A cell therapeutic drug for treating a disease associated with steatosis

Abstract

This invention discloses a cell therapy drug for treating diseases related to steatosis. The cell therapy drug comprises endothelial progenitor cells or endothelial cells derived from iPSC differentiation. This invention demonstrates that intravenous injection of iPSC-derived endothelial progenitor cells into an animal model of fatty liver disease can treat fatty liver disease by reducing steatosis in liver tissue. The research findings of this invention provide a novel treatment approach for clinical treatment of diseases related to steatosis, especially fatty liver disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular, the present application relates to a cell therapy drug for treating fatty liver disease. BACKGROUND

[0002] Fatty liver disease (FLD) refers to the condition of abnormal increase of fat accumulation in the liver, which is mainly divided into alcoholic fatty liver disease (AFLD) and non-alcoholic fatty liver disease (NAFLD). NAFLD is one of the most common liver diseases at present, which is mainly characterized by hepatocyte steatosis, and is often accompanied by the occurrence of metabolic syndrome such as obesity, type 2 diabetes, hypertension and dyslipidemia during the course of the disease. In the absence of a history of continuous or recent heavy drinking, the use of drugs that promote fat formation, and the absence of specific genetic diseases, the presence of hepatocyte simple steatosis (>5%) in the liver can be diagnosed as NAFLD. The severity thereof can range from simple fat accumulation (simple fatty liver) to fatty hepatitis with inflammation (NASH), and even develop into cirrhosis and hepatocellular carcinoma.

[0003] Currently, the treatment of fatty liver disease mainly focuses on controlling risk factors and preventing disease progression. Changing lifestyle, losing weight, increasing physical activity, and improving dietary habits are the basis for treating NAFLD. Losing 5% of body weight can significantly improve liver steatosis (EASL-EASD-EASO Clinical Practice Guidelines on the management of metabolic dysfunction-associated steatotic liver disease (MASLD) [J]. J Hepatol, 2024, 81(3): 492-542.); drug therapy, there is no approved drug specifically for NAFLD at present, but some drugs such as vitamin E, insulin sensitizers (such as metformin) and anti-inflammatory drugs (such as corticosteroids) have shown certain effects in clinical trials (Xiang Y, Shen L, Xue Y, et al. Efficacy and safety of diacerein monotherapy in adults with obesity: A randomized, double-blind, placebo-controlled trial [J]. Diabetes Obes Metab, 2024.); surgical treatment, for patients with fatty liver disease caused by severe obesity, if lifestyle intervention and drug therapy are ineffective, weight loss surgery can be considered, which can significantly reduce body weight and improve the condition of fatty liver disease (Yang Jialin, Peng Yong. Research progress of weight loss surgery in the treatment of non-alcoholic fatty liver disease [J]. China Modern General Surgery Progress, 2021, 24(1): 51-53, 57.); But the adjustment of lifestyle requires the patient to adhere to it for a long time and may take effect slowly; Drug therapy may be accompanied by adverse reactions and must be carried out under the guidance of professional medical personnel; Surgical treatment involves high risk and high cost. Therefore, it is urgent to develop new drugs to treat fatty liver disease and provide more treatment options for patients with fatty liver disease. SUMMARY

[0004] In order to solve the problems existing in the prior art, the inventors of the present application have made extensive and in-depth research and for the first time found that iPSC differentiated endothelial progenitor cells have the effect of treating fatty liver disease, especially non-alcoholic fatty liver disease, providing a new treatment method for the treatment of fatty liver disease.

[0005] Based on this, the present application provides the following technical solutions:

[0006] A pharmaceutical composition for treating, preventing, alleviating, and / or ameliorating a disease associated with steatosis is provided in a first aspect of the present invention.

[0007] Further, the pharmaceutical composition comprises iPSC-differentiated endothelial progenitor cells or endothelial cells.

