WO2018069422A1

WO2018069422A1 - Methods and pharmaceutical compositions for the treatment of non-alcoholic fatty liver disease

Info

Publication number: WO2018069422A1
Application number: PCT/EP2017/076005
Authority: WO
Inventors: Pierre-Emmanuel RAUTOU; Chantal BOULANGER-ROBERT
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université Paris Descartes; Assistance Publique-Hôpitaux De Paris (Aphp); Université Paris Diderot - Paris 7
Priority date: 2016-10-12
Filing date: 2017-10-12
Publication date: 2018-04-19

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of non-alcoholic fatty liver disease. The inventors showed that autophagy is defective in liver sinusoidal endothelial cells (LSECs) from patients with non-alcoholic steatohepatitis (NASH) and contributes to the main features of this disease. In particular, the present invention relates to a method of treating a non-alcoholic fatty liver disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent capable of restoring autophagy in the subject's liver sinusoidal endothelial cells.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF NON-ALCOHOLIC FATTY LIVER DISEASE

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of non-alcoholic fatty liver disease.

BACKGROUND OF THE INVENTION:

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease worldwide with a reported prevalence ranging from 6-33%, depending on the studied population. It encompasses a spectrum of liver manifestations ranging from simple steatosis (also known as non-alcoholic fatty liver, NAFL) to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis, which may ultimately progress to hepatocellular carcinoma. NAFLD is strongly associated with components of the metabolic syndrome, mainly obesity and type 2 diabetes mellitus. NAFLD patients are at increased risk of liver-related as well as cardiovascular mortality. Current paradigm suggests a benign course for NAFL whereas NASH is considered to be the progressive phenotype. Although previously under-recognized accumulating evidence suggests that NAFL may also progress, suggesting a higher number of patients at risk than previously appreciated. Liver-biopsy remains the gold standard for definitive diagnosis, but the majority of patients can be diagnosed accurately by non-invasive methods. Approved therapies for NAFLD are still lacking and lifestyle modifications aiming at weight-loss remain the mainstay of NAFLD treatment. Putative treatment can be directed against various pathophysiological aspects of NAFLD and includes management of obesity, insulin resistance, hyperlipidemia and oxidative stress, suppression of inflammation and modulation of gut bacteria.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of non-alcoholic fatty liver disease. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors show that autophagy is defective in liver sinusoidal endothelial cells (LSECs) from patients with NASH and contributes to the main features of this disease. Agents capable of restoring endothelial autophagy thus represents an attractive strategy for NASH treatment. The first object of the present invention relates to a method of treating a non-alcoholic fatty liver disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent capable of restoring autophagy in the subject's liver sinusoidal endothelial cells.

As used herein, the term "non-alcoholic fatty liver disease" has its general meaning in the art and is intended to refer to the spectrum of disorders resulting from an accumulation of fat in liver cells in individuals with no history of excessive alcohol consumption. In the mildest form, NAFLD refers to hepatic steatosis. The term NAFLD is also intended to encompass the more severe and advanced form non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and virus- induced (e.g., HIV, hepatitis) fatty liver disease. The term "NASH", as used herein, collectively refers to the state where the liver develops a hepatic disorder (e.g., inflammation, ballooning, fibrosis, cirrhosis, or cancer), or the state where the liver may induce such a pathological condition, and "NASH" is distinguished from "simple steatosis"; i.e., a condition in which fat is simply accumulated in the liver, and which does not progress to another hepatic-disorder-developing condition.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

In some embodiments, the subject suffers from obesity. The term "obesity" refers to a condition characterized by an excess of body fat. The operational definition of obesity is based on the Body Mass Index (BMI), which is calculated as body weight per height in meter squared (kg/m²). Obesity refers to a condition whereby an otherwise healthy subject has a BMI greater than or equal to 30 kg/m², or a condition whereby a subject with at least one co-morbidity has a BMI greater than or equal to 27 kg/m². An "obese subject" is an otherwise healthy subject with a BMI greater than or equal to 30 kg/m² or a subject with at least one co-morbidity with a BMI greater than or equal 27 kg/m². In Asian and Asian-Pacific countries, including Japan, "obesity" refers to a condition whereby a subject with at least one obesity-induced or obesity- related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg/m². An "obese subject" in these countries refers to a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg/m².

