WO2020046941A1 - Crystalline forms of substituted dihydroindene-4-carboxamide compounds and methods of preparing and using same - Google Patents

Abstract

The present invention includes crystalline solid forms of certain substituted dihydroindene-4-carboxamide compounds, and compositions comprising the same, that can be used to treat or prevent hepatitis B virus (HBV) and/or hepatitis D virus (HDV) infection in a patient. In certain embodiments, the compounds and compositions of the invention inhibit and/or reduce HBsAg secretion.

Description

TITLE OF THE INVENTION

Crystalline Forms of Substituted Dihydroindene-4-Carboxamide Compounds and

Methods of Preparing and Using Same

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/723,823, filed August 28, 2018, which application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Hepatitis B is one of the world’s most prevalent diseases. Although most individuals resolve the infection following acute symptoms, approximately 30% of cases become chronic. 350-400 million people worldwide are estimated to have chronic hepatitis B, leading to 0.5-1 million deaths per year, due largely to the development of hepatocellular carcinoma, cirrhosis and/or other complications. Hepatitis B is caused by hepatitis B virus (HBV), a noncytopathic, liver tropic DNA virus belonging to Hepadnaviridae family.

A limited number of drugs are currently approved for the management of chronic hepatitis B, including two formulations of alpha-interferon (standard and pegylated) and five nucleoside/nucleotide analogues (lamivudine, adefovir, entecavir, telbivudine, and tenofovir) that inhibit HBV DNA polymerase. At present, the first-line treatment choices are entecavir, tenofovir and/or peg-interferon alfa-2a. However, peg-interferon alfa-2a achieves desirable serological milestones in only one third of treated patients, and is frequently associated with severe side effects. Entecavir and tenofovir require long-term or possibly lifetime administration to continuously suppress HBV replication, and may eventually fail due to emergence of drug-resistant viruses.

Hepatitis D virus (HDV) is a small circular enveloped RNA virus that can propagate only in the presence of HBV. In particular, HDV requires the HBV surface antigen protein to propagate itself. Infection with both HBV and HDV results in more severe complications compared to infection with HBV alone. These complications include a greater likelihood of experiencing liver failure in acute infections and a rapid progression to liver cirrhosis, with an increased chance of developing liver cancer in chronic infections. In combination with hepatitis B, hepatitis D has the highest mortality rate of all the hepatitis infections. The routes of transmission of HDV are similar to those for HBV. Infection is largely restricted to persons at high risk of HBV infection, particularly injecting drug users and persons receiving clotting factor concentrates.

Currently, there is no effective antiviral therapy available for the treatment of acute or chronic type D hepatitis. Interferon-alfa given weekly for 12 to 18 months is the only licensed treatment for hepatitis D. Response to this therapy is limited, as only about one- quarter of patients is serum HDV RNA undetectable 6 months post therapy.

Much research has been dedicated to the identification of novel agents that can be used to effectively treat and/or prevent HBV and/or HDV infection in a subject. Such agents should be easily and reproducibly prepared in large scale, so that they can be used to treat large number of patients infected with, or at risk of being infected with, HBV and/or HDV. There is thus a need to identify scalable synthetic routes for those anti-HBV and/or anti-HDV antiviral agents. The present invention addresses this need.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of illustrative embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, certain illustrative embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1 comprises a powder X-ray diffraction (PXRD) diffractogram of (l-methyl-lH- l,2,4-triazol-3-yl)methyl (S)-(4-((3-chloro-4-fluorophenyl)carbamoyl)-7-fluoro-2,3-dihydro- lH-inden-l-yl)carbamate, herein referred to as (I), Crystalline Form 1 recorded in transmission mode.

FIG. 2 comprises a thermogravimetric Fourier transform infrared (TG-FTIR) spectrum of (I) Crystalline Form 1 recorded at 10 K/min.

FIG. 3 comprises a Differential Scanning Calorimetry (DSC) spectrum of (I) Crystalline Form 1 recorded at 10 K/min in a hermetic aluminum pan.

FIGs. 4A-4B comprise a Dynamic Vapor Sorption (DVS) spectrum (FIG. 4 A) and an isothermal graph (FIG. 4B) of (I) Crystalline Form 1.

FIG. 5 comprise a light microscopy image of (I) Crystalline Form 1.