[0008] The term "endothelial progenitor cell" as used herein means a cell whose differentiation is directed to endothelial cells. Endothelial progenitor cells can be confirmed by analyzing the expression pattern of transcription factors or cell surface antigens. For example, the expression pattern of transcription factors or cell surface antigens is measured alone or in combination, wherein the expression level thereof is not detected or low (even if it is detected before induction of differentiation), and is significantly increased after induction of differentiation. Markers effective for confirming endothelial progenitor cells include UBXN11, LRRC75A, MMACHC, FXYD5, VMP1, KRT18, YIPF2, CNP, SPATA21, SAP30L AS1, SLC12A6, ANP32E, CTNNB1, VIM, CNN3, RPL8, YWHAH, SDC4, HIST1H4I, SRPX2, DNASE1, MYO19, SH3KBP1, ASAP1, RNF24, SPNS2, S100A11, SOX4, RNF214, PLP2, PIH1D2, SGPL1, ELK3, F3, UGDH, RBMS2, FMNL3, RPL6, ATP2A1, EFCAB10, SLC12A2, UCKL1 AS1, ARHGAP18, RPE, SFT2D2, CXCL16, PAICS, ZBTB11 AS1, CLIC1, CERS5, CCDC85C, DHRS7, CTRL, TYMS, SPCS1, IFITM3, RPS27, RPL34, MZF1 AS1, TRAM1, SUPT7L, NOL9, MFSD11, PLD4, NQO1, NEAT1, GNAS, LDHA, UNKL, LDHB, CENPK, C1orf174, HSP90B1, TCF7L2, LENG8, IQGAP3, ANKRD13D, NPC1, MYH11, ATP2B4, FANCD2, TNRC6B, DDX17, TIRAP, HERC4, DSTN, FOXP3, ABL2, KDELR2, CCDC50, PARK7, CAPN2, ACAP1, SLC13A4, SEPT11, RAPH1, KRT8, LMO3, CIRBP, CYP20A1, LY6E, FAM69A, PRSS3, MEST, NPB, CLMP, MSN, SUCLA2, PCMTD1, PSMC3, TOMM7, GCHFR, MEIS1, TRIAP1, USP34, SPAG1, RPL39L, PDK1, LARS, MAP4K4, QTRT1, CLK1, TSC22D1 AS1, IL1B, MKRN2OS, ARL16, TSPAN13, HMGCS1, PSAP, SLC44A2, TNFRSF10B, MSC, ENG, TLDC2, TRIM41, CPM, CREB3L2, NSUN6, AHCY, NCBP1, N4BP2L2, RAB31, RNF149, SRPK2, SBDS, NAMPT, AES, SERPINF1, RPS27L, HAUS2, AP4B1 AS1, KDM2B, USP6NL, TONSL, FBLIM1, SLC31A2, ADGRL1, EFHC1, SMAD9, SLC7A11, LPIN1, DDAH2, PID1, SCAF11, BBS1, PCYOX1, CD55, PMEL, TOR1B, UBQLN1, DUSP5, CUEDC2, FKBP5, LMF2, LYRM7, NSG1, SAT2, LMAN1, NFX1, COMMD1, CCPG1, NINJ1, RNASEH1, SPATA5, CENPW, DNAJC4, PTTG1, TMEM109, MCTS1, TMEM63A, FBN1, TPM1, MAVS, ADPRHL2, CTSK, INSIG1, C16orf74, MAZ, MEG3, RFTN2, AGAP6, GPC6, PDLIM5, SLC25A5 AS1, RSL1D1, FAM219B, CFLAR, GNPTAB, PIGU, ITGB3BP, NTM, ZNF106, RPRD1B, ETS1, ITGAE, ECSCR, SND1, TRIO, ANGPTL1, SEC61G, CDR2, CRMP1, PNISR, OXLD1, PRKACA, NFKBIZ, MORN2, RAB3D, CRKL, MSL1, TMPO, GALNT2, CRELD2, ETS2, ALKBH4, PHF19, POLH, TRRAP, NET1, ADGRE5, GAS6, VEZF1, BMPR2, SMAD3, GFOD1, RABGAP1L, SLC39A7, TCTN1, CSRNP1, LRRN3, TNRC6A, MFAP2, TMEM98, SPATC1L, LINC00476, CENPQ, CSTF3, SLC8A1, E2F7, RPL7L1, ZFHX4, TXNDC11, STK4, STX16, TMEM11, PTK6, ZFAS1, U2AF2, TRIM16, ZBTB7A, P3H3, TMCO3, C15orf39, ACVRL1, SHQ1, GNS, SPRY1, DIAPH2, DLC1, TCF3, FAM229A, PTGES3L, RRP7A, MGRN1, GIT2, UBE2T, CSGALNACT2, EFEMP2, CSNK1E, MAGED2, C5AR1, ARHGAP45, CDKN2B AS1,TMEM234,RNF213,SIDT2,TEX2,MCM3,TMEM67,ZKSCAN1,SLC2A1,MAD2L1,MGLL,NR3C1,PHGDH,SYT1,RERE,KIF1B,N CAPD3,NADK,FOXRED2,GNB5,TMEM204,USP24,KIF20B,GADD45B,ZNF135,TACC3,FOSB,ACOT9,EBF1,MYO1E,PPP1R10,NF KBIB,ROBO4,TPGS1,CFD,KIF22,EIF4A3,RNF14,NPAT,STX11,CDCA7,ST6GALNAC4,RBL1,NIF3L1,GLCE,CDCA7L,RASA3, STK10,EDNRA,ICA1L,DCTN5,GNG2,PTPN2,BST1,BMP1,RPS6KA2,SEC11C,TRMU,H1F0,STXBP5L,AP5B1,STAM,MCM2,ALDH3 B1,SLC23A2,TNFAIP3,KLF7,DIP2C,KMT2B,GALNT7,NUAK2,PLXNA4,MRI1,FLNB,MBOAT2,TMEM136,AP3M2,ACSS2,AARS, DUS3L,NGLY1,ZNF274,ICAM1,FAM129A,PPP6R1,TIE1,NUMBL,PLXND1,ENC1,CCDC142,BCAN,PRR15,FLRT2,NUCB2,TARS 2,POLB,RAB24,KBTBD6,SLC38A4,MAP2,PMP22,TOP3A,VAMP2,UBTD2,PLK4,MAPK8IP3,APBA2,TBC1D17,ZNF444,PLEKHG 4B,MTOR,STIL,GBA,IFT88,B9D1,RAMP1,S1PR3,SHISA9,MOCS3,CEP104,TIAM1,KIF15,ZNF793,ZNF865,ABCB10,ANKHD1 EIF4EBP3, CMPK2, SYTL3, SHROOM1, SPATA6, PPP2R3B, TNFSF13B, FKBP7, CNTROB, PEX11A, LHFPL2, DOK3, ARMC9, RANBP10, ZNF561, SCAMP5, ARHGAP39, DENND3, ATP9A , DCPS, ZNF302, ARHGAP9, IP6K1, ADAM22, EMCN, CYFIP2, WDR90, PPM1M, C1QTNF6, DACH1, BMF, SETD4, TBC1D9, PIK3IP1, P4HTM, CD101, SCPEP1, TLDC1, GTPBP10, TAP2. The isolation, purification, in vitro culture, and characterization of endothelial progenitor cells were described in Hill et al., N. Engl. J. Med.: 593-600 (2003); Assmus et al., Circulation 106: 3009-16 (2002); Wang et al., J. Am. Coll. Cardiol. 4949: 1566-71 (2007); and Kalka et al., PNAS 97: 3422-7 (2000), the contents of which are incorporated herein by reference in their entirety.

