono, monocytes; Neu, neutrophils; EC, endothelial cells; DC, dendritic cells; MP , macrophages; Hepa, hepatocytes; HSC, hepatic stellate cells. (B) Comparison of frequencies of different cell populations in control or Yap1 fl/fl Lyz2-Cre + mice. (C) Prediction and scoring of cell-cell interactions based on the ligand/receptor principle.
  • Figure 4 (A) Quantification of the frequency of Ctgf + Vcam1 + ECs in healthy and cirrhotic human livers from a public database (http://www.livercellatlas.mvm.ed.ac.uk.).
  • (FH) IFN- ⁇ treatment suppressed CTGF and VCAM1 mRNA expression in mouse liver sections after 1 injection of HUVECs and CCl4 (n 4 mice/group). Scale bar, 100 ⁇ m.
  • Figure 5 (A) Schematic diagram of the screening strategy based on the activity of the 8 ⁇ GTIIC-luc reporter gene. (B) Luciferase assay for Flu treatment was performed in Raw264.7 or HepG2 cells. (C) Western blot analysis of DRD2 expression in hepatocytes and macrophages isolated from WT mice. (D) Immunofluorescence demonstrates that DRD2 is selectively highly expressed in macrophages (F4/80) but not in hepatocytes (HNF-4 ⁇ ) and correlates with altered levels of YAP. Scale bar, 100 ⁇ m.
  • E,F,G,H Flu induced YAP phosphorylation and upregulated interferon ⁇ 1 (Ifnb1) in Raw264.7 cells (E,G) or peritoneal macrophages (F,H).
  • Figure 7 (A,B) KEGG enrichment of RNA-sequencing data from CD45 + CD11b + F4/80 + liver macrophages from WT or Drd2 fl/fl Lyz2-Cre + mice after 4 CCl injections Pathway analysis and scatterplots.
  • FC fold change.
  • (E) Ki67 staining assessed hepatocyte proliferation in WT, Yap1 fl/fl Lyz2-Cre + and Drd2 fl/fl Lyz2-Cre + mice after 7 CCl injections (n 3 mice/group). Scale bar, 50 ⁇ m.
  • (FJ) Antibody blockade of IFNAR1 signaling reversed attenuation of liver fibrosis in myeloid-specific DRD2 or YAP deficient mice (n 3 or 4 mice/group). Sirius red and Masson staining (F,G), liver hydroxyproline (H ) and VCAM1 and Ki67 expression (I, J). Scale bar, 100 ⁇ m.
  • FIG. 8 (A) Schematic summarizing the experimental setup for assessing the efficacy of the DRD2 antagonist Flu in the minipig NASH model induced by Western diet and chemical insult.
  • Figure 9 Data quantified as percentage of F4/80 + cells relative to visual hepatocytes in control and cirrhotic human liver sections as shown in Figure 1A. All results are shown as mean ⁇ SD. *p ⁇ 0.05;**p ⁇ 0.01.
  • Figure 11 (A) qRT-PCR analysis of YAP mRNA in wild type (WT) and Yap1 fl/fl Lyz2-Cre + BMDMs. (B) Western blot analysis of YAP expression in wild-type and Yap1 fl/fl Lyz2-Cre + BMDMs.
  • Figure 23 (A) qRT-PCR analysis of DRD2 mRNA in WT and Drd2 fl/fl Lyz2-Cre + BMDMs. (B) Immunoblot analysis of DRD2 expression in WT and Drd2 fl/fl Lyz2-Cre + BMDMs.
  • Figure 26 Ki67 staining to assess hepatocyte proliferation in WT, Yap1 fl/fl Lyz2-Cre + or Drd2 fl/fl Lyz2-Cre + mice after chronic injury of CCl4 with or without antibody blockade of IFNAR1 signaling .
  • Scale bar 50 ⁇ m.