FIGs. 6A-6B comprise Raman spectra of (I) Crystalline Form 1. FIG. 6A shows a broad spectral width, and FIG. 6B shows a zoomed-in view of the fingerprint region of the Raman spectrum. FIGs. 7A-7H comprise PXRD diffractograms of the polymorph screen resulting materials. FIG. 7A shows the spectra of room temperature Exps. 1-10. FIG. 7B shows the spectra of 5 °C Exps. 11-15. FIG. 7C shows the spectra of 50 °C Exps. 16-20. FIG. 7D shows the spectrum of the lyophilized (I) material (Exp. 21). FIG. 7E shows the spectra of Exps. 22-28 where the lyophilized (I) was resuspended. FIG. 7F shows the spectra of Exps. 31 and 32. FIG. 7G shows the spectra of Exps. 33 and 34. FIG. 7H shows the spectrum of Crystalline Form 1 (top) overlay ed with the spectrum of Exp. 29 (bottom), wherein a mixture of crystal forms was present in the solid recovered in Exp. 29.

FIG. 8A comprises an 'fl NMR spectrum of material collected from Exp. 29. Peak listing (in ppm): 8.483, 8.112, 8.103, 8.089, 8.080, 7.860, 7.831, 7.774, 7.758, 7.746, 7.743, 7.734, 7.729, 7.720, 7.713, 7.704, 7.698, 7.690, 7.491, 7.461, 7.430, 7.236, 7.206, 7.177,

5.349, 5.328, 5.118, 5.077, 5.061, 3.907, 3.906, 3.685, 3.671, 3.663, 3.654, 3.641, 3.357,

3.333, 3.292, 3.276, 3.265, 3.247, 3.105, 3.081, 2.573, 2.567, 2.561, 2.555, 2.548, 2.490,

2.463, 1.994, 1.973, 1.967, 1.951, 1.945, 1.843, 1.833, 1.821, 1.809, 1.799, 1.418.

Integration of peaks (left to right, as shown at bottom of spectrum): 1.000, 0.943, 1.024, 0.900, 2.056, 1.084, 1.031, 0.047, 0.037, 1.062, 2.015, 3.024, 0.557, 1.054, 1.075, 0.950, 0.062, 1.038, 0.525, 0.406, 0.203.

FIG. 8B comprises 'H NMR spectra of material collected from Crystalline Form 1 overlay ed with the material collected from Exp. 29.

FIG. 9 is a PXRD diffractogram of the materials collected from Exp. 29 (bottom) and Exp. 30 (top).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates, in certain aspects, to developable forms of certain substituted compounds that are useful to treat and/or prevent HBV and/or HDV infection and related conditions in a subject. In certain embodiments, the compounds were originally described in PCT/IB2018/000387, filed March 20, 2018, which is incorporated herein in its entirety by reference.

In certain embodiments, the present invention provides developable forms of (1- methyl-lH-l,2,4-triazol-3-yl)methyl (S)-(4-((3-chloro-4-fluorophenyl)carbamoyl)-7-fluoro- 2,3-dihydro-lH-inden-l-yl)carbamate (I), or a salt or solvate thereof:

In other embodiments, the present invention provides polymorphs of (I) free base (which can include any solvates thereof).

As described herein, a crystalline form of (I) free base (Crystalline Form 1) was identified, as well as a range of crystallization conditions for the formation of the crystalline form. Free base (I) was found to crystallize from solvents such as acetonitrile,

tetrahydrofuran (THF), 2-methyl tetrahydrofuran, acetone, isopropanol, methanol, ethanol, toluene, ethyl acetate, t-butyl methyl ether, dichloromethane, THF/water, acetone/water, and methanol/water. A second crystalline form of (I) (Crystalline Form 2) was also observed alongside Crystalline Form 1 after slow solvent evaporation of a solution of (I) in THF.

Compounds

In certain embodiments, the at least one compound of the invention is a component of a pharmaceutical composition further including at least one pharmaceutically acceptable carrier.

The compounds contemplated in the invention may possess one or more stereocenters, and each stereocenter may exist independently in either the (R) or (S) configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms. The compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In certain embodiments, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In other embodiments, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereoisomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography. Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to ¾ ¾ ⁿC, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵0, ¹⁷0, ¹⁸0, ³²P, and ³⁵S. In certain embodiments, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In other embodiments, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet other embodiments, substitution with positron emitting isotopes, such as ⁿC, ¹⁸F, ¹⁵0 and ¹³N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.