[0009] Endothelial progenitor cells and their progeny can be cryopreserved until needed using any method known in the art. (See, for example, U.S. Patent No. 5,071,741, PCT International Patent Applications WO93 / 14191, WO95 / 07611, WO96 / 27287, WO96 / 29862 and WO98 / 14058, Karlsson et al., 65 Biophysical J. 2524) 2536 (1993)). EPCs can be suspended in an isotonic solution containing a specific cryopreservative, preferably cell culture medium. Such cryopreservatives include dimethyl sulfoxide (DMSO), glycerol, etc. These cryopreservatives are used at concentrations of 5-15% (e.g., 8-10%). The cells are gradually frozen to temperatures from -10°C to -150°C (e.g., -20°C to -100°C, or -70°C to -80°C).

[0010] In some implementations, iPSCs can be derived from somatic cells, induced from somatic cells. These somatic cells can be stem cells or mature cells. These cells can be referred to as "donor cells." Adult stem cells are undifferentiated cells distributed throughout the body. They can proliferate through cell division to replenish dead cells and regenerate damaged tissue. Adult or somatic stem cells have been identified in many organs and tissues, including the brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, intestine, liver, ovarian epithelium, and testes. They are believed to reside in specific regions within each tissue, known as "stem cell nests," and provide a cell source only for that tissue. Types of adult stem cells include hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, and skin stem cells. If mature cells are harvested as donor cells, they can come from any tissue, organ, body fluid, or bodily secretions. Therefore, cells can be skin cells, hair follicle cells, blood cells, cells extracted from urine, or cells collected from any tissue or organ, including but not limited to bones, teeth, dental tissue, heart, lungs, brain, pancreas, liver, kidneys, bladder, uterus, intestines, stomach, gallbladder, muscles, fat, testes, mucous membranes, eyes, foreskin, prostate, spleen, or any other tissue.

[0011] In this invention, any of many standard techniques well known to those skilled in the art can be used to induce iPSC differentiation into endothelial progenitor cells. For example, iPSC differentiation into endothelial progenitor cells can be induced by adding specific growth factors to the culture medium or by adjusting the composition of the culture medium (such as adding vascular endothelial growth factor VEGF or endothelial cell differentiation-promoting factor FGF-2, etc.). In a specific embodiment of this invention, the endothelial progenitor cells from which iPSC differentiation originate are derived from Cenogenes Regenerative Medicine Technology (Beijing) Co., Ltd.

[0012] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0013] Endothelial progenitor cells or endothelial cells derived from iPSC differentiation can be formulated together with a carrier in a suitable morphology, which is a pharmaceutically acceptable carrier commonly used in cell therapy. Therefore, the pharmaceutical compositions described above in this invention comprise endothelial progenitor cells or endothelial cells and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a composition that is physiologically permissible and generally does not cause gastrointestinal disturbances, dizziness, or other allergic reactions or similar reactions when administered to humans. Examples of pharmaceutically acceptable carriers include, for example, water, suitable oils, physiological saline, water-soluble glucose, and non-oral delivery carriers such as ethylene glycol, and may further include stabilizers and preservatives. Suitable stabilizers are antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives are benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. For other pharmaceutically acceptable carriers, please refer to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0014] In some implementations, solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc. Such solid dosage forms can be prepared by mixing one or more excipients into a mixture, for example, by mixing starch, calcium carbonate, sucrose or lactose, gelatin, etc. Furthermore, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid dosage forms for oral administration include suspensions, internal solutions, emulsions, syrups, etc. Besides widely used water and liquid paraffin as simple diluents, they may also contain various excipients, such as humectants, sweeteners, flavorings, preservatives, etc.

