TECHNICAL FIELD

The present invention relates to the field of medicine, in particular to the treatment of cholestatic or fibrotic diseases.

BACKGROUND

Abnormal and exaggerated deposition of extracellular matrix is the hallmark of all fibrotic diseases, including liver, pulmonary, kidney or cardiac fibrosis. The spectrum of affected organs, the progressive nature of the fibrotic process, the large number of affected persons, and the absence of effective treatment pose an enormous challenge when treating fibrotic diseases.

In an attempt to propose new therapeutic strategies for the treatment of fibrotic diseases, the inventors found that 2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate (Nitazoxanide—NTZ), a synthetic antiprotozoal agent or its deuterated derivatives or its active metabolite 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide (Tizoxanide, known as TZ) in combination with statins show synergistic antifibrotic activities. Moreover, the evaluation of NTZ combined to a statin in a liver injury model revealed its capacity to reduce circulating bile acid concentration, thus reflecting the synergistic potential of this combination to treat both cholestatic (such as PBC and PSC) and fibrotic diseases.

NTZ, first described in 1975 (Rossignol and Cavier 1975), was shown to be highly effective against anaerobic protozoa, helminths, and a wide spectrum of microbes including both anaerobic and aerobic bacteria (Rossignol and Maisonneuve 1984; Dubreuil, Houcke et al. 1996; Megraudd, Occhialini et al. 1998; Fox and Saravolatz 2005; Pankuch and Appelbaum 2006; Finegold, Molitoris et al. 2009). It was first studied in humans for the treatment of intestinal cestodes (Rossignol and Maisonneuve 1984) and it is now licensed in the United States (Alinia®, Romark laboratories) for the treatment of diarrhea caused by the protozoan parasitesCrystosporidium parvumandGiardia intestinalis. NTZ has also been widely commercialized in Latin America and in India where it is indicated for treating a broad spectrum of intestinal parasitic infections (Hemphill, Mueller et al. 2006). The proposed mechanism of action by which NTZ exerts its antiparasitic activity is through the inhibition of pyruvate:ferredoxin oxidoreductase (PFOR) enzyme-dependent electron transfer reactions that are essential for anaerobic metabolism (Hoffman, Sisson et al. 2007). NTZ also exhibited activity againstMycobacterium tuberculosis, which does not possess a homolog of PFOR, thus suggesting an alternative mechanism of action. Indeed, the authors showed that NTZ can also act as an uncoupler disrupting membrane potential and intra-organism pH homeostasis (de Carvalho, Darby et al. 2011).

The pharmacological effects of NTZ are not restricted to its antiparasitic or antibacterial activities and in recent years, several studies revealed that NTZ can also confer antiviral activity (Di Santo and Ehrisman 2014; Rossignol 2014). NTZ interferes with the viral replication by diverse ways including a blockade in the maturation of hemagglutinin (influenza) or VP7 (rotavirus) proteins, or the activation of the protein PKR involved in the innate immune response (for a review, see (Rossignol 2014)). NTZ was also shown to have broad anticancer properties by interfering with crucial metabolic and prodeath signaling pathways (Di Santo and Ehrisman 2014).

Statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) are commonly prescribed as medications for the treatment of hypercholesterolemia and for the prevention of cardiovascular disease.

