Application of PF-06260933 in preparation of medicine for treating biliary atresia

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of PF-06260933 in preparation of a medicine for treating biliary atresia. The invention provides an application of an HGK inhibitor PF-06260933 in preparation of a medicine for treating biliary atresia. A BA organ and biliary duct ligation (BDL) induced mouse model is constructed, PF-06260933 is adopted to inhibit pro-inflammatory and pro-fibrosis related signal channels of biliary duct epithelial cells, and the improvement effect of PF-06260933 on biliary duct atresia pathological phenotypes is observed. Results show that the PF-06260933 treatment not only can significantly improve inflammatory fibrosis phenotypes of BA organs, but also can significantly improve mouse liver functions, relieve liver inflammation and fibrosis, and effectively reverse pathogenic activation phenotypes of bile duct epithelial cells. Compared with the traditional niberous surgery or non-specific immunosuppression therapy, the method has the advantages of remarkable progress and huge clinical application potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of PF-06260933 in preparation of a drug for treating biliary atresia. BACKGROUND

[0002] Biliary atresia (BA) is a severe hepatobiliary system disease that occurs in the neonatal period, and its core pathological feature is the progressive fibro-inflammatory obstruction of intrahepatic and extrahepatic bile ducts. This obstruction prevents the normal excretion of bile from the liver into the intestine, leading to cholestasis, progressive biliary cirrhosis, and ultimately end-stage liver failure. The current standard clinical treatment for BA is a hepatoenterostomy, which reconstructs the bile excretion channel to alleviate disease progression, but a significant portion of children still experience liver fibrosis that cannot be effectively controlled, ultimately requiring liver transplantation as the only means of saving their lives.

[0003] Biliary epithelial cells (BECs) are the main attack targets and core participants in the pathological process of BA. During disease development, BECs gradually lose their normal physiological functions and cell morphology, and instead acquire a phenotype with mesenchymal cell characteristics and exhibit strong pro-inflammatory and pro-fibrotic functions. Mesenchymalized BECs recruit and activate a large number of immune cells around the bile duct by actively secreting various pro-inflammatory cytokines and chemokines, thereby continuously amplifying local inflammatory reactions. At the same time, they also promote the development of liver fibrosis by highly expressing extracellular matrix components and integrin signaling molecules. The abnormal phenotype of pathogenic BECs is an important driving force for the destruction of bile duct structure, occlusion of the lumen, and progressive fibrosis of the liver.

[0004] Current clinical treatment options for biliary atresia are very limited, mainly relying on invasive palliative surgery and end-stage liver transplantation, and there is a lack of targeted drugs that can fundamentally block disease progression. Given the important role of BECs in regulating BA bile duct inflammation and fibrosis, there is an urgent need to develop innovative therapies targeting pathogenic BECs in order to improve the prognosis of BA children and reduce the demand for liver transplantation. BA-derived bile duct organoids can highly mimic the complex pathology of the disease, support individualized research, and provide a stable and reliable 3D platform in vitro, showing great potential in the field of drug screening and development. In addition, the bile duct ligation (BDL) mouse model can highly mimic the clinical pathological features of BA, including cholestasis, liver function damage, progressive bile duct inflammation, and liver fibrosis. The use of BA patient-derived bile duct organoids and BDL mouse models can help explore effective therapeutic drugs for BA.

[0005] In the prior art, the clinical treatment of BA mainly relies on surgical procedures such as hepatoenterostomy. However, the surgery is a palliative treatment and cannot fundamentally curb the progression of liver fibrosis in all children. A large number of children still have to accept liver transplantation as the only rescue treatment due to the continuous progression of the disease. The fundamental defect of the existing treatment strategy is the lack of targeted drugs that can directly intervene in the core pathological mechanism of the disease. The core pathological driving force of biliary atresia is the pathogenic transformation of BECs, and the persistent cholangitis and fibrosis mediated by them are the key link leading to bile duct obstruction and liver failure. The existing surgical procedures cannot effectively block this pathological process at the cellular level.

[0006] Therefore, there is a need to invent a drug that can target the key pathogenic link of biliary atresia. SUMMARY

[0007] In order to solve the above technical problems, the present application provides a new use of a drug that can target the key pathogenic link of biliary atresia, especially a drug that can effectively inhibit or reverse the pathogenic phenotype of cholangiocytes, thereby blocking the driven cholangitis and liver fibrosis process, to make up for the deficiency of the existing surgical treatment, delay or avoid the progression of the disease to end-stage liver disease, and ultimately improve the clinical prognosis of children and reduce the demand for liver transplantation.

[0008] In order to achieve the above-mentioned purposes of the present application, the technical solutions adopted by the present application are as follows: The present application provides the use of PF-06260933 in the preparation of a drug for treating biliary atresia.

