WO2021016543A1

WO2021016543A1 - Antiviral combinations of thiazolide compounds

Info

Publication number: WO2021016543A1
Application number: PCT/US2020/043455
Authority: WO
Inventors: Jean-Francois Rossignol
Original assignee: Romark Laboratories L.C.
Priority date: 2019-07-25
Filing date: 2020-07-24
Publication date: 2021-01-28

Abstract

The present disclosure relates to treatment of hepatitis B virus infection, including treatment of chronic hepatitis B, with a thiazolide compound, which may be, for example, nitazoxanide, tizoxanide or a combination thereof, and at least one medicament selected from Tenofovir Disoproxil Fumarate (TDF), Tenofovir Alafenamide (TAF) and Entecavir (ETV).

Description

ANTIVIRAL COMBINATIONS OF THIAZOLIDE COMPOUNDS

RELATED APPLICATION

[0001] The present application claims priority to U.S. provisional application No.

62/878,518 filed July 25, 2019, which is incorporated by reference in its entirety.

FIELD

[0002] The present application generally relates to an antiviral field and more specifically for uses of a thiazolide compound, such as nitazoxanide, in combination with another antiviral compound against hepatitis B virus.

SUMMARY

[0003] One embodiment is a method of treating a chronic hepatitis B infection comprising administering to a subject in need a thereof an effective amount of a thiazolide compound, which is selected from the group consisting of nitazoxanide, tizoxanide or a combination thereof.

[0004] Another embodiment is a method of treating a hepatitis B virus infection comprising administering to a subject in need thereof an anti-hepatitis B effective amount of a) a thiazolide compound, which is selected from the group consisting of nitazoxanide (NTZ), tizoxanide (TIZ) or a combination thereof and b) at least one medicament selected from tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF) and entecavir (ETV).

FIGURES

[0005] FIG 1 reports moderately synergistic to additive interactions between NTZ and

TDF.

[0006] FIG. 2 reports moderately synergistic to antagonistic interactions between NTZ and ETV. [0007] FIG. 3 shows a design for the study of Example 2.

[0008] FIG. 4 shows a design for the study of Example 3.

DETAILED DESCRIPTION

[0009] Related Documents

[0010] The following documents, which are all incorporated by reference, may be useful for understanding the present disclosure: U.S. patents nos. 9,351,937; 9,126,992; 9,107,913;

9,023,877; 8,895,752; 8,846,727; 8,772,502; 8,633,230; 8, 524, 278;8, 124,632; 7,645,783;

7,550,493; 7,285,567; 6,117,894; 6,020,353; 5,968,961; 5,965,590; 5,935,591; 5,886,013;

5,859,038; 5,856,348; 5,387,598; U.S. patent application publications nos. 2015-025768; 2014- 0065215; 2012-0294831; 2016-0243087; PCT publication no. W02016077420; J. Biol. Chem., 2009 Oct 23; 284(43): 29798-29808; Antiviral Research, 110(2014): 94-103; Biochim

Biophys Acta., 2003 Jul ll;1614(l):73-84.

[0011] Definition of Terms

[0012] Unless otherwise specified,“a” or“an” means“one or more.”

[0013] AFP stands for Alpha-Fetoprotein.

[0014] ALT stands for Alanine Aminotransferase.

[0015] AST stands for Aspartate Aminotransferase.

[0016] AUC stands for the area under the curve, which is the definite integral in a plot of drug concentration in blood plasma vs. time.

[0017] BID stands for Twice Daily.

[0018] BUN stands for Blood Urea Nitrogen. [0019] cccDNA stands for Covalently Closed Circular Viral DNA.

[0020] CHB stands for Chronic Hepatitis B.

[0021] Cmax is a maximum concentration that a drug achieves in a specified compartment or test area of the body after the drug has been administrated.

[0022] CRF stands for Case Report Form.

[0023] CTP stands for Child-Turcotte-Pugh.

[0024] Ctrough is a trough concentration, i.e. the lowest concentration reached by a drug before the next dose is administered.

[0025] DNA stands for Deoxyribonucleic Acid.

[0026] EDC stands for Electronic Data Collection.

[0027] ETV stands for Entecavir.

[0028] GCP stands for Good Clinical Practice.

[0029] GGT stands for Gamma Glutamyl Transferase.

[0030] HBcAg stands for HBV Core Antigen.

[0031] HBeAg stands for Hepatitis B e Antigen.

[0032] HBsAg stands for Hepatitis B Surface Antigen.

[0033] HBV stands for Hepatitis B Virus.

[0034] HCC stands for Hepatocellular Carcinoma.

[0035] HCV stands for Hepatitis C Virus. [0036] HDL stands for High-Density Lipoprotein.

[0037] HDV stands for Hepatitis D Virus.

[0038] HTV stands for Human Immunodeficiency Virus.

[0039] ICF stands for Informed Consent Form.

[0040] ICH stands for International Conference on Harmonization.

[0041] IgM stands for Immunoglobulin M.

[0042] INR stands for International Normalized Ratio.

[0043] IRB stands for Institutional Review Board.

LMV stands for Lamivudine.

[0045] NTZ stands for Nitazoxanide.

[0046] PT stands for Prothrombin Time.

[0047] QD stands for Once Daily.

[0048] SAE stands for Serious Adverse Events.

[0049] TAF stands for Tenofovir Alafenamide.

[0050] TDF stands for Tenofovir Disoproxil Fumarate.

[0051] TIZ stands for tizoxanide.

[0052] Disclosure

[0053] A thiazolide compound, such as nitazoxanide or tizoxanide, may be used for treating hepatitis B virus (HBV) infection, including for treating chronic hepatitis B. [0054] The thiazolide compound may be nitazoxanide (1, see formula below) or a pharmaceutically acceptable salt thereof. Nitazoxanide is approved by the U.S. Food Drug

Administration for the treatment of diarrhea caused by Giardia lamblia or Cryptosporidium parvum. Nitazoxanide may be commercially available as ALINIA^® for Oral Suspension and ALINIA Tablets

[0055] In some embodiments, the thiazolide compound may be tizoxanide or its pharmaceutically acceptable salt, also shown below. Tizoxanide (2, see formula below) is an active metabolite of nitazoxanide.

O

(NTZ, 1) TIZ, 2)

[0056] In some embodiments, nitazoxanide and tizoxanide may be used together as a combination.

[0057] Thiazolide compounds may be synthesized, for example, according to published procedures of U.S. patents nos. 3,950,351 and 6,020,353, PCT W02006042195A1 and US2009/0036467A. Other suitable thiazolide compounds are disclosed in U.S. Pat. Nos. 7,645,783, 7,550,493, 7,285,567, 6,117,894, 6,020,353, 5,968,961, 5,965,590, 5,935,591, and

5,886,013.

[0058] In some embodiments, the thiazolide compound may be administered in a daily dose ranging from 600 mg to 1800 mg. For example, the daily dose may be about 600 mg or about 1200 mg or about 1800 mg. The daily dose may be administered in one, two, three, four or more administering events.

[0059] In some embodiments, the thiazolide compound may be administered orally. In some embodiments, the thiazolide compound may be administered as a solid oral formulation.

[0061] Yet in some embodiments, the thiazolide compound may be administered as a liquid oral formulation.

[0062] In some embodiments, the thiazolide compound may be administered orally as an extended release solid formulation such as the ones disclosed in U.S. patents Nos. 8,524,278; 9,351,937; 9,827,227 as well US patent application publications Nos. 2010-0209505; 2014- 0065215; 2016-0243087; 2018-0085353, each of which is incorporated herein by reference in its entirety. For example, in some embodiments, the thiazolide compound may be administered orally in the form of a solid oral dosage form comprising: (a) a first portion comprising a first quantity of the thiazolide compound, such as nitazoxanide, tizoxanide or a combination thereof, in a controlled release formulation; and (b) a second portion comprising a second quantity of the thiazolide compound, such as nitazoxanide, tizoxanide or a combination thereof, in an immediate release formulation. Such the solid oral dosage form may be, for example, a bilayer tablet comprising: (a) a first layer comprising a first quantity of the thiazolide compound in a controlled release formulation; and (b) a second layer comprising a second quantity of the thiazolide compound in an immediate release formulation.

[0063] In many embodiments, a thiazolide compound such as nitazoxanide, tizoxanide or a combination thereof may be used for treating a chronic HB V infection.

[0064] In certain embodiments, a thiazolide compound such as nitazoxanide, tizoxanide or a combination thereof may be used for treating a chronic HBV infection in an HBeAg- negative chronic hepatitis B patient. For example, a patient may be HBeAg-negative with undetectable HBV DNA or low levels of HBV DNA in serum after treatment with an anti-HBV medicament, which may be tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF) or entecavir (ETV). [0065] Yet in certain other embodiments, a thiazolide compound, such as nitazoxanide, tizoxanide or a combination thereof, alone or in combination with an anti-HBV medicament, which may be TDF, TAF or ETV, may be used for treating a chronic HBV infection in an HBeAg-positive chronic hepatitis B patient.

[0066] For example, a patient may be HBeAg-positive with HBV DNA in serum after treatment with an anti-HBV medicament, which may be TDF, TAF or ETV. Alternatively, a patient may be HBeAg-positive with HBV DNA in serum having never been treated with an anti-HBV medicament. The treatment of an HBeAg-positive patient with a thiazolide compound, alone or in combination with another anti-HBV medicament, such as TAF, TDF or ETV, may achieve at least one of the following outcomes: loss of HBeAg in serum, reduction of HBV DNA, improvement of inflammation or necrosis of the liver and/or loss of HBsAg.

[0067] During the initial period of administration of the thiazolide compound, such as nitazoxanide and/or tizoxanide, the patient may be also administered with at least one additional medicament such as TDF, TAF or ETV. For example, if a patient achieved an HBeAg-negative status with low or undetectable serum HBV DNA after treatment with TDF, then TDF may also administered to the patient during the initial period of administration of the thiazolide compound. If a patient achieved an HBeAg-negative status with low or undetectable serum HBV DNA after treatment with TAF, then TAF may also be administered to the patient during the initial period of administration of the thiazolide compound. If a patient achieved an HBeAg-negative status with low or undetectable serum HBV DNA after treatment with ETV, ETV may also be administered to the patient during the initial period of administration of the thiazolide compound.