  • FIG 30 Sirius red staining, ⁇ -SMA or type I collagen expression in IFN ⁇ - CCl4 (1-4) (A) or IFN ⁇ - CCl4 (4-7) models (B) were quantified.
  • the IFN ⁇ -CCl 4 (1-4) model evaluates the effect of IFN- ⁇ on liver fibrosis at the early stage (four injections) of CCl 4 chronic injury.
  • Embodiment one material and method
  • TBIL total bilirubin
  • DBIL direct bilirubin
  • IBIL indirect bilirubin
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • GGT glutamyl transpeptidase
  • mice were maintained in a specific pathogen free (SPF) environment.
  • Yap1 fl/fl mice (Stock No. 032192, Jackson Laboratory, Bar Harbor, ME) and Drd2 fl/fl mice (Stock No. 020631, Jackson Laboratory) were crossed with the Lyz2-Cre transgene to obtain Yap1 fl/fl -Lyz2, respectively -Cre + and Drd2 fl/fl -Lyz2-Cre + mice.
  • Mice were genotyped using polymerase chain reaction (PCR) with tail DNA as template.
  • PCR polymerase chain reaction
  • liver fibrosis was induced by intraperitoneal injection of 1.6 g/kg of CCl 4 to mice every 3 days for 7 times; or corn oil was used as a control to less induce liver damage. Two days after the last injection, mice were sacrificed for analysis.
  • liver fibrosis was also induced by extrahepatic cholestasis by bile duct ligation (BDL) [ref.
  • mice were anesthetized with isoflurane via an inhaler. The control group received a sham operation consisting of exposure but no ligation. Twenty days after surgery, mice were analyzed. To generate the NASH model [Ref. S2], mice were fed a Western diet containing 21.1% fat, 41% sucrose and 1.25% cholesterol (w/w) and a diet containing 23.1 g/L d-fructose and 18.9 g/L d- Glucose high glucose solution, continued for 20 weeks, and treated with 0.32g/kg of CCl 4 , intraperitoneal injection, once a week.
  • a Western diet containing 21.1% fat, 41% sucrose and 1.25% cholesterol (w/w) and a diet containing 23.1 g/L d-fructose and 18.9 g/L d- Glucose high glucose solution, continued for 20 weeks, and treated with 0.32g/kg of CCl 4 , intraperitoneal injection, once
  • mice were injected peritoneally with Flu (MCE, catalog HY-A0081, 1 mg/kg), IFN- ⁇ protein (Sino Biological, catalog 50708-MCCH, 20 ug/kg), K- 7174 (25 mg/kg) or anti-mouse IFNAR-1 antibody (BioLegend, MAR1-5A3, 5 mg/kg).
  • Flu MCE, catalog HY-A0081, 1 mg/kg
  • IFN- ⁇ protein Seo Biological, catalog 50708-MCCH, 20 ug/kg
  • K- 7174 25 mg/kg
  • anti-mouse IFNAR-1 antibody BioLegend, MAR1-5A3, 5 mg/kg
  • Miniature pig model Bama miniature pigs were purchased from Chengdu Dashuo Biotechnology Co., Ltd. and raised in Chengdu Dashuo Experimental Animal Center. Each piglet was individually housed in a single cage. Male minipigs were used in NASH studies [Refs S2-S4]. Minipigs were fed a high-fat diet (containing 2% cholesterol and 30% fat [w/w]) and high-sugar water (2.31% fructose and 1.89% glucose) and injected every 3 days with 20% CCl4 dissolved in corn oil ( 0.25ml/kg).
  • a high-fat diet containing 2% cholesterol and 30% fat [w/w]
  • high-sugar water 2.31% fructose and 1.89% glucose
  • the treatment group was injected with Flu (0.11 mg/kg, 5% dimethyl sulfoxide (DMSO)) every 3 days, while the control group was injected with the same volume of normal saline (containing 5% DMSO). The experiment lasted 6 months, and liver biopsies were taken to test the effect of Flu treatment.
