WO2019121764A1

WO2019121764A1 - Medicament for the treatment of liver fibrosis and cirrhosis of liver

Info

Publication number: WO2019121764A1
Application number: PCT/EP2018/085625
Authority: WO
Inventors: Jochen Hampe; Raghavan THANGAPANDI; Mario BROSCH; Clemens Schafmeyer
Original assignee: Technische Universitaet Dresden; Christian Albrechts Universitaet Kiel
Current assignee: Technische Universitaet Dresden; Christian Albrechts Universitaet Kiel
Priority date: 2017-12-21
Filing date: 2018-12-18
Publication date: 2019-06-27
Anticipated expiration: 2020-06-21

Abstract

The present invention concerns a medicament for use in the treatment and/or prevention of liver fibrosis and/or of cirrhosis of liver. The medicament is an inhibitor of GPR55 and is effective in the treatment of and preferably also in the prevention of liver fibrosis and/or of cirrhosis of liver, independent of the presence of inflammation in the liver.

Description

New PCT application Medicament for the treatment of liver fibrosis and cirrhosis of liver-Technische Universitat Dresden Christian-Albrechts-Universitat zu Kiel

Medicament for the treatment of liver fibrosis and cirrhosis of liver

The present invention concerns a medicament for use in the treatment and/or prevention of liver fibrosis and/or of cirrhosis of liver. The medicament is effective in the treatment of and preferably also in the prevention of liver fibrosis and/or of cirrhosis of liver, independent of the presence of inflammation in the liver. The medicament can be for use in the treatment and/or prevention of liver fibrosis and/or of cirrhosis of liver in the presence of inflammation, e.g. inflammation caused by viral infection, e.g. by hepatitis B virus, by hepatitis C virus, or for use in the treatment of and/or prevention of liver fibrosis and/or of cirrhosis of liver in the absence of inflammation, e.g. alcoholic liver disease (ALD), non-alcoholic liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), for example in obese patients or in case of liver intoxication, e.g. caused by overdosage of a pharmaceutical compound or drug abuse.

State of the art

Huang et al., Dig. Liver Dis, 43 (3), 188-193 (2011) describe that influencing the

endocannabinoid system has been suggested for treating liver fibrosis. Buch et al, Nature Genetics, 47 (22), 1443-1448 (2015) describe a genome-wide study on the association with alcohol-related liver cirrhosis and identified variants in the MBOAT7 and TM6SF2 genes and confirmed the SNP rs738409 in the PNPLA3 gene as risk factors. This SNP is regarded as a candidate functional variant that influences the expression of MBOAT7, which has been implicated in anti-inflammatory processes. In rat liver microsomes, MBOAT7 catalyses the transfer of fatty acid between phospholipids and lysophospholipids, which are regarded as potent drivers of hepatic inflammation. Quoting Moreno -Naverrete et al.,“The I- a-lysophosphatidylinositol/GPR55 system and its potential role in human obesity”, Diabetes 61, 281-291 (2012), Buch et al. mention that a key product of the MBOAT7 activity in the microsomes is sensed by GPR55, a lysophosphatidylinositol (LPI) receptor having cannobinoid sensitivity, thus providing a link between MBOAT7 and the role of the endocannabinoid system in hepatic extracellular matrix remodelling. Buch et al. describe it as a hypothetical mechanism by which the identified variant of MBO AT7 may modulate inflammation-driven liver fibrogenesis in alcohol-related liver injury.

Thabet et al., Nature Communications 7:12757 (2016) (DOI : l0.l038/ncommsl2757) describe the SNP rs64l728 in the MBOAT7 gene as a risk factor of liver inflammation and transition to fibrosis in chronic hepatitis C.

Arifin and Falasca, Metabolites 6, 6 (2016) (DOI: l0.3390/metabo6010006) describe that the mRNA encoding the human receptor GPR55 was found in certain brain regions, adipose tissue, testis, myometrium, tonsil, adenoid and spleen. GPR55 is described as an atypical cannabinoid receptor, and as a receptor of the agonist lysophospholipid LPI.

Kotsikorou et al, Biochemistry 2013, 52, 9456-9469, describe a screening process for identifying antagonists of GPR55.

Currently, liver fibrosis can be countered by liver transplantation or by treatment an inflammation underlying the fibrosis or by reducing the intake of alcohol, of intoxicating compounds, or by reducing intake of fat or reducing body weight, but no direct curative treatment for liver fibrosis is known.