[0068] The additional medicament, such as TDF, TAF or ETV, may be administered in an effective amount approved by a regulatory authority, such as the United States Food and Drug Administration, for treating HBV infection, including for treating chronic hepatitis B. The additional medicament, such as TDF, TAF or ETV, may be administered separately,

sequentially, or simultaneously with the thiazolide compound. [0069] TDF is approved by the FDA for treating chronic hepatitis B. TDF may be available in the following formulations: ISO mg TDF tablet, which is equivalent to 123 mg

Tenofovir Disoproxil (TD); 200 mg TDF tablet, which is equivalent to 163 mg TD; 250 mg TDF tablet, which is equivalent to 204 mg TD; 300 mg TDF tablet, which is equivalent to 245 mg TD; an oral powder, which contains 40 mg TDF (33mg TD) per 1 g of powders. The FDA

recommended oral dose may be 8mg TDF/kg of body weight (up to 300 mg TDF) once daily administered as oral powder or tablet without regard to food. For oral powder in pediatric patients at least 2 years of age, FDA recommended daily dosage ranges from 2 g to 7.5 g of powder (80 mg to 300 mg TDF). FDA recommended TDF dosage for adult patients and pediatric patients at least 12 years of age (35 kg or more) may be 300 mg once daily. This dosage may be reduced for adults with renal impairments, such as adults with creatinine clearance levels of lower than 50 mL/min. For example, for adults with creatinine clearance from 30 to 49 mL/min, 300 mg TDF may be administered only every 48 hours; for adults with creatinine clearance of from 10 to 29 mL/min, 300 mg TDF may be administered only every 72 to 96 hours; for hemodialysis patients, 300 mg TDF may be administered every 7 days or after a total of approximately 12 hours of dialysis.

[0070] TAF is approved by the FDA for treating chronic hepatitis B virus infection. TAF may be available as a 25 mg tablet of tenofovir alafenamide, which is equivalent to 28 mg of TAF. The FDA recommended dosage may taking the 25 mg tablet once daily, preferably with food.

[0071] Entecavir is approved by the FDA for treating chronic hepatitis B infection.

Entacavir may be available as the following formulations: oral solution containing 0.05 mg of entecavir per milliliter; 0.5 mg tablet; 1.0 mg tablet. The FDA recommended dose of entecavir is 0.5 mg once daily for nucleoside-treatment-nai^'ve patients at least 16 years of age with compensated liver disease and 1 mg once daily for patients at least 16 years of age with decompensated liver disease or a history of hepatitis B viremia while receiving lamivudine or known lamivudine or telbivudine resistance mutations. This dosage may be reduced for adults with renal impairments, such as adults with creatinine clearance levels of lower than 50 mL/min. For example, for adults with creatinine clearance from 30 to 49 mL/min, the usual daily dose of ETV may be cut in half or the usual daily dose may be administered once every 48 hours. For adults with creatinine clearance of from 10 to 29 mL/min, the usual daily dose may be reduced to 30% of the usual dose or the usual daily dose may be administered every 72 hours. For hemodialysis patients or patients with creatinine clearance < 10 mL/min, the usual daily dose may be reduced to 10% of the usual dose or the usual daily dose may be administered every 7 days. For oral solution, FDA recommended daily dosage in pediatric patients at least 2 years of age and weighing at least 10 kg ranges from 3 to 20 mL of solution depending on weight and prior treatment experience.

[0072] The thiazolide compound, such as nitazoxanide, tizoxanide or a combination thereof, alone or in combination with at least one additional medicament, such as TDF, TAF or ETV, may be administered for at least 12 weeks, at least 24 weeks, at least 48 weeks, at least 72 weeks, at least 96 weeks and/or until the patient has no detectable HBsAg in serum.

[0073] In some embodiments, a patient may have prior to administering of the thiazolide compound a serum of HBsAg level greater than 100 IU/mL and administering the thiazolide compound, such as nitazoxanide, tizoxanide or a combination thereof, alone or in combination with at least one additional medicament, such as TDF, TAF or ETV, may reduce the serum HBsAg level in the patient or suppress the serum HBsAg level in the patient below a detectable level while also suppressing serum HBV DNA below a detectable level. For example, an HBeAg-negative chronic hepatitis B patient may have prior to administering of the thiazolide compound a serum of HBsAg level greater than 100 IU/mL and administering the thiazolide compound, such as nitazoxanide, tizoxanide or a combination thereof, alone or in combination with at least one additional medicament, such as TDF, TAF or ETV, may reduce the serum HBsAg level in the patient or suppress the serum HBsAg level in the patient below a detectable level while also suppressing serum HBV DNA below a detectable level. Quantitative serum HBsAg levels may be determined, for example, by a chemiluminescent microparticle

immunoassay such as the ARCHITECT HBsAg QT assay by Abbott Laboratories (lower limit of detection = 0.05 IU/mL) or the Elecsys^® HBsAg P quant P assay by Roche Diagnostics (lower limit of detection = 0.1 IU/mL). The detection of HBsAg may also be determined by conventional enzyme immunoassay using 96 well plates such as the Monalisa HBsAg ULTRA assay by Bio-Rad Laboratories or the EΊΊ-MAK-4 assay by DiaSorin. Quantitative HBV DNA levels may be determined by a Real-Time Polymerase Chain Reaction (RT-PCR) technique such as the cobas^® HBV test for use on the cobas^® 6800/8800 Systems by Roche Diagnostics or the Abbott RealTime HBV Viral Load assay by Abbott Molecular, Inc. The reduction of the serum HBsAg level or suppression of the serum HBsAg level may be statistically significant compared to that observed when patients are administered a placebo.

[0074] If the serum HBsAg and HBV DNA levels are suppressed in the patient below detectable levels, the thiazolide compound may be administered to the patient alone or in combination with at least one additional medicament such as TDF, TAF or ETV for an additional period to ensure the levels of suppression are maintained. Such additional period may be at least 12 weeks, at least 24 weeks, at least 36 weeks or at least 48 weeks.

[0075] In some embodiments, administering of the thiazolide compound, such as nitazoxanide, tizoxanide or a combination thereof, alone or in combination with at least one additional medicament, such as TDF, TAF or ETV, may result in one or more of the following effects in a patient:

[0076] a) suppression of HBV DNA in the patient. In some embodiments, such suppression may be accompanied with an HBeAg loss, i.e., with a serum HBeAg level in the patient being below the detectable level. In some embodiments, such suppression of HBV DNA may be sustained over a period of time after the end of administering of thiazolide compound. Such period of time may be for example, 24 weeks. The sustained suppression may be accompanied with an HBsAg loss, i.e. with a serum HBsAg level in the patient being below a detectable level.

[0077] b) an HBeAg loss; [0078] c) HBeAg seroconversion, which is determined by the presence of antibodies to HBeAg using, for example, an enzyme linked immunosorbent assay (ELISA) such as the CD Anti-HBeAb ELISA kit by Creative Diagnostics or by electrochemiluminescence immunoassay such as the Elecsys^® Anti-HBe assay by Roche Diagnostics or by chemiluminescent

microparticle immunoassay such as the ARCHITECT Anti-HBe assay

[0079] d) an HBsAg loss;

[0080] e) HBsAg seroconversion, which is determined by the presence of antibodies to HBsAg using, for example, an enzyme linked immunosorbent assay (ELISA) such as the CD Anti-HBs ELISA (Quantitative) kit by Creative Diagnostics or by electrochemiluminescence immunoassay such as the Elecsys^® Anti-HBs P assay by Roche Diagnostics or by

chemiluminescent microparticle immunoassay such as the ARCHITECT Anti-HBs assay;

[0081] f) clinical remission of liver disease, which may be determined by normal serum biochemical tests for liver function including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) or the absence of symptoms such as jaundice, dark urine, abdominal pain and swelling, nausea, vomiting and fatigue; or

[0082] g) an improvement in a fibrosis status of the patient, which may be determined by microscopic examination of tissue samples collected during liver biopsy or via a change in one or more fibrosis scores, such as Fibrosis 4 score or Fibroscan^® test.

[0083] Such effect(s) may be statistically significant compared to that observed when patients are administered a placebo.

[0084] Fibrosis 4 score is disclosed, for example, in Kim et al. Liver Int. 2010

Apr;30(4):546-53 and Shah et al. Clin Gastroenterol Hepatol. 2009 Oct;7(10): 1104-12.

Fibroscan^® test is disclosed, for example, in Ganne-Carrie N; Ziol M; de Ledinghen V; et al.

[2006] Hepatology. 44 (6): 1511-7 and Jung, Kyu Sik; Kim, Seumg Up (2012) Clinical and Molecular Hepatology. 18 (2): 163-73. [0085] In some embodiments, a thiazolide compound, such nitazoxanide, tizoxanide or a mixture thereof, may be administered as a salt, such as a pharmaceutically acceptable salt.

[0086] When a thiazolide compound, such as a nitazoxanide, tizoxanide or a combination thereof, is administered together with TDF or TAF, a plasma concentration, such as Cmax, Ctrough and/or AUC, of tizoxanide in the subject may be at least 3% or at least 4% or at least 5% or at least 6% or at least 8% or at least 9% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 40% or at least 50% or at least 100% or at least 200% or at least 300% or at least 400% or at least 500% or at least 600% or at least 700% or at least 800% or at least 900% or at least 1000% of a respective plasma concentration, such as Cmax, Ctrough and/or AUC, of tenofovir.

[0087] When a thiazolide compound, such as a nitazoxanide, tizoxanide or a combination thereof, is administered together with ETV, a plasma concentration, such as Cmax, Ctrough and/or AUC, of tizoxanide in the subject may be at least 200% or at least 300% or at least 400% or at least 500% or at least 600% or at least 700% or at least 800% or at least 900% or at least 1000% or at least 2000% or at least 3000% or at least 5000% or at least 10000% or at least 20000% or at least 30000% or at least 40000% or at least 50000% or at least 60000% or at least 70000% or at least 80000% or at least 90000% or at least 100000% of a respective plasma concentration, such as Cmax, Ctrough and/or AUC, of entecavir.

[0088] The term“salt” may be used in its broadest sense. For example, the term“salt” includes hydrogen salts and hydroxide salts with ions of the present compound. In some embodiments, the term salt may be a subclass referred to as pharmaceutically acceptable salts, which are salts of the present compounds having a pharmacological activity and which are neither biologically nor otherwise undesirable. In all embodiments, the salts can be formed with acids, such as, without limitation, hydrogen, halides, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate,

glucoheptanoate, glycero-phosphate, hemi sulfate, heptanoate, hexanoate, hydrochloride hydrobromide, hydroiodide, 2-hydroxyethane sulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, thiocyanate, tosylate, and imdecanoate. In all embodiments, the salts can be formed with bases, such as, without limitation, hydroxide, ammonium salts, alkali metal salts such as lithium, sodium and potassium salts, alkaline earth metal salts such as calcium, magnesium salts, aluminum salts, salts with organic bases such as ammonia, methylamine, diethylamine, ethanolamine, dicyclohexylamine, N-methylmorpholine, N-methyl-D-glucamine, and salts with amino acids such as arginine and lysine. Basic nitrogen- containing groups can be quartemized with agents including lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aralkyl halides such as benzyl and phenethyl bromides.