  • Flu 0.11 mg/kg, 5% dimethyl sulfoxide (DMSO)
  • RAW264.7 cells, HEK293T cells and HepG2 cells were cultured in supplemented with 10% (v/v) fetal bovine serum (FBS) (catalogue number 10099-141) (GIBCO, USA), 100 U/ml penicillin and 100 ⁇ g/ml Streptomycin in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, USA), 37°C, 5% CO2 in a humidified incubator.
  • FBS fetal bovine serum
  • DMEM Dulbecco's modified Eagle's medium
  • EMOA mouse hemangioendothelioma cells
  • Primary endothelial cell culture system Primary endothelial cell culture system (PriMed-iCell-002).
  • BMDMs bone marrow-derived macrophages
  • GPCR protein compound library screening For screening experiments in HEK293T cells, a GPCR/G protein compound library (489 compounds dissolved in DMSO, 10 mM) was obtained from MCE (MedChemExpress Inc.). The sequence of the YAP/TAZ reactive promoter was previously described and cloned into the pGL3-basic vector to construct the 8 ⁇ GTIIC-Luc reporter [ref. S6]. HEK293T cells were transfected with plasmids with 8 x GTIIC-Luc reporter or PGL3-promoter (as a reference for normalization of transfection efficiency). After 24 hours, cells were plated on blank clear-bottom 96-well plates and grown overnight.
  • drugs were transferred from library stock plates (10 mM in DMSO) to plates containing 10 [mu]M cells. After another 24 hour incubation, the cells in the 96-well plate were lysed and tested for luciferase activity. Luminescence was detected according to the protocol of the Bright-Glo luciferase detection system (Promega). Experiments in HepG2 cells were performed with a similar procedure.
  • the 8 ⁇ GTIIC-Luc sequence was cloned into the pEZX-LvGA01 lentiviral vector (GeneCopoeia) to construct the Lv-8 ⁇ GTIIC-Luc reporter (contains 2 reporter genes: for testing Gaussia luciferase (Gluc) of the YAP-responsive promoter and secreted alkaline phosphatase (SEAP) normalized according to transfection efficiency as a reference). Screening of lentiviral particles and stable cell lines were prepared as before. Luminescence was detected according to the protocol of the Secrete-Pair TM Dual Luminescence Detection Kit (GeneCopoeia).
  • shRNA sequence information used in the present invention is as follows: NC, 5'-TTCTCCGAACGTGTGTCACGTTTC-3' (SEQ ID NO: 1); shDRD2-1, 5'-CCACTACAACTATGCCAT-3' (SEQ ID NO: 2), shDRD2-2, 5 '-GACCAGAATGAGTGTATCATT-3' (SEQ ID NO:3), shDRD2-3, 5'-CAGGATTCACTGTGACATCTT-3' (SEQ ID NO:4); shYAP-1, 5'-CCACCAAGCTAGATAAAGAAA-3' (SEQ ID NO:5 ), shYAP-2, 5'-GCGGTTGAAACAACAGGAATT-3' (SEQ ID NO: 6), shYAP-3, 5'-CTGGTCAAAGATACT
  • the LV2 plasmid containing the shRNA sequence and the helper plasmids (pMD2.G and pSPAX2) encoding the backbone structure were co-transfected into HEK293T cells by using lipofectamine 6000 transfection reagent (Biyotime, China).
  • the above "shRNA sequence” actually refers to the DNA sequence corresponding to the shRNA (that is, the base U involved in the RNA sequence is replaced by the base T), and this naming method is established by those skilled in the art.
  • the desired shRNA sequence and its corresponding DNA sequence first design the desired shRNA sequence and its corresponding DNA sequence; then synthesize the DNA sequence corresponding to the shRNA and connect it to a vector (such as a plasmid) and transfect it into a host cell; finally, the desired shRNA will be expressed in the host cell expression and exert its gene silencing function. After 48 hours, the supernatant was collected and filtered through a 0.22 ⁇ M membrane. The supernatant was diluted 1:4 and then incubated with Raw 264.7 cells for 48 hours at 37°C, 5 ⁇ g/ml polybrene.
