COMBINATION THERAPY HAVING ANTIOXYDANT PROPERTIES

Abstract

The present invention relates to novel uses of nitazoxanide, or analogues thereof.

Claims

1-10. (canceled)

11. A method of treating a disease involving oxidative stress comprising the administration, to a subject in need of treatment, a combination product comprising: (i) elafibranor, a metabolite of elafibranor, a pharmaceutically acceptable salt of elafibranor, or a pharmaceutically acceptable salt of a metabolite of elafibranor, and (ii) a compound of formula (I) or a pharmaceutically acceptable salt thereof: ##STR00014## in which R represents a O—R1 group or an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or a moiety of formula (A): ##STR00015## wherein R′ represents an (C1-C6)alkyl group, an (C2-C6)alkenyl group, an (C2-C6)alkynyl group, a (C3-C14)cycloalkyl group, (C3-C14)cycloalkyl(C1-C6)alkyl group, a (C3-C14)cycloalkyl(C1-C6)alkenyl group, a (C3-C14)cycloalkenyl group, a (C3-C14)cycloakenyl(C1-C6)alkyl group, a (C3-C14)cycloalkenyl(C2-C6)alkenyl group, a (C3-C14)cycloalkenyl(C2-C6)alkynyl group; R″ and R′″, independently, represent a hydrogen atom, an (C1-C6)alkyl group, or a nitrogen protecting group or a pharmaceutically acceptable salt thereof; R1 represents a hydrogen atom or a (C1-C6)alkylcarbonyl group; and R2 represents a halogen or a NO2 group.

12. The method according to claim 11, wherein the disease is selected from the group consisting of neurological disorders, central nervous system disorders, metabolic conditions, cardiovascular diseases, cataract, atherosclerosis, ischemia, myocardial ischemia, ischemic brain damage, lung ischemia-reperfusion injury, scleroderma, stroke, inflammation, inflammatory bowel disease, rheumatoid arthritis, respiratory diseases, autoimmune diseases, liver diseases, kidney diseases, skin conditions, infections and cancers.

13. The method according to claim 11, wherein the disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, tardive dyskinesia, epilepsy, acute diseases of the central nervous system, spinal cord injuries, brain trauma, obesity, insulin resistance, dyslipidemia, impaired glucose tolerance, high blood pressure, atherosclerosis, diabetes, type 1 or type 2 diabetes, metabolic syndrome, human immunodeficiency virus-induced oxidative stress, influenza virus-induced oxidative stress, HBV-induced oxidative stress, hepatitis C virus-induced oxidative stress, encephalomyocarditis virus-induced oxidative stress, respiratory syncytial virus-induced oxidative stress, dengue virus-induced oxidative stress, NAFLD-associated oxidative stress, NAFLD-associated oxidative stress with liver fibrosis, NASH-associated oxidative stress, NASH-associated oxidative stress with liver fibrosis, NASH-associated oxidative stress with liver cirrhosis, myocardial ischemia, ischemic brain damage, lung ischemia-reperfusion injury, scleroderma, stroke, inflammatory bowel disease and rheumatoid arthritis.

14. The method according to claim 11, wherein component (i) is elafibranor or a pharmaceutically acceptable salt thereof.

15. The method according to claim 11, wherein component (ii) is selected from Nitazoxanide (NTZ), Tizoxanide (TZ), a pharmaceutically acceptable salt of NTZ and a pharmaceutically acceptable salt of TZ.

16. The method according to claim 11, wherein component (i) is elafibranor or a pharmaceutically acceptable salt thereof and component (ii) is NTZ or a pharmaceutically acceptable salt thereof.

17. The method according to claim 11, wherein said combination product is a pharmaceutical composition comprising component (i) and component (ii).

18. The method according to claim 11, wherein said combination product is a kit comprising: a first pharmaceutical composition comprising component (i); and a second pharmaceutical composition comprising component (ii).

19. The method according to claim 11, wherein component (i) and component (ii) are formulated for oral administration.

20. The method according to claim 11, wherein component (i) and component (ii) are formulated in a pill, a tablet or a suspension for oral ingestion.

Description

DESCRIPTION OF THE FIGURES

[0083] FIG. 1 is a graph representing the levels of 4-HNE as quantified by immunochemistry on liver samples from 6 week-old C57BL/6 mice fed a control (CSAA) diet, CDAA+1% CHOL (CDAA/c) diet, or CDAA/c diet supplemented with NTZ 100 mg/kg/day alone, ELA 1 mg/kg/day alone or combined NTZ 100 mg/kg/day/ELA 1 mg/kg/day for 12 weeks.