[0089] The terms“therapeutically acceptable salt,” and“pharmaceutically acceptable salt,” as used herein, represent both salts and zwitterionic forms of the compounds of the present invention which are water or oil-soluble or dispersible; which are suitable for treatment of diseases without undue toxicity, irritation, and allergic response; which are commensurate with a reasonable benefit/risk ratio; and which are effective for their intended use. The salts can be prepared during the final isolation and purification of the compounds or separately by reacting the appropriate compound in the form of the free base with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, L-ascorbate, aspartate, benzoate, benzene sulfonate (besylate), bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate, hemi sulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethane sulfonate (isethionate), lactate, maleate, malonate, DL-mandelate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, phosphonate, picrate, pivalate, propionate, pyroglutamate, succinate, sulfonate, tartrate, L-tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate,

bicarbonate, para-toluenesulfonate (p-tosylate), and imdecanoate. Also, basic groups in the compounds of the present invention can be quatemized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides.

Examples of acids which can be employed to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric. Salts can also be formed by coordination of the compounds with an alkali metal or alkaline earth ion. Hence, the present invention contemplates sodium, potassium, magnesium, and calcium salts of the compounds of the compounds of the present invention and the like.

[0090] Basic addition salts can be prepared during the final isolation and purification of the compounds by reacting a carboxyl, phenol or similar group with a suitable base such as a metal hydroxide, carbonate, or bicarbonate, or with ammonia or an organic primary, secondary, or tertiary amine. The cations of therapeutically acceptable salts include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as nontoxic quaternary amine cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N- dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, andN,N’- dibenzyl ethyl enediamine. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.

[0091] In some embodiments, the thiazolide compound may be administered as a part of a pharmaceutical composition. The pharmaceutical composition may include in addition to the thiazolide compound may include a carrier, such as a pharmaceutically acceptable carrier. The term“carrier” may be used in its broadest sense. For example, the term“carrier” refers to any carriers, diluents, excipients, wetting agents, buffering agents, suspending agents, lubricating agents, adjuvants, vehicles, delivery systems, emulsifiers, disintegrants, absorbents,

preservatives, surfactants, colorants, flavorants, and sweeteners. In some embodiments, the carrier may be a pharmaceutically acceptable carrier, a term narrower than carrier, because the term pharmaceutically acceptable carrier” means a non-toxic that would be suitable for use in a pharmaceutical composition. Actual dosage levels of active ingredients in the pharmaceutical compositions may vary so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient.

[0092] The pharmaceutical compositions may be administered systemically, for example, in an oral formulation, such as a solid oral formulation. For example, it may be in the physical form of a powder, tablet, capsule, lozenge, gel, solution, suspension, syrup, or the like. In some embodiments, the pharmaceutical composition may be in a form of a formulation disclosed in U.S. patents nos. 8,524,278 and 9,351,937. Such formulation may, for example, include a controlled release portion, which includes a thiazolide compound, such as nitazoxanide and/or tizoxanide; and an immediate release portion, which contains a thiazolide compound, such as nitazoxanide and/or tizoxanide. These compositions may be administered in a single dose or in multiple doses which are administered at different times.

[0093] In some embodiments, the total amount of a thiazolide compound, such as nitazoxanide and/or tizoxanide, in the composition may be about 60% to 75% by weight of the composition. The composition may be formulated for immediate release, controlled release or sustained release. The compositions may contain one or more additional pharmaceutically acceptable additives or excipients. These excipients are therapeutically inert ingredients that are well known and appreciated in the art. As used herein, the term“inert ingredient” may refer to those therapeutically inert ingredients that are well known in the art of pharmaceutical manufacturing, which can be used singly or in various combinations, and include, for example, diluents, disintegrants, binders, suspending agents, glidants, lubricants, fillers, coating agents, solubilizing agent, sweetening agents, coloring agents, flavoring agents, and antioxidants. See, for example, Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa.

[0094] Examples of diluents or fillers include, but are not limited to, starch, lactose, xylitol, sorbitol, confectioner’s sugar, compressible sugar, dextrates, dextrin, dextrose, fructose, lactitol, mannitol, sucrose, talc, microcrystalline cellulose, calcium carbonate, calcium phosphate dibasic or tribasic, dicalcium phosphaste dehydrate, calcium sulfate, and the like. The amount of diluents or fillers may be in a range between about 2% to about 15% by weight of the entire composition.

[0095] Examples of disintegrants include, but are not limited to, alginic acid, methacrylic acid DVB, cross-linked PVP, microcrystalline cellulose, sodium croscarmellose, crospovidone, polacrilin potassium, sodium starch glycolate, starch, including com or maize starch,

pregelatinized starch and the like. Disintegrant(s) typically represent about 2% to about 15% by weight of the entire composition.

[0096] Examples of binders include, but are not limited to, starches such as potato starch, wheat starch, com starch; microcrystalline cellulose; celluloses such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose (HPMC), ethyl cellulose, sodium carboxy methyl cellulose; natural gums like acacia, alginic acid, guar gum; liquid glucose, dextrin, povidone, syrup, polyethylene oxide, polyvinyl pyrrolidone, poly-N-vinyl amide, polyethylene glycol, gelatin, poly propylene glycol, tragacanth, and the like. The amount of binders) is about 0.2% to about 14% by weight of the entire composition.

[0097] Examples of glidants include, but are not limited to, silicon dioxide, colloidal anhydrous silica, magnesium trisilicate, tribasic calcium phosphate, calcium silicate, magnesium silicate, colloidal silicon dioxide, powdered cellulose, starch, talc, and the like. The amount of glidant(s) is about 0.01% to about 0.3% by weight of the entire composition.

[0098] Examples of lubricants include, but are not limited to, magnesium stearate, aluminum stearate, calcium stearate, zinc stearate, stearic acid, polyethylene glycol, glyceryl behenate, mineral oil, sodium stearyl fumarate, talc, hydrogenated vegetable oil and the like. The amount of lubricant(s) is about 0.2% to about 1.0% by weight of the entire composition.

[0099] The compositions may contain a binder that is a low-viscosity polymer.

Examples of low-viscosity polymers include, but are not limited to, low-viscosity hydroxypropyl methylcellulose polymers such as those sold by Dow Chemical under the tradename “MethoceL™” (e.g., Methocel E50LV™, Methocel K100LVR™, and Methocel F50LVR™) and low-viscosity hydroxyethylcellulose polymers. The low-viscosity polymer is typically present at about 10% to about 20%, or about 10% to about 15%, or preferably about 12%, of the total weight of the entire composition, or, in those embodiments having controlled release and immediate release portions, the low-viscosity polymer in the controlled release portion is typically present at about 15% to about 20%, preferably about 18%, of the weight of the controlled release portion.

[0100] The compositions may further comprise a coating material. The coating material is typically present as an outer layer on the dosage form that completely covers the formulation. For example, in some embodiments, the dosage form is an oral tablet in which the controlled release portion forms a first layer of the tablet and the immediate release portion forms a second layer that is deposited on top of the first layer to form a core tablet. In such embodiments, e.g., the coating material can be in the form of an outer coating layer that is deposited on top of the core tablet. The coating material typically is about 1% to about 5% by weight of the

composition, and may comprise hydroxypropylmethylcellulose and/or polyethylene glycol, and one or more excipients selected from the group comprising coating agents, opacifiers, taste- masking agents, fillers, polishing agents, coloring agents, antitacking agents and the like.

Examples of film-coating substances and methods for using such coating substances are well known to those of skill in the art.

[0101] In some embodiments, the thiazolide compound, such as nitazoxanide and/or tizoxanide, may be administered in a pharmaceutical formulation that contains both the thiazolide compound, such as nitazoxanide and/or tizoxanide, and another anti-HBV

medicament, such as TDF, TAF or ETV. Such pharmaceutical formulation may be, for example, an oral formulation, such as a capsule or a tablet.

[OlOZj When a pharmaceutical formulation contains a thiazolide compound, such as a nitazoxanide, tizoxanide or a combination thereof, together with TDF or TAF, when administered to a subject, such as a human being, the formulation may provide a plasma concentration, such as Cmax, Ctrough and/or AUC, of tizoxanide being at least 3% or at least 4% or at least 5% or at least 6% or at least 8% or at least 9% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 40% or at least 50% or at least 100% or at least 200% or at least 300% or at least 400% or at least 500% or at least 600% or at least 700% or at least 800% or at least 900% or at least 1000% of a respective plasma concentration, such as Cmax, Ctrough and/or AUC, of tenofir.

[0103] When a pharmaceutical formulation contains a thiazolide compound, such as a nitazoxanide, tizoxanide or a combination thereof, together with ETV, when administered to a subject, such as human being, the formulation may provide a plasma concentration, such as Cmax, Ctrough and/or AUC, of tizoxanide being at least 200% or at least 300% or at least 400% or at least 500% or at least 600% or at least 700% or at least 800% or at least 900% or at least 1000% or at least 2000% or at least 3000% or at least 5000% or at least 10000% or at least 20000% or at least 30000% or at least 40000% or at least 50000% or at least 60000% or at least 70000% or at least 80000% or at least 90000% or at least 100000% of a respective plasma concentration, such as Cmax, Ctrough and/or AUC, of entecavir.

[0104] Embodiments described herein are further illustrated by, though in no way limited to, the following working examples.

EXAMPLE 1

[0105] Studies to Evaluate Antiviral Activity of Combinations of Nitazoxanide with

Tenofovir Disoproxil Fumarate or Entecavir

Summary

[0106] In this study, the antiviral activity of nitazoxanide (NTZ) in combination with tenofovir disoproxil fumarate (TDF) or entecavir (ETV) in inhibiting replication of HBV in cell cultures was evaluated. Synergistic interactions were observed between NTZ and TDF and between NTZ and ETV.

Introduction

[0107] The present studies evaluate the activity of NTZ in combination with TDF or ETV in inhibiting replication of HBV in cell cultures.

Materials and Methods

[0108] Materials. TDF and ETV were purchased from Moraveck Biochemicals, Inc. (La

Brea, CA, USA). NTZ was provided by Romark Laboratories, L.C. (Tampa, FL, USA).

[0109] Antiviral assays. HBV antiviral assays were conducted as previous described.2 Briefly, confluent cultures of 2.2.15 cells were maintained on 96-well flat-bottomed tissue culture plates (confluence in this culture system is required for active, high levels of HBV replication equivalent to that observed in chronically-infected individuals.2,3 Cultures were treated with nine consecutive daily doses of the test compounds. HBV DNA levels were assessed by quantitative dot blot hybridization 24 h after the last treatment.

[0110] Combination drug treatments for were performed as previously described.4,5 For these assays, the relative ratio of the two drugs in combination were held constant through a dilution series. The first ratios used (1 :3 NTZ/TDF and 10:1 NTZ/ETV) were based on delivering an equipotent (not equimolar) dose. Then combinations with decreasing ratios of NTZ (1:10 and 1 :30 for NTZ/TDF; 3 : 1 and 1 : 1 for NTZ/ETV) were tested to evaluate antiviral activity across a range of ratios. Evaluation of drug interactions in the combination treatments was conducted against the corresponding monotherapies in the same experiments using

Calcusyn^® (Biosoft, Inc., Cambridge, UK).