  • MOI vesicular stomatitis virus
  • liver macrophages and hepatocytes Liver tissue was finely minced with scissors and treated with 2 mg/ml collagenase A and DNase as previously described (with some modifications) [ref. S8]. Cell-to-cell contacts were broken using an 18G syringe, and the cell suspension was filtered and centrifuged. Then, the cell pellet was washed once with DPBS and treated with erythrocyte lysis reagent, and the cells were washed, centrifuged and resuspended in DPBS.
  • NPCs were centrifuged and washed by 1-2 mL of MACS buffer (Miltenyi, 130-091-221), and centrifuged and resuspended in 90 ⁇ L of MACS buffer. Then, NPCs were incubated with anti-F4/80 magnetic beads (Miltenyi, 130-110-443) and incubated at 4°C for 15 minutes with shaking. F4/80 + cells were labeled with anti-F4/80 magnetic beads and collected using LS MACS columns (Miltenyi, 130-042-401 ) and separators. Wash the column three times with 3 mL of MACS buffer to remove excess beads and unlabeled cells. After removing the column from the magnetic field, magnetically retained F4/80 + cells were eluted as selected cells.
  • RNA quantification and sequencing Total cellular RNA was isolated using TRIzol reagent following standard protocols. One-step qRT-PCR was performed with QuantiTect SYBR Green RT-PCR kit (Qiagen, Germany) using Applied Biosystems 7500 real-time thermal cycler (Applied Biosystems, USA). The sequences of RT-PCR primers used in the present invention are shown in Table 3. Target gene and GAPDH transcript levels were determined by the ⁇ CT method. According to the standard procedure, the total cellular RNA of mouse liver macrophages sorted by flow cytometry was isolated with TRIzol reagent, and sent to Beijing Genomics Institute (BGI) for RNA sequencing analysis.
  • BGI Beijing Genomics Institute
  • Transcriptome libraries were constructed using BGISEQ-500RS RNA-Seq (quantitative). Align the sequenced reads (Sequenced reads) with the mouse (Mus musculus) reference genome (GRCm38.p5) with HISAT2 (version 2.1.0) [Reference S10], and use HTSeq-count (version 0.9.1) Aligned reads were quantified for mRNA expression [ref. S11]. DESeq2 (R/Bioconductor package) was used to determine differentially expressed genes between samples [ref. S12]. Pathway enrichment analysis and Gene Ontology (Gene Ontology) analysis were performed using KOBAS 3.0 (available online: http://kobas.cbi.pku.edu.cn/).
  • Single-cell RNA sequencing and analysis According to the procedure of Guangzhou Gideo Biotechnology Co., Ltd., NPCs were loaded into the GemCode instrument (10 ⁇ Genomics) to generate single-cell gel beads in emulsion (Gel Bead in emulsion, GEMs). Briefly, PCR tubes containing GEMs are placed on the PCR machine to generate cDNA. According to the 10 ⁇ Genomics single-cell 3' library construction V3 process, 35 ⁇ L cDNA obtained by reverse transcription was added to 65 ⁇ L cDNA amplification mixture to amplify the cDNA. Library quality detection was performed using a high-sensitivity DNA detection kit (Agilent Technologies).
  • ABI Step One Plus real-time PCR system (Life Technologies) was used for quantification and pooling, and sequencing was performed according to the PE150 mode of illuminaNovaseq.
  • 5269 cells from wild-type NPCs passed the quality control threshold of >1000 transcripts and 4731 cells from Yap1 fl/fl -Lyz2-Cre+ NPCs passed. All datasets were analyzed and processed by the R 3.6.1 Seurat package [S8].