There are currently 7 unique prescribed statins that include pravastatin, simvastatin, and lovastatin (which are naturally-derived from fungal fermentation) and a second group of chemically synthesized statins composed of fluvastatin, atorvastatin, rosuvasatin and pitavastatin. Although all statins contain a dihydroxy-heptanoic acid HMG-CoA like moiety, which competes for binding to HMG-CoA reductase, each statin is unique and exhibits significant differences in chemical structure, potency (ex. 1050 for HMG-CoA reductase inhibition), tissue penetration and retention, half-life, metabolism and elimination, drug-drug interactions, and safety. The mechanisms involved in the beneficial effects of statins on the prevention of cardiovascular disease have been largely attributed to the ability of these agents to inhibit cholesterol biosynthesis. Owing to the fact that 60-70% of serum cholesterol is derived from hepatic biosynthesis and that HMG-CoA reductase is the crucial, rate-limiting enzyme in the cholesterol biosynthetic pathway, it is not surprising that inhibition of this enzyme results in a dramatic reduction in circulating LDL-cholesterol. Moreover, reduction of LDL-cholesterol leads to upregulation of hepatic LDL receptors and increase of LDL clearance. Both clinical and experimental data suggest that the sum of benefits from statin therapy may extend well beyond their favorable effects on serum cholesterol levels. These cholesterol-independent effects, described as pleiotropic effects of statins, are related to the reduced formation of isoprenoids. Indeed, inhibition of HMG-CoA reductase results not only in deprivation of intracellular mevalonate but also several downstream isoprenoid derivatives including farnesyl pyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). Both FPP and GGPP are required for posttranslational prenylation of a number of proteins (approximately 2% of total cellular proteins (Wang, Liu et al. 2008)). Protein isoprenylation enables proper subcellular localization and trafficking of intracellular molecules. For example, non-isoprenylated GTPases remain cytosolic whereas isoprenylated GTPases harbour a FPP or GGPP lipid attachment that permits insertion and anchorage into the cell membrane, and subsequently participate in signal transduction. Therefore, inhibiting isoprenylation results in the inactivation of the small GTPases (ex Rho, Ras, Rac and Cdc42) which are essential in many cellular events (intracellular signal transduction, cellular proliferation, inflammation, motility (for a review see (McFarlane, Muniyappa et al. 2002; Zhou and Liao 2009; Yeganeh, Wiechec et al. 2014; Kavalipati, Shah et al. 2015)). Since it has been demonstrated that the Rho GTPase and its target protein Rock are involved in the activation/differentiation of fibroblasts into myofibroblasts (Ji, Tang et al. 2014), a key event in the fibrotic process, several studies were conducted with statins to evaluate their antifibrotic properties in different pathological models. Simvastatin was shown to reduce the expression of fibrotic markers in both human and rat HSC and to confer antifibrotic properties in various animal models of fibrosis (Rombouts, Kisanga et al. 2003; Watts, Sampson et al. 2005; Wang, Zhao et al. 2013; Marrone, Maeso-Diaz et al. 2015). As well, pitavastatin (Miyaki, Nojiri et al. 2011) and fluvastatin (Chong, Hsu et al. 2015) were able to reduce fibrosis in the CDAA diet-induced NAFLD/NASH model. These beneficial effects of statins are not restricted to liver fibrosis. Indeed, it was demonstrated that atorvastatin was significantly potent against bleomycin-induced lung fibrosis (Zhu, Ma et al. 2013) while simvastatin was shown to inhibit expression of fibrotic markers in fibroblasts derived from human fibrotic lung (Watts, Sampson et al. 2005).

In this invention, using a phenotypic screening assay to identify potential antifibrotic agents, it was discovered that NTZ or its deuterated derivatives or its active metabolite TZ, in combination with a statin, interferes, in an additive or synergistic manner, with the activation of myofibroblasts. This effect was totally unexpected in view of the properties previously reported for these molecules. Combination of NTZ or its deuterated derivatives or TZ with a specific statin appears as a potent therapeutic strategy for diverse types of fibrotic diseases. Moreover, the evaluation of NTZ or derivatives thereof in combination with a specific statin revealed an unexpected synergistic capacity to reduce circulating bile acid concentration, thus reflecting its potential to treat both cholestatic diseases (such as PBC and PSC) and fibrotic diseases.

SUMMARY OF INVENTION

The present invention relates to a synergistic combination comprising (i) [2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate (NTZ), a deuterated derivative of NTZ (NTZ-D), 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide (TZ), or Tizoxanide glucuronide (TZG) and (ii) at least one statin. This combination is useful in a method for the treatment of a cholestatic or fibrotic disease.

Therefore, the present invention relates to a synergistic combination of(i) NTZ, NTZ-D, TZ or TZG, or a pharmaceutically acceptable salt of NTZ, NTZ-D, TZ or TZG; and(ii) a statin.

