Nitazoxanide and thiazolide for the treatment of diseases associated with oxidative stress

文档序号：1894218 发布日期：2021-11-26 浏览：20次中文

阅读说明：本技术 用于治疗氧化应激相关疾病的硝唑尼特和噻唑化物 (Nitazoxanide and thiazolide for the treatment of diseases associated with oxidative stress ) 是由尼古拉斯·斯坦科维奇-瓦伦丁克林内·富卡尔佩吉·帕罗什罗伯特·瓦尔恰克于 2020-04-09 设计创作，主要内容包括：本发明涉及硝唑尼特或其类似物的新用途。(The invention relates to a new application of nitazoxanide or analogues thereof.)

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, for use in a method of treating oxidative stress associated with a disease:

wherein:

r1 represents a hydrogen atom, a deuterium atom, a halogen atom, (C6-C14) an aryl group, a heterocyclic group, (C3-C14) a cycloalkyl group, (C1-C6) an alkyl group, a sulfonyl group, a sulfoxide group, (C1-C6) an alkylcarbonyl group, (C1-C6) an alkoxy group, a carboxylic acid ester group, a nitro group (NO2), an amino group (NH2), (C1-C6) an alkylamino group, an amide group, (C1-C6) an alkylamide group, or (C1-C6) a dialkylamide group;

r2 represents a hydrogen atom, a deuterium atom, an NO2 group, (C6-C14) an aryl group, a heterocyclic group, a halogen atom, (C1-C6) an alkyl group, (C3-C14) a cycloalkyl group, (C2-C6) an alkynyl group, (C1-C6) an alkoxy group, (C1-C6) an alkylthio group, (C1-C6) an alkylcarbonyl group, (C1-C6) an alkylcarbonylamino group, (C6-C14) an arylcarbonylamino group, a carboxylic acid or carboxylic acid ester group, an amide group, (C1-C6) an alkylamide group, (C1-C6) a dialkylamide group, NH₂A group, or a (C1-C6) alkylamino group;

or R1 and R2 together with the carbon atom to which they are attached form a substituted or unsubstituted 5-to 8-membered cycloalkyl, heterocyclic or aryl group;

r3, R4, R5, R6, and R7 represent, identically or differently, a hydrogen atom, a deuterium atom, a halogen atom, a hydroxyl group, (C1-C6) an alkylcarbonyl group, (C1-C6) an alkyl group, (C1-C6) an alkoxy group, (C1-C6) an alkylthio group, (C1-C6) an alkylcarbonyloxy group, (C6-C14) an aryloxy group, (C6-C14) an aryl group, a heterocyclic group, (C3-C14) a cycloalkyl group, a NO2 group, a sulfonamidoalkyl group, an NH2 group, an amino (C1-C6) alkyl group, (C1-C6) an alkylcarbonylamino group, a carboxylic acid group, a carboxylate group, or an R9 group;

r9 represents an O-R8 group, or an amino acid selected from 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):

wherein R' represents a (C1-C6) alkyl group, (C2-C6) alkenyl group, (C2-C6) alkynyl group, (C3-C14) cycloalkyl group, (C3-C14) cycloalkylalkyl group, (C3-C14) cycloalkyl (C2-C6) alkenyl group, (C3-C14) cycloalkenyl group, (C3-C14) cycloalkenyl (C1-C6) alkyl group, (C3-C14) cycloalkenyl (C2-C6) alkenyl group, or (C3-C14) cycloalkenyl (C2-C6) alkynyl group; wherein R 'and R' independently represent a hydrogen atom, a (C1-C6) alkyl group, or a nitrogen protecting group; and is

R8 represents a hydrogen atom, a deuterium atom, a glucuronyl group orA group in which R8a, R8b and R8c, which are the same or different, represent a hydrogen atom or a deuterium atom.

2. The compound for use according to claim 1, wherein the oxidative stress is associated with a disease selected from the group consisting of: neurological disorders such as central nervous system disorders, metabolic disorders, cardiovascular diseases, cataracts, atherosclerosis, ischemia such as myocardial ischemia, ischemic brain injury, pulmonary ischemia-reperfusion injury, scleroderma and stroke, inflammation such as inflammatory bowel disease, rheumatoid arthritis, respiratory diseases, autoimmune diseases, liver diseases, kidney diseases, skin disorders, infections and cancer.

3. A compound of formula (I) or a pharmaceutically acceptable salt thereof, for use in a method of treating a disease involving oxidative stress:

wherein:

or R1 and R2 together with the carbon atom to which they are attached form a substituted or unsubstituted 5-to 8-membered cycloalkyl, heterocyclic or aryl group;

R8 represents a hydrogen atom, a deuterium atom, a glucuronyl group orA group in which R8a, R8b and R8c, which are the same or different, represent a hydrogen atom or a deuterium atom.

4. A compound for use according to claim 3 for the treatment of a disease selected from the group consisting of: neurological disorders such as central nervous system disorders, metabolic disorders, cardiovascular diseases, cataracts, atherosclerosis, ischemia such as myocardial ischemia, ischemic brain injury, pulmonary ischemia-reperfusion injury, scleroderma and stroke, inflammation such as inflammatory bowel disease, rheumatoid arthritis, respiratory diseases, autoimmune diseases, renal diseases and skin disorders.

5. The compound for use according to claim 3 or 4, wherein the disease is selected from the group consisting of: alzheimer's disease, parkinson's disease, huntington's disease, tardive dyskinesia, epilepsy, acute disorders of the central nervous system such as spinal cord injury and/or brain trauma, obesity, insulin resistance, dyslipidemia, impaired glucose tolerance, hypertension, atherosclerosis and diabetes such as type 1 or type 2 diabetes, metabolic syndrome, myocardial ischemia, ischemic brain injury, pulmonary ischemia-reperfusion injury, scleroderma, stroke, inflammatory bowel disease and rheumatoid arthritis.

6. The compound for use according to any one of claims 1 to 5, wherein the compound is selected from nitazoxanide, tizoxanide and pharmaceutically acceptable salts thereof.

7. The compound for use according to any one of claims 1 to 6, wherein the compound is NTZ or a pharmaceutically acceptable salt thereof.

8. The compound for use according to any one of claims 1 to 7, wherein the compound is formulated for oral administration.

9. The compound for use according to any one of claims 1 to 8, wherein the compound is formulated as a pill, tablet or suspension for oral ingestion.

Technical Field

The invention relates to a new application of nitazoxanide or analogues thereof.