The compounds described herein, and other related compounds having different substituents can be synthesized using techniques and materials described herein and in PCT/IB2018/000387 and as described, for example, in Fieser & Fieser’s Reagents for Organic Synthesis, Vol. 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Vol. 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Vol. 1-40 (John Wiley and Sons, 1991), Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989), March, Advanced Organic Chemistry 4^th Ed., (Wiley 1992); Carey & Sundberg, Advanced Organic Chemistry, 4^th Ed., Vols. A and B (Plenum 2000,2001), and Green & Wuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all of which are incorporated by reference for such disclosure). General methods for the preparation of compound as described herein are modified by the use of appropriate reagents and conditions, for the introduction of the various moieties found in the formula as provided herein.

The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 'H or ¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatography such as high pressure liquid chromatography (HPLC), gas chromatography (GC), gel-permeation chromatography (GPC), or thin layer chromatography (TLC). Combination Therapies

Non-limiting examples of one or more additional agents useful for treating HBV and/or HDV infections include: (a) reverse transcriptase inhibitors; (b) capsid inhibitors; (c) cccDNA formation inhibitors; (d) RNA destabilizes; (e) oligomeric nucleotides targeted against the HBV genome; (f) immunostimulators, such as checkpoint inhibitors (e.g., PD-L1 inhibitors); and (g) GalNAc-siRNA conjugates targeted against an HBV gene transcript.

(a) Reverse Transcriptase Inhibitors

In certain embodiments, the reverse transcriptase inhibitor is a reverse-transcriptase inhibitor (NARTI or NRTI). In other embodiments, the reverse transcriptase inhibitor is a nucleotide analog reverse-transcriptase inhibitor (NtARTI or NtRTI).

Reported reverse transcriptase inhibitors include, but are not limited to, entecavir, clevudine, telbivudine, lamivudine, adefovir, and tenofovir, tenofovir disoproxil, tenofovir alafenamide, adefovir dipovoxil, (li?,2i?,3i?,5i?)-3-(6-amino-9i/-9-purinyl)-2-fluoro-5- (hydroxymethyl)-4-methylenecyclopentan-l-ol (described in U.S. Patent No. 8,816,074, incorporated herein in its entirety by reference), emtricitabine, abacavir, elvucitabine, ganciclovir, lobucavir, famciclovir, penciclovir, and amdoxovir.

Reported reverse transcriptase inhibitors further include, but are not limited to, entecavir, lamivudine, and (li?,2i?,3i?,5i?)-3-(6-amino-9i/-9-purinyl)-2-fluoro-5- (hydroxymethyl)-4-methylenecyclopentan-l-ol.

Reported reverse transcriptase inhibitors further include, but are not limited to, a covalently bound phosphoramidate or phosphonamidate moiety of the above-mentioned reverse transcriptase inhibitors, or as described in for example U.S. Patent No. 8,816,074, US Patent Application Publications No. US 2011/0245484 Al, and US 2008/0286230A1, all of which incorporated herein in their entireties by reference.

Reported reverse transcriptase inhibitors further include, but are not limited to, nucleotide analogs that comprise a phosphoramidate moiety, such as, for example, methyl (((( 1 //.3//.4//.5//)-3-(6-amino-9//-purin-9-yl)-4-riuoro-5-hydroxy-2-methylenecyclopentyl) methoxyXphenoxy) phosphoryl)-(D or L)-alaninate and methyl ((((li?,2i?,3i?,4i?)-3-fluoro-2- hydro\y-5-methylene-4-(6-o\o- 1.6-dihydro-9//-purin-9-yl)cyclopentyl)methoxy)(phenoxy) phosphoryl)-(D or L)-alaninate. Also included are the individual diastereomers thereof, which include, for example, methyl ((R)-((( l//.3//.4//.5//)-3-(6-amino-9//-purin-9-yl)-4-riuoro-5- hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-(D or L)-alaninate and methyl ((S)-((( 1 //.3//.4//.5//)-3-(6-amino-9//-purin-9-yl)-4-riuoro-5-hydro\y-2- methylenecyclopentyl) methoxy)(phenoxy)phosphoryl)-(D or L)-alaninate.

Reported reverse transcriptase inhibitors further include, but are not limited to, compounds comprising a phosphonamidate moiety, such as, for example, tenofovir alafenamide, as well as those described in U.S. Patent Application Publication No. US 2008/0286230 Al, incorporated herein in its entirety by reference. Methods for preparing stereoselective phosphoramidate or phosphonamidate containing actives are described in, for example, U.S. Patent No. 8,816,074, as well as U.S. Patent Application Publications No. US 2011/0245484 Al and US 2008/0286230 Al, all of which incorporated herein in their entireties by reference.