[0009] The chemical name of PF-06260933 is 5-(4-Chlorophenyl)-[3,3'-bipyridine]-6,6'-diamine, which can reduce atherosclerotic inflammation and reduce thrombosis by inhibiting MAP4K4 (HGK), and also can reduce blood-brain barrier damage in mice and reduce intestinal ischemia / reperfusion injury. There is no report on its use in the treatment of BA disease.

[0010] The structural formula of PF-06260933 is as follows: .

[0011] In some embodiments of the present application, the PF-06260933 includes pharmaceutically acceptable salts thereof.

[0012] In some embodiments of the present application, the pharmaceutically acceptable salt includes acid addition salts and base addition salts.

[0013] "Pharmaceutically acceptable acid addition salt" refers to salts of the free base which retain the biological effectiveness and properties of the free base and which are not biologically or otherwise undesirable, formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor 10 sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane 1, 2 disulfonic acid, ethanesulfonic acid, 2 hydroxyethanesulfonic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2 oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid and the like.

[0014] "Pharmaceutically acceptable base addition salt" refers to those salts of the free acids which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable. These salts are prepared from inorganic and organic bases. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0015] In particular, the pharmaceutically acceptable salts of PF-06260933 include PF-06260933 dihydrochloride.

[0016] In some embodiments of the present application, the biliary atresia comprises viral-induced biliary atresia or biliary stasis-induced biliary atresia.

[0017] In some embodiments of the present application, the virus comprises a herpes virus, a cytomegalovirus, a rotavirus, or a reovirus.

[0018] In some embodiments of the present application, the liver disease caused by biliary atresia comprises liver fibrosis or cirrhosis.

[0019] In some embodiments of the present application, the biliary atresia is caused by viral infection in neonatal period, infant period or childhood.

[0020] In some embodiments of the present application, the drug comprises pharmaceutically acceptable excipients.

[0021] In some embodiments of the present application, the pharmaceutically acceptable excipients comprise at least one of solvent, binder, disintegrant, filler, lubricant, wetting agent, tonicity adjusting agent, stabilizer, glidant, flavoring agent, preservative, suspending agent, coating material, fragrance, anti-adhesion agent, integrating agent, penetration enhancer, pH adjusting agent, buffer, plasticizer, surfactant, antifoaming agent, thickening agent, inclusion agent, humectant, absorbent, diluent, flocculating agent and deflocculating agent, filter aid, release retardant, carrier.

[0022] The above pharmaceutically acceptable excipients are generally recognized for this purpose and as inactive ingredients of a medicament. A compilation of pharmaceutically acceptable excipients can be found in Handbook of Pharmaceutical Excipients, 2ndEdition, Edited by A. Wade and P. J. Weller; published by American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994; in the tool book of Chinese Pharmacopoeia-Pharmaceutical Excipients, etc.

[0023] In some embodiments of the present application, the dosage form of the drug comprises a gastrointestinal administration dosage form, or a non-gastrointestinal administration dosage form.

[0024] In some embodiments of the present application, the gastrointestinal administration dosage form comprises at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drop, chewable tablet. In some embodiments of the present application, the non-gastrointestinal administration dosage form comprises at least one of injection administration dosage form, respiratory administration dosage form, skin administration dosage form, mucosa administration dosage form, cavity administration dosage form.

[0025] In some embodiments of the present application, the dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults.

[0026] In some embodiments of the present application, the administration subject of the drug is a mammal. In some embodiments of the present application, the mammal comprises human.

[0027] In some embodiments of the present application, the human comprises a child or an adult.

[0028] In some embodiments of the present application, the child is a neonate within 28 days of birth, an infant under 1 year old, a toddler between 1-3 years old, a toddler between 3-6 years old, or a child between 6 years old and 18 years old.

[0029] In some embodiments of the present application, the adult is a female adult in gestation, a female adult in perinatal period, or a female adult in lactation.

[0030] In some embodiments of the present application, the drug comprises other active ingredients for treating biliary atresia.

[0031] The beneficial effects of the present application are: The present application provides the use of HGK inhibitor PF-06260933 in the preparation of a drug for treating biliary atresia. By constructing BA organoids and mouse models induced by bile duct ligation (BDL), PF-06260933 is used to inhibit the pro-inflammatory and pro-fibrotic related signaling pathways of bile duct epithelial cells, and the improvement effect on the pathological phenotype of biliary atresia is observed. The results show that PF-06260933 treatment can not only significantly improve the inflammatory fibrosis phenotype of BA organoids, but also significantly improve the liver function of mice, reduce liver inflammation and fibrosis, and effectively reverse the pathogenic activation phenotype of bile duct epithelial cells. Not only can it effectively improve the clinical symptoms and pathological changes of BA mice in phenotype, but also can accurately target pathogenic bile duct epithelial cells, which has a significant progress and great clinical application potential compared with traditional palliative surgery or non-specific immunosuppressive therapy. BRIEF DESCRIPTION OF DRAWINGS

[0032] The present application will be further described below in conjunction with the drawings and examples, wherein: Figure 1 PF-06260933 on the pathological gene expression of BA organoids, wherein: HC: healthy control group; BA: biliary atresia group; D1: biliary atresia drug intervention for 1 day; D3: biliary atresia drug intervention for 3 days; D5: biliary atresia drug intervention for 5 days.