[0111] The antiviral studies utilized dilution steps ranging from as little as 2-fold to as much as 10-fold, comprised of 4-8 titration points, with 3-6 replicates each. Calculations of potency were based on data from two or more evaluations. EC₅₀ and EC₉₀ values were calculated by linear regression analysis (MS EXCEL®, QuattroPro^®) using data combined from all treated cultures.^2,6 Standard deviations for EC₅₀ and EC₉₀ values were calculated from the standard errors generated by the regression analyses. EC₅₀ and EC₉₀ are drug concentrations at which a 2-fold, or a 10-fold depression of intracellular HBV DNA (relative to the average levels in untreated cultures), respectively, was observed. For combination treatments, EC₅₀ and EC₉₀ are presented for the first compound listed. The molar ratio of the compounds in each combination is also indicated.

RESULTS

[0112] EC

_50s and EC_90s for NTZ, TDF and ETV alone are presented in Table 1. Results of combination studies are presented in Figures 1 and 2.

Table 1: Potency (mM) of NTZ, TDF and ETV against HBV Replication

[0113] In Figures 1 and 2, analysis of combination therapies was performed using

Calcusyn™ software (Biosoft, Inc., Cambridge, UK). Two types of evaluations are presented.

The left-hand panels present CI-Fa (Combination Index-Fraction (of virus) affected) plots). For these plots, a combination index [Cl] greater than 1.0 indicates antagonism and a Cl less than 1.0 indicates synergism. Evaluations of synergy, additivity (summation), or antagonism at different levels of virus inhibition (e.g. 5% (Fa=0.05) to 99% (Fa=0.99)) are provided by the plotted lines and points. Dotted lines denoting 1.96 standard deviations for significance evaluations can be added but are not included in this example for clarity of presentation. The right-hand panels present conservative isobolograms. For these plots, ED₅₀, ED75, and ED₉₀ (50%, 75%, and 90% effective antiviral dose) values for the combination treatments are displayed as single points. Three lines radiating out from the axes denote the expected (e.g. additive) EDC₅₀, EDC75, and EDC₉₀ values for drug combinations as calculated from the monotherapies. ED₅₀, ED₇₅, and ED₉₀ values for the combinations that plot to the left (e.g. less than) of the corresponding lines indicate synergy, and values plotting to the right (e.g. greater than) of the corresponding lines indicate antagonism.

CONCLUSION

[0114] Moderately synergistic interactions were observed between NTZ and TDF at concentration ratios of 1 :3 and 1:10 NTZ:TDF. When the NTZ concentration was decreased further so that the ratio of NTZ:TDF was 1 :30, the interaction was no longer synergistic, but simply additive in effect. A moderately synergistic interaction was observed between NTZ and ETV tested in combination at a ratio of 10:1 NTZ:ETV. As the ratio of NTZ:ETV declined, however, less favorable interactions were observed. At a ratio of 3:1 NTZ:ETV, the interaction was moderately synergistic to antagonistic, and at a ratio of 1:1 NTZ:ETV, the interaction between the two drugs was antagonistic.

REFERENCES

1. Korba et al Antivir Res. 2008; 77 : 56-63.

2. Korba et al Antivir Res. 1992; 19:55-70.

3. Sells et al J Virol 1988; 62:2836-2844.

4. Korba, BE. Antivir Res. 1996; 29:49-51.

5. Iyer et al Antivir Agents Chemother. 2004; 48:2199-2205.

6. Okuse et al Antivir Res. 2005; 65 :23-34. EXAMPLE 2

[0115] RANDOMIZED DOUBLE-BLIND STUDY OF NITAZOXANIDE

COMPARED TO PLACEBO IN SUBJECTS WITH HB e Ag-NEGATIVE CHRONIC

HEPATITIS B VIROLOGICALLY SUPPRESSED FOR AT LEAST TWELVE MONTHS ON TENOFOVIR DISOPROXIL FUMARATE, TENOFOVIR ALAFENAMIDE OR

ENTECAVIR

STUDY SYNOPSIS

[0116] Title: Randomized Double-Blind Study of Nitazoxanide (NTZ) Compared to

Placebo in Subjects with HBeAg-negative Chronic Hepatitis B (CHB) Virologically Suppressed for at Least Twelve Months on Tenofovir Disoproxil Fumarate (TDF), Tenofovir Alafenamide (TAF) or Entecavir (ETV)

[0117] Indication: Treatment of HBeAg-negative CHB

[0118] Design: Randomized double-blind placebo-controlled dose-range-finding trial

[0119] Number of Patients : 48

[0120] Population: Adults (³21 years) with HBeAg-negative CHB virologically suppressed for at least 12 months on TDF, TAF or ETV

[0121] Randomization: 1:1:1:1 (12 patients per group)

[0122] Study Dose and Administration:

[0123] Group 1: Placebo

[0124] Group 2: 600 mg NTZ once daily

[0125] Group 3: 600 mg NTZ twice daily [0126] Group 4: 900 mg NTZ twice daily

[0127] All subjects will continue TDF, TAF or ETV therapy throughout the study.

[0128] NTZ 300 mg extended release tablets and matching placebo tablets will be administered in double-blind fashion orally with food.

[0129] After 12 weeks, subjects will continue treatment with the same treatment regimen if they have achieved >0.5 logio decline in qHBsAg from baseline to week 12. Stopping rules are provided based upon HBsAg response.

[0130] Objectives: To evaluate safety, effectiveness, and pharmacokinetic- pharmacodynamic (PK/PD) relationships associated with three different NTZ treatment regimens added to TDF, TAF or ETV in treating CHB.

[0131] Primary Efficacy Parameter: Mean change in qHBsAg from baseline to week 12

[0132] Secondary Efficacy Parameters:

[0133] 1. Sustained HBV DNA suppression with HBsAg loss 24 weeks after end of treatment with NTZ and TDF, TAF or ETV

[0134] 11 Change in qHBsAg from baseline to different time points on treatment

[0135] in. HBsAg loss, HBsAg seroconversion

[0136] IV. HBV DNA suppression

[0137] v. Change from baseline in Fibrosis 4 (FIB-4) and Fibroscan~ score

[0138] Safety Parameters: Adverse events, laboratory safety tests [0139] Biological Samples: Blood and urine samples collected at screening, baseline

(Day 0), day 3, weeks 1, 2, 4, 8, 12, 16, 20, 24 and every 12 weeks thereafter during treatment and at off-treatment follow-up weeks 4, 8, 12, 24 and 48.

[0140] Figure 3 depicts the study design.

1.0 BACKGROUND

Chronic Hepatitis B (CHB)

[014.1] Hepatitis B virus (HBV) is a partially double-stranded DNA virus belonging to the Hepadnaviridae family of viruses.

[0142] CHB infection remains a major public health problem, affecting more than 250 million people worldwide. These patients have a higher risk of developing severe liver diseases such as cirrhosis, liver failure, or hepatocellular carcinoma. (World Health Organization, 2018)

[0143] Currently available therapies for CHB include nucleoside/nucleotide reverse transcriptase inhibitors (Nrtls) and pegylated interferon. These therapies do not eliminate the covalently closed circular viral DNA (cccDNA), which functions as a nonreplicative

minichromosome and persists throughout the lifespan of infected hepatocytes, and therefore, the goals of treatment are sustained suppression of HBV DNA and remission of liver disease. While six Nrtls have been approved by the U.S. FDA for treatment of CHB (TDF, TAF, ETV, lamivudine, adefovir and telbivudine), TDF, TAF and ETV are the preferred Nrtls for treatment. (Lok et al, 2016; Terrault et al, 2018)

[0144] Response rates for patients with HBeAg-negative CHB enrolled in randomized clinical trials of TDF, TAF or ETV are as follows (Terrault et al, 2018):

HBV DNA suppression (<60 IU/mL) 90-93%

ALT normalization 76-88%

Loss ofHBsAg < 1% [0145] Long-term administration ofNrtls is standard of care for HBe-Ag-negative CHB because it reduces liver-related complications by suppressing viral replication. The best and safest stopping rule is HBV DNA suppression with loss ofHBsAg. Loss ofHBsAg is associated with reduced risk of hepatic decompensation and improved survival. (Terrault et al, 2016; Sarin et al, 2015; European Association for the Study of the Liver, 2017) In this context, a treatment for CHB that could induce loss ofHBsAg leading to functional cure is needed.

1.2 Nitazoxanide (NTZ)

[0146] NTZ is a thiazolide anti-infective medicament.

[0147] Alinia^® (NTZ) for Oral Suspension (patients 1 year of age and older) and Alinia (NTZ) Tablets (patients 12 years and older) are marketed in the United States for the treatment of diarrhea caused by Giardia lamblia or Cryptosporidium parvum.

1.2.2 Overview of Clinical Experience with NTZ

[0148] NTZ has been marketed for diarrheal disease caused by Giardia or

Cryptosporidium in the United States since 2003 and in Latin America since 1996. It is estimated that more than 300 million patients have been exposed to NTZ worldwide. No drug- related serious adverse events have been reported during post-marketing experience with NTZ.

[0149] Phase 2 and 3 clinical studies have been conducted in approximately 5,600 subjects to evaluate the safety and efficacy of NTZ. During these studies, no drug-related serious adverse events have been observed. The side effects have been usually of a mild transient nature, and less than 1% of subjects have discontinued therapy because of an adverse event. The most common adverse events reported in clinical trials include abdominal pain, chromaturia, diarrhea, dizziness, headache, nausea and vomiting and did not differ significantly from those of placebo except for chromaturia which was reported by 4 to 5% of subjects and is attributed to urinary excretion of NTZ metabolites. Clinical chemistry and hematology obtained before and after treatment have not revealed any abnormalities attributable to the test drug. 1.3 Rationale for the Study

[0150] There is a need for a new treatment of CHB that provides functional cure with suppression of HBV DNA and loss of HBsAg. This study will evaluate the effect of different doses ofNTZ on quantitative HBsAg in an effort to identify a dosage regimen for further development with the objective of inducing loss of HBsAg.

2.0 STUDY OBJECTIVES

[0151] This randomized controlled trial is designed to evaluate safety, effectiveness and pharmacokinetic-pharmacodynamic (PK/PD) relationships associated with three different NTZ treatment regimens added to TDF, TAF or ETV in treating CHB.

3.0 STUDY DESIGN

[0152] The study is a randomized double-blind placebo-controlled study. Forty-eight

[48] subjects are selected according to the inclusion and exclusion criteria listed below. Upon enrollment in the study, subjects are randomized to one of the following treatment groups (12 patients per group):

[0153] Group 1: PLACEBO

[0154] Subjects take three placebo tablets twice daily.

[0155] Group 2: 600 me NTZ OD

[0156] Subjects take two NTZ 300 mg extended release tablets and one placebo tablet in the morning and three placebo tablets in the evening.

[0157] Group 3: 600 me NTZ BID

[0158] Subjects take two 300 mg NTZ 300 mg extended release tablets and one placebo tablet twice daily. [0159] Group 4: 900 mg NTZ BID

[0160] Subjects will take three NTZ 300 mg extended release tablets twice daily.