(b) Capsid Inhibitors

As described herein, the term“capsid inhibitor” includes compounds that are capable of inhibiting the expression and/or function of a capsid protein either directly or indirectly.

For example, a capsid inhibitor may include, but is not limited to, any compound that inhibits capsid assembly, induces formation of non-capsid polymers, promotes excess capsid assembly or misdirected capsid assembly, affects capsid stabilization, and/or inhibits encapsidation of RNA (pgRNA). Capsid inhibitors also include any compound that inhibits capsid function in a downstream event(s) within the replication process (e.g., viral DNA synthesis, transport of relaxed circular DNA (rcDNA) into the nucleus, covalently closed circular DNA (cccDNA) formation, virus maturation, budding and/or release, and the like). For example, in certain embodiments, the inhibitor detectably inhibits the expression level or biological activity of the capsid protein as measured, e.g., using an assay described herein. In certain embodiments, the inhibitor inhibits the level of rcDNA and downstream products of viral life cycle by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.

Reported capsid inhibitors include, but are not limited to, compounds described in International Patent Applications Publication Nos WO 2013006394, WO 2014106019, and WO2014089296, all of which incorporated herein in their entireties by reference.

Reported capsid inhibitors also include, but are not limited to, the following compounds and pharmaceutically acceptable salts and/or solvates thereof: Bay -41-4109 (see Int’l Patent Application Publication No. WO 2013144129), AT-61 (see Int’l Patent

Application Publication No. WO 1998033501; and King, et al, 1998, Antimicrob. Agents Chemother. 42(12):3179-3186), DVR-01 and DVR-23 (see Int’l Patent Application

Publication No. WO 2013006394; and Campagna, et al, 2013, J. Virol. 87(l2):693l, all of which incorporated herein in their entireties by reference.

In addition, reported capsid inhibitors include, but are not limited to, those generally and specifically described in U.S. Patent Application Publication Nos. US 2015/0225355, US 2015/0132258, US 2016/0083383, US 2016/0052921 and Int’l Patent Application Publication Nos. WO 2013096744, WO 2014165128, WO 2014033170, WO 2014033167, WO

2014033176, WO 2014131847, WO 2014161888, WO 2014184350, WO 2014184365, WO 2015059212, WO 2015011281, WO 2015118057, WO 2015109130, WO 2015073774,

WO 2015180631, WO 2015138895, WO 2016089990, WO 2017015451, WO 2016183266, WO 2017011552, WO 2017048950, WO2017048954, WO 2017048962, WO 2017064156 and are incorporated herein in their entirety by reference.

(c) cccDNA Formation Inhibitors

Covalently closed circular DNA (cccDNA) is generated in the cell nucleus from viral rcDNA and serves as the transcription template for viral mRNAs. As described herein, the term“cccDNA formation inhibitor” includes compounds that are capable of inhibiting the formation and/or stability of cccDNA either directly or indirectly. For example, a cccDNA formation inhibitor may include, but is not limited to, any compound that inhibits capsid disassembly, rcDNA entry into the nucleus, and/or the conversion of rcDNA into cccDNA. For example, in certain embodiments, the inhibitor detectably inhibits the formation and/or stability of the cccDNA as measured, e.g., using an assay described herein. In certain embodiments, the inhibitor inhibits the formation and/or stability of cccDNA by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.

Reported cccDNA formation inhibitors include, but are not limited to, compounds described in Int’l Patent Application Publication No. WO 2013130703, and are incorporated herein in their entirety by reference.

In addition, reported cccDNA formation inhibitors include, but are not limited to, those generally and specifically described in U.S. Patent Application Publication No. US 2015/0038515 Al, and are incorporated herein in their entirety by reference.

(d) RNA Destabilizer

As used herein, the term“RNA destabilizer” refers to a molecule, or a salt or solvate thereof, that reduces the total amount of HBV RNA in mammalian cell culture or in a live human subject. In a non-limiting example, an RNA destabilizer reduces the amount of the RNA transcript(s) encoding one or more of the following HBV proteins: surface antigen, core protein, RNA polymerase, and e antigen. In certain embodiments, the RNA destabilizer reduces the total amount of HBV RNA in mammalian cell culture or in a live human subject by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.