[0033] Figure 2 PF-06260933 on the secretion of inflammatory factors of BA organoids.

[0034] Figure 3 PF-06260933 on the liver function index of mice.

[0035] Figure 4 PF-06260933 on the liver tissue pathology of mice.

[0036] Figure 5 Results of the effect of PF-06260933 on the pathological phenotype of cholangiocytes.

[0037] Figure 6 Results of the effect of PF-06260933 on the expression of various proinflammatory and profibrotic genes in cholangiocytes. DETAILED DESCRIPTION

[0038] The concept and the technical effects produced by the present application will be described below in conjunction with the embodiments so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0039] The main experimental materials of the present application are as follows: HGK inhibitor: PF-06260933 (MedChemExpress, item number HY-19562).

[0040] Solvent control (Vehicle): the same solvent formula as the working solution of PF-06260933, but without active ingredients.

[0041] EpCAM microsphere magnetic bead sorting kit (MACS).

[0042] Example 1: Effect of PF-06260933 on pathological gene expression of BA organoids.

[0043] 1. Experimental method 1) Cholangiocyte organoid culture Fresh liver tissue of a patient with biliary atresia was taken and cut into single cell suspension after enzymatic digestion.

[0044] EpCAM-positive cholangiocytes were separated by magnetic activated cell sorting (MACS) using human EpCAM microspheres.

[0045] About 100,000 sorted cells were resuspended in Matrigel and seeded in a 24-well plate in a dome shape.

[0046] After polymerization at 37℃, the organoid culture solution containing growth factors was covered.

[0047] 2) Dosing regimen The organoids were divided into three groups: healthy control group, BA model group, and BA+PF-06260933 treatment group.

[0048] Treatment groups were added with 50 μM PF-06260933 (optimal concentration determined by concentration gradient experiment) in culture medium.

[0049] Intervention was continued for 1 day, 3 days, 5 days.

[0050] 3) Observation index and detection method Transcriptome sequencing (RNA-seq): compare gene expression profiles of each group of organoids, focusing on the analysis of EMT characteristics, leukocyte chemotaxis, and changes in the activity of extracellular matrix formation pathways.

[0051] Multiple cytokine detection: collect organoid culture supernatant and detect the levels of CCL2, CXCL5, GM-CSF, IFN-γ, IL-1β, IL-8, and other pro-inflammatory factors.

[0052] 2, Experimental results The results of the effect of PF-06260933 on the pathological gene expression of BA organoids are shown in Figure 1 Compared with the BA model group, the mesenchymalization (EMT) characteristics, pro-inflammatory characteristics, and extracellular matrix formation activity in the organoids of the drug treatment group were significantly inhibited. This indicates that the HGK inhibitor PF-06260933 can significantly improve the pathological phenotype of BA organoids.

[0053] The results of the effect of PF-06260933 on the secretion of inflammatory factors by BA organoids are shown in Figure 2 The secretion of various pro-inflammatory cytokines by organoids was significantly down-regulated after drug intervention, indicating that the HGK inhibitor PF-06260933 can significantly inhibit the expression of various pro-inflammatory cytokines and improve the inflammatory characteristics of BA bile ducts.

[0054] Example 2: Effect of PF-06260933 on mouse liver function 1, Experimental method 1) Animal grouping and model construction Grouping: 4-week-old mice were randomly divided into 3 groups: Sham group (Sham): only received open surgery without bile duct ligation.

[0055] Bile duct ligation group (BDL+Vehicle): received bile duct ligation surgery and intraperitoneal injection of solvent control every day from the 7th day after surgery.

[0056] Bile duct ligation treatment group (BDL+PF-06260933): received bile duct ligation surgery and intraperitoneal injection of PF-06260933 (10 mg / kg) every day from the 7th day after surgery.

[0057] Model construction (bile duct ligation, BDL): After isoflurane anesthesia, the mice were fixed and the abdomen was prepared for skin disinfection. An incision was made along the midline of the abdomen, the abdominal cavity was opened, and the common bile duct was exposed.