[0161] NTZ and placebo tablets are administered orally with food. All subjects continue

TDF, TAF or ETV therapy throughout the study.

[0162] After 12 weeks, subjects will continue treatment with the same treatment regimen if they have achieved >0.5 logio decline in qHBsAg from baseline to week 12. Subjects will discontinue treatment with the investigational medication (NTZ and/or placebo) due to:

Adverse events

Withdrawn consent

Noncompliance

Subject fails to achieve >0.5 logio decline in qHBsAg from baseline to week 12, >1 logio decline from baseline to week 24, >2 logio decline from baseline to week 48, >3 logio decline from baseline to week 72 or HBsAg loss at week 96.

Subject experiences HBsAg loss with HBV DNA suppression maintained on treatment for 24 weeks (consolidating therapy). TDF, TAF or ETV therapy will also be discontinued.

[0163] Subjects return to the clinic at day 3, weeks 1, 2, 3, 4, 8, 12, 16, 20, 24 and every 12 weeks thereafter during the treatment period. Blood samples are collected at each visit for laboratory and/or serology tests. A blood sample for pharmacokinetics are also collected at every visit during the treatment period.

[0164] Subjects return to the clinic for off-treatment follow-up 4, 8 and 12 weeks following the end of treatment with investigational medication (NTZ and/or placebo). In the event that a subject has sustained HBV DNA suppression with HBsAg loss at the week 12 follow-up visit, that subject returns for additional follow-up visits 24 and 48 weeks after the end of treatment.

[0165] Study design : This study may evaluate whether the combination of NTZ plus TDF, TAF or ETV is superior to TDF, TAF or ETV alone in reducing quantitative serum HBsAg.

[0166] Dose and duration of treatment : The dose of TDF, TAF or ETV used in this study is the standard dose approved by the FDA and other regulatory authorities. Extended release tablets of nitazoxanide administered twice daily may be used in order to achieve steady state plasma concentrations of tizoxanide above the concentrations required to inhibit HBsAg in cell cultures. A 600 mg once daily regimen may also be used to evaluate effectiveness. Two different doses (600 mg and 900 mg) may be used in twice daily regimens.

4.0 SUBJECT SELECTION

[0167] The criteria for inclusion and exclusion are defined below:

4.1 Inclusion Criteria

[0168] 1. Age ³21 years

[0169] 2. CHB virus infection (serum HBsAg-positive for at least 6 months or serum HBsAg-positive and negative immunoglobulin M (IgM) antibodies to HBV core antigen

(IgM ant-HBc))

[0170] 3. HBeAg-negative

[0171] 4. Virologically suppressed (HBV DNA < lower limit of quantitation) for

>12 months on TDF, TAF or ETV therapy

[0172] 5. Quantitative HBsAg >100 IU/mL [0173] 6. ALT below 1.5 times the upper limit of normal

[0174] 7. Able to comply with the study requirements

4.2 Exclusion Criteria

[0175] 1. Unable to take oral medications

[0176] 2. Females who are pregnant, breast-feeding or not using birth control. A double barrier method, oral birth control pills administered for at least 2 monthly cycles prior to study drug administration, an IUD, or medroxyprogesterone acetate administered

intramuscularly for a minimum of one month prior to study drug administration are acceptable methods of birth control for inclusion into the study. In addition, female patients should have a baseline pregnancy test and should agree to continue an acceptable method of birth control for the duration of the study (including follow-up) if sexually active.

[0177] 3. Any investigational drug therapy within 30 days prior to enrollment

[0178] 4. Other causes of liver disease

[0179] 5. Co-infection with human immunodeficiency virus (HTV), hepatitis C virus (HCV) or hepatitis D virus (HDV) based on an enzyme immunoassay (EIA)

[0180] 6. History of alcoholism or with an alcohol consumption of > 40 g per day

[0181] 7. Clinically unstable

[0182] 8. Any concomitant condition that, in the opinion of the investigator would preclude evaluation of response or make it unlikely that the contemplated course of therapy and follow-up could be completed

[0183] 9. History of hypersensitivity or intolerance to NTZ or any of the excipients comprising the NTZ tablets [0184] 10. Hepatocellular carcinoma

[0185] 11 Decompensated liver disease including history of ascites, bleeding esophageal varices, portal hypertension or hepatic encephalopathy

[0186] 12. FibroScan^® score >7 or history of cirrhosis on liver biopsy

[0187] 13. Creatinine clearance <65 ml/minute (by the Cockcroft-Gault equation using ideal body weight)

[0188] 14. History of clinically relevant psychiatric disease, seizures, central nervous system dysfunction, severe pre-existing cardiac, renal, pathologic bone fracture or other risk factors for osteoporosis, hematological disease or medical illness that in the investigator’s opinion might interfere with therapy

[0189] 15. Malignant disease within 3 years of trial entry

[0190] 16. Rheumatological conditions, inflammatory bowel disease or psoriasis requiring or anticipated to require biological/immunosuppressive therapies

5.0. RANDOMIZATION

[0191] At the baseline visit, subjects are randomized to one of the following treatment groups:

[0192] Group 1: PLACEBO - Subjects receive three placebo tablets twice daily with food in addition to continuing TDF, TAF or ETV therapy.

[0193] Group 2: 600 mg NTZ QD - Subjects receive two NTZ 300 mg extended release tablets and one placebo tablet in the morning and three placebo tablets in the evening in addition to continuing TDF, TAF or ETV therapy. [0194] Group 3: 600 mg NTZ BID - Subjects receive two NTZ 300 mg extended release tablets and one placebo tablet twice daily with food for 12 weeks in addition to continuing TDF, TAF or ETV therapy.

[0195] Group 4: 900 mg NTZ BID - Subjects receive three NTZ 300 mg extended release tablets twice daily with food for 12 weeks in addition to continuing TDF, TAF or ETV therapy.

6.0 EVALUATIONS DURING TREATMENT

[0196] Subjects return to the clinic for evaluation at day 3, and weeks 1, 2, 4, 8, 12, 16, 20, 24 and every 12 weeks thereafter during treatment (visit window ±3 days), and the following evaluations are performed:

[0197] 1. Brief physical examination including body weight and vital statistics

[0198] 2. Urine pregnancy test for all females of child-bearing potential (except for day 3)

[0199] 3. Review concomitant medications

[0200] 4. Collection of blood sample for qHBV DNA, qHBsAg, anti-HBs, HBeAg and anti-HBe

[0201] 5. Collection of blood sample for laboratory safety: hemoglobin, hematocrit, complete blood count, (total and differential), platelet count, prothrombin time, AST, ALT,

GGT, alkaline phosphatase, bilirubin total/direct, albumin, BUN, creatinine, sodium, potassium, chloride, AFP, PT/INR (except for day 3)

[0202] 6. Collection of blood sample for pharmacokinetics (before the morning dose) [0203] 7. Collection of urine sample for routine urinalysis (glucose, protein and blood) (except for day 2 and day 4)

[0204] 8. FibroScan^® testing (at end of treatment only)

[0205] 9. Review of adverse events/side effects

[0206] 10. Review of compliance, collection of any unused medications from the previous 4 weeks, and dispense 4-week supply of study medication.

6.4 Follow-up after end of treatment with NTZ/placebo

[0207] Subjects return to the clinic for follow-up evaluation 4, 8 and 12 weeks after the end of treatment with the investigational medication (NTZ/placebo). In the event that a subject has sustained HBV DNA suppression with HBsAg loss at the week 12 follow-up visit, that subject will also return for additional follow-up visits 24 and 48 weeks after the end of treatment. The following evaluations are performed during each follow-up visit:

[0208] 1. Brief physical examination including body weight and vital statistics

[0209] 2. Review concomitant medications

[0210] 3. Collection of blood sample for qHBV DNA, qHBsAg, anti-HBs, HBeAg and anti-HBe

[0211] 4. Collection of blood sample for laboratory safety: hemoglobin, hematocrit, complete blood count, (total and differential), platelet count, prothrombin time, AST, ALT,

GGT, alkaline phosphatase, bilirubin total/direct, albumin, BUN, creatinine, sodium, potassium, chloride, AFP, PT/INR

[0212] 5. Collection of urine sample for routine urinalysis (glucose, protein and blood) [0213] 6. FibroScan^® testing (at end of follow-up only)

[0214] 7. Review of adverse events/side effects

[0215] All adverse events must be followed up until they resolve or have stabilized (even beyond the follow-up visit dates provided above).

6.6 Study Medication Discontinuation and Withdrawal Criteria

[0216] Treatment with the investigational medication (NTZ/placebo) is discontinued for individual subjects for the following reasons:

[0217] 1. Subject withdraws consent.

[0218] 2. A subject must discontinue study medication for the following medical or administrative reasons [reasons for discontinuing must be recorded in the case report form

(CRF)]:

Adverse event(s) deemed sufficiently severe to require study

discontinuation;

Violation of eligibility criteria;

Material deviation from the treatment plan specified in the protocol; Progression of the disease which in the opinion of the investigator precludes further participation; and/or

Women who become pregnant.

[0219] Subject is:

HBsAg-positive with < 0.5 logio decline in qHBsAg from baseline at week 12

HBsAg-positive with < 1 logio decline in qHBsAg from baseline at week

24 HBsAg-positive with < 2 logio decline in qHBsAg from baseline at week

48

HBsAg-positive with < 3 logio decline in qHBsAg from baseline at week

72

HBsAg-positive at week 96

[0220] 4. HBsAg loss with HBV DNA suppression maintained on treatment for 24 weeks (consolidating therapy).

[0221] IMPORTANT: TDF, TAF or ETV therapy are only discontinued after a subject experiences treatment success (sustained HBV DNA suppression with HBsAg loss for 24 weeks on treatment). Otherwise, subjects will continue TDF, TAF or ETV therapy after discontinuing the investigational medication (NTZ/placebo).

6.8 Virology Testing and Plan for Monitoring Resistance

[0222] All virologic assessments is performed using an FDA-approved or FDA-cleared assays. Quantitative HBsAg are determined using the ARCHITECT HBsAg assay by Abbott.

8.0 STATISTICAL CONSIDERATIONS

8.1 Sample Size Calculation

[0223] The study may evaluate the safety and efficacy of three different NTZ treatment regimens compared to placebo as add-on therapy to TDF, TAF or ETV in reducing quantitative HBsAg (in order to select a dose for future clinical trials).