[0015] In some implementations, formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, oils, freeze-dried formulations, suppositories, etc. Non-aqueous solvents and suspending agents can use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable lipids such as ethyl oleate, etc. The base of suppositories can use Witepsol, polyethylene glycol, Tween 61, cocoa butter, laurates, glycerin, gelatin, etc.

[0016] In some embodiments, the pharmaceutical composition of the present invention may have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, oils, lyophilized agents, and suppositories. The base of the suppository may be Witepsol, polyethylene glycol, Tween 61, cocoa butter, laurates, glycerin, gelatin, etc.

[0017] Furthermore, the diseases associated with fatty degeneration include fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia.

[0018] Furthermore, the diseases associated with fatty degeneration are selected from fatty liver diseases.

[0019] In a specific embodiment of the present invention, the applicant has experimentally demonstrated that endothelial progenitor cells derived from iPSC differentiation can improve the symptoms of steatosis in mice with fatty liver disease. Therefore, the pharmaceutical composition comprising endothelial progenitor cells or endothelial cells derived from iPSC differentiation described in this invention can treat, prevent, alleviate, and / or relieve diseases associated with steatosis, including fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia, wherein the symptom is steatosis.

[0020] Furthermore, the pharmaceutical composition further includes any one or more of the following functional ingredients:

[0021] 1) Components that maintain the activity of endothelial progenitor cells or endothelial cells;

[0022] 2) Components that promote the proliferation of endothelial progenitor cells or endothelial cells;

[0023] 3) Components that promote the differentiation of endothelial progenitor cells.

[0024] Furthermore, the functional ingredients include serum substitutes, non-essential amino acids, glutamine, stabilized dipeptides of L-alanyl-L-glutamine, growth factors, or any combination thereof.

[0025] Furthermore, the functional ingredients include KOSR, MSC serum-free additive, UltraserTMG, glycine, and L... alanine, L Asparagine, L Aspartic acid, L Glutamic acid, L Proline, L Serine, VEGF, bFGF, EGF, TGFβ, PDGF or any combination thereof.

[0026] As an alternative, the pharmaceutical composition of the present invention comprises endothelial progenitor cells or secretions of endothelial cells derived from iPSC differentiation.

[0027] The secretion may be the culture supernatant of endothelial progenitor cells or endothelial cells derived from iPSC differentiation, or it may be secretory factors of endothelial progenitor cells or endothelial cells derived from iPSC differentiation contained in purified culture supernatant.

[0028] In this invention, the term "culture supernatant" refers to a culture medium obtained by culturing cells (e.g., endothelial progenitor cells or endothelial cells of this invention) that does not contain the cells themselves. Therefore, a culture supernatant suitable for use in this invention can be obtained, for example, by separating and removing cellular components after culturing. This culture supernatant can also undergo other processing, such as centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze-drying, dilution, desalting, preservation, etc.

[0029] A second aspect of the invention provides the use of iPSC-derived endothelial progenitor cells or endothelial cells in the preparation of medicaments for the treatment, prevention, mitigation, and / or relief of diseases associated with fatty degeneration.

[0030] Furthermore, the diseases associated with fatty degeneration include fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia.

[0031] Furthermore, the diseases associated with fatty degeneration are selected from fatty liver diseases.

[0032] Furthermore, the amount of endothelial progenitor cells or endothelial cells derived from iPSC differentiation in a unit dose is 1 × 10⁻⁶. 4 ~1×10 6 cells.

[0033] Preferably, the amount of endothelial progenitor cells or endothelial cells derived from iPSC differentiation in a unit dose is 1 × 10⁻⁶. 6 cells.

[0034] Furthermore, the drug can be used directly or in the form of a pharmaceutical composition.

[0035] Furthermore, the pharmaceutical composition includes endothelial progenitor cells or endothelial cells derived from iPSC differentiation.

[0036] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0037] Furthermore, the dosage forms of the drug include non-gastrointestinal dosage forms and / or gastrointestinal dosage forms.

[0038] Furthermore, the non-gastrointestinal dosage forms include injectable dosage forms, cavity dosage forms, mucosal dosage forms, and / or skin dosage forms.

[0039] Furthermore, the gastrointestinal dosage forms include tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, suspensions, syrups, and / or drops.

[0040] Preferably, the dosage form of the drug is selected from injectable dosage forms.