Reported RNA destabilizers include compounds described in U.S. Patent No.

8,921,381, as well as compounds described in U.S. Patent Application Publication Nos. US 2015/0087659 and US 2013/0303552, all of which are incorporated herein in their entireties by reference.

In addition, reported RNA destabilizers include, but are not limited to, those generally and specifically described in Int’l Patent Application Publication Nos. WO 2015113990, WO 2015173164, US 2016/0122344, WO 2016107832, WO 2016023877, WO 2016128335, WO 2016177655, WO 2016071215, WO 2017013046, WO 2017016921, WO 2017016960, WO 2017017042, WO 2017017043, WO 2017102648, WO 2017108630, WO 2017114812, WO 2017140821, WO 2018085619, and are incorporated herein in their entirety by reference.

(e) Oligomeric Nucleotides Targeted Against the HBV Genome

Reported oligomeric nucleotides targeted against the HBV genome include, but are not limited to, Arrowhead- ARC-520 (see U.S. Patent No. 8,809,293; and Wooddell et al, 2013, Molecular Therapy 2l(5):973-985, all of which incorporated herein in their entireties by reference).

In certain embodiments, the oligomeric nucleotides can be designed to target one or more genes and/or transcripts of the HBV genome. Oligomeric nucleotide targeted to the HBV genome also include, but are not limited to, isolated, double stranded, siRNA molecules, that each include a sense strand and an antisense strand that is hybridized to the sense strand. In certain embodiments, the siRNA target one or more genes and/or transcripts of the HBV genome.

(f) Immunostimulators

Checkpoint Inhibitors

As described herein, the term“checkpoint inhibitor” includes any compound that is capable of inhibiting immune checkpoint molecules that are regulators of the immune system (e.g., stimulate or inhibit immune system activity). For example, some checkpoint inhibitors block inhibitory checkpoint molecules, thereby stimulating immune system function, such as stimulation of T cell activity against cancer cells. A non-limting example of a checkpoint inhibitor is a PD-Ll inhibitor.

As described herein, the term“PD-L1 inhibitor” includes any compound that is capable of inhibiting the expression and/or function of the protein Programmed Death-Ligand 1 (PD-L1) either directly or indirectly. PD-L1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a type 1 transmembrane protein that plays a major role in suppressing the adaptive arm of immune system during pregnancy, tissue allograft transplants, autoimmune disease, and hepatitis. PD-L1 binds to its receptor, the inhibitory checkpoint molecule PD-l (which is found on activated T cells, B cells, and myeloid cells) so as to modulate activation or inhibition of the adaptive arm of immune system. In certain embodiments, the PD-L1 inhibitor inhibits the expression and/or function of PD-L 1 by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.

Reported PD-L1 Inhibitors include, but are not limited to, compounds recited in one of the following patent application publications: US 2018/0057455; US 2018/0057486; WO 2017/106634; WO 2018/026971; WO 2018/045142; WO 2018/118848; WO 2018/119221; WO 2018/119236; WO 2018/119266; WO 2018/119286; WO 2018/121560; WO

2019/076343; WO 2019/087214; and are incorporated herein in their entirety by reference.

(g) GalNAc-siRNA Conjugates Targeted Against an HBV Gene Transcript

“GalNAc” is the abbreviation for N-acetylgalactosamine, and“siRNA” is the abbreviation for small interfering RNA. An siRNA that targets an HBV gene transcript is covalently bonded to GalNAc in a GalNAc-siRNA conjugate useful in the practice of the present invention. While not wishing to be bound by theory, it is believed that GalNAc binds to asialoglycoprotein receptors on hepatocytes thereby facilitating the targeting of the siRNA to the hepatocytes that are infected with HBV. The siRNA enter the infected hepatocytes and stimulate destruction of HBV gene transcripts by the phenomenon of RNA interference.

Examples of GalNAc-siRNA conjugates useful in the practice of this aspect of the present invention are set forth in published international application PCT/CA2017/050447 (PCT Application Publication number WO/2017/177326, published on October 19, 2017) which is hereby incorporated by reference in its entirety.

A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-E_max equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to elsewhere herein may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to elsewhere herein are the concentration-effect curve, isobologram curve and combination index curve, respectively.