[0084] FIG. 2 shows representative images of 4-HNE staining for each mice group described in the legend of FIG. 1 (Magnification ×300).

[0085] FIG. 3 shows the Nrf2-ARE-related transcriptional activity induced by TZ, ELA or the combination thereof. DL-Sulforaphane (DLS) was used as a positive control of induction. Data are shown as mean, ###: p<0.001, ***: p<0.001.

EXAMPLES

Evaluation of Elafibranor, Nitazoxanide and the Combination of Elafibranor+Nitazoxanide in a Chronic CDAA+1% Cholesterol Model of Fibrosing NASH (12 Weeks)

Material & Methods

Experimental Design

[0086] Given the prominent role of oxidative stress in NASH pathogenesis, we evaluated NTZ capacity to prevent redox homeostasis dysregulation in the CDAA/c diet induced NASH model.

[0087] The choline-deficient and L-amino acid-defined (CDAA) diet lacks choline, which is essential for hepatic β-oxidation and very low density lipoprotein production, and is believed to induce hepatocellular steatosis. Subsequently, lipid peroxidation and oxidative stress lead to lobular inflammation, comprehensively resulting in fibrosis.

[0088] In the current study, the preventive effects of NTZ 100 mg/kg/day, ELA 1 mg/kg/day and the combination of both were assessed in a murine model. 6 week-old male C57Bl/6J mice were fed a control (CSAA) diet (n=8), CDAA+1% cholesterol diet (n=12), or CDAA+1% cholesterol diet supplemented with NTZ 100 mg/kg/day (n=8), ELA 1 mg/kg/day (n=8) or combined drugs (NTZ 100 mg/kg/day coadministered with ELA 1 mg/kg/day (n=8)) for 12 weeks. The food was purchased from Ssniff® company (Soest, Germany). Nitazoxanide (Interchim, Ref #RQ550), elafibranor (Genfit) or both compounds were incorporated by Ssniff® into CDAA+1% chol diet in powder form to the required dose.

[0089] The body weight and the food intake were monitored twice per week. On the last day of treatment, mice were sacrificed after a 6 h fasting period. The liver was rapidly excised for transcriptomic and histological studies.

[0090] All animal procedures were performed according to standard protocols and in accordance with the standard recommendations for the proper care and use of laboratory animals.

Transcriptomic Studies

RNA Extraction

[0091] Hepatic Total RNA was isolated using Nucleospin® 96 Kit (Macherey Nagel) following manufacturer's instructions. 150 ng of total RNA were reverse transcribed in cDNA using M-MLV-RT (Moloney Murine Leukemia Virus Reverse Transcriptase) (Invitrogen cat #28025) in presence of RT buffer 1× (Invitrogen cat #P/NY02321), 1 mM DTT (Invitrogen cat #P/NY00147), 0.5 mM dNTPs (Promega), 200 ng pdN6 (Roche cat #11034731001) and 40 U of Ribonuclease inhibitor (Promega cat #N2515).

RNA-Sequencing:

[0092] Upon measurement of RNA samples concentration by nanodrop, the quality was assessed using bioanalyser. Libraries were prepared using the Illumina TruSeq stranded mRNA LT kit and mRNA were sequenced using a NextSeq 500 device (paired-end sequence, 2×75 bp), with a High Output flow cell.

RNA-Seq Data Analysis:

[0093] Reads were cleaned using Trimmomatic v.0.36 with the following parameters: SLIDINGWINDOW:5:20 LEADING:30 TRAILING:30 MINLEN:60. Then reads were aligned on the genome reference (Mus musculus GRCm38.90) with rnacocktail using hisat2 v.2.1.0 as aligner with default parameters.

[0094] A count table was produced using featureCounts v1.5.3 with default parameters.

[0095] To identify differentially expressed genes (DE genes), we used R (version 3.4.3) and the DESEq2 library (v. 1.18.1). Gene annotations were retrieved using the AnnotationDbi library (v. 1.40.0). Briefly, the count matrix produced by FeatureCounts was analyzed by the DESeqDataSetFromMatrix( ) function followed by the DEseq( ) function from the DESeq2 library. For each condition (ie comparison NTZ+CDAA/c vs CDAA/c), the fold change and the p-value were retrieved using the results( ) function from DESeq2. The different tables were merged using the Ensembl ID as a key.