[0041] In some embodiments, the dosage form of the pharmaceutical composition of the present invention is a dosage form that is advantageous for administration, prepared by conventional methods, including but not limited to: non-gastrointestinal dosage forms and gastrointestinal dosage forms. Specific examples include but are not limited to: aqueous injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder inhalers, sustained-release agents, and controlled-release agents.

[0042] In some embodiments, the injectable dosage form includes, but is not limited to, various injectable formulations such as intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and intracavitary injection.

[0043] In some implementations, the cavity drug delivery dosage form includes, but is not limited to: suppositories, aerosols, effervescent tablets, drops, pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.

[0044] In some embodiments, the mucosal drug delivery dosage forms include, but are not limited to: eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, adhesive tablets, and films.

[0045] In some embodiments, the skin delivery dosage forms include, but are not limited to: topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.

[0046] A third aspect of the present invention provides a method for reducing and / or alleviating fatty degeneration of hepatocytes, cardiomyocytes or renal tubular epithelial cells in vitro.

[0047] Furthermore, the method includes contacting hepatocytes, cardiomyocytes, or renal tubular epithelial cells with endothelial progenitor cells or endothelial cells derived from iPSC differentiation, or contacting hepatocytes, cardiomyocytes, or renal tubular epithelial cells with secretions from endothelial progenitor cells or endothelial cells derived from iPSC differentiation.

[0048] In some embodiments, fatty degeneration primarily occurs in hepatocytes, cardiomyocytes, or renal tubular cells. In a specific embodiment of this invention, the inventors have experimentally demonstrated that endothelial progenitor cells or endothelial cells derived from iPSC differentiation can reduce fatty degeneration in liver tissue; therefore, the endothelial progenitor cells or endothelial cells derived from iPSC differentiation or their secretions described in this invention can alleviate and / or relieve fatty degeneration in hepatocytes, cardiomyocytes, or renal tubular epithelial cells.

[0049] A fourth aspect of the present invention provides a method for evaluating whether a drug to be screened has therapeutic, preventive, alleviating and / or relieving effects on diseases related to fatty degeneration.

[0050] Furthermore, the method includes using endothelial progenitor cells or endothelial cells derived from iPSC differentiation as positive agents to evaluate the efficacy of the drugs to be screened.

[0051] Furthermore, the diseases associated with fatty degeneration include fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia.

[0052] Preferably, the disease associated with fatty degeneration is selected from fatty liver disease.

[0053] The fifth aspect of the present invention provides a method for treating diseases related to fatty degeneration.

[0054] Furthermore, the method includes administering to the subject an effective amount of the iPSC differentiation-derived endothelial progenitor cells or endothelial cells, the secretions of the iPSC differentiation-derived endothelial progenitor cells or endothelial cells, or the pharmaceutical composition described above.

[0055] Furthermore, the diseases associated with fatty degeneration include fatty liver disease, coronary heart disease, myocardial infarction, kidney disease, diabetes, and hyperlipidemia.

[0056] Preferably, the disease associated with fatty degeneration is selected from fatty liver disease.

[0057] The "therapeutic effective amount" will be the amount of an active agent capable of preventing or at least slowing down (alleviating) a medical condition such as fatty degeneration. The dosage and administration of the cellular or pharmaceutical compositions disclosed herein can be determined by those skilled in the art of clinical pharmacology or pharmacokinetics. The effective amount of the active agent of the invention used in treatment will depend, for example, on the treatment objective, route of administration, and the patient's condition. Accordingly, the therapist may need to adjust the dosage and change the route of administration as needed to achieve the best therapeutic effect.

[0058] The terms “subject” and “patient” are used interchangeably. This term includes, but is not limited to, humans, non-human animals such as non-human primates like chimpanzees and other ape and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domesticated subjects such as dogs and cats; laboratory animals, including rodents such as mice, rats, and guinea pigs; and so on. This term does not indicate a specific age or sex. Therefore, it is intended to encompass adult and newborn subjects, as well as fetuses, regardless of male or female. The term “subject” also includes living organisms susceptible to conditions or disease states as generally disclosed (but not limited to) throughout this specification. Examples of subjects include humans, dogs, cats, cattle, goats, and mice, including transgenic species.

[0059] When used for treatment, the "dosing regimen" can vary depending on a variety of factors, including the activity of the specific composition used, the patient's age, weight, general health condition, sex and diet, timing of administration, route of administration, excretion rate, drug combination, and the severity of the specific disease to be prevented or treated. Although the dosage of the pharmaceutical composition can vary depending on the patient's condition and weight, the severity of the disease, the form of the drug, and the route and duration of administration, it can be appropriately selected by those skilled in the art, and administration can be once daily or several times daily. This dosage is not intended to limit the scope of the invention in any way. The pharmaceutical compositions of the present invention can be formulated in the form of pills, sugar-coated tablets, capsules, liquids, gels, syrups, pastes, or suspensions.