In some embodiments, the agent is a polynucleotide that encodes for a polypeptide capable of restoring autophagy in the subject's liver sinusoidal endothelial cells. Typically, the polynucleotide encodes for an ATG5, ATG7 o Beclin-1 polypeptide.

As used herein, the term "ATG5" has its general meaning in the art and refers to autophagy protein 5 which is a protein that in humans is encoded by the ATG5 gene. Exemplary polynucleotides sequences include NM_001286106, NM_001286107, NM_001286108, NM 001286111, NM_004849, NM_053069 or NM 001314013 NCBI reference sequences.

As used herein, the term "ATG7" has its general meaning in the art and refers to autophagy protein 5 which is a protein that in humans is encoded by the ATG7 gene. Exemplary polynucleotides sequences include NM 001136031, NM 001144912, NM 006395, NM_001253717, NM_001253718 or NM_028835 NCBI references sequences.

As used herein, the term "Beclin-1" has its general meaning in the art and refers to the protein which in humans is encoded by the BECN1 gene. Exemplary polynucleotides sequences include NM_001313998, NM 001313999, NM 001314000, NM_003766, and NM_019584. In some embodiments, the polynucleotide of the present invention is included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. So, a further object of the invention relates to a vector comprising a nucleic acid encoding for a ELA polypeptide of the invention. Typically, the vector is a viral vector which is an adeno-associated virus (AAV), a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" means a vector derived from an adeno- associated virus serotype, including without limitation, AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines. In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g.. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species. Typically, the polynucleotide or the vector of the present invention include "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Another nucleic acid sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3'- direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters". In some embodiments, the polynucleotide of the present invention expresses specifically the polypeptide in the subject's liver sinusoidal endothelial cells.

According to the invention, the agent of the present invention is administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Typically the agent of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1: A defect in autophagy occurs in liver sinusoidal endothelial cells (LSECs) in the liver of patients with NASH. Quantification of autophagic vacuoles in LSECs in human liver biopsies from control patients, patients with bland steatosis or with NASH. Controls n=7 ; Steatosis: n=4 ; NASH: n=12. *p<0,05. Results are shown as median ± IQR.

Figure 2: IL-6 and TNF-a decrease autophagy in liver sinusoidal endothelial cells. (A)

Immunoblotting of TSECs treated with IL-6 at lOpg/mL and 50pg/mL for 24 hrs. (B) Immunoblotting of TSECs treated with TNF-a at lpg/mL and lOpg/mL. for 24 hrs. N=5 or 6 independent experiments per group*p<0.05. Results are shown as mean ± SEM.

Figure 3: The lack of autophagy in LSEC alters the macroscopic appearance of the liver in high fat diet (HFD) fed mice. Atg51ox/lox and Atg51ox/lox-VE-CadCre+ mice were fed a chow or a high fat diet (HFD) for 16 weeks. Quantification of granular appearance of the liver of WT (Atg51ox/lox) or endothelial autophagy deficient mice (Atg51ox/lox-VE-CadCre+) fed a chow diet (CD) or a high fat diet (HFD) for 16 weeks.

Figure 4: Deficiency in endothelial autophagy does not change hepatic injuries and steatosis in mice fed a high fat diet (HFD). Atg51ox/lox and Atg51ox/lox-VE-CadCre+ mice were fed a chow or a high fat diet (HFD) for 16 weeks. Intra-hepatic cholesterol (A) and triglycerides (B). (C) Histological evaluation of hepatic steatosis. (D) Serum AST and ALT (E) levels. Atg51ox/lox, chow diet: n=6 ; Atg51ox/lox, HFD : n=9 ; Atg51ox/lox-VE-CadCre+, chow diet: n=9 ; Atg51ox/lox-VE-CadCre+ HFD: n=14. *p<0,05. **p<0.01. ns : not significant. Results are shown as median ± IQR.

Figure 5: Deficiency in endothelial autophagy increases hepatic inflammation in high fat diet (HFD) fed mice. Atg51ox/lox and Atg51ox/lox-VE-CadCre+ mice were fed a chow or a high fat diet (HFD) for 16 weeks. Hepatic monocyte chemoattractant protein- 1 (MCP-1) and chemokine (C-C motif) ligand 5 (CCL5) gene expression (A and B) and VCAM-1 protein expression (C). CD : n=6 ; Atg51ox/lox, HFD : n=9 ; Atg51ox/lox-VE-CadCre+, CD : n=9 ; Atg51ox/lox-VE-CadCre+ HFD : n=14. *p<0,05. **p<0,01. ns : not significant. Results are shown as median ± IQR.