Background

[2- [ (5-Nitro-1, 3-thiazol-2-yl) carbamoyl ] phenyl ] acetate (or nitazoxanide, or NTZ), first published in 1975 (Rossgnol and Cavier 1975), is an approved drug in the United states for the treatment of diarrhea caused by the protozoan parasites Cryptosporidium parvum (Crystosporidium parvum) and Giardia intestinalis (Giardia intestinalis). NTZ has also been commercialized in latin america and india where it is indicated for the treatment of a broad spectrum of intestinal parasitic infections. The proposed mechanism of action of NTZ to exert its antiparasitic activity is through inhibition of Pyruvate Ferredoxin Oxidoreductase (PFOR) dependent electron transfer reactions, which are essential for anaerobic metabolism (Hoffman, Sisson et al 2007). NTZ also exhibits activity against Mycobacterium tuberculosis (which does not have a homologue of PFOR), thus suggesting an additional mechanism of action. In fact, the authors show that NTZ can also act as an uncoupler to disrupt membrane potential and pH homeostasis in organisms (de Carvalho, Darby et al 2011).

The pharmacological effects of NTZ are not limited to its antiparasitic or antibacterial activity, and in recent years, several studies have revealed that NTZ can also interfere with viral replication to confer antiviral activity by a variety of means, including blocking the maturation of the hemagglutinin (influenza) or VP7 (rotavirus) proteins, or activating the protein PKR involved in the innate immune response (for review see Rossignol 2014). NTZ has also been shown to have anti-cancer properties by interfering with critical metabolic and pro-death signaling pathways (Di Santo and Ehrisman 2014).

The applicant has recently also shown that NTZ has anti-fibrotic properties (WO2017178172) and is currently evaluating its effect on populations suffering from NASH-induced stage 2 or 3 fibrosis.

Disclosure of Invention

The inventors herein show that NTZ has antioxidant properties, which opens up new therapeutic opportunities.

The present invention results from the surprising observation made by the inventors that NTZ activates the expression of a different glutathione S-transferase (GST) gene. GST is a family of enzymes that play an important role in detoxification by catalyzing the binding of many hydrophobic and electrophilic compounds to reduced glutathione. GST has a particular role in protecting cells from reactive oxygen species and peroxidation products. Their activation can therefore be advantageously used to protect cells, tissues and organs against oxidative stress.

Complementary analysis of hepatic transcriptomic signatures induced by NTZ revealed enrichment of a subset of genes (including GST enzymes) under the control of Nrf2, Nrf2 being a transcriptional primary regulator of redox homeostasis in cells. In vitro assays demonstrate the ability of TZ (an active metabolite of NTZ) to induce the Nrf2-ARE signaling pathway at a functional level. Furthermore, TZ has been demonstrated to be able to maintain GSH (reduced glutathione) pool in human hepatocytes under oxidative stress, GSH being the most abundant antioxidant in the liver. Taken together, these data demonstrate the unexpected antioxidant capacity of NTZ.

Accordingly, one aspect of the present invention relates to a compound of formula (I) as defined below, including NTZ and analogues thereof, or pharmaceutically acceptable salts of compounds of formula (I), for use as an antioxidant. In a particular embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is used for its liver antioxidant properties.

Another aspect of the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, for use in a method of treating a disease involving oxidative stress. Diseases involving oxidative stress are well known to those skilled in the art and reference may be made to many other sources including de Araujo et al (de Ara jo, Martins et al 2016). For example, subjects that may benefit from the present invention include, but are not limited to, those suffering from nervous system disorders such as central nervous system disorders, metabolic disorders, cardiovascular diseases, cataracts, atherosclerosis, ischemia such as myocardial ischemia, ischemic brain injury, pulmonary ischemia-reperfusion injury, scleroderma and stroke, inflammation such as inflammatory bowel disease, rheumatoid arthritis, respiratory diseases, autoimmune diseases, renal diseases, and skin disorders.

The term "treating" refers to the curative or prophylactic treatment of a disease in a subject in need thereof. Treatment includes administering a compound of the invention to a subject having an alleged disease to prevent, cure, delay, reverse or slow the progression of the disease, thereby improving the condition of the subject. Accordingly, the present invention also relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, for use in a method of preventing, curing, delaying, reversing or slowing the progression of a disease. The compounds of the invention may also be administered to a subject that is healthy or at risk of developing disease. The subject to be treated is a mammal, preferably a human. The subject to be treated according to the present invention may be selected based on several criteria associated with the particular disease for which treatment is sought, such as previous drug treatment, associated pathology, genotype, exposure to risk factors, viral infection, and based on detection of any biomarkers associated with the disease.

In addition, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, for use in a method of treating oxidative stress associated with a disease, in particular a disease selected from nervous system disorders such as central nervous system disorders, metabolic disorders, cardiovascular diseases, cataracts, atherosclerosis, ischemia, such as myocardial ischemia, ischemic brain injury, pulmonary ischemia-reperfusion injury, scleroderma and stroke, inflammation, such as inflammatory bowel disease, rheumatoid arthritis, respiratory diseases, autoimmune diseases, liver diseases, kidney diseases, skin disorders, infections and cancer.

Neurological disorders include, but are not limited to, alzheimer's disease, parkinson's disease, huntington's disease, tardive dyskinesia, epilepsy, and acute disorders of the central nervous system such as spinal cord injury and/or brain trauma.

Metabolic disorders include, but are not limited to, obesity, insulin resistance, dyslipidemia, impaired glucose tolerance, hypertension, atherosclerosis, and diabetes such as type 1 or type 2 diabetes. Metabolic disorders also include metabolic syndrome.

In a particular embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is used in a method of treating infection-induced oxidative stress, such as virus-induced oxidative stress, in particular 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 and dengue virus-induced oxidative stress.

The invention also relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, for use in a method of treating oxidative stress associated with liver disease. Accordingly, the compounds of formula (I) are useful in methods of treating oxidative stress associated with liver disease. In particular, the subject to be treated may suffer from cirrhosis, non-alcoholic fatty liver disease (NAFLD), NAFLD with liver fibrosis, non-alcoholic steatohepatitis (NASH), NASH with liver fibrosis or NASH with cirrhosis. Accordingly, the present invention also relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, for use in a method of treating oxidative stress associated with cirrhosis, NAFLD-related oxidative stress, NAFLD-associated oxidative stress associated with liver fibrosis, NASH-associated oxidative stress associated with liver fibrosis or NASH-associated oxidative stress with cirrhosis. In another specific embodiment, the subject to be treated has NAFLD, NAFLD with liver fibrosis, NASH or NASH with liver fibrosis. Thus, in a particular embodiment of the invention, a compound of formula (I) or a pharmaceutically acceptable salt thereof is used in a method of treating NAFLD-related oxidative stress, NAFLD-associated oxidative stress associated with liver fibrosis, NASH-associated oxidative stress or NASH-associated oxidative stress.