The combination of the invention may be in the form of a pharmaceutical composition or of a kit-of-parts.

Furthermore, the components of the synergistic combination of the invention may be administered simultaneously, sequentially and separately.

In a particular embodiment, component (i) of the synergistic combination is NTZ or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the invention, said at least one statin is selected in the group consisting of mevastatin, cerivastatin, pitavastatin, fluvastatin, simvastatin, atorvastatin, lovastatin, rosuvastatin, and pravastatin. In another particular embodiment, the statin is selected in the group consisting of pitavastatin, fluvastatin, simvastatin and atorvastatin. In another particular embodiment, the statin is selected in the group consisting of pitavastatin, fluvastatin and simvastatin.

Furthermore, the synergistic combination of the invention may further comprise at least one therapeutically active agent with known antifibrotic activity selected from pirfenidone or receptor tyrosine kinase inhibitors (RTKIs) such as nintedanib, sorafenib and other RTKIs, or angiotensin II (AT1) receptor blockers, or CTGF inhibitor, or any antifibrotic compound susceptible to interfere with the TGF and BMP-activated pathways including activators of the latent TGF complex such as MMP2, MMP9, THBS1 or cell-surface integrins, TGF receptors type I (TGFBRI) or type II (TGFBRII) and their ligands such as TGFβ, activin, inhibin, nodal, anti-Müllerian hormone, GDFs or BMPs, auxiliary co-receptors (also known as type III receptors), or components of the SMAD-dependent canonical pathway including regulatory or inhibitory SMAD proteins, or members of the SMAD-independent or non-canonical pathways including various branches of MAPK signaling, TAK1, Rho-like GTPase signaling pathways, phosphatidylinositol-3 kinase/AKT pathways, TGFβ-induced EMT process, or canonical and non-canonical Hedgehog signaling pathways including Hh ligands or target genes, or any members of the WNT, or Notch pathways which are susceptible to influence TGFβ signaling.

Alternatively, the synergistic combination according to the invention may further comprise at least one therapeutically active agent selected from JAK/STAT inhibitors and other anti-inflammatory agents and/or immunosuppressant agents. For example, the therapeutically active agent may be selected from glucocorticoids, NSAIDS, cyclophosphamide, nitrosoureas, folic acid analogs, purine analogs, pyrimidine analogs, methotrexate, azathioprine, mercaptopurine, ciclosporin, myriocin, tacrolimus, sirolimus, mycophenolic acid derivatives, fingolimod and other sphingosine-1-phosphate receptor modulators, monoclonal and/or polyclonal antibodies against such targets as proinflammatory cytokines and proinflammatory cytokine receptors, T-cell receptor and integrins.

The present invention also relates to the synergistic combination according to the invention, for use as a medicament.

Furthermore, the invention relates to the synergistic combination described herein, for use in a method for treating a fibrotic disorder. In a particular embodiment, the fibrotic disorder is selected in the group consisting of liver, gut, kidney, skin, epidermis, endodermis, muscle, tendon, cartilage, heart, pancreas, lung, uterus, nervous system, testis, penis, ovary, adrenal gland, artery, vein, colon, intestine (e.g. small intestine), biliary tract, soft tissue (e.g. mediastinum or retroperitoneum), bone marrow, joint, eye and stomach fibrosis. In a further particular embodiment, the fibrotic disorder is selected in the group consisting of liver, kidney, skin, epidermis, endodermis, muscle, tendon, cartilage, heart, pancreas, lung, uterus, nervous system, testis, ovary, adrenal gland, artery, vein, colon, intestine (e.g. small intestine), biliary tract, soft tissue (e.g. mediastinum or retroperitoneum), bone marrow, joint and stomach fibrosis. In a further particular embodiment, the fibrotic disorder is selected in the group consisting of liver, gut, lung, heart, kidney, muscle, skin, soft tissue, bone marrow, intestinal, and joint fibrosis. In yet another embodiment the fibrotic disorder is selected in the group consisting of non-alcoholic steatohepatitis (NASH), pulmonary fibrosis, idiopathic pulmonary fibrosis, skin fibrosis, eye fibrosis (such as capsular fibrosis), endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis (a complication of coal workers' pneumoconiosis), proliferative fibrosis, neoplastic fibrosis, lung fibrosis consecutive to chronic inflammatory airway disease (COPD, asthma, emphysema, smoker's lung, tuberculosis), alcohol or drug-induced liver fibrosis, liver cirrhosis, infection-induced liver fibrosis, radiation or chemotherapeutic-induced fibrosis, nephrogenic systemic fibrosis, Crohn's disease, ulcerative colitis, keloid, old myocardial infarction, scleroderma/systemic sclerosis, arthrofibrosis, some forms of adhesive capsulitis, chronic fibrosing cholangiopathies such as primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC), biliary atresia, familial intrahepatic cholestasis type 3 (PFIC3), peri-implantational fibrosis and asbestosis.