[0224] A student’s t-test with a 0.05 two-sided significance level has 90% power to detect the difference between a mean change of 0.5 logio for one treatment group and no change for the other treatment group when sigma (common standard deviation) is 0.33 and the sample size in each group is 10. A sample size of 12 per group was selected in order to allow for the possibility of up to 2 non-evaluable subjects per group (due to drop-out, etc.). Efficacy Variables

[0225] Primary Efficacy Parameter: Change in qHBsAg from baseline to week 12

[0226] Secondary Efficacy Parameters:

[0227] r. Sustained suppression of HBV DNA with HBsAg loss for 24 weeks after the end of treatment

[0228] rr. Change in qHBsAg from baseline to different time points on treatment

[0229] rrr HBsAg loss

[0230] rv. HBsAg seroconversion

[0231] v. HBV DNA suppression

[0232] vi. Change from baseline in Fibrosis 4 (FIB-4) score

[0233] vrr. Change from baseline in FibroScan^® score

[0234] Response Definitions

[0235] HBsAg Loss: HBsAg below lower limit of detection

[0236] HBsAg seroconversion: Loss of HBsAg and gain of anti-HBs

[0237] HBV DNA suppression: HBV DNA less than lower limit of quantitation, target not detected

8.4.1 Efficacy Analyses

[0238] Efficacy analyses is based on a population consisting of all patients randomized to the study (“intent-to-treat” population). In the event that there are significant numbers of patients who fail to complete the study, violate the protocol, or are noncompliant with the study medication, efficacy analyses will also be conducted for a population excluding these patients (“per protocol population”).

[0239] Primary efficacy analyses are conducted as follows:

[0240] For each NTZ treatment group, compare the mean change in quantitative HBsAg from baseline to week 12 to that of the placebo treatment group.

[0241] Secondary efficacy analyses are conducted as follows:

[0242] For each of the three NTZ groups, compare the proportion of subjects with sustained suppression of HBV DNA and HBsAg loss 24 weeks after the end of treatment to the proportion for the placebo treatments group.

[0243] For each NTZ dose group and for each study time point, compare the change in qHBsAg from baseline to the change in qHBsAg for the placebo treatment group.

[0244] For each of the three NTZ groups and for each study time point, compare the proportion of subjects with HBsAg loss to the proportion for the placebo treatments group.

[0245] For each of the three NTZ groups and for each study time point, compare the proportion of subjects with HBsAg seroconversion to the proportion for the placebo treatments group.

[0246] For each of the three NTZ groups and for each study time point, compare the proportion of subjects with quantifiable HBV DNA at baseline who experienced HBV DNA suppression to the proportion for the placebo treatments group.

[0247] For each NTZ dose group and for each study time point, compare the mean change in Fibrosis 4 (FIB-4) score from baseline to the change for the placebo treatment group. [0248] For each NTZ dose group, compare the mean change in FibroScan^® score from baseline to end of treatment and to end of off-treatment follow-up to the change for the placebo treatment group.

[0249] Means are compared by two-sided student’s t tests, a = 0.05. Non-par am etric data are analyzed using the Wilcoxon rank sum (Mann-Whitney) test. Proportions are compared using a two-sided Fisher’s Exact test, a = 0.05.

LIST OF REFERENCES

Anderson VR, Curran MP. Drugs 2007; 67:1947-1967.

European Association for the Study of the Liver. J Hepatol 2017; 67:370-398.

Korba BE, et al. Antivir Res 2008; 77: 56-63.

Lok A SF, et al. Hepatol 2016; 63:284-306.

Romark, L.C. Alinia (nitazoxanide) prescribing information. April 2017.

Rossignol JF. Antiviral Res 2014; 110:94-103.

Sarin SK, et al. Hepatol Int 2016; 10:1-98.

Sekiba K, et al. Cell Mol Gastroenterol Hepatol 2019; in press.

TerraultNA, et al. Hepatol, 2016; 63:261-283.

Terrault NA et al. Hepatol 2018; 67:1560-98.

World Health Organization. Hepatitis B Fact Sheet. July 18, 2018. Accessed at:

http://www.who.int/news-room/fact-sheets/detail/hepatitis-b. Accessed date: February 5, 2019. EXAMPLE 3

[0250] RANDOMIZED, DOUBLE-BLIND, ACTIVE-CONTROLLED, DOSE-

RANGING TRIAL TO EVALUATE EFFICACY AND SAFETY OF NITAZOXANIDE IN

THE TREATMENT OF SUBJECTS WITH CHRONIC HEPATITIS B

[0251] STUDY SYNOPSIS

[0252] Title of Study: Randomized, Double-Blind, Active-Controlled, Dose Ranging

Trial to Evaluate Efficacy and Safety of Nitazoxanide in the Treatment of Subjects with Chronic Hepatitis B

[0253] Objectives: The primary objective of the study is to evaluate the effect of NTZ monotherapy administered at three different doses or NTZ in combination with TAF for 12 weeks compared to TAF monotherapy on mean change in qHBsAg from baseline to week 12.

[0254] Secondary objectives include evaluation of (i) sustained HBsAg loss with HBV DNA suppression 24 weeks after end of treatment; (ii) change in qHBsAg from baseline to different time points on treatment; (iii) HBsAg loss; (iv) HBsAg seroconversion; (v) HBeAg loss; (vi) HBeAg seroconversion; (vii) HBV DNA suppression; (viii) ALT normalization; (ix) change from baseline in Fibrosis 4 (FIB-4) score; and (x) change from baseline in Fibroscan score.

[0255] Methodology: Subjects with CHB will be screened for eligibility within 30 days of the baseline visit (Day 1). At the baseline visit (Day 1) subjects will be randomized 1 : 1 : 1 : 1 : 1 to the following treatment groups:

[0256] Group 1 : 600 mg NTZ once daily for 12 weeks

[0257] Group 2: 600 mg NTZ twice daily for 12 weeks

[0258] Group 3: 900 mg NTZ twice daily for 12 weeks [0259] Group 4: 600 mg NTZ twice daily + 25 mg TAF once daily for 12 weeks

[0260] Group 5: 25 mg TAF once daily for 12 weeks

[0261] Subjects will continue treatment until they reach a stopping rule/milestone for treatment failure (determined based on changes in qHBsAg and qHBV DNA), achieve HBsAg loss with HBV DNA suppression for 24 weeks on treatment (consolidating therapy), or until withdrawal due to withdrawn consent, adverse event, pregnancy, violation of eligibility criteria, material deviation from the treatment plan specified in the protocol or disease progression which in the opinion of the investigator precludes further participation. All subjects will return to the clinic at baseline (Day 1), Day 3, Week 1, 2, 4, 8, 12, 16, 20, 24 and every 12 weeks thereafter during the treatment period. Each subject will return for off-treatment follow-up visits at Weeks 4, 8 and 12 following the end of treatment. Subjects with HBsAg loss at the Week 12 off- treatment follow-up visit will return to the clinic at Weeks 24 and 48 following end of treatment for off-treatment follow-up to evaluate durability of response. On-treatment and off-treatment follow-up visits will include physical examination, blood and urine sample collection, review of concomitant medications and adverse events, study medication dispensing and accountability (as applicable). FibroScan^® will be performed at baseline, the end-of-treatment visit, and at the last off-treatment follow-up visit. Ultrasonography for HCC will be performed at screening and every six months during the study.

[0262] Number of patients may be 120, including 60 HBeAg-negative and 60 HBeAg- positive subjects.

[0263] Diagnosis and main criteria for inclusion:

[0264] Inclusion Criteria

[0265] 1. Age >21 years [0266] 2. CHB virus infection (serum HBsAg-positive for at least 6 months or serum HBsAg-positive and negative immunoglobulin M (IgM) antibodies to HBV core antigen

(IgM ant-HBc))

[0267] 3. HBV DNA > 3 logio copies/mL

[0268] 4. Quantitative HBsAg >100 IU/mL

[0269] 5. Able to comply with the study requirements

[0270] Exclusion Criteria

[0271] 1. Unable to take oral medications

[0272] 2. Females who are pregnant, breast-feeding or not using birth control. A double barrier method, oral birth control pills administered for at least 2 monthly cycles prior to study drug administration, an IUD, or medroxyprogesterone acetate administered

[0273] 3. Any investigational drug therapy within 30 days prior to enrollment

[0274] 4. Other causes of liver disease

[0275] 5. Co-infection with human immunodeficiency virus (HTV), hepatitis C virus (HCV) or hepatitis D virus (HDV) based on an enzyme immunoassay (EIA)

[0276] 6. History of alcoholism or with an alcohol consumption of > 40 g per day

[0277] 7. Clinically unstable [0278] 8. Any concomitant condition that, in the opinion of the investigator would preclude evaluation of response or make it unlikely that the contemplated course of therapy and follow-up could be completed

[0279] 9. History of hypersensitivity or intolerance to NTZ or any of the excipients comprising the NTZ tablets

[0280] 10. Hepatocellular carcinoma

[0281] 11. Decompensated liver disease including history of ascites, bleeding esophageal varices, portal hypertension or hepatic encephalopathy

[0282] 12. FibroScan^® score >11 or history of cirrhosis on liver biopsy

[0283] 13. Creatinine clearance <65 ml/minute (by the Cockcroft-Gault equation using ideal body weight)

[0284] 14. History of clinically relevant psychiatric disease, seizures, central nervous system dysfunction, severe pre-existing cardiac, renal, pathologic bone fracture or other risk factors for osteoporosis, hematological disease or medical illness that in the investigator’s opinion might interfere with therapy

[0285] 15. Malignant disease within 3 years of trial entry

[0286] 16. Rheumatological conditions, inflammatory bowel disease or psoriasis requiring or anticipated to require biological/immunosuppressive therapies

[0287] Test and reference product, dosage and mode of administration:

[0288] Test product: NTZ 300 mg tablets

[0289] Reference product: TAF 25 mg tablets

[0290] All study medication is to be taken orally with food. [0291] Table 2

[0292] Duration of treatment: Between 12 - 120 weeks.

[0293] Criteria for evaluation:

[0294] Efficacy:

[0295] Primary endpoint: Mean change in qHBsAg from baseline to week 12

[0296] Secondary endpoints: Proportion with sustained HBsAg loss with HBV DNA suppression 24 weeks after end of treatment; change in qHBsAg from baseline to different time points on treatment; proportion with HBsAg loss; proportion with HBsAg seroconversion;

proportion with HBeAg loss, proportion with HBeAg seroconversion, proportion with HBV DNA suppression; proportion with ALT normalization; change from baseline in Fibrosis 4 (FIB- 4) score; and change from baseline in Fibroscan^® score. [0297] Safety parameters: Adverse events and laboratory safety tests.

[0298] Figure 4 depicts the study design.

2 INTRODUCTION

2.1 Chronic Hepatitis B (CHB)

[0299] Hepatitis B virus (HBV) is a partially double-stranded DNA virus belonging to the Hepadnaviridae family of viruses.

[0300] CHB infection remains a major public health problem, affecting more than 250 million people worldwide. These patients have a higher risk of developing severe liver diseases such as cirrhosis, liver failure, or hepatocellular carcinoma. (World Health Organization, 2018)

[0301] Currently available therapies for CHB include nucleoside/nucleotide reverse transcriptase inhibitors (Nrtls) and pegylated interferon. These therapies do not eliminate the covalently closed circular viral DNA (cccDNA), which functions as a nonreplicative

minichromosome and persists throughout the lifespan of infected hepatocytes, and therefore, the goals of treatment are sustained suppression of HBV DNA and remission of liver disease. While six Nrtls have been approved by the U.S. FDA for treatment of CHB (TDF, TAF, ETV, lamivudine, adefovir and telbivudine), TDF, TAF and ETV are the preferred Nrtls for treatment (Lok et al. 2016, Terrault et al. 2018).