The present invention also relates to a method for treating oxidative stress associated with liver disease, wherein the method comprises administering to a subject in need of treatment for liver disease a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In a particular embodiment of the method of the invention, the subject suffers from cirrhosis, non-alcoholic fatty liver disease (NAFLD), NAFLD with liver fibrosis, non-alcoholic steatohepatitis (NASH), NASH with liver fibrosis or NASH with cirrhosis.

In a particular embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is used in a method of treating oxidative stress associated with cancer, in particular liver cancer, more particularly hepatocellular carcinoma (HCC).

The present invention also relates to a method of treating cancer, in particular liver cancer, e.g. hepatocellular carcinoma-associated oxidative stress, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

The compounds used in the present invention are of formula (I):

wherein:

or R1 and R2 together with the carbon atom to which they are attached form a substituted or unsubstituted 5-to 8-membered cycloalkyl, heterocyclic or aryl group;

wherein R' represents a (C1-C6) alkyl group, (C2-C6) alkenyl group, (C2-C6) alkynyl group, (C3-C14) cycloalkyl group, (C3-C14) cycloalkylalkyl group, (C3-C14) cycloalkyl (C2-C6) alkenyl group, (C3-C14) cycloalkenyl group, (C3-C14) cycloalkenyl (C1-C6) alkyl group, (C3-C14) cycloalkenyl (C2-C6) alkenyl group or (C3-C14) cycloalkenyl (C2-C6) alkynyl group; wherein R 'and R' independently represent a hydrogen atom, a (C1-C6) alkyl group, or a nitrogen protecting group; and is

R8 represents a hydrogen atom, a deuterium atom, a glucuronyl group orA group in which R8a, R8b and R8c, which are the same or different, represent a hydrogen atom or a deuterium atom.

In another particular embodiment, the compounds of formula (I) are as follows:

r1 represents a hydrogen atom, a deuterium atom, a halogen atom, (C6-C14) an aryl group, a heterocyclic group, (C3-C14) a cycloalkyl group, (C1-C6) an alkyl group, a sulfonyl group, a sulfoxide group, (C1-C6) an alkyl groupCarbonyl groups, (C1-C6) alkoxy groups, carboxylic acid groups, carboxylic ester groups, NO₂A group, an NH2 group, (C1-C6) alkylamino group, amide group, (C1-C6) alkylamide group, or (C1-C6) dialkylamide group;

or R1 and R2 together with the carbon atom to which they are attached form a substituted or unsubstituted 5-to 8-membered cycloalkyl, heterocyclic or aryl group;

r3 represents a hydrogen atom, a deuterium atom, a halogen atom, an O-R8 group, (C1-C6) an alkylcarbonyl group, (C1-C6) an alkyl group, (C1-C6) an alkoxy group, (C1-C6) an alkylthio group, (C1-C6) an alkylcarbonyloxy group, (C6-C14) an aryloxy group, (C6-C14) an aryl group, a heterocyclic group, (C3-C14) a cycloalkyl group, NO2, a sulfonamidoalkyl group, an NH2 group, an amino (C1-C6) alkyl group, (C1-C6) an alkylcarbonylamino group, a carboxylic acid group, a ester group selected from 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):

r8 represents a hydrogen atom, a deuterium atom, orA group in which R8a, R8b and R8c, which are the same or different, represent a hydrogen atom or a deuterium atom; and is

R4, R5, R6 and R7 represent, identically or differently, a hydrogen atom, a deuterium atom, a halogen atom, a hydroxyl group, (C1-C6) an alkylcarbonyl group, (C1-C6) an alkyl group, (C1-C6) an alkoxy group, (C1-C6) an alkylthio group, (C1-C6) an alkylcarbonyloxy group, (C6-C14) an aryloxy group, (C6-C14) an aryl group, a heterocyclic group, (C3-C14) a cycloalkyl group, NO2, a sulfonamido (C1-C6) alkyl group, an NH2 group, an amino (C1-C6) alkyl group, (C1-C6) an alkylcarbonylamino group, a carboxylic acid ester group, 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):

wherein R' represents a (C1-C6) alkyl group, a (C2-C6) alkenyl group, a (C2-C6) alkynyl group, a (C3-C14) cycloalkyl group, a (C3-C14) cycloalkyl (C1-C6) alkyl group, a (C3-C14) cycloalkyl (C1-C6) alkenyl group, a (C3-C14) cycloalkenyl group, a (C3-C14) cycloalkenyl (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, a (C1-C6) alkyl group, or a nitrogen protecting group.

In a particular embodiment, in the compounds of formula (I) of the present invention:

the alkyl group may be a substituted or unsubstituted (C1-C6) alkyl group, in particular a substituted or unsubstituted (C1-C4) alkyl group;

the alkynyl group may be a substituted or unsubstituted (C2-C6) alkynyl group;

the cycloalkyl group may be a substituted or unsubstituted (C3-C14) cycloalkyl group

The alkoxy group may be a substituted or unsubstituted (C1-C6) alkoxy group, such as a substituted or unsubstituted (C1-C4) alkoxy group;

the alkylthio group can be a substituted or unsubstituted (C1-C6) alkylthio group, such as a substituted or unsubstituted (C1-C4) alkylthio group;

the alkylamino group can be a (C1-C6) alkylamino group, such as a (C1-C4) alkylamino group;

the dialkylamino group can be a (C1-C6) dialkylamino group, such as a (C1-C4) dialkylamino group;

the aryl group can be a substituted or unsubstituted (C6-C14) aryl group, such as a substituted or unsubstituted (C6-C14) aryl group;

the heterocyclic group may be a substituted or unsubstituted heterocycloalkyl or heteroaryl group.

Nitrogen protecting Groups are well known to the person skilled in the art, for example those described in the literature, as for example in the book "protecting Groups in Greens' Organic Synthesis" (Wuts and Greene 2007).

In one embodiment, the compound of formula (I) is a compound of formula (II):

wherein R9 represents a hydrogen atom, a deuterium atom, an O-R8 group (R8 is as defined above), or an amino acid selected from 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):

wherein R' represents a (C1-C6) alkyl group, a (C2-C6) alkenyl group, a (C2-C6) alkynyl group, a (C3-C14) cycloalkyl group, a (C3-C14) cycloalkyl (C1-C6) alkyl group, a (C3-C14) cycloalkyl (C1-C6) alkenyl group, a (C3-C14) cycloalkenyl group, a (C3-C14) cycloalkenyl (C1-C6) alkyl group, a (C3-C14) cycloalkenyl (C2-C6) alkenyl group, or a (C3-C14) cycloalkenyl (C2-C6) alkynyl group; wherein R 'and R' independently represent a hydrogen atom, a (C1-C6) alkyl group, or a nitrogen protecting group.