Objective of the invention

It is an objective of the invention to provide to provide a medicament for the treatment of liver fibrosis, preferably for the treatment of liver fibrosis and/or of cirrhosis of liver, which treatment does not necessarily comprise a treatment of the cause of liver fibrosis, e.g. a treatment which is not necessarily coupled with a treatment of the causative agent of the liver fibrosis, which causative agent may e.g. be a virus or toxic substance like alcohol or drug abuse.

Description of the invention

The present invention achieves the objective by the features of the claims and especially by providing compounds, especially inhibitors of GPR55, for use as a medicament in the treatment and/or prevention of liver fibrosis and/or of cirrhosis of liver. Treatment and/or prevention of liver fibrosis and/or of cirrhosis of liver is achieved by administration of an inhibitor of GPR55 to a patient who is suspected of having liver fibrosis or cirrhosis of liver, who is diagnosed as having liver fibrosis or cirrhosis of liver or who is considered to have an increased risk of developing liver fibrosis or cirrhosis of liver.

GPR55 is encoded by the GPR55 gene, in humans by nucleotides 230907318 to 230961066 of chromosome 2, in mouse by nucleotides 85938318 to 85961007 of chromosome 1. GPR55 is a class A G-protein-coupled receptor and has been shown to be activated by L-a- lysophopsphatidylinosito 1.

Surprisingly, it was found that administration of an inhibitor of GPR55 reduces and/or prevents liver fibrosis and/or of cirrhosis of liver independent from the presence or absence of inflammation. Accordingly, it is assumed that the inhibition of GPR55 in the treatment and/or prevention of liver fibrosis and/or of cirrhosis of liver acts via another mechanism which is independent from inflammation and which therefore is effective also in the treatment of and/or prevention of liver fibrosis caused by non- inflammatory causes, e.g. alcoholic liver disease, non-alcoholic liver disease (NAFLD), for example non-alcoholic steatohepatitis (NASH) in obese patients, or in case of liver intoxication. Further, the treatment using an inhibitor of GPR55 is independent of the presence of a mutation in the MBOAT7 and TM6SF2 and in the PNPLA3 genes, e.g. independent from the SNP rs738409 in the PNPLA3 gene. Accordingly, the medicament can be used in patients having the wild-type allele of the MBOAT7 and TM6SF2 and the PNPLA3 genes, and/or in a human, also termed patient, having a mutation in at least one of the MBOAT7 and TM6SF2 and the PNPLA3 genes. Preferably, the compounds, which especially are inhibitors of GPR55, are for use in patients having a mutation in at least one of the genes MBOAT7, TM6SF2 and/or PNPLA3, wherein the mutation preferably reduces activity of the gene product, e.g. in comparison to the activity of the product of the wild-type gene, which patients are considered to have an increased risk of developing liver fibrosis. An exemplary mutation of the MBOAT7 encoding gene that was found to reduce activity is the SNP rs64l738, which was found to result in lower expression of the MBOAT7 gene product, and to lead to severe NAFLD.

The use of an inhibitor of GPR55 in the treatment of liver fibrosis and/or of cirrhosis of liver can be independent from the treatment of the causative agent of the liver fibrosis, which causative agent may e.g. be a virus or toxic substance like alcohol or drug abuse. Preferably, the use of an inhibitor of GPR55 in the treatment of liver fibrosis is coupled with the treatment of the causative agent of the liver fibrosis and/or of cirrhosis of liver, which causative agent may e.g. be a virus and or abuse of a toxic substance like alcohol in the case of alcoholic liver disease, or drug abuse or obesity in the case of non-alcoholic liver disease. The treatment of the causative agent of the liver fibrosis and/or of cirrhosis of liver can e.g. be the use of an antiviral agent for treatment of the viral infection or the prevention of further intake of the toxic substance, or a diet suitable in the prevention of non-alcoholic liver disease.

The administration of an inhibitor of GPR55 also in the presence of inflammation reduces and/or prevents liver fibrosis, e.g. inflammation caused by viral infection.

It is an advantage of the use of the inhibitor of GPR55 that the inhibition of GPR55 does essentially not have significant detrimental side-effects, as e.g. evidenced by the mild neurological symptoms occurring in the experimental GPR55 knock-out mouse.