According to a particular embodiment of the invention, the cholestestatic disease is selected in the group consisting of primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), intrahepatic cholestasis of pregnancy, progressive familial intrahepatic cholestasis, biliary atresia, cholelithiasis, infectious cholangitis, cholangitis associated with Langerhans cell histiocytosis, Alagille syndrome, nonsyndromic ductal paucity, drug-induced cholestasis, and total parenteral nutrition-associated cholestasis. In a particular embodiment, the cholestatic disease is PBC.

According to a particular embodiment, in each aspects and embodiments described herein, NTZ, NTZ-D or TZ, or a pharmaceutically acceptable salt of NTZ; NTZ-D or TZ is used.

DESCRIPTION OF THE FIGURES AND TABLES

Abbreviations Used in the Figures, in the Tables, and in the Text:

α-SMA: alpha Smooth Muscle Actin

ATORVA: Atorvastatin

BDL: Bile Duct Ligation

BMP: Bone Morphogenetic Protein

cDNA: Complementary Deoxyribonucleotide acid

COL1 A1: Collagen, type 1, Alpha 1

CDAA: Choline-Deficient L-Amino Acid

CDAAc: Choline-Deficient L-Amino Acid-defined diet supplemented with cholesterol

CHOL: cholesterol

CSAA: Choline Supplemented L-Amino Acid-defined

DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine

DMSO: Dimethyl sulfoxide

DTT: Dithiothreitol

ELISA: Enzyme-Linked Immunosorbent Assay

EMT: Epithelial-mesenchymal transition

EOB: Excess Over Bliss

FBS: Fetal Bovine Serum

FDA: Food and Drug Administration

FLUVA: Fluvastatin

FPP: Farnesyl Pyrophosphate

GDF: Growth Differentiation Factors

Hh: Hedgehog

GGPP: Geranylgeranlpyrophosphate

HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme A

hHSC: Human Hepatic Stellate Cells

HSC: Hepatic Stellate Cells

IC50: Half maximal Inhibitory Concentration

InMyoFib: Intestinal Myofibroblasts

MMP2: Matrix Metallopeptidase 2

MMP9: Matrix Metallopeptidase 9

μl: microliter

LDL: Low Density Lipoprotein

LOVA: Lovastatin

NHLF: Normal Human Lung Fibroblasts

NTZ: Nitazoxanide

PBC: Primary Biliary Cholangitis

PBS: Phosphate Buffer Saline

PITA: Pitavastatin

PSC: Primary Sclerosing Cholangitis

qPCR: Quantitative Polymerase Chain Reaction

pMol: picomoles

PRAVA: Pravastatin

rhFGF: recombinant human basic Fibroblast Growth Factor

ROSU: Rosuvastatin

RNA: Ribonucleic Acid

RT: Reverse Transcriptase

SIMVA: Simvastatin

SmBM: Smooth Muscle cell Basal Medium

SteCGS: Stellate Cell Growth Supplement

STeCM: Stellate Cell Medium

TBA: Total Bile Acids

TGFβ1: Tumor Growth Factor beta 1

TGFBRI: TGF type I receptor

TGFBRII: TGF type II receptor

THBS1: Thrombospondine 1

TMB: Tetramethylbenzidine

TZ: Tizoxanide

TZG: Tizoxanide glucuronide