In one particular embodiment, the compound of formula (I) is selected from:

-NTZ：

-Tizoxanide (TZ):

and

-glucuronide Tizoxanide (TZG):

in another embodiment, the compound of formula (II) is

R8a, R8b and R8c, which are the same or different, represent a hydrogen atom or a deuterium atom; and/or

R1, R3, R4, R5 and R6 represent, identically or differently, a hydrogen atom or a deuterium atom, with the proviso that R1, R2, R8a, R8b, R8c, R3, R4, R5 and R6 are not simultaneously hydrogen atoms.

In a particular embodiment, the compound of formula (I) is [ (5-nitro-1, 3-thiazol-2-yl) carbamoyl ] phenyl (d3) acetate, 2- [ (5-nitro-1, 3-thiazol-2-yl) carbamoyl ] phenyl (d2) acetate; or 2- [ (5-nitro-1, 3-thiazol-2-yl) carbamoyl ] phenyl (d1) acetate.

In another particular embodiment, the compound of formula (I) is 2-amino-3, 3-dimethylbutyrate 2- (5-nitrothiazol-2-ylcarbamoyl) phenyl ester, in particular (S) -2-amino-3, 3-dimethylbutyrate 2- (5-nitrothiazol-2-ylcarbamoyl) phenyl ester, or a pharmaceutically acceptable salt thereof, such as the hydrochloride salt of the formula (RM 5061):

in another particular embodiment, the compound of formula (I) is 2- (5-nitrothiazol-2-ylcarbamoyl) phenyl 2-amino-3-methylpentanoate, in particular 2- (5-nitrothiazol-2-ylcarbamoyl) phenyl (2S,3S) -2-amino-3-methylpentanoate, or a pharmaceutically acceptable salt thereof, for example the hydrochloride of the formula (RM 5066):

in another particular embodiment, the compound of formula (I) is 2-amino-3, 3-dimethylbutyrate 2- (5-chlorothiazol-2-ylcarbamoyl) phenyl ester, in particular (S) -2-amino-3, 3-dimethylbutyrate 2- (5-chlorothiazol-2-ylcarbamoyl) phenyl ester, or a pharmaceutically acceptable salt thereof, such as the hydrochloride of the formula (RM 5064):

in another particular embodiment, the compound of formula (I) is 2- (5-chlorothiazol-2-ylcarbamoyl) phenyl 2-amino-3-methylpentanoate, in particular 2- (5-chlorothiazol-2-ylcarbamoyl) phenyl (2S,3S) -2-amino-3-methylpentanoate, or a pharmaceutically acceptable salt thereof, for example the hydrochloride of the formula (RM 5065):

in a particular embodiment, the compound of formula (I) is NTZ, TZ, TZG or a pharmaceutically acceptable salt thereof. In another particular embodiment, the compound of formula (I) is NTZ or TZ or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the compound of formula (I) is NTZ or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the present invention relates to a method of treating oxidative stress associated with liver disease, wherein the method comprises administering to a subject in need of treatment for liver disease a therapeutically effective amount of NTZ and/or TZ or a pharmaceutically acceptable salt thereof. In a particular embodiment, the subject is administered NTZ or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the present invention relates to a method of treating liver fibrosis-associated oxidative stress, wherein said method comprises administering to a subject in need of treatment of liver fibrosis a therapeutically effective amount of NTZ and/or TZ or a pharmaceutically acceptable salt thereof. In a particular embodiment, the subject is administered NTZ or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the present invention relates to a method of treating cirrhosis-related oxidative stress, wherein the method comprises administering to a subject in need of treatment of cirrhosis a therapeutically effective amount of NTZ and/or TZ or a pharmaceutically acceptable salt thereof. In a particular embodiment, the subject is administered NTZ or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the present invention relates to a method of treating NAFLD-associated oxidative stress, wherein the method comprises administering to a subject in need of treatment of NAFLD a therapeutically effective amount of NTZ and/or TZ or a pharmaceutically acceptable salt thereof. In a particular embodiment, the subject is administered NTZ or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the present invention relates to a method of treating NAFLD associated with liver fibrosis associated oxidative stress, wherein the method comprises administering to a subject in need of treatment of NAFLD associated with liver fibrosis a therapeutically effective amount of NTZ and/or TZ or a pharmaceutically acceptable salt thereof. In a particular embodiment, the subject is administered NTZ or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the present invention relates to a method of treating NASH-related oxidative stress, wherein the method comprises administering to a subject in need of treatment for NASH a therapeutically effective amount of NTZ and/or TZ or a pharmaceutically acceptable salt thereof. In a particular embodiment, the subject is administered NTZ or a pharmaceutically acceptable salt thereof.

In another particular embodiment, the present invention relates to a method of treating NASH with liver fibrosis related oxidative stress, wherein said method comprises administering to a subject in need of treatment for NASH with liver fibrosis a therapeutically effective amount of NTZ and/or TZ or a pharmaceutically acceptable salt thereof. In a particular embodiment, the subject is administered NTZ or a pharmaceutically acceptable salt thereof.

The synthesis of NTZ or an analogue may be performed, for example, as described in (Rossignol and Cavier 1975), or by any other synthetic means known to the person skilled in the art.

The compound of formula (I) may be included in a pharmaceutical composition together with a pharmaceutically acceptable carrier. These pharmaceutical compositions may also contain one or more excipients or vehicles that are acceptable in a pharmaceutical environment (e.g., saline solutions, physiological solutions, isotonic solutions, and the like that are compatible with pharmaceutical use and are well known to those of ordinary skill in the art). These compositions may also comprise one or several agents or media selected from dispersing agents, solubilizing agents, stabilizing agents, preservatives and the like. The agents or vehicles which can be used for these formulations (liquid and/or injection and/or solid) are in particular methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, liposomes, etc. The compositions may be for enteral or parenteral administration. For example, the compounds of formula (I) may be formulated for oral, intravascular (e.g., intravenous or intraarterial), intramuscular, intraperitoneal, subcutaneous, transdermal or intranasal administration. The composition may be in solid or liquid dosage form. Illustrative formulations include, but are not limited to, injectable suspensions, oral suspensions, gels, oils, ointments, pills, tablets, suppositories, powders, gel caps, capsules, aerosols, ointments, creams, patches, or galenic forms or means of devices to ensure prolonged and/or slow release. For such formulations, agents such as cellulose, carbonate or starch may be advantageously used.

The compounds of formula (I) may be formulated as pharmaceutically acceptable salts, in particular as acid or base salts, which are compatible with pharmaceutical use. Salts of the compounds of formula (I) include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium salts and alkylated ammonium salts. These salts may be obtained during the final purification step of the compound or by incorporating the salts into a previously purified compound.