[0060] In some embodiments, the dosage of the iPSC-derived endothelial progenitor cells or endothelial cells is not less than 1 × 10⁻⁶. 4 cells / mL (e.g., not less than 1×10⁻⁶) 4 cells / ml, not less than 3 × 10 4 cells / ml, not less than 5 × 10 4 cells / ml, not less than 7 × 10 4 cells / ml, not less than 1×10 5 cells / ml, not less than 3 × 10 5 cells / ml, not less than 5 × 10 5 cells / ml, not less than 7 × 10 5 cells / ml, not less than 1×10 6 cells / ml, not less than 3 × 10 6 cells / ml, not less than 5 × 10 6 cells / ml, not less than 7 × 10 6 cells / ml, not less than 1×10 6 cells / ml, not less than 3 × 10 6 cells / ml, not less than 5 × 10 6 cells / ml, not less than 7 × 10 6 cells / ml, not less than 1×10 6 cells / ml, not less than 3 × 10 6 cells / ml, not less than 5 × 10 6 cells / ml, not less than 7 × 10 6 cells / ml, not less than 1×10 6 cells / ml, not less than 3 × 10 6 cells / ml, not less than 5 × 10 6 cells / ml, not less than 7 × 10 6 cells / ml, not less than 1×10 10 cells / ml, not less than 3 × 10 10 cells / ml, not less than 5 × 1010 1 / ml or not less than 7×10 10 cells / ml, for example 1×10 5 1×10 8 7×10 5 7×10 6 1×10 6 5×10 6 per ml.

[0061] In other embodiments, the dosage of the iPSC-derived endothelial progenitor cells or endothelial cells is not less than 1 × 10⁻⁶. 3 Units / kg (e.g., not less than 1×10) 3 Units / kg, not less than 3×10 3 Units / kg, not less than 5×10 3 Units / kg, not less than 7×10 3 Units / kg, not less than 1×10 4 Units / kg, not less than 3×10 4 Units / kg, not less than 5×10 4 Units / kg, not less than 7×10 4 Units / kg, not less than 1×10 5 Units / kg, not less than 3×10 5 Units / kg, not less than 5×10 5 Units / kg, not less than 7×10 5 Units / kg, not less than 1×10 6 Units / kg, not less than 2×10 6 Units / kg, not less than 5×10 6 Units / kg, not less than 7×10 6 Units / kg, not less than 1×10 7 Units / kg, not less than 3×10 7 Units / kg, not less than 5×10 7 Units / kg, not less than 7×10 7 Units / kg, not less than 1×10 8 Units / kg, not less than 3×10 8 Units / kg, not less than 5×10 8 Units / kg, not less than 7×10 8 Units / kg, not less than 1×10 9 Units / kg, not less than 3×10 9 Units / kg, not less than 5×10 9 Units / kg, not less than 7×10 9 Units / kg, not less than 1×10 10Units / kg, not less than 3×10 10 Units / kg, not less than 5×10 10 Units / kg or not less than 7×10 10 per kg, for example 1×10 5 1×10 8 7×10 5 7×10 6 1×10 6 5×10 6 per kg.

[0062] The iPSC-derived endothelial progenitor cells or endothelial cells of this invention can be administered alone or in combination with other therapies. Co-administration of the cells of this invention with other therapies can be performed simultaneously or sequentially. Single or multiple doses are possible. Importantly, the minimum possible amount sufficient to achieve maximum efficacy without side effects should be used, taking all factors into account.

[0063] Advantages and beneficial effects of the present invention:

[0064] This invention is the first to discover that endothelial progenitor cells derived from iPSC differentiation can be used to treat fatty liver disease, especially non-alcoholic fatty liver disease, providing a new treatment approach for fatty liver disease. Attached Figure Description

[0065] Figure 1 This is a graph showing the changes in body weight of mice in each group during the experiment;

[0066] Figure 2 These are HE staining images of the livers of mice in each group;

[0067] Figure 3 This is a graph showing the scoring results of the degree of fatty degeneration, inflammatory cell infiltration, macrophage proliferation and necrosis in each group of mice. Detailed Implementation

[0068] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0069] Example 1: Study on the therapeutic effect of iPSC-EPCs on non-alcoholic fatty liver disease in db / db mice

[0070] 1. Experimental materials

[0071] (1) Test sample and reference sample

[0072] Test sample

[0073] Name: iPSC-EPCs (marked with DIR)

[0074] Batch number: 2024412

[0075] Specifications: 100 μL / vial

[0076] Concentration: 1×10 8 cells / mL

[0077] Storage conditions: 2~8℃

[0078] Shelf life: Use within 4 hours

[0079] Storage conditions and shelf life after opening: Single use only.

[0080] Provided by: Chenuo Regenerative Medicine Technology (Beijing) Co., Ltd.

[0081] Manufacturer: Chenno Regenerative Medicine Technology (Beijing) Co., Ltd.

[0082] Reference standard / solvent

[0083] Name: Physiological saline containing 3% human serum albumin

[0084] Specifications: 100 μL / vial

[0085] Storage conditions: 2~8℃

[0086] Shelf life: Use within 4 hours

[0087] Storage conditions and shelf life after opening: Single use only.