Inhibitors of GPR55, also referred to as antagonists of GPR55, can be determined by the process described by Kotsikorou et al, Biochemistry 2013 (loc. cit). Exemplary compounds for use in the treatment of or in the prevention of liver fibrosis and/or of cirrhosis of liver, which preferably are inhibitors of GPR55, are comprised in the group comprising or consisting of 5 -phenyl-3 - { 1 - [ 1 -(/ -tolyl)cyclopropanc-carbonyl]pipcridin-4-yl } -1,3,4- oxadiazol-2(37 )-one (CID23612552), {furan-2-yl[4-(2-methyl-5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3-_<i]pyrimidin-4-yl)piperazin-l-yl]methanone} (CID 1434953), (A- j 4-[/V-(3, 4-dimcthy 1 isoxazo 1-5-yl )sulfamoy 1] phenyl [-6, 8-dimcthyl-2-(pyridin-2- yl)quinoline-4-carboxamide) (CID1261822), CID16020046, 3-(2-hydroxybenzyl)-5- isopropyl-8-mcthyl-2//-chromcn-2-onc, tetrahydromagnolol, magnolol, and combinations of at least two of these. Further, inhibitors of GPR55 can be selected from structural analogues of lysophosphatidylinositol (LPI), like e.g. CID 16020046.

The invention is now described in greater detail by way of examples and with reference to the figures, which show in

Fig. 1 a graph on the proportion of MAF in human patients without diagnosed liver fibrosis (F0, left col.) and with diagnosed liver fibrosis (Fl-4, right col.) both for no inflammation (INF 0) and for inflammation present (INF 1),

Fig. 2 shows cell numbers in wild-type and in MBOAT7 knock-out mice, in a) of macrophage, in b) of monocytes, in c) of CD8+ T-cells, in d) of CD4+ T-cells, and in e) of neutrophils,

- Fig. 3 a) shows levels of collagen type I and III, determined by Pikrosirius staining of liver tissue, in b) relative hydroxyproline levels in liver tissue of wild-type (WT) and of MBOAT7 knock-out (KO) mice, and in c) a Pikrosirius stain of liver tissue of an MBOAT7 knock-out mouse and in d) a Pikrosirius stain of liver tissue of a wild-type mouse,

- Fig. 4 shows relative RNA levels, in a) of TGFP transcripts, in b) of a-SMA

transcripts, in c) of Sphkl transcripts, and in d) of Coll A transcripts in liver tissue of wild-type (WT) and of MBOAT7 knock-out (KO) mice,

Fig. 5 shows relative RNA levels, in a) for IL6 transcripts, in b) for ILl-b (ILl-b) transcripts, in c) for TNFa (TNFa) transcripts, in d) for IFNy (IFNg) transcripts in liver tissue of wild-type (WT) and of MBOAT7 knock-out (KO) mice, and

Fig. 6 a) the relative transcripts of SPKH1 encoding mRNA, Fig. 6 b) the relative transcripts of COL la encoding mRNA, Fig. 6 c) the relative transcripts of a-SMA encoding mRNA, and in Fig. 6 d) the relative transcripts of TGFP encoding mRNA,

- Fig. 7 shows levels of phosphatidyl inositol compounds in liver of wild-type mice (left col.) and of MBOAT7 knock-out mice (right col.),

- Fig. 8 shows levels of phosphatidyl inositol compounds in liver for NASH (left col.) and NAFLD (center col.) and for patients healthy persons (ho, right col.),

Fig. 9 shows formulae of exemplary compounds for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver, which are exemplary and preferred inhibitors of GPR55. The examples show that reduction of the activity of MBOAT7, e.g. represented by a knock out of the MBOAT7 encoding gene, results in increased occurrence of liver fibrosis. The knock-out of MBOAT7 is not correlated with inflammation, providing an example for liver fibrosis which is independent from inflammation and not caused by an infectious agent, e.g. not caused by viral infection.

In the examples, liver fibrosis was induced by the knock-out of the MBOAT7 encoding gene and by a high- fat, choline-deficient, methionine supplemented diet. Further, the examples show that the inhibition of GPR55 reduces or prevents liver fibrosis in the experimental genetic knock-out of the MBOAT7 encoding gene and in wild-type mice by way of administration of the exemplary GPR55 inhibitor CID 16020046.

In human patients, a correlation of presence of the mutation rs64l738 of the MBOAT7 gene, which was found to result in lower expression of the MBOAT7 gene product, with liver fibrosis was found, both for patients without inflammation (INF 0), and for patients with inflammation (INF 1). The result is depicted in Fig. 1, showing that at least for this mutation, the risk for liver fibrosis is independent from presence of inflammation.

Example: Effect of GPR55 inhibitor on wild-type mice and on MBOAT7 knock-out mice under conditions promoting liver fibrosis

MBOAT7 knock-out mice were generated by crossing Mboat7^fl/fl mice, obtained from MRC Harwell, London, with C57LB1/6 Alb Cre mice, obtained from Jacksons Laboratory, giving the deletion of the MBOAT7 gene. The deletion of the MBOAT7 gene in hepatocytes was confirmed by genotyping using PCR and by quantitative reverse PCR for transcripts of MBOAT7.