According to a particular embodiment, the composition of the invention comprises at least one compound of formula (I) as active ingredient, together with acceptable excipients. For example, the composition of the invention comprises a combination of two compounds of formula (I) NTZ and TZ as active ingredients.

The frequency and/or dosage of administration can be adjusted by one of ordinary skill in the art with respect to the subject to be treated, the pathology, the form of administration, and the like. In general, the compounds of formula (I), in particular NTZ or a pharmaceutically acceptable salt thereof, may be administered at a dose of between 0.01 mg/day and 4000 mg/day, for example between 50 mg/day and 2000 mg/day, in particular between 100 mg/day and 1000 mg/day, more in particular between 500 mg/day and 1000 mg/day.

In another preferred embodiment, the compound of formula (I), preferably NTZ or a pharmaceutically acceptable salt thereof, is administered in the form of a pill or tablet intended for oral ingestion. In another particular embodiment, the compound of formula (I), preferably NTZ or a pharmaceutically acceptable salt thereof, is administered in the form of a suspension intended for oral ingestion.

In another aspect, the present invention relates to a method of treating a disease comprising administering NTZ or a pharmaceutically acceptable salt thereof, wherein NTZ is administered at a dose between 500 mg/day and 1000 mg/day, wherein the disease is selected from alzheimer's disease, parkinson's disease, huntington's disease, tardive dyskinesia, epilepsy, acute diseases of the central nervous system such as spinal cord injury and/or brain trauma, obesity, insulin resistance, dyslipidemia, impaired glucose tolerance, hypertension, atherosclerosis and diabetes such as type 1 or type 2 diabetes, metabolic syndrome, oxidative stress induced by human immunodeficiency virus, oxidative stress induced by influenza virus, oxidative stress induced by HBV, oxidative stress induced by hepatitis c virus, oxidative stress induced by encephalomyocarditis virus, oxidative stress induced by respiratory syncytial virus, oxidative stress induced by dengue virus, oxidative stress induced by influenza virus, oxidative stress induced by HBV, oxidative stress induced by hepatitis c virus, and/or a pharmaceutically acceptable salt thereof, Liver cirrhosis-related oxidative stress, NAFLD-related oxidative stress with liver fibrosis, NASH-related oxidative stress with liver fibrosis, myocardial ischemia, ischemic brain injury, pulmonary ischemia-reperfusion injury, scleroderma, stroke, inflammatory bowel disease, and rheumatoid arthritis.

In another aspect, the present invention relates to a method of treating a disease comprising administering NTZ or a pharmaceutically acceptable salt thereof, wherein NTZ is administered at a dose between 500 mg/day and 1000 mg/day, wherein the disease is selected from alzheimer's disease, parkinson's disease, huntington's disease, tardive dyskinesia, epilepsy, acute diseases of the central nervous system such as spinal cord injury and/or brain trauma, obesity, insulin resistance, dyslipidemia, impaired glucose tolerance, hypertension, atherosclerosis and diabetes such as type 1 or type 2 diabetes, metabolic syndrome, oxidative stress induced by human immunodeficiency virus, oxidative stress induced by influenza virus, oxidative stress induced by HBV, oxidative stress induced by hepatitis c virus, oxidative stress induced by encephalomyocarditis virus, oxidative stress induced by respiratory syncytial virus, oxidative stress induced by dengue virus, oxidative stress induced by influenza virus, oxidative stress induced by HBV, oxidative stress induced by hepatitis c virus, and/or a pharmaceutically acceptable salt thereof, NAFLD-associated oxidative stress, NAFLD-associated oxidative stress with liver fibrosis, NASH-associated oxidative stress with liver fibrosis, myocardial ischemia, ischemic brain injury, pulmonary ischemia-reperfusion injury, scleroderma, stroke, inflammatory bowel disease, and rheumatoid arthritis.

If necessary, it can be administered once daily or even several times daily. The duration of treatment will depend on the particular disease to be treated. For example, the administration may be performed during one or more days, such as at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least seven days. Alternatively, the administration may be for at least one week, at least two weeks, at least four weeks. For chronic diseases, administration for more than 4 weeks may be considered, e.g. at least one month, two months, three months, four months, five months, six months or more than six months, e.g. at least one year or several years. In some cases, the combination product of the invention may be administered during the lifetime of the subject.

The invention is further described with reference to the following non-limiting examples.

Drawings

FIG. 1: long-term oral administration of NTZ contributes to antioxidant defense mechanisms.

C57BL/6 mice, 6 weeks old, were fed Control (CSAA) feed, CDAA + 1% cholesterol (CDAA/C) feed or CDAA/C feed supplemented with NTZ 100 mg/kg/day for a period of 12 weeks. After sacrifice, 4-HNE levels were determined by immunochemistry and quantified (panel a). Representative images of 4-HNE staining of each group are shown on panel B (magnification x 300).

FIG. 2: chronic oral administration of NTZ induced hepatic expression of GSTA1(a) and GSTA2(B) at the mRNA level.

C57BL/6 mice, 6 weeks old, were fed Control (CSAA) feed, CDAA + 1% cholesterol (CDAA/C) feed or CDAA/C feed supplemented with NTZ 100 mg/kg/day for a period of 12 weeks. After sacrifice, liver levels of liver GSTa mRNA were analyzed by RNAseq and count levels were determined.

FIG. 3: chronic oral administration of NTZ induced hepatic expression of GSTA4 at the mRNA level.

C57BL/6 mice, 6 weeks old, were fed Control (CSAA) feed, CDAA + 1% cholesterol (CDAA/C) feed or CDAA/C feed supplemented with NTZ 100 mg/kg/day for a period of 12 weeks. After sacrifice, liver levels of GSTA4 mRNA were analyzed by RNAseq and normalized count levels were determined.

FIG. 4: genes induced by NTZ differences were significantly enriched in Nrf2 target genes. C57BL/6 mice, 6 weeks old, were fed Control (CSAA) feed, CDAA + 1% cholesterol (CDAA/C) feed or CDAA/C feed supplemented with NTZ 100 mg/kg/day for a period of 12 weeks. After sacrifice, transcriptomes were analyzed by RNAseq. The proportion of Nrf2 target genes in the RNA-seq identified whole transcriptome (27636 genes) was calculated and compared to the proportion of Nrf2 target genes in the gene set differentially expressed under NTZ + CDAA/c versus CDAA/c conditions.

FIG. 5: TZ induces Nrf2-ARE (antioxidant response element) signaling in human hepatocytes. Luciferase activity mediated by Antioxidant Response Element (ARE) was measured in HepG2 cells treated with TZ. DL-Sulforaphane (DL-Sulforaphane, DLS) was used as a reference compound.