[0302] Response rates for patients with HBeAg-negative CHB enrolled in randomized clinical trials of TDF, TAF or ETV are as follows (Terrault et al. 2018):

[0303] HBV DNA suppression (<60 IU/mL)90-93%

[0304] ALT normalization 76-88%

[0305] Loss of HBsAg < 1% [0306] Long-term administration ofNrtls is standard of care for HBe-Ag-negative CHB because it reduces liver-related complications by suppressing viral replication. The best and safest stopping rule is HBV DNA suppression with loss ofHBsAg. Loss ofHBsAg is associated with reduced risk of hepatic decompensation and improved survival. (Terrault et al. 2016, Sarin et al. 2015, European Association for the Study of the Liver 2017) In this context, a treatment for CHB that could induce loss ofHBsAg leading to functional cure is needed.

2.2 Nitazoxanide (NTZ)

[0307] NTZ is a thiazolide anti-infective medicament.

[0308] Alinia^® (NTZ) for Oral Suspension (patients 1 year of age and older) and Alinia (NTZ) Tablets (patients 12 years and older) are marketed in the United States for the treatment of diarrhea caused by Giardia lamblia or Cryptosporidium parvum. Alinia for Oral Suspension and Alinia.

2.2.2 Overview of Clinical Experience with NTZ

[0309] NTZ has been marketed for diarrheal disease caused by Giardia or

[0310] Phase 2 and 3 clinical studies have been conducted in approximately 5,600 subjects to evaluate the safety and efficacy of NTZ. During these studies, no drug-related serious adverse events have been observed. The side effects have been usually of a mild transient nature, and less than 1% of subjects have discontinued therapy because of an adverse event. The most common adverse events reported in clinical trials include abdominal pain, chromaturia, diarrhea, dizziness, headache, nausea and vomiting and did not differ significantly from those of placebo except for chromaturia which was reported by 4 to 5% of subjects and is attributed to urinary excretion of NTZ metabolites. Clinical chemistry and hematology obtained before and after treatment have not revealed any abnormalities attributable to the test drug.

2.3 Rationale for the Study

[0311] There is a need for a new treatment of CHB that provides functional cure with suppression of HBV DNA and loss of HBsAg. This study will evaluate the effect of different doses of NTZ on quantitative HBsAg in an effort to identify a dosage regimen for further development with the objective of inducing loss of HBsAg. The study will also evaluate the effects of NTZ and combination regimens on quantitative HBeAg and HBV DNA.

3 STUDY OBJECTIVES

[0312] This randomized controlled trial is designed to evaluate efficacy and safety of NTZ monotherapy or in combination with TAF compared to TAF monotherapy in treatment of CHB.

4 STUDY DESIGN

4.1 Study Design Overview

[0313] This is a randomized, double-blind, active-controlled, dose-ranging study of NTZ monotherapy and NTZ in combination of TAF compared to TAF monotherapy in the treatment of subjects with CHB. One hundred twenty (120) subjects including 60 HBeAg-negative and 60 HBeAg-positive will be randomized 1 : 1 : 1 : 1 : 1 to one of the treatment groups in Table 3. Any other CHB antiviral therapy will be discontinued prior to enrolling in the study.

[0314] Table 3. Treatment Groups and Assignment

[0315] Subjects will continue treatment until they reach a stopping rule/milestone for treatment failure (determined based upon qHBsAg and qHBV DNA), achieve HBsAg loss with HBV DNA suppression for 24 weeks on treatment (consolidating therapy) or until withdrawn consent, adverse event, pregnancy, violation of eligibility criteria, material deviation from the treatment plan specified in the protocol or disease progression which in the opinion of the investigator precludes further participation (see section 7 for discontinuation rules). Subjects may be treated with study intervention for up to a maximum of 120 weeks.

[0316] Subjects will return to the clinic at Day 3, Weeks 1, 2, 4, 8, 12, 16, 20, 24 and every 12 weeks thereafter during the treatment period. Blood samples will be collected at each visit for laboratory and/or serology tests. A blood sample for pharmacokinetics will also be collected at every visit during the treatment period.

[0317] Subjects will return to the clinic for off-treatment follow-up 4, 8 and 12 weeks following the end of treatment with investigational medication (NTZ and/or TAF). In the event that a subject has sustained HBsAg loss at the week 12 follow-up visit, that subject will return for additional follow-up visits 24 and 48 weeks after the end of treatment.

[0318] The study will be unblinded after all subjects have completed 12 weeks of treatment and complete data is collected through that time point for analysis of safety and the primary efficacy parameter.

4.3 Dose and Duration of Treatinent

[0319] The dose of TAF used in this study is the standard dose approved by the FDA and other regulatory authorities. The doses of NTZ were selected based upon safety, tolerability and pharmacokinetics data from prior clinical experience with nitazoxanide. The study will use extended release tablets administered twice daily in order to achieve steady state plasma concentrations of tizoxanide above the concentrations required to inhibit HBsAg in cell cultures. The study will also use a 600 mg once daily regimen to evaluate effectiveness. The study will use two different doses (600 mg and 900 mg) in twice daily regimens.

[0320] The duration of treatinent to the primary endpoint (12 weeks) may provide a relatively quick determination regarding whether NTZ induces a meaningful reduction of qHBsAg. Subjects will be allowed to continue treatment beyond 12 weeks if they are showing progressive declines in qHBsAg or HBV DNA or experience HBsAg loss HBV DNA

suppression. The stopping rules require discontinuation of the investigational medication in the event that a subject fails to achieve progressive improvement in qHBsAg or qHBV DNA during treatment. Subjects achieving HBsAg loss and HBV DNA suppression will continue treatment with the investigational medication until they have maintained HBsAg loss and HBV DNA suppression for 24 weeks on treatment (consolidating therapy). The extended duration of treatment for subjects showing response to treatment may allow subjects to continue treatment if they are experiencing benefit with respect to qHBsAg.

5 STUDY POPULATION

5.1 Inclusion Criteria

[0321] 1. Age ³21 years

[0322] 2. CHB virus infection (serum HBsAg-positive for at least 6 months or serum HBsAg-positive and negative immunoglobulin M (IgM) antibodies to HBV core antigen

(IgM ant-HBc))

[0323] 3. HBV DNA > 3 logio copies/mL

[0324] 4. Quantitative HBsAg >100 IU/mL [0325] 5. Able to comply with the study requirements

5.2 Exclusion Criteria

[0326] 1. Unable to take oral medications

[0327] 2. Females who are pregnant, breast-feeding or not using birth control. A double barrier method, oral birth control pills administered for at least 2 monthly cycles prior to study drug administration, an IUD, or medroxyprogesterone acetate administered

[0328] 3. Any investigational drug therapy within 30 days prior to enrollment

[0329] 4. Other causes of liver disease

[0330] 5. Co-infection with human immunodeficiency virus (HTV), hepatitis C virus (HCV) or hepatitis D virus (HDV) based on an enzyme immunoassay (EIA)

[0331] 6. History of alcoholism or with an alcohol consumption of > 40 g per day

[0332] 7. Clinically unstable

[0333] 8. Any concomitant condition that, in the opinion of the investigator would preclude evaluation of response or make it unlikely that the contemplated course of therapy and follow-up could be completed

[0334] 9. History of hypersensitivity or intolerance to NTZ or any of the excipients comprising the NTZ tablets

[0335] 10. Hepatocellular carcinoma [0336] 11. Decompensated liver disease including history of ascites, bleeding esophageal varices, portal hypertension or hepatic encephalopathy

[0337] 12. FibroScan^® score >11 or history of cirrhosis on liver biopsy

[0338] 13. Creatinine clearance <65 ml/minute (by the Cockcroft-Gault equation using ideal body weight)

[0339] 14. History of clinically relevant psychiatric disease, seizures, central nervous system dysfunction, severe pre-existing cardiac, renal, pathologic bone fracture or other risk factors for osteoporosis, hematological disease or medical illness that in the investigator’s opinion might interfere with therapy

[0340] 15. Malignant disease within 3 years of trial entry

[0341] 16. Rheumatological conditions, inflammatory bowel disease or psoriasis requiring or anticipated to require biological/immunosuppressive therapies

6 STUDY INTERVENTION

6.1 Study Intervention(s) Administration

6.1.1 Study Intervention Description

[0342] NTZ 300 mg extended release tablets for use in this study are manufactured by Romark Global Pharma, Manati, Puerto Rico. Each tablet is a yellow, round, convex, film- coated, bi-layer tablet for oral administration, each tablet contains 300 mg of NTZ and the inactive ingredients.

[0343] TAF 25 mg tablets were manufactured by Gilead Sciences. Each tablet is a yellow, round, film-coated tablet, debossed with“GST’ on one side of the tablet and“25” on the other side. [0344] Placebo tablets designed to match the NTZ and TAF tablets were manufactured by Romark Global Pharma, Manati, Puerto Rico.

6.1.2 Dosing and Administration

[0345] Study medication will be administered orally twice daily with food. Tablets will be administered by treatment group as shown in Table 4.

[0346] Table 4. Study Intervention Administration by Treatment Group

7. STUDY INTERVENTION DISCONTINUATION AND SUBJECT

DISCONTINUATION/WITHDRAWAL

7.1 Discontinuation of Study Intervention

[0347] Treatment will be discontinued for individual subjects for the following reasons:

[0348] 1. Subject is: [0349] HBsAg-positive, HBV DNA-positive, and has < 0.5 logio decline in qHBsAg and < 1 logio decline in qHBV DNA from baseline at week 12

[0350] HBsAg-positive, HBV DNA-positive, and has < 1 logio decline in qHBsAg and < 2 logio decline in qHBV DNA from baseline at week 24

[0351] HBsAg-positive, HBV DNA-positive, and has < 2 logio decline in qHBsAg and < 3 decline in qHBV DNA from baseline at week 48

[0352] HBsAg-positive, HBV DNA-positive, and has < 3 logio decline in qHBsAg and

< 4 logio decline in qHBV DNA from baseline at week 72

[0353] HBsAg-positive at week 96

[0354] HBsAg loss with HBV DNA suppression maintained on treatment for 24 weeks

[0355] 2. Adverse event(s) deemed sufficiently severe to require study medication discontinuation

[0356] 3. Discontinuation from study intervention does not mean discontinuation from the study, and remaining study procedures should be completed as indicated by the study protocol.

9. STATISTICAL CONSIDERATIONS

9.1 Sample Size Calculation

[0357] The study may evaluate the efficacy and safety of three different NTZ

monotherapy regimens and one NTZ+TAF combination regimen compared to TAF monotherapy as in reducing quantitative HBsAg (in order to select a dose for future clinical trials).