  • the membrane was blotted with the specific primary antibody for 1 hour and washed 3 times for 5 minutes with Tris buffered saline solution containing 0.1% Tween-20 (v/v). It was then incubated with horseradish peroxidase-conjugated secondary antibody for 45 minutes and washed 5 times for 5 minutes with Tris-buffered saline containing 0.1% Tween 20 (v/v). Proteins were visualized and quantified by ImageJ software using chemiluminescence with Clarity Western ECL substrate (Bio-rad, USA).
  • Example 2 YAP levels increase in macrophages of human cirrhotic liver and mouse fibrotic liver
  • YAP expression in macrophages in cirrhotic patients human liver samples from non-cirrhotic or cirrhotic patients were immunofluorescently stained for YAP and F4/80. observed that YAP expression was significantly increased in human cirrhotic liver samples. In particular, YAP expression induced during cirrhosis was found to be partially colocalized with F4/80 + macrophages ( FIG. 1A , FIGS. 29A-29C , FIG. 9 ).
  • liver fibrosis was induced by repeated intraperitoneal injections of carbon tetrachloride (CCl4 ) injury [Ref. 2].
  • Liver fibrosis was assessed by immunohistochemistry of ⁇ -smooth muscle actin ( ⁇ -SMA) and morphometric analysis of Sirius red.
  • ⁇ -SMA ⁇ -smooth muscle actin
  • Sirius red morphometric analysis of Sirius red.
  • ⁇ -SMA and Sirius red were gradually enhanced, and liver fibrosis was evident after 4 CCl injections (Fig. 1B), which is consistent with what was reported in [Ref. 2] .
  • Example 3 Myeloid-specific YAP deficiency alleviates liver fibrosis in mice and liver fibrosis in NASH model
  • Example 4 Genetic deletion of YAP in macrophages activates type I interferon signaling after chronic liver injury
  • CD45 + CD11b + F4/80 + macrophages were isolated from myeloid Yap-deficient mice or control mice after 4 injections of CCl4 and analyzed.
  • RNA sequencing analysis 416 differentially expressed genes were identified (P ⁇ 0.05, fold change cut-off >1.5).
  • Gene Ontology (GO) enrichment analysis revealed six major enriched pathways, including 'wound healing' and 'response to interferon beta (IFN- ⁇ )' (Fig. 2G).
  • IFN- ⁇ interferon beta
  • Fig. 2G Genes related to class I interferon signaling were upregulated in Yap-deficient macrophages, including Ifnb1, Ifi27I2a, Ifi44, Isg15, etc.
  • IFN- ⁇ protein is mainly produced by macrophages and is increased in the liver of myeloid Yap-deleted mice during the early stages of CCl4 -induced chronic injury, but at later stages (during which, HSCs marked by desmin, an important source of IFN- ⁇ protein) did not increase (Fig. 2I-2K, Fig. 12A-12B). Immunofluorescence staining revealed that hepatic IFN- ⁇ protein expression was significantly increased in bone marrow Yap-deleted macrophages after 1 or 3 injections of CCl4 (Fig. 2K). Treatment with IFN- ⁇ attenuated liver fibrosis after chronic CCl4 injury, confirming its anti-fibrotic function (Fig. 2L-2M, Fig.
  • IFN- ⁇ mainly exerts this activity during the early stages of CCl4- induced chronic injury, rather than after fibrosis has already occurred (Fig. 30A-30B).
  • Example 5 Single-cell analysis identifies altered cell populations caused by YAP deficiency in macrophages
  • Fig. 3C the prediction of cellular crosstalk based on the ligand/receptor principle also indicated the possibility of high-level interactions between macrophages and ECs.
  • Specific markers for macrophages (Adgre1, Clec4f, Macro and Trem2) and ECs (Pecam1 and Cdh5) were determined (Fig. 3D-3E).