FIG. 6: in human hepatocytes exposed to the oxidative stress inducer menadione, TZ prevents Glutathione (GSH) depletion.

Intracellular GSH levels were monitored by thiol tracers (thiolracker Violet dye) in menadione-stressed hepatocytes in the presence or absence of TZ. N-acetylcysteine (NAC) was used as a positive control.

Detailed Description

Examples

Evaluation of nitazoxanide in a Long-term CDAA + 1% Cholesterol model of fibrotic NASH (12 weeks)

Design of experiments

In view of the prominent role of oxidative stress in the pathogenesis of NASH, we evaluated the ability of NTZ to prevent redox homeostasis disorders in the CDAA/c feed-induced NASH model.

Choline deficiency and L-amino acid defined (CDAA) feed is deficient in choline, which is essential for liver β -oxidation and very low density lipoprotein production, and is thought to induce liver cell steatosis. Subsequently, lipid peroxidation and oxidative stress cause lobular inflammation, leading extensively to fibrosis.

In the current study, the prophylactic effect of NTZ at 100 mg/kg/day was evaluated in a murine model. C57Bl/6J male mice, 6 weeks old, were fed either Control (CSAA) diet (n-8), CDAA + 1% cholesterol diet (n-12) or CDAA/C + 1% cholesterol diet supplemented with NTZ 100 mg/kg/day for 12 weeks. The foodstuff is purchased fromCompany (Soest, germany). Nitazoxanide (Interchim, Ref # RQ550) is prepared by mixingIncorporated as a powder added to CDAA + 1% cholesterol feed to the required dosage.

Body weight and food intake were monitored twice weekly. On the last day of treatment, mice were sacrificed after a 6 hour fasting period. Liver was rapidly excised for transcriptomics and histology studies.

All animal procedures were performed according to standard protocols and according to standard recommendations for proper care and use of laboratory animals.

Transcriptomics studies

RNA extraction

Use of96 kit (Macherey Nagel), following the manufacturer's instructions, total liver RNA was isolated. In the presence of RT buffer 1X (Invitrogen, Cat. No. P/NY02321), 1mM DTT (Invitrogen, Cat. No. P/NY00147), 0.5mM dNTP (Promega), 200ng pdN6(Roche, Cat. No. 11034731001) and 40U ribonuclease inhibitor (Promega, Cat. No. N2515), 150ng of total RNA was reverse transcribed to cDNA using M-MLV-RT (Moloney murine leukemia virus reverse transcriptase) (Invitrogen, Cat. No. 28025).

RNA sequencing:

after measuring the concentration of the RNA sample by nanodrop, the amount thereof was evaluated using a bioanalyzer. Libraries were prepared using Illumina TruSeq stranded mRNA LT kit and mRNA sequenced using NextSeq 500 equipment (end-paired sequences, 2x75 bp) with high throughput flow channels.

RNA-seq data analysis:

the readings were cleaned using trimmatic v.0.36 with the following parameters: SLIDNGWINDOW: 5:20LEADING:30TRAILING:30 MINLENEN: 60. The reads were then aligned on the reference genome (Mus musculus grcm38.90) with default parameters using rnaccoktail, using hisat2 v.2.1.0 as an aligner.

The count table is generated with default parameters using featureCounts v1.5.3.

To identify differentially expressed genes (DE genes), we used the R (version 3.4.3) and DESEq2 libraries (version 1.18.1). Gene annotations were retrieved using the AntotationDbi library (version 1.40.0). Briefly, the count matrix (count matrix) generated by featurepools was analyzed by the desqdatasetfrommatrix () function from the DESeq2 library followed by the DESeq () function. For each condition (i.e., comparing NTZ + CDAA/c to CDAA/c), the fold change and p-value are retrieved using the results () function in DESeq 2. The different tables are merged using Ensembl ID as key.

To assess the effect of Nrf2 on NTZ-induced transcriptome remodeling, a table listing Nrf2 target genes was generated by combining information from Hayes and McMahon and Jung and Kwak (Hayes and McMahon 2009; Jung and Kwak 2010), mouse Nfe2l2 target genes from the trruit database (https:// www.grnpedia.org/TRRUST /), Nrf2 Pathway from Wikipathway (https:// www.wikipathways.org/index. php/Pathway: WP2884), and ChIP-seq data from chord et al (chord, Campbell et al 2012). This list of Nrf2 target genes was used to identify Nrf2 target genes in either the RNA-seq identified whole transcriptome (27636 genes) or the set of genes differentially expressed under NTZ + CDAA/c versus CDAA/c conditions (the differentially expressed genes are considered to meet the criteria of at least | fold change versus CDAA/c | >1.4 fold and modulated p-value < 0.01). The ratio between the whole gene bank and the Nrf2 target gene was calculated and expressed as a percentage. To determine whether the proportion of Nrf2 target gene observed under NTZ + CDAA/c versus CDAA/c conditions is different from that observed in the identified whole transcriptome, a chi-square test was performed.

Histology

After sacrifice, liver samples were processed for histological analysis and examination as follows.

Tissue embedding and sectioning

Slicing liver firstFixation in 4% formalin solution for 40 hours, followed by several dehydration steps in ethanol (successive baths in 70, 80, 95 and 100% ethanol). The liver pieces were then incubated in three xylene baths, then two liquid paraffin baths (58 ℃). Then put the liver slices into a small shelf for useFill gently to cover the tissue completely. Then, the tissue sample was sliced to a thickness of 3 μm. Sections were prepared for Immunohistochemistry (IHC).

Immunohistochemical assay: 4-HNE (4-hydroxynonenal)

Immunohistochemical assays were performed using an immunoperoxidase protocol. The sections were dewaxed at 58 ℃ in a xylene bath (2X 3 min). Samples were hydrated with ethanol (100%, 95% and 70% continuous baths) for 3 minutes each and immersed in 1xPBS (2 × 5 minutes). Subsequently, endogenous peroxidase was blocked with H2O2 solution (0.3% H2O2 in distilled water) for 30 minutes, followed by three 5-minute washes in 1x PBS. In addition, heat-mediated antigen retrieval was performed with citrate buffer at pH 6.0 for 40 minutes at 95 ℃. To block non-specific binding, 1x PBS solution containing 3% normal goat serum and 0.1% Triton was added for 60 minutes. Subsequently, the tissue was incubated with 4-HNE primary antibody overnight at 4 ℃ and washed with 1 × PBS (3 × 5 min). The tissues were incubated with HRP secondary antibody for 1 hour at room temperature and then washed with 1x PBS (3 x 5 min). The slides were then exposed to peroxidase substrate 3,3' -Diaminobenzidine (DAB) for 15 minutes and washed with tap water. Finally, the stained sample was counterstained with Mayer hematoxylin for 3 minutes, washed with tap water (2 minutes), and the tissue was dehydrated in ethanol and xylene.