[0358] A student’s t-test with a 0.05 two-sided significance level will have 90% power to detect the difference between a mean change of 0.5 logio for one treatment group and no change for the other treatment group when sigma (common standard deviation) is 0.33 and the sample size in each group is 10. A sample size of 12 per group was selected in order to allow for the possibility of up to 2 non-evaluable subjects per group (due to drop-out, etc.).

9.2 Efficacy Variables

[0359] Primary Efficacy Parameter: Mean change in qHBsAg from baseline to week 12

[0360] Secondary Efficacy Parameters:

[0361] 1. Sustained HBsAg loss with suppression of HBV DNA for 24 weeks after the end of treatment

[0362] 11. Change in qHBsAg from baseline to different time points on treatment

[0363] in HBsAg loss

[0364] IV. HBsAg seroconversion

[0365] v. HBeAg loss

[0366] vi. HBeAg seroconversion

[0367] Vll. HBV DNA suppression

[0368] viii. ALT normalization

[0369] IX. Change from baseline in Fibrosis 4 (FIB-4) score

[0370] x. Change from baseline in FibroScan^® score

[0371] 9.3 Response Definitions

[0372] HBsAg Loss: HBsAg below lower limit of detection

[0373] HBsAg seroconversion: Loss of HBsAg and gain of anti-HBs [0374] HBeAg Loss: HBeAg below lower limit of detection

[0375] HBeAg seroconversion: Loss of HBeAg and gain of anti-HBe

[0376] HBV DNA suppression: HBV DNA less than lower limit of quantitation, target not detected

[0377] ALT normalization: ALT < upper limit of normal

9.4. Statistical Methodology

[0378] The statistical methodology is described briefly below.

9.4.1. Efficacy Analyses

[0379] The study will be unblinded for analysis of efficacy and safety data after all subjects have completed 12 weeks of treatment and complete data is collected and locked through that time point.

[0380] Efficacy analyses will be based on a population consisting of all patients randomized to the study (“intent-to-treat” population). In the event that there are significant numbers of patients who fail to complete the study, violate the protocol, or are noncompliant with the study medication, efficacy analyses will also be conducted for a population excluding these patients (“per protocol population”).

[0381] Primary efficacy analyses will be conducted as follows:

[0382] For each NTZ monotherapy or combination treatment group, compare the mean change in qHBsAg from baseline to week 12 to that of the TAF monotherapy group.

[0383] Secondary efficacy analyses will be conducted as follows: [0384] For each NTZ monotherapy or combination treatment group, compare the proportion of subjects with sustained suppression of HBV DNA and HBsAg loss 24 weeks after the end of treatment to the proportion for the TAF monotherapy group.

[0385] For each NTZ monotherapy or combination treatment group and for each study time point, compare the change in qHBsAg from baseline to the change in qHBsAg for the TAF monotherapy group.

[0386] For each NTZ monotherapy or combination treatment group and for each study time point, compare the proportion of subjects with HBsAg loss to the proportion for the TAF monotherapy group.

[0387] For each NTZ monotherapy or combination treatment group and for each study time point, compare the proportion of subjects with HBsAg seroconversion to the proportion for the TAF monotherapy group.

[0388] For each NTZ monotherapy or combination treatment group and for each study time point, compare the proportion of subjects with HBeAg loss to the proportion for the TAF monotherapy group (HBeAg-positive cohort only).

[0389] For each NTZ monotherapy or combination treatment group and for each study time point, compare the proportion of subjects with HBeAg seroconversion to the proportion for the TAF monotherapy group (HBeAg-positive cohort only).

[0390] For each NTZ monotherapy or combination treatment group and for each study time point, compare the proportion of subjects with quantifiable HBV DNA at baseline who experienced HBV DNA suppression to the proportion for the TAF monotherapy group.

[0391] For each NTZ monotherapy or combination treatment group and for each study time point, compare the proportion of subjects with ALT > upper limit of normal at baseline who experienced ALT normalization to the proportion for the TAF monotherapy group. [0392] For each NTZ monotherapy or combination treatment group and for each study time point, compare the mean change in Fibrosis 4 (FIB-4) score from baseline to the change for the TAF monotherapy group.

[0393] For each NTZ monotherapy or combination treatment group, compare the mean change in FibroScan^® score from baseline to end of treatment and to end of off-treatment followup to the change for the TAF monotherapy group.

[0394] Means will be compared by two-sided student’s t tests, a = 0.05. Non-parametric data will be analyzed using the Wilcoxon rank sum (Mann-Whitney) test. Proportions will be compared using a two-sided Fisher’s Exact test, a = 0.05.

9.4.2 Safety Analyses

[0395] All randomized patients who receive the study medication will be assessed for drug safety. Safety analyses will be done descriptively.

References

Anderson VR, Curran MP. Drugs 2007; 67:1947-1967.

European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017; 67:370-398.

Korba BE, et al. AntivirRes 2008; 77:56-63.

Lok A SF, et al. Hepatol 2016; 63:284-306.

Romark, L.C. Alinia (nitazoxanide) prescribing information. April 2017.

Rossignol JF. Antiviral Res 2014; 110:94-103.

Sarin SK, et al. Hepatol Int 2016; 10:1-98. Sekiba K, et al. Cell Mol Gastroenterol Hepatol 2019; in press.

TerraultNA, et al. Hepatol, 2016; 63:261-283.

Terrault NA et al. Hepatol 2018; 67: 1560-98.

World Health Organization. Hepatitis B Fact Sheet. July 18, 2018. Accessed at:

http://www.who.int/news-room/fact-sheets/detail/hepatitis-b. Accessed date: February 5, 2019.

* * *

[0396] Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention.

[0397] All of the publications, patent applications and patents cited in this specification are incorporated herein by reference in their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a chronic hepatitis B infection comprising administering to a subject in need a thereof an effective amount of a thiazolide compound, which is selected from the group consisting of nitazoxanide, tizoxanide or a combination thereof.

2. The method of claim 1, wherein a daily dose of the administered thiazolide compound is from 600 mg to 1800 mg.

3. The method of claim 1, wherein a daily dose of the administered thiazolide compound is about 600 mg.

4. The method of claim 1, wherein a daily dose of the administered thiazolide compound is about 1200 mg.

5. The method of claim 1, wherein a daily dose of the administered thiazolide compound is about 1800 mg.

6. The method of any one of the preceding claims, wherein the thiazolide compound is administered orally in the form of an extended release solid formulation.

7. The method of any one the preceding claims, wherein the thiazolide compound is orally administered in the form of a solid oral dosage form comprising: (a) a first portion comprising a first quantity of the thiazolide compound in a controlled release formulation; and (b) a second portion comprising a second quantity of the thiazolide compound in an immediate release formulation.

8. The method of any one of the preceding claims, wherein the thiazolide compound is administered orally as a bilayer tablet comprising: (a) a first layer comprising a first quantity of the thiazolide compound in a controlled release formulation; and (b) a second layer comprising a second quantity of the thiazolide compound in an immediate release formulation.

9. The method of any one of the preceding claims, wherein the subject is an HBeAg- positive chronic hepatitis B patient.

10. The method of any one of claims 1-8, wherein the subject is an HBeAg-negative chronic hepatitis B patient.

11. The method of claim 10, wherein the subject has been treated with at least one of Tenofovir Disoproxil Fumarate (TDF), Tenofovir Alafenamide (TAF) or Entecavir (ETV) prior to said administering.

12. The method of claim 11, wherein the subject has been treated with Tenofovir Disoproxil Fumarate (TDF) prior to said administering.

13. The method of claim 11, wherein the subject has been treated with Tenofovir Alafenamide (TAF) prior to said administering.

14. The method of claim 11, wherein the subject has been treated with Entecavir (ETV) prior to said administering.

15. The method of any one of claims 10-14, wherein the subject has been the HBeAg- negative chronic hepatitis B patient for at least 12 months prior to said administering.

16. The method of any one of the preceding claims further comprising administering to the subject at least one of Tenofovir Disoproxil Fumarate (TDF), Tenofovir Alafenamide (TAF) or Entecavir (ETV) during a period of the administering of the thiazolide compound.

17. The method of claim 16, wherein Tenofovir Disoproxil Fumarate (TDF) is administered to the subject during the period of the administering of the thiazolide compound.

18. The method of claim 16, wherein Tenofovir Alafenamide (TAF) is administered to the subject during the period of the administering of the thiazolide compound.

19. The method of claim 17 or 18, wherein the administering of the thiazolide compound and the administering the Tenofovir Disoproxil Fumarate or the Tenofovir

Alafenamide are such that a plasma concentration of tizoxanide in the subject is at least 3 % of a plasma concentration of tenofir in the subject.

20. The method of claim 16, wherein Entecavir (ETV) is administered to the subject during the period of the administering of the thiazolide compound.

21. The method of claim 20, wherein the administering of the thiazolide compound and the administering the Entecavir are such that a plasma concentration of tizoxanide in the subject is at least 200 % of a plasma concentration of entecavirin the subject.

22. The method of any one of the preceding claims, wherein the thiazolide compound is administered to the subject for at least 12 weeks.

23. The method of any one of the preceding claims, wherein the thiazolide compound is administered to the subject for at least 24 weeks.

24. The method of any one of the preceding claims, wherein the thiazolide compound is administered to the subject for at least 48 weeks.

25. The method of any one of the preceding claims, wherein the thiazolide compound is administered to the subject for at least 72 weeks.

26. The method of any one of the preceding claims, wherein the thiazolide compound is administered to the subject for at least 96 weeks.

27. The method of any one of the preceding claims, wherein prior to the

administering of the thiazolide compound the subject has a serum HBsAg level greater than 100 IU/mL after said administering the serum HBsAg level is reduced.

28. The method of claim 27, wherein after said administering the serum HBsAg level is suppressed below a detectable level.

29. The method of claim 28, further comprising administering to the subject the thiazolide compound after the serum HBsAg level in the subject has been suppressed below the detectable level.

30. The method of claim 29, wherein the thiazolide compound is administered for at least 12 weeks after the serum HBsAg level in the subject has been suppressed below the detectable level.

31. The method of claim 29, wherein the thiazolide compound is administered for at least 24 weeks after the serum HBsAg level in the subject has been suppressed below the detectable level.

32. The method of any one of the preceding claims, wherein the administering the thiazolide compound results in one or more of the following: a) sustained HBV DNA

suppression in the subject; b) HBeAg loss in the subject; c) HBeAg seroconversion in the subject; d) HBsAg loss in the subject; e) HBsAg seroconversion in the subject; f) clinical remission of liver disease; and g) an improvement in a fibrosis status of the subject.

33. A method of treating a hepatitis B virus infection comprising administering to a subject in need thereof an anti-hepatitis B effective amount of a) a thiazolide compound, which is selected from the group consisting of nitazoxanide, tizoxanide or a combination thereof and b) at least one medicament selected from Tenofovir Disoproxil Fumarate (TDF), Tenofovir

Alafenamide (TAF) and Entecavir (ETV).