In the analyses, for comparison wild-type mice (WT) were treated identically and in parallel.

When fed on a CHOW diet, these MBOAT7 knock-out mice and wild-type (WT) mice C57LBF6 were analysed for the effect of the MBOAT7 knock-out (KO) on inflammation. Fig. 2 in a) to e) shows that in comparison to wild-type, the MBOAT7 knock-out did not significantly influence presence of immune cells, exemplified by macrophage, monocytes, CD8+ T-cells and CD4+ T-cells and neutrophils in the liver tissue. The knock-out of MBOAT7 increased fibrosis in mice fed on a NASH diet as shown by the increase of positive area in Pikrosirius staining of liver tissue, indicating collagen I and III, as depicted in Fig. 3 a). The analysis of relative hydroxyproline content of liver tissue in the MBOAT7 knock-out mice (KO) showed a significant increase, as depicted in Fig. 3b). A pikrosirius stain of liver tissue from a MBOAT7 knock-out mouse is shown in Fig. 3 c), and from a wild-type mouse in Fig. 3 d), showing increased characteristics of liver fibrosis in the MBOAT7 knock-out mouse only.

Also, the results of the analysis of TGFp, aSMA, Sphkl and CollA, which are depicted in Fig. 4 a) to d), show significantly increased levels in MBOAT7 knock-out (KO) mice compared to wild-type (WT), indicating significantly increased formation of liver fibrosis (NASH diet) due to the MBOAT7 knock-out.

The results depicted in Fig. 5 of the relative RNA levels a) for IL6 transcripts, b) for ILl-b transcripts, c) for TNFa transcripts, and d) for IFNy transcripts in liver tissue of wild-type and of MBOAT7 knock-out mice show that the knock-out of MBOAT7 did not significantly influence inflammation markers when mice were on NASH diet.

These results demonstrate that the inactivation of MBOAT7 by the knock-out does not result in inflammation of the liver, excluding inflammation as a cause for liver fibrosis in these knock-out mice. Accordingly, it is presently concluded that the effect of the inhibitor of GPR55 against liver fibrosis is not based on influencing the inflammatory pathway, but is independent from presence of inflammation in liver fibrosis.

The MBOAT7 knock-out (KO) mice and wild-type (WT) mice were treated with 0.5 mg/kg CID 16020046 (obtained from Tocris bioscience, UK), which is a known inhibitor of GPR55, and for comparison in parallel treated with 1% DMSO. The analytical results are depicted in Fig. 6, showing that administration of the inhibitor of GPR55 results in a reduced

transcription of genes indicating fibrosis. WT designates the wild-type, KO designates the MBOAT7 knock-out, and _P designates animals that received placebo, Inh designates animals that received the GPR55 inhibitor CID 16020046. In detail, the transcription of SPKH1 was essentially not affected by the GPR55 inhibitor, but transcriptions of COL1 A, of a-SMA, and of TGFP were reduced by administration of a GPR55 inhibitor. These results indicated that administration of an inhibitor of GPR55 both in wild-type mice and in the fibrosis-prone GPR55 gene knock-out mice under conditions that in wild-type mice induce liver fibrosis, reduces or inhibits the development of liver fibrosis to a significant extent.

Phosphatidyl inositol (PI) lipid compounds were analysed using liver tissue samples. The analyses of the phosphatidyl inositol (PI) lipid compounds in the liver of the MBOAT7 knock-out mice and wild-type mice showed that the lack of MBOAT7 affects the levels of some PI species, especially results in a decreased level of lysophosphatityl inositol (PI 38:4:0). Fig. 7 depicts the analytical results for the wild-type (left col.) and for the MBOAT7 knock-out (right col.).

When analysing the phosphatidyl inositol (PI) lipid compounds in the liver of patients diagnosed for non-alcoholic liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) patients, it was found that in comparison to healthy persons (ho), both NASH and NAFLD patients have significantly lower levels of lysophosphatidyl inositol (PI[38:4]) , as depicted in Fig. 8. In Fig. 8, for each PI, the left column indicates NASH patients, the centre column indicates NAFLD patients, and the right column indicates healthy (ho) persons. For the inhibitor of GPR55 for use in the treatment or prevention of liver fibrosis and/or cirrhosis of liver, the person to be treated preferably has a reduced level of lysophosphatidyl inositol, e.g. in a blood or in a liver sample.