Histology

[0096] At sacrifice, liver samples were processed for histological analysis and examined as follows.

Tissue Embedding and Sectioning

[0097] The liver slices were first fixed for 40 hours in formalin 4% solution followed by several dehydration steps in ethanol (successive baths at 70, 80, 95 and 100% ethanol). The liver pieces were subsequently incubated in three xylene baths followed by two baths in liquid paraffin (58° C.). Liver pieces were then put into small racks that were gently filled with Histowax® to completely cover the tissue. Then, tissue samples were thicked in 3 μm sections. Sections were prepared for immunohistochemistry (IHC).

Immunohistochemistry Assay: 4-HNE (4-Hydroxynonenal)

[0098] Immunohistochemistry assay was performed by using an immunoperoxidase protocol. Sections were dewaxed at 58° C. and in xylene baths (2×3 min). The specimens were hydrated with ethanol (successive baths at 100%, 100%, 95% and 70%) (3 min each) and submerged in 1×PBS (2×5 min). Subsequently, endogenous peroxidase was blocked with H.sub.2O.sub.2 solution (0.3% H.sub.2O.sub.2 in distilled water) for 30 min, followed by three washes in 1×PBS for 5 min. Furthermore, heat mediated antigen retrieval was performed with citrate buffer at pH 6.0 for 40 min at 95° C. To block nonspecific binding, 1×PBS solution with 3% normal goat serum and 0.1% Triton was added for 60 min. Subsequently, the tissues were incubated with primary 4-HNE antibody overnight at 4° C. and rinsed with 1×PBS (3×5 min). The tissues were incubated with HRP secondary antibody for 1 h at room temperature and then rinsed with 1×PBS (3×5 min). Slides are then revelated with the peroxidase substrate 3,3′-diaminobenzidine ((DAB) for 15 min, and rinsed with tap water. Finally, the stains were counterstained with Mayer hematoxylin for 3 min and rinsed with tap water (2 min) and tissues were dehydrated in ethanol and xylene.

4-HNE IHC Analysis:

[0099] The histological examinations and scoring were performed blindly. Images were acquired using Panoramic 250 Flash II digital slide scanner (3DHistech). Scoring: seven randomly selected fields from each section were examined and analyzed in QuantCenter software. 4-HNE accumulation was calculated as 4-HNE-positive area/total selected fields area.

ARE Reporter-HepG2 Cell Line

[0100] The ARE Reporter-Hep G2 cell line is designed to monitor Nrf2 antioxidant response pathway. The cell contains a firefly luciferase gene under the control of ARE (antioxidant response element) stably integrated into Hep G2 cells.

[0101] ARE reporter-HepG2 cells (BPS Bioscience, Inc., San Diego, cat #60513) were cultured following manufacturer's instructions. After thawing (BPS thaw medium 1K, cat #60187), cells were cultured in growth-medium (BPS growth medium 1K, cat #79533) and subsequently plated at a density of 40 000 cells per well in a 96-well microplate in 45 μL of assay medium (BPS thaw medium). TZ, ELA and DL-Sulforaphane (Sigma cat #54441) were dissolved in DMSO and diluted into assay medium. 5 μl of dilution were added on cells to reach a final concentration of 1 μM for TZ and 3 μM for ELA. DL-Sulforaphane was used as a positive control at the dose of 3 μM. After 18 h exposure, luciferase activity was determined. 50 μL of One-Step Luciferase assay system (BPS cat #60690) were added per well and after ˜15 min of rocking at room temperature, luminescence was measured using a luminometer.

[0102] Fold induction over DMSO above 2 and with a p-value<0.05 was considered as significant.

Statistical Analysis

[0103] Statistical analyses were performed using Prism Version 7, as follows:

In Vivo Studies

[0104] CSAA vs CDAA/c groups were compared by a Student t-test (#: p<0.05; ##: p<0.01; ###: p<0.001) or by a Mann-Whitney test ($: p<0.05; $$: p<0.01; $$$: p<0.001).

[0105] NTZ, ELA or ELA/NTZ treated group were compared to CDAA/c+1% chol diet using One-way ANOVA and uncorrected Fisher's LSD post-hoc (* p<0.05, **p<0.01, ***p<0.001) or using Kruskal-Wallis test and uncorrected Dunn's test post-hoc. (§ p<0.05, § § p<0.01, § § § p<0.001).