4-HNE IHC analysis:

histological examination and scoring was performed blindly. Images were acquired using a Pannoramic 250Flash II digital slide scanner (3 DHistech). And (3) scoring: seven randomly selected fields per slice were examined and analyzed in QuantCenter software. 4-HNE accumulation was calculated as 4-HNE positive area/total selection field area.

Intracellular GSH detection:

tizoxanide (interchem; cat # RP253) was dissolved in DMF (Sigma; cat # 227056). Menadione (Sigma; catalog No. M2518) and N-acetylcysteine (Sigma; catalog No. A9165) were dissolved in water.

Hep G2 cells (ECACC) were plated at a density of 20000 cells/well in 100. mu.l DMEM (Gibco; Cat. No. 41965-039) supplemented with 1% P/S (Gibco; Cat. No. 15140-122), 1% glutamine (Gibco; Cat. No. 25030-024) in 96-well microplates. The complete medium contained 10% SVF (Gibco; Cat. No. 10270-106). The next day, the medium was removed and replaced with 100. mu.L DMEM (Gibco; Cat. No. 31053-028), without phenol red and SVF but supplemented with 1% P/S (Gibco; Cat. No. 15140-122), 1% glutamine (Gibco; Cat. No. 25030-024). Serum deprived HepG2 was preincubated with TZ or N-acetylcysteine (NAC) for 1 hour, followed by exposure to Menadione (MND) for 2 hours. Compounds were dissolved in their respective above-mentioned media and diluted in the deprivation-type medium. Mu.l of 20 Xdilutions were added to the cells to reach final concentrations of TZ 1. mu. M, NAC 10mM and MND 100. mu.M, respectively. The cellular levels of reduced glutathione were subsequently monitored using ThiolTracker Violet dye (Invitrogen, cat # T10096). Briefly, cells were washed twice with DPBS (Invitrogen, Cat. No. 14287-080), then incubated at 37 ℃ for 30 minutes with 100. mu.l of a pre-warmed ThioTracker Violet dye solution prepared in DPBS according to the supplier's instructions, and fluorescence was measured (Ex: 404nm, Em: 526 nm).

ARE reporter System-HepG 2 cell line

ARE reporter system-HepG 2 cells (BPS Bioscience, inc., San Diego, catalog No. 60513) were cultured according to the manufacturer's instructions. After thawing (BPS thaw medium 1, catalog No. 60187), cells were cultured in growth medium (BPS growth medium 1K, catalog No. 79533) and subsequently plated at a density of 40000 cells/well in 45 μ l assay medium (BPS thaw medium) in 96-well microplates. TZ (Interchim; Cat. No. RP253) & DL-sulforaphane (Sigma; Cat. No. S4441) was dissolved in DMSO and diluted into assay medium. Mu.l of 10 Xdilution was added to the cells to give final concentrations of TZ and DL-sulforaphane (DL-sulfofo) of 1. mu.M and 3. mu.M, respectively. After 18 hours of exposure, luciferase activity was determined. Mu.l of One-Step luciferase assay system (BPS catalog No. 60690) was added to each well and after shaking at room temperature for-15 minutes, luminescence was measured using a luminometer.

Statistical analysis

The results of the experiment are expressed as mean ± SEM and plotted as a histogram. Statistical analysis was performed using Prism version 7 as follows:

in vivo data

CSAA and CDAA + 1% cholesterol groups were compared by student's t test (#: p < 0.05; #: p < 0.01; #: p <0.001) or by Mann-Whitney test ($: p < 0.05; $: p < 0.01; $: p < 0.001).

The NTZ-treated group was compared with the CDAA + 1% cholesterol feed group by student's t test (#: p < 0.05; #: p < 0.01; #: p <0.001) or by Mann-Whitney test ($: p < 0.05; $: p < 0.01; $: p < 0.001).

In vitro data

For the ARE luciferase reporter system assay, the treatment effect was compared to the medium effect by student's t-test (#: p < 0.05; #: p < 0.01; #: p < 0.001).

For intracellular GSH detection, menadione conditions were compared to unstimulated conditions by the Mann-Whitney test ($: p < 0.05; $: p < 0.01; $: p < 0.001). The treatment effects were compared to the medium effects by student's t test (#: p < 0.05; #: p < 0.01; #: p <0.001) or by Mann-Whitney test ($: p < 0.05; $: p < 0.01; $: p < 0.001).

Results

Oxidative stress is an important pathophysiological mechanism of NASH (Koruk, Taysi et al 2004; Masarone, Rosato et al 2018) and it is widely recognized that 4-HNE is an aldehyde peroxide product of unsaturated fatty acids, an indicator of oxidative stress (Takeuchi-Yolomoto, Noto et al 2013).

Thus, as shown in figure 1, significant increases in intrahepatic 4-HNE levels were observed in the CDAA/c group compared to CSAA feed, while significantly lower levels of 4-HNE in the group exposed to NTZ in parallel to the CDAA/c regimen, indicating a protective effect of NTZ on oxidative stress-induced lipid peroxidation.

To further study the antioxidant stress effect of NTZ, transcriptomics analysis was performed on liver samples. As shown in fig. 2, the levels of hepatic GSTA1 transcript (panel a) and GSTA2 (panel B) transcript were significantly induced in the CDAA/c group compared to the CSAA group, reflecting the implementation of antioxidant defense mechanisms. Interestingly, comparing the group receiving NTZ + CDAA/c with the CDAA/c protocol alone, significant induction of expression was observed for both enzymes, suggesting an improvement in the defense against oxidative stress. In fact, GSTA is well known as a detoxification enzyme, allowing elimination of HNE by binding to glutathione. The level of hepatic GSTA4 mRNA was also analyzed (fig. 3), as this enzyme is considered a key participant in HNE detoxification, and it has higher activity in binding HNE to GSH than other isoenzymes (Awasthi, Ramana et al 2017). As for the other GSTs, in comparing the CDAA/c group with the CSAA group, a significant induction of GSTA4 transcript levels was observed, and mice fed with the NTZ + CDAA/c regimen exhibited significantly higher GSTA4 levels compared to the CDAA/c group, supporting the discovery that NTZ promotes a defense mechanism against oxidative stress.