  • Single-cell t-distributed stochastic neighborhood embedding (tSNE) analysis of ECs revealed five cell subpopulations, among which EC subpopulation 1 (EC1) showed higher expression of the pro-fibrotic genes Ctgf and Vcam1 (Fig. 3F–H, Fig. 16, Figure 17). Therefore, the inventors decided to focus on determining the crosstalk between macrophages and EC populations.
  • other cell types including HSCs, were also affected after myeloid-specific Yap deletion (Fig. 3I-3J).
  • Example 6 Crosstalk between YAP-deficient macrophages and endothelial cells affects liver fibrosis
  • Fig. 4A Human liver samples from non-cirrhotic or cirrhotic patients were co-stained for CTGF or VCAM1 with the endothelial marker CD31. In cirrhotic liver samples, the expression of CTGF and VCAM1 was significantly increased in CD31 + ECs (Fig. 4B-4C).
  • CTGF and VCAM1 in endothelial cells were highly upregulated after 1 or 3 CCl4 injections, which was reversed by myeloid-specific Yap deletion (Fig. 4D, Fig. 18).
  • Immunofluorescence staining showed that macrophages mainly aggregated with CTGF + VCAM1 + sinus endothelial cells marked by LYVE-1 (lymphatic endothelial cell receptor 1) after 1 injection of CCl4 (Fig. 4E, Fig. 19).
  • Example 7 High-throughput GPCR ligand library screening to determine compounds that selectively antagonize YAP that promotes fibrosis in macrophages
  • the inventors conducted a screening experiment with a GPCR protein compound library and found that Fluphenazine dihydrochloride (Flu), a classic dopamine receptor D2 (DRD2) antagonist, can effectively target and block dopamine receptor body D2 for therapeutic use), was one of the most potent compounds at reducing YAP activity (Fig. 5A). Furthermore, Flu decreased the signal of YAP response in macrophage Raw264.7 but not in hepatocyte HepG2 (Fig. 5B). Western blotting of proteins isolated from mouse liver macrophages or hepatocytes confirmed that DRD2 was selectively highly expressed in macrophages (Fig. 5C).
  • Example 8 DRD2 antagonism or myeloid-specific Drd2 deletion alleviates liver fibrosis
  • Drd2 fl/fl Lyz2-Cre + mice exhibited mild liver fibrosis and attenuated liver injury after repeated injections of CCl 4 (Fig. 6D, Fig. 6E). In both cholestatic injury [ref.
  • Example 9 Targeting the DRD2-YAP axis in macrophages to inhibit fibrosis by stimulating type I IFN responses
  • Immunofluorescence staining revealed that myeloid Drd2 deletion upregulated IFN- ⁇ expression after 1 or 3 injections of CCl 4 , and suppressed CTGF or VCAM1 in ECs and enhanced liver function following CCl 4 -induced chronic injury. Proliferation of cells (Fig. 7C-E).
  • Type I interferons act through a coreceptor consisting of IFNAR1 and IFNAR2.
  • IFNAR1 neutralizing antibodies MAR1-5A3
  • corresponding IgG isotype controls were tested in mice with bone marrow-specific deletion of Drd2 or Yap (Fig. 7F).
  • IFNAR1 neutralizing antibody Treatment with an IFNAR1 neutralizing antibody successfully inhibited genes downstream of type I interferon signaling, including Ifit1, Oas2, Mx1, Isg15, and Ifit3 (Figure 24), without affecting liver YAP levels ( Figure 25).
  • This treatment with IFNAR1-neutralizing antibodies reversed the attenuation of liver fibrosis, upregulation of endothelial VCAM1 expression, and enhanced hepatocyte proliferation observed during CCl4 -induced chronic injury in myeloid Drd2- and Yap-deficient mice ( Figure 7G -J, Figure 26, Figure 27).
  • the Flu-treated group showed a lower degree of liver fibrosis compared with the control group, and Oil Red O staining of liver sections showed that the Flu-treated group had less lipid deposition ( FIG. 8B ).
  • the deposition of ⁇ -SMA and collagen I, as well as the amount of hepatic hydroxyproline were all decreased ( FIGS. 8C-8D ).