[0088] Provided by: Chenuo Regenerative Medicine Technology (Beijing) Co., Ltd.

[0089] Manufacturer: Chenno Regenerative Medicine Technology (Beijing) Co., Ltd.

[0090] (2) Experimental materials and equipment

[0091] The reagents and instruments used in the in vivo efficacy experiment are shown in Tables 1 and 2.

[0092] Table 1. Reagents used in in vivo experiments

[0093]

[0094] Table 2. Instruments and equipment used in in vivo experiments

[0095]

[0096] (3) Experimental animals and feeding

[0097] Animals

[0098] Species & strain: db / db mice; db / m mice

[0099] Grade: SPF grade

[0100] Number and sex of animals: A total of 16 male db / db mice and 5 db / m mice were ordered.

[0101] Supplier: Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.

[0102] Production license number: SCXK(Zhe)2019-0004

[0103] Animal quality certificate number: 20240311Abbb0105000608.

[0104] Feeding conditions

[0105] The animals were housed in intelligent individually ventilated cages (IVCs). The cage specifications were: 362 mm × 154 mm × 135 mm, with no more than 5 animals per cage. Artificial lighting, light:dark = 12 h:12 h. The animals were kept free to eat, drink and move throughout the feeding process.

[0106] Feed

[0107] Growth and reproduction feed for SPF rats and mice, produced by Beijing Keao Xieli Feed Co., Ltd. The batch number was 200663313, the production license number was SCXK(Jing)2019-0003, and the quality certificate number was 1112622000023997. Comprehensive refilling was carried out 1-2 times a week. Fast the animals for about 4-5 h before sacrifice.

[0108] Drinking water

[0109] Watsons drinking water, and the water bottles were changed 1-2 times a week.

[0110] Litter

[0111] Shaving litter, produced by Chenfu Yidianyuan Litter Processing Factory in Dachang Hui Autonomous County. The batch number was 20200622, and the production license number was SCXK(Ji)2017-001. It was changed once a week, and when there were abnormal situations such as water leakage or soiling in the cage, it should be changed at any time.

[0112] Identification of experimental animals

[0113] Ear tags were used to mark the animals, and the ear tags of the animals in this cage were shown on the cage card.

[0114] Label of experimental cage

[0115] Cage labeling: Before grouping, use temporary cage cards to mark the animal cages. After grouping, indicate the experiment number, cage number, dosage, animal group, breed / strain, sex, animal number, experiment supervisor, and experiment start and end dates on cage cards of different colors.

[0116] 2. Experimental Methods

[0117] 3. (1) Model building and grouping

[0118] The db / db mouse has a defect in the leptin receptor gene and is a typical rodent model of diabetes, exhibiting symptoms such as overeating, obesity, hyperlipidemia, insulin resistance, and hepatic steatosis. It is a suitable animal model to mimic human metabolic syndrome-related NAFLD.

[0119] Grouping method: All animals were weighed and their blood glucose was measured in a clean bench. Sexual behavior tests were conducted and the number of erections was recorded. Based on weight and blood glucose, the animals were randomly grouped using a random number method. The group with the smallest difference in average value among the groups was selected as the final group. The specific dosing regimen is shown in Table 3.

[0120] Table 3 Dosing Regimen

[0121]

[0122] (2) Detection indicators

[0123] General observation: After grouping, observations were conducted twice a week until the end of the experiment. The observations included the drug administration site, the animals' mental state, diet, and exercise.

[0124] Weight measurement: After grouping, the mice were weighed twice a week using an electronic balance until the end of the experiment.

[0125] Sample collection: 0.5-1.0 mL of blood was collected via the abdominal aorta before euthanasia, centrifuged at 4000 r for 10 min, and the serum was separated and stored at -80℃ for later use.

[0126] (3) Euthanasia of laboratory animals

[0127] When the experiment ends or a humane end point is reached, the animals are euthanized using CO2.

[0128] Gross Anatomy

[0129] Gross observation: All animals found to be dead, euthanized due to near death, or euthanized according to plan on day 17 in this experiment underwent systematic dissection. Liver tissue was harvested for subsequent operations and fixation.

[0130] During gross dissection, observe the animal's body surface and orifices, cranial cavity, thoracic cavity, abdominal cavity, and their contents for any abnormalities. Record the gross dissection findings and take photographs.

[0131] (4) Histopathological examination

[0132] Organ weight: The final body weight and liver weight of all animals scheduled for euthanasia were measured after fasting, and the organ coefficient was calculated.

[0133] Histopathology: Liver tissue was fixed, dehydrated, and embedded to prepare paraffin blocks. The surface of the paraffin blocks was trimmed using a rotary microtome to fully expose the tissue samples, and then sections were prepared with a thickness of 3-5 μm. After sectioning, the sections were placed in a 65℃ incubator for 30-60 minutes. HE staining was performed: Hematoxylin and eosin were used to stain the tissue sections and graft samples, and the histopathology was observed under a microscope.