As a consequence of these observations in the patients and in the mice experiments, it is currently assumed that the inhibitor of GPR55 used in the treatment or prevention of liver fibrosis and/or cirrhosis of liver acts by way of affecting the level of lysophosphatidyl inositol, preferably increasing its level, by inhibiting GPR55.

Claims

1. Inhibitor of GPR55 for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver.

2. Inhibitor of GPR55 for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to claim 1 , characterized by the absence of inflammation in the liver fibrosis.

3. Inhibitor of GPR55 for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to one of the preceding claims, characterized by the presence of or increased risk for alcoholic liver disease (ALD), non-alcoholic liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

4. Inhibitor of GPR55 for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to claim 1, characterized by the presence of inflammation in the liver.

5. Inhibitor of GPR55 for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to one of the preceding claims, characterized by a reduced level of lysophosphatidyl inositol (PI[38 :4]) in the person to be treated in comparison to the level of lysophosphatidyl inositol in a healthy person.

6. Inhibitor of GPR55 for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to one of the preceding claims, wherein the inhibitor is selected from the group comprising 5-phcnyl-3- { 1 -[ 1 -(/ tolyl)cyclopropanc- carbonyl]piperidin-4-yl}-l,3,4-oxadiazol-2(377)-one (CID23612552), (furan-2-yl[4- (2 -methyl-5, 6,7, 8-tetrahydrobenzo[4,5]thieno[2,3-_<i]pyrimidin-4-yl)piperazin-l- yljmethanone} (CID1434953), (A-{4-[A-(3,4-dimethylisoxazol-5- yl)sulfamoyl]phenyl}-6,8-dimethyl-2-(pyridin-2-yl)quinoline-4-carboxamide)

(CID 1261822), CID 16020046, 3-(2-hydroxybcnzy l)-5-isopropy 1-8-mcthy 1-2/7- chromen-2-one, tetrahydromagnolol, magnolol, structural analogues of

lysophosphatidylinositol, and combinations of at least two of these.

7. Inhibitor of GPR55 for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to one of the preceding claims, characterized by formulation of the inhibitor for intravenous or intra-oral administration of the inhibitor.

8. Inhibitor of GPR55 for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to one of the preceding claims, characterized in that the use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver is for a human having haploid or diploid a wild-type allele of at least one of the genes comprised of MBOAT7, of TM6SF2 and/or of PNPLA3, or having haploid or diploid a mutant allele resulting in reduced activity of at least one gene product of the genes comprised of MBOAT7, of TM6SF2 and/or of PNPLA3.

9. Compound for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to one of the preceding claims, wherein the compound is selected from the group comprising 5-phcnyl-3- { 1 -[ 1 -(/ -tolyl)cyclopropanc-carbonyl]pipcridin-4- yl}-l,3,4-oxadiazol-2(37 )-one (CID23612552), {furan-2-yl[4-(2-methyl-5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3-_<i]pyrimidin-4-yl)piperazin-l-yl]methanone}

(CID1434953), (A-{4-[A-(3,4-dimethylisoxazol-5-yl)sulfamoyl]phenyl}-6,8-dimethyl- 2-(pyridin-2-yl)quinoline-4-carboxamide) (CID1261822), CID16020046, 3-(2- hydroxybcnzyl)-5-isopropyl-8-mcthyl-2/7-chromcn-2-onc, tetrahydromagnolol, magnolol, structural analogues of lysophosphatidylinositol, and combinations of at least two of these.

10. Compound for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to claim 9, characterized by the absence of inflammation in the liver.

11. Compound for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to claim 9, characterized by the presence of inflammation in the liver

12. Compound for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to one of claims 9 to 11, characterized by the presence of alcoholic liver disease (ALD), non-alcoholic liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

13. Compound for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to one of claims 9 to 12, characterized by a reduced level of lysophosphatidyl inositol (PI[38 :4]) in the person to be treated in comparison to the level of lysophosphatidyl inositol in a healthy person.

14. Compound for use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver according to one of claims 9 to 13, characterized by the use in the treatment or prevention of liver fibrosis and/or of cirrhosis of liver for a human having haploid or diploid a wild-type allele of at least one of the genes comprised of MBOAT7, of TM6SF2 and/or of PNPLA3, or having haploid or diploid a mutant allele resulting in reduced activity of at least one gene product of the genes comprised of MBOAT7, of TM6SF2 and/or of PNPLA3.

15. Method of treatment or for the prevention of fibrosis and/or of cirrhosis of liver,

characterized by administration of an inhibitor or a compound according to one of the preceding claims to a patient diagnosed or suspected of having or being at risk of developing fibrosis and/or of cirrhosis of liver.