[0058] For BDL and treatment group mice, the common bile duct was double-ligated with sutures and the bile duct was disconnected between the two ligation lines. For Sham group mice, only the common bile duct was isolated, but not ligated and disconnected; the abdominal incision was sutured layer by layer. After the operation, all mice were recovered in a warm environment and had free access to water and food.

[0059] 2) Dosing regimen Dosing began on the 7th day after surgery and continued for 7 days.

[0060] BDL + PF-06260933 group: intraperitoneal injection of 10 mg / kg PF-06260933 daily.

[0061] BDL + Vehicle group and Sham group: intraperitoneal injection of an equal volume of solvent control daily.

[0062] 3) Sample collection On the 14th day after surgery (i.e., 7 days after dosing), the following operations were performed: Orbital blood was collected from the mice, and after standing and centrifugation, serum was separated for liver function testing.

[0063] The mice were euthanized and dissected.

[0064] Part of the left lobe of the liver tissue was fixed in 4% PFA for subsequent paraffin embedding, sectioning, and histological staining (HE, Sirius red).

[0065] The remaining liver tissue was handled on ice, and EpCAM+ bile duct epithelial cells were sorted by magnetic beads for subsequent RNA-seq transcriptome sequencing analysis.

[0066] 4) Observation indicators and detection methods Liver function testing: Biochemical analyzer was used to detect liver function indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and direct bilirubin (DBIL) in mouse serum.

[0067] Histopathological examination: HE staining: Observe the liver tissue structure, bile duct proliferation, and the degree of inflammatory cell infiltration.

[0068] Sirius red staining: Evaluate liver collagen fiber deposition, quantify fibrosis area, and perform fibrosis scoring.

[0069] Bile duct epithelial cell transcriptome analysis: RNA extraction, library construction and RNA-seq were performed on the sorted EpCAM+ cells.

[0070] Bioinformatics analysis of sequencing data, including differential expression gene (DEG) analysis, gene set variation analysis (GSVA), focusing on the changes in mesenchymalization, pro-inflammatory response, and extracellular matrix formation pathway activity.

[0071] 2. Experimental results The effects of PF-06260933 on mouse liver function are shown in Figure 3 Compared with the BDL+Vehicle group, the serum liver function indicators of the BDL+PF-06260933 treatment group of mice were significantly reduced (P<0.05), indicating that the HGK inhibitor PF-06260933 can effectively improve the liver function of mice with biliary atresia.

[0072] The effects of PF-06260933 on mouse liver histopathology are shown in Figure 4 HE staining and Sirius red staining showed that the inflammatory infiltration and fibrosis of the livers of mice in the treatment group were significantly reduced.

[0073] The results of the improvement of PF-06260933 on the pathological phenotype of cholangiocytes are shown in Figure 5 Compared with the Sham group, the mesenchymalization, pro-inflammatory response, and extracellular matrix formation activity of cholangiocytes in the BDL+Vehicle group were significantly increased. Compared with the BDL+Vehicle group, the above pathogenic pathway activity of cholangiocytes in the BDL+PF-06260933 treatment group was significantly down-regulated. Further indicating that the HGK inhibitor can directly improve the pathological state of cholangiocytes and reverse their pro-inflammatory and pro-fibrotic phenotypes.

[0074] The results of the inhibition of PF-06260933 on the expression of various pro-inflammatory and pro-fibrotic genes in cholangiocytes are shown in Figure 6 The HGK inhibitor can directly inhibit the expression of various pro-inflammatory and pro-fibrotic related genes in cholangiocytes of BA mice.

[0075] The embodiments of the application are described in detail above in combination with the drawings, but the application is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the purpose of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of PF-062609333 in the manufacture of a medicament for treating biliary atresia.

2. The use of claim 1, wherein: the PF-062609333 comprises pharmaceutically acceptable salts thereof.

3. The use of claim 1, wherein: the biliary atresia comprises viral-induced biliary atresia or biliary stasis-induced biliary atresia.

4. The use of claim 3, wherein: the virus comprises herpes virus, cytomegalovirus, rotavirus, or reovirus.

5. The use of claim 1, wherein: the medicament comprises pharmaceutically acceptable excipients.

6. The use of claim 5, wherein: the pharmaceutically acceptable excipients comprise at least one of solvents, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, aromatics, antiadherents, integrating agents, permeation promoters, pH adjusting agents, buffers, plasticizers, surfactants, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculating and deflocculating agents, filtration aids, release retarders, carriers.

7. The use of claim 6, wherein: the dosage form of the medicament comprises a parenteral dosage form or a non-parenteral dosage form.

8. The use of claim 7, wherein: the subject of administration of the medicament comprises a mammal; preferably, the mammal comprises a human.

9. The use of claim 8, wherein: the human comprises a child or an adult.

10. The use of claim 1, wherein: the medicament comprises other active ingredients for treating biliary atresia.

Images