[0134] (5) Statistical analysis

[0135] For measurement data such as body weight, organ weight, and coefficients, the following methods were used for statistical analysis:

[0136] The Kolmogorov-Smirnov method should be used for normality testing, the Levene median method for homogeneity of variance testing, and a one-way ANOVA should be performed. If the normality and homogeneity of variance tests fail, then the nonparametric Kruskal-Wallis test is required.

[0137] If the ANOVA result is significant (P≤0.05), then the Dunnett t-test should be used for multiple comparisons; if the ANOVA result is not significant (P>0.05), then the statistical analysis is terminated.

[0138] If the Kruskal-Wallis test result is significant (P≤0.05), then the Mann-Whitney test is used for multiple comparisons; if the Kruskal-Wallis test result is not significant (P>0.05), then the statistical analysis ends.

[0139] 3. Experimental Results

[0140] (1) General observation

[0141] General observations showed that no abnormalities were observed in any group of mice during the experimental observation period.

[0142] (2) Weight

[0143] Weight monitoring results showed that the weights of wild-type control mice (db / m) on D0 and D17 were 30.32±1.24g and 29.48±1.11g, respectively, remaining relatively stable; the weights of model control mice on D0 and D17 were 60.80±3.95g and 62.18±2.54g, respectively, showing steady weight gain; the weights of iPSC-EPCs group on D0 and D17 were 60.28±7.23g and 56.89±7.33g, respectively, showing a gradual decreasing trend. The weight changes of each group are shown in the table below. Figure 1 .

[0144] (3) Gross anatomical observation and organ coefficients

[0145] Gross visual observation of the organs revealed no visible abnormalities in any organ of the wild-type control group mice (db / m). All db / db mice exhibited abnormally enlarged liver tissue, while other organs showed no visible abnormalities. Compared to the model control group, the liver surface of the iPSC-EPCs group was smoother and redder in color; other organs showed no visible changes compared to the model control group.

[0146] Liver weight results showed that, compared with wild-type control mice (db / m), the liver weight of model control mice (db / db) was significantly increased (P<0.0001). Compared with the model control group, the liver weight of animals in the iPSC-EPCs group was significantly decreased (P<0.05).

[0147] Liver coefficient results showed that, compared with wild-type control mice (db / m), the liver coefficient of model control mice (db / db) was significantly increased (P<0.0001). Compared with the model control group, the liver coefficient of iPSC-EPCs group was significantly decreased (P<0.05).

[0148] Table 4. Liver weight and liver coefficient at the end point (D17) for each group of animals.

[0149]

[0150] Note: # indicates a comparison between the model control group and the wild-type control group, with P < 0.0001; # indicates a comparison between the iPSC-EPCs group and the model control group, with P < 0.05.

[0151] (4) Histopathological examination

[0152] HE staining was performed on the liver tissues of mice in each group for pathological observation, and the results are as follows: Figure 2As shown, no pathological changes were observed in the wild-type control group (db / m). The model control group (db / db) mice showed severe steatosis in their livers, along with a small amount of inflammatory cell infiltration and macrophage proliferation, and occasional punctate necrosis. Compared with the model control group, the iPSC-EPCs group showed milder steatosis, and no significant difference in the degree of inflammatory cell infiltration, macrophage proliferation, and punctate necrosis.

[0153] The degree of fatty degeneration, inflammatory cell infiltration, macrophage proliferation, and necrosis in each group of animals was scored, and the results are as follows: Figure 3 As shown, the fatty degeneration score of the model control group was significantly higher than that of the wild-type control group (P<0.0001); the fatty degeneration score of the iPSC-EPCs group was significantly lower than that of the model control group (P<0.01).

[0154] In summary, under the conditions of this experiment, iPSC-EPCs (1×10⁻⁶) 6 A single intravenous injection of iPSC-EPCs (cells / mouse) into db / db mice reduced mouse body weight, liver weight, liver coefficient, and the degree of fatty degeneration in the liver, indicating that iPSC-EPCs have the potential to treat fatty liver disease.

[0155] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of iPSC-derived endothelial progenitor cells in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.

2. The application according to claim 1, characterized in that, The drug can be used directly or in the form of a pharmaceutical composition.

3. The application according to claim 2, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

4. The application according to claim 1, characterized in that, The dosage forms of the drug include non-gastrointestinal dosage forms and gastrointestinal dosage forms.

5. The application according to claim 4, characterized in that, The non-gastrointestinal dosage forms include injectable dosage forms, cavity dosage forms, mucosal dosage forms, or skin dosage forms.

6. The application according to claim 4, characterized in that, The gastrointestinal dosage forms include tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, suspensions, syrups, or drops.

7. The application according to claim 1, characterized in that, The dosage form of the drug is selected from injectable dosage forms.

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