  • Flu treatment significantly promoted hepatocyte proliferation in NASH minipig livers (Fig. 8E).
  • the present invention found that YAP levels in macrophages were increased in both human and mouse livers with hepatic fibrosis, and demonstrated through a series of experiments that genetic and pharmacological targeting of DRD2 in hepatic macrophages can alleviate Liver fibrosis.
  • the present invention also found that the pro-fibrotic dopamine receptor D2 (DRD2)-YAP axis is specifically present in liver macrophages, and after the initiation of liver injury, macrophages modulate the DRD2/YAP/IFN signaling axis. Anti-fibrotic function.
  • DRD2 or YAP genes in hepatic macrophages also understood as genetic targeting of the YAP pathway in hepatic macrophages
  • IFN type I interferon
  • Pro-fibrotic CTGF connective tissue growth factor
  • VCAM1 vascular cell adhesion factor 1
  • endothelial cell subsets ie improved liver fibrosis.
  • YAP-IFN signaling in macrophages affects liver fibrosis at least in part by regulating the above-mentioned endothelial cell subsets that exhibit pro-fibrotic molecular phenotypes.
  • this endothelial cell subset might further recruit pro-fibrotic immune cells or stimulate CTGF-dependent fibroblast activation via VCAM1.
  • drugs targeting DRD2 in liver macrophages may be potential drugs for treating liver fibrosis.
  • the present invention found that the profibrotic function of YAP in macrophages may be triggered by dopamine receptor D2 (DRD2). It was found that selective dopamine receptor D2 (DRD2) antagonists (also understood as pharmacological targeting of the YAP pathway in hepatic macrophages) administered to a large animal (minipig) NASH model recapitulating human pathology Blocks YAP in macrophages (but not hepatocytes) and selectively inhibits YAP-dependent pro-fibrotic functions of macrophages. Such DRD2 antagonists (preferably Flu) have been shown to effectively block fibrosis, restore liver structure, and promote liver regeneration in large animal (minipig) NASH preclinical models.
  • DRD2 antagonists preferably Flu
  • DRD2 antagonists are currently actually used to block bone, brain and lung metastasis of tumor cells and to treat diseases involving the central nervous system such as schizophrenia.
  • DRD2 antagonists of the present invention block the effects of liver fibrosis, providing additional insight into their therapeutic effects and uses.
  • the DRD2 antagonism (pharmacological and genetic targeting of DRD2 in macrophages) involved in the present invention induces type I interferon signaling through the YAP pathway, selectively targets liver macrophages, and effectively promotes The liver regenerated and avoided fibrosis, and even reversed it (summarized in Figure 31).
  • the above-mentioned anti-fibrosis effect of DRD2 antagonism has been demonstrated in both rodent and large animal models.
  • VCAM-1 is a TGF-beta1 inducible gene upregulated in idiopathic pulmonary fibrosis. Cellular signaling 2015; 27(12):2467-73.
  • Interferon regulatory factor 3 and type I interferons are protective in alcoholic liver injury in mice by way of crosstalk of parenchymal and myeloid cells. Hepatology 2011;53(2):649-60.
  • Torres-Rosas R et al. Dopamine mediates vagal modulation of the immune system by electroacupuncture. Nature medicine 2014; 20(3):291-5.

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Abstract

L'invention concerne l'utilisation d'un inhibiteur de DRD2 dans la préparation d'un médicament pour le traitement de maladies associées à la fibrose hépatique. Dans un mode de réalisation, l'inhibiteur de DRD2 est la fluphénazine (Flu) et un sel pharmaceutiquement acceptable de celle-ci.
PCT/CN2022/107831 2021-08-18 2022-07-26 Utilisation d'inhibiteur de drd2 dans la préparation d'un médicament pour le traitement de maladies associées à la fibrose hépatique WO2023020212A1 (fr)

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