Figure 6: Deficiency in endothelial autophagy increases liver fibrosis in mice fed a high fat diet (HFD). Atg51ox/lox and Atg51ox/lox-VE-CadCre+ mice were fed a chow or a high fat diet (HFD) for 16 weeks. Hepatic gene expression of transforming growth factor beta- 1 (Tgfp-l) and collagen 1 2 (A and B), and hepatic protein expression of a-smooth muscle actin (a-SMA) (C). (D) Sirius red staining. Quantification. Atg51ox/lox, chow diet: n=6 ; Atg51ox/lox, HFD : n=9 ; Atg51ox/lox-VE-CadCre+, chow diet : n=9 ; Atg51ox/lox-VE- CadCre+ HFD : n=14. *p<0.05. **p<0.01. ns : not significant. Results are shown as median ± IQR.

EXAMPLE (figures 1 to 6):

Background and aims: Non-alcoholic steatohepatitis (NASH) is the liver manifestation of the metabolic syndrome and consists on excessive lipids accumulation in the liver (steatosis), hepatic injury and inflammation, with or without fibrosis. NASH has the potential for cirrhosis and its complications such as hepatocellular carcinoma and may require liver transplantation.

Autophagy is a cellular conserved process involved in the degradation of dysfunctional proteins and organelles. While the role of autophagy in hepatocytes and inflammatory cells in chronic liver diseases has been analyzed, the role of autophagy in liver sinusoidal endothelial cells (LSECs) is unknown. Our aim was to analyze the potential implication of autophagy in LSECs in NASH.

Methods: Quantification of autophagic vacuoles in LSECs from healthy controls (n=4) and from patients with bland steatosis (n=7) or NASH (n=10) was performed using electron microscopy. Then, we tested the effect of the two inflammatory cytokines IL-6 and TNFa (lOpg/mL and Ipg/mL, respectively, i.e. concentrations present in the portal venous blood of patients with NASH), on the level of autophagy of transformed liver sinusoidal endothelial cells (TSECs) exposed to shear stress. Finally, we analyzed the effect of the deficiency in autophagy specifically in endothelial cells using Atg51ox/lox-VECadherinCre+ mice (n=13) compared to littermate controls (Atg51ox/lox; n=8) all fed a high fat diet. NASH features were analyzed.

Results: LSECs from patients with NASH contained twice less autophagic vacuoles than those from healthy controls (p=0.01) and from patients with bland steatosis (p=0.01). Using cultured TSEC, we showed that IL-6 or TNFa decreased autophagy level in sinusoidal endothelial cells (reduction of LC3II/GAPDH ratio by 35%, p=0.04, and 45% p=0.03, respectively). More particularly, exposure of transformed sinusoidal endothelial cells (TSEC) to Interleukin-6 (IL-6) or TNF-alpha at concentrations similar to those reported in the portal vein of patients with metabolic syndrome induced a decrease in autophagy level. As compared to littermate controls, mice deficient in endothelial autophagy fed a high fat diet had more frequently a nodular surface of the liver (11% vs 78%, respectively; p=0.0001). They also had a higher liver expression of inflammatory molecules (3 fold more Mcp-1 mR A, p=0.03; 1.5 fold more Rantes mRNA, p=0.03; 3 fold more VCAM-1 protein expression, p=0.04), more activation of hepatic stellate cells (a-SMA protein expression: 0.01 vs 0.1 arbitrary units, p=0.03) and more liver fibrosis (3 fold more liver Collagenela2 mRNA expression, p=0.007; 3 fold more Tgfpl mRNA expression, p=0.03; 1.5 fold more Picrosirius staining, p=0.01).

Conclusion: Autophagy is defective in LSECs from patients with NASH. Inflammatory cytokines founded in the portal blood of patients could be responsible for this defect. The lack of autophagy in LSECs contributes to the development of the main features of this disease. Activators of endothelial autophagy are an attractive strategy for NASH treatment.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method of treating a non-alcoholic fatty liver disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent capable of restoring autophagy in the subject's liver sinusoidal endothelial cells.