In Vitro Studies

ARE Reporter Assay:

[0106] DMSO and DL-Sulforaphane (DLS) groups were compared by a Student t-test (#: p<0.05; ##: p<0.01; ###: p<0.001). Treated groups were compared with DMSO group as well as between them using One-way ANOVA and uncorrected Fisher's LSD post-hoc (* p<0.05, **p<0.01, ***p<0.001)

Results

[0107] 6 week-old C57BL/6 mice were fed a control (CSAA) diet, CDAA+1% CHOL (CDAA/c) diet, or CDAA/c diet supplemented with NTZ 100 mg/kg/day alone, ELA 1 mg/kg/day alone or combined NTZ 100 mg/kg/day/ELA 1 mg/kg/day for 12 weeks.

[0108] After the sacrifice, the hepatic levels of SOD1; SOD2, GPX, CAT, GSTA1, GTA2, GSTA4 transcripts were analyzed by RNAseq and the count levels were determined and data were normalized over the CDAA/c values. The results show that ELA and NTZ induced respectively a different subset of antioxidant genes in the liver that surprisingly led to a complementary signature when both drugs are combined.

[0109] 4-HNE, a peroxidized aldehyde product of unsaturated fatty acids, is considered as a relevant indicator of oxidative stress (Takeuchi-Yorimoto, Noto et al. 2013). Our results show that an increase of intrahepatic 4-HNE levels is observed in the CDAA/c group in comparison with the CSAA group as shown in FIG. 1 and FIG. 2. Surprisingly, the levels of 4-HNE in the group that was exposed to the ELA/NTZ combination was reduced with an unexpected amplitude (−79%) in comparison with the CDAA/c group. Moreover, this therapeutic effect of the combination is higher than the effect obtained with each agent administered alone, showing an unexpected anti-oxidative stress effect of the combination of NTZ and ELA.

[0110] To further investigate the anti-oxidative stress effect of this combination, transcriptomic analyses were conducted on liver samples.

[0111] ELA (1 mg/kg/day) significantly induced the expression of a subset of antioxidant genes (SOD, Cat, GPX1) considered as the 1st line defense antioxidants meanwhile NTZ (100 mg/kg/day) significantly induced the expression of GST enzymes that are involved in detoxification of peroxidized products, a step that is downstream in the antioxidative signaling pathway. Interestingly, the results have shown that ELA/NTZ combination significantly induced the expression of several subsets of genes involved in different stages of the defense against oxidative stress, suggesting a more complete antioxidant defense mechanism when ELA and NTZ are co-administered in comparison with the result obtained with ELA or NTZ alone.

[0112] Since Nrf2 is a transcriptional master regulator of intracellular redox homeostatis (Hayes and Dinkova-Kostova 2014), we evaluated the effect of TZ (the active metabolite of NTZ), ELA and the combination thereof on the Nrf2-ARE-mediated transcription in human hepatocytes (FIG. 3). Unexpectedly, TZ, but not ELA, induces a significant activation of Nrf2-antioxidant pathway compared to the untreated condition and this induction is significantly higher with the combination compared to each agent alone demonstrating that a higher beneficial effect is obtained with the combination.

[0113] Altogether, these data suggest a stronger and more complete therapeutic response against oxidative stress when ELA/NTZ or ELA/TZ are combined together.

REFERENCES

[0114] de Araújo, R., D. Martins, et al. (2016). “Oxidative Stress and Disease—from the edited volume: A Master Regulator of Oxidative Stress—The Transcription Factor Nrf2, Edited by Jose Antonio Morales-Gonzalez, Angel Morales-Gonzalez and Eduardo Osiris Madrigal-Santillan.” [0115] Hayes, J. D. and A. T. Dinkova-Kostova (2014). “The Nrf2 regulatory network provides an interface between redox and intermediary metabolism.” Trends Biochem Sci 39(4): 199-218. [0116] Rossignol, J. F. and R. Cavier (1975). DE2438037A1—2-Benzamido-5-nitrothiazoles, S.P.R.L. Phavic, Belg. 11 pp. [0117] Takeuchi-Yorimoto, A., T. Noto, et al. (2013). “Persistent fibrosis in the liver of choline-deficient and iron-supplemented L-amino acid-defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after choline supplementation.” Toxicol Appl Pharmacol 268(3): 264-277.