In mice and humans, GSTA1, GSTA2, and GSTA4 are described as genes positively regulated by Nrf2, Nrf2 being a key regulator of cellular redox status (Hayes and Dinkova-Kostova 2014). Thus, we compared the proportion of Nrf2 target gene observed in the sequenced whole transcriptome with the proportion of Nrf2 gene in the gene induced by NTZ treatment (obtained by comparison of NTZ + CDAA/c characteristics with CDAA/c conditions). Unexpectedly, significant enrichment of Nrf2 characteristics was induced by NTZ treatment (fig. 4). Although the Nrf2 target gene accounted for 1.3% of the identified full transcriptome in the RNA-seq data, Nrf2 regulated genes were found to account for greater than 8% of all differentially expressed genes in NTZ-treated mice.

To confirm the efficacy of NTZ to activate the Nrf2 antioxidant pathway at the functional level, Antioxidant Response Element (ARE) -mediated luciferase activity was measured in HepG2 cells treated with TZ (an active metabolite of NTZ). Under basal conditions, Nrf2 is retained in the cytoplasm by binding to the cytoskeletal protein Keap1, whereas when exposed to oxidative stress or other ARE activators, Nrf2 is released from Keap1 and translocates to the nucleus where it can bind to the ARE, resulting in expression of antioxidant and phase II enzymes that protect the cell from oxidative damage. As shown in fig. 5, TZ exposure resulted in a significant increase in ARE-mediated transcription, reflecting its ability to induce Nrf2 translocation to the nucleus and associated ARE signaling in human hepatocytes. Taken together, these data indicate that NTZ can induce a defense mechanism against oxidative stress, and that part of this potential mechanism is dependent on Nrf 2.

In complement to these analyses, we evaluated the effect of TZ on GSH (reduced glutathione) content, which is the most potent antioxidant in the liver, in hepatocytes exposed to the oxidative stress inducer Menadione (MND) (Al-Busafi, Bhat et Al 2012). As shown in fig. 6, MND exposure as expected caused a significant reduction in cellular GSH. TZ treatment completely prevented this MND-induced reduction. This result confirms the antioxidant properties of the active metabolite TZ of NTZ.

Taken together, all these results demonstrate that NTZ and/or its metabolite TZ may provide therapeutic benefit in oxidative stress related diseases.

Reference to the literature

Al-Busafi, S.A., M.Bhat et Al (2012), "Antioxidant therapy of non-alcoholic steatohepatitis in nonalcoholic steatohepatitis". Hepatitis research and treatment 2012: 947575-.

Awasthi, Y.C., K.V.ramana et al (2017). "Regulation roles of glutathione-S-transferase and 4-hydroxynonenal in stress-mediated signaling and toxicity" (regulation roles of glutathione-S-transferases and 4-hydroxynenal in stress-mediated signaling and toxicity) ".

Chord, b.n., m.r.campbell et al (2012), "identification of novel NRF2 regulatory genes by ChIP-Seq: effect on retinoid X receptor alpha (Identification of novel NRF2-regulated genes by ChIP-Seq: influen on dependent X receptor alpha). Nucleic Acids Res 40(15):7416-7429.

de Ara jo, r, d. martins et al (2016) oxidative stress and disease-from edited books: the major Regulator of Oxidative Stress-The Transcription Factor Nrf2(Oxidative Stress and Disease-from The edited volume: A Master Regulator of Oxidative Stress-The Transcription Factor Nrf2), edited by Jose antioxidant Morales-Gonzalez, antigen Morales-Gonzalez and Eduardo Osirisis major-Santillan.

de Carvalho, L.P.S., C.M.Darby et al (2011), "Nitazoxanide disrupts the membrane potential and intracellular pH homeostasis of M.tuberculosis (Nitazoxanide dis membrane potential and intracellular pH homeosis of Mycobacterium tuberculosis),. ACS Med.chem.Lett.2 (copyright (C)2015 American Chemical Society, ACS. retained all rights): 849. sup.854.

Di Santo, n. and j.ehrmim (2014), "functional view of nitazoxanide as a potential anticancer drug". mutat. res., fundam. mol. mech. mutagen.768 (copyright (C)2015 American Chemical Society (ACS). reserve all rights): 16-21.

Hayes, J.D. and A.T. Dinkova-Kostov (2014). "Nrf 2 regulatory network provides an interface between redox and intermediary metabolism (The Nrf2 regulatory network services an interface between redox and intermediary metabolism)," Trends Biochem Sci 39(4): 199-.

Hayes, j.d., and m.mcmahon (2009). "NRF 2 and KEAP1 mutations: permanently activate adaptive responses in cancer (NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer)'. Trends Biochem Sci 34(4): 176-.

Hoffman, P.S., G.Sisson et al (2007), "Antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductase of Helicobacter pylori, specific anaerobes and parasites as well as Campylobacter jejuni (Antiparasitic drug azoxanide inhibitors of Helicobacter pylori, selected anaerobic bacteria and parasitides, and Campylobacter jejuni)," Antimicrobe.Agents Chemother. 51 (copyright (C)2015, American society of chemistry (ACS). Retention all: 868- & 876.

Jung, K.A. and M.K.KWak (2010), "Nrf 2 system as a potential target for developing indirect antioxidants (The Nrf2 systems as a potential target for The development of index antioxidants), Molecules 15(10): 7266-.

Koruk, M., S.Taysi et al (2004). "Oxidative stress and enzymatic antioxidant status in patients with non-alcoholic steatohepatitis with tissues with nonalcoholic stephanistic pat.," an Clin Lab Sci 34(1):57-62.

Masarone, M., V.Rosato et al (2018). "Role of Oxidative Stress in the Pathophysiology of non-alcoholic Fatty Liver Disease" (Role of Oxidative Stress in Pathology of Nonalcoholic Fatty Liver Disease) ". Oxid Med Cell Longev 2018:9547613.

Rossignol, j. -F. (2014.) "nitazoxanide: a broad-spectrum Antiviral agent (Nitazoxanide: A first-in-class broad-spectrum Antiviral agent) was first created, Antiviral Res.110 (copyright (C)2015 American Chemical Society (ACS), all rights reserved) 94-103.

Rossignol, j.f. and r.cavier (1975) DE2438037a 1-2-benzamide-5-nitrothiazole (DE2438037a1-2-Benzamido-5-nitrothiazoles), s.p.r.l. Phavic, belg.11 pp.

Takeuchi-Yoimoto, A., T.Noto et al (2013). "Persistent fibrosis in the liver of Choline-deficient and iron-supplemented L-amino acid-defined feed-induced nonalcoholic steatohepatitis rats due to sustained oxidative stress after choline supplementation (Persistent fibrosis in the liver of Choline-deficient and iron-supplemented L-amino acid-defined diet-induced nonalcoholic steatohepatitis ratio from connecting oxidative stress after supplementation)".

Wuts, p.g.m. and t.w.greene (2007), Greene's Protective Groups in Organic Synthesis, Protective Groups in Organic Synthesis in greens, fourth edition, John Wiley & Sons.

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