Methods

The invention provides a method of treating or preventing hepatitis virus infection in a subject. In certain embodiments, the infection comprises hepatitis B virus (HBV) infection. In other embodiments, the infection comprises hepatitis D virus (HDV) infection. In yet other embodiments, the method comprises administering to the subject in need thereof a therapeutically effective amount of at least one compound of the invention. In yet other embodiments, the compound of the invention is the only antiviral agent administered to the subject. In yet other embodiments, the at least one compound is administered to the subject in a pharmaceutically acceptable composition. In yet other embodiments, the subject is further administered at least one additional agent useful for treating the hepatitis virus infection. In yet other embodiments, the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitor; capsid inhibitor;

cccDNA formation inhibitor; RNA destabilizer; oligomeric nucleotide targeted against the HBV genome; immunostimulator, such as checkpoint inhibitor (e.g, PD-L1 inhibitor); and GalNAc-siRNA conjugate targeted against an HBV gene transcript. In yet other

embodiments, the subject is co-administered the at least one compound and the at least one additional agent. In yet other embodiments, the at least one compound and the at least one additional agent are coformulated.

The invention further provides a method of inhibiting and/or reducing HBV surface antigen (HBsAg) secretion either directly or indirectly in a subject. In certain embodiments, the method comprises administering to the subject in need thereof a therapeutically effective amount of at least one compound of the invention. In other embodiments, the at least one compound is administered to the subject in a pharmaceutically acceptable composition. In yet other embodiments, the compound of the invention is the only antiviral agent

administered to the subject. In yet other embodiments, the subject is further administered at least one additional agent useful for treating HBV and/or HDV infection. In yet other embodiments, the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitor; capsid inhibitor; cccDNA formation inhibitor; RNA destabilizer; oligomeric nucleotide targeted against the HBV genome;

immunostimulator; and GalNAc-siRNA conjugate targeted against an HBV gene transcript.

In yet other embodiments, the subject is co-administered the at least one compound and the at least one additional agent. In yet other embodiments, the at least one compound and the at least one additional agent are coformulated.

In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

Pharmaceutical Compositions and Formulations

The invention provides pharmaceutical compositions comprising at least one compound of the invention or a salt or solvate thereof, which are useful to practice methods of the invention. Such a pharmaceutical composition may consist of at least one compound of the invention or a salt or solvate thereof, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one compound of the invention or a salt or solvate thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combinations of these. At least one compound of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

In certain embodiments, the pharmaceutical compositions useful for practicing the method of the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 1,000 mg/kg/day.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous, or another route of administration. A composition useful within the methods of the invention may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically- based formulations.

In certain embodiments, the compositions of the invention are part of a

pharmaceutical matrix, which allows for manipulation of insoluble materials and

improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions. By way of example, a

pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes.

The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit.

As used herein, a“unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the invention and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMBUMIN®), solubilized gelatins (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

Formulations may be employed in admixtures with conventional excipients, /. e.. pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring, and/or fragrance-conferring substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic, anxiolytics or hypnotic agents. As used herein,“additional ingredients” include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.

The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and any combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05-0.5% sorbic acid.

The composition may include an antioxidant and a chelating agent that inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alpha- tocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. The chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or weting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol,

polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants. Known preservatives include, but are not limited to, methyl, ethyl, or «-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.

Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an“oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, ionic and non-ionic surfactants, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e.. such as with a

physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdermal patch, as known to those skilled in the art.

Administration/Dosing

The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present invention to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 0.01 mg/kg to 100 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

The compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as, but not limited to, type and severity of the disease being treated, and type and age of the animal.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease or disorder in a patient.

In certain embodiments, the compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account.

Compounds of the invention for administration may be in the range of from about 1 pg to about 7,500 mg, about 20 pg to about 7,000 mg, about 40 pg to about 6,500 mg, about 80 p g to about 6,000 mg, about 100 p g to about 5,500 mg, about 200 p g to about 5,000 mg, about 400 p g to about 4,000 mg, about 800 p g to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments there-in-between.

In some embodiments, the dose of a compound of the invention is from about 0.5 pg and about 5,000 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

In certain embodiments, the present invention is directed to a packaged

pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second

pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

The term“container” includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake. For example, in certain embodiments, the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound’s ability to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.

Administration

Routes of administration of any of the compositions of the invention include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g, trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees, liquids, drops, capsules, caplets and gelcaps. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, a paste, a gel, toothpaste, a mouthwash, a coating, an oral rinse, or an emulsion. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic, generally recognized as safe (GRAS) pharmaceutically excipients which are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.

Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Patents Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation. Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. The capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin from animal-derived collagen or from a hypromellose, a modified form of cellulose, and manufactured using optional mixtures of gelatin, water and plasticizers such as sorbitol or glycerol. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

For oral administration, the compounds of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents; fillers; lubricants; disintegrates; or wetting agents. If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY® film coating systems available from Colorcon, West Point, Pa. ( e.g ., OPADRY® OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and

OPADRY® White, 32K18400). It is understood that similar type of film coating or polymeric products from other companies may be used.

A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients.

Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surface-active agents include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalbne cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, com starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using“wet” granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e., having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e., drug) by forming a solid dispersion or solid solution.

U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) will melt.

The present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds useful within the methods of the invention, and a further layer providing for the immediate release of one or more compounds useful within the methods of the invention. Using a wax/pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.

Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non- aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl para-hydroxy benzoates or sorbic acid). Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.

Parenteral Administration

As used herein,“parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold in devices such as patient-controlled analgesia (PCA) devices. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e.. powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non toxic parenterally acceptable diluent or solvent, such as water or l,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form in a recombinant human albumin, a fluidized gelatin, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Topical Administration

An obstacle for topical administration of pharmaceuticals is the stratum comeum layer of the epidermis. The stratum comeum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes comified and living cells. One of the factors that limit the penetration rate (flux) of a compound through the stratum comeum is the amount of the active substance that can be loaded or applied onto the skin surface. The greater the amount of active substance which is applied per unit of area of the skin, the greater the concentration gradient between the skin surface and the lower layers of the skin, and in turn the greater the diffusion force of the active substance through the skin. Therefore, a formulation containing a greater concentration of the active substance is more likely to result in penetration of the active substance through the skin, and more of it, and at a more consistent rate, than a formulation having a lesser concentration, all other things being equal.

Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

Enhancers of permeation may be used. These materials increase the rate of penetration of drugs across the skin. Typical enhancers in the art include ethanol, glycerol monolaurate, PGML (polyethylene glycol monolaurate), dimethylsulfoxide, and the like. Other enhancers include oleic acid, oleyl alcohol, ethoxy diglycol, laurocapram,

alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositions of the invention may contain liposomes. The composition of the liposomes and their use are known in the art (i.e., U.S. Patent No. 6,323,219).

In alternative embodiments, the topically active pharmaceutical composition may be optionally combined with other ingredients such as adjuvants, anti-oxidants, chelating agents, surfactants, foaming agents, wetting agents, emulsifying agents, viscosifiers, buffering agents, preservatives, and the like. In other embodiments, a permeation or penetration enhancer is included in the composition and is effective in improving the percutaneous penetration of the active ingredient into and through the stratum comeum with respect to a composition lacking the permeation enhancer. Various permeation enhancers, including oleic acid, oleyl alcohol, ethoxy diglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known to those of skill in the art. In another aspect, the composition may further comprise a hydrotropic agent, which functions to increase disorder in the structure of the stratum comeum, and thus allows increased transport across the stratum comeum. Various hydrotropic agents such as isopropyl alcohol, propylene glycol, or sodium xylene sulfonate, are known to those of skill in the art.

The topically active pharmaceutical composition should be applied in an amount effective to affect desired changes. As used herein“amount effective” shall mean an amount sufficient to cover the region of skin surface where a change is desired. An active compound should be present in the amount of from about 0.0001% to about 15% by weight volume of the composition. For example, it should be present in an amount from about 0.0005% to about 5% of the composition; for example, it should be present in an amount of from about 0.001% to about 1% of the composition. Such compounds may be synthetically-or naturally derived.

Buccal Administration

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) of the active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, may have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein. The examples of formulations described herein are not exhaustive and it is understood that the invention includes additional modifications of these and other formulations not described herein, but which are known to those of skill in the art.

Rectal Administration

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.

Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e.. about 20 °C) and which is liquid at the rectal temperature of the subject (i.e., about 37 °C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.

Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790.

Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO 97/47285, WO 93/18755, and WO 90/11757. Controlled Release Formulations and Drug Delivery Systems:

In certain embodiments, the compositions and/or formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

In certain embodiments of the invention, the compounds useful within the invention are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, include a delay of from about 10 minutes up to about 12 hours.

The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration. Definitions

As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, separation science, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.

In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

In this document, the terms“a,”“an,” or“the” are used to include one or more than one unless the context clearly dictates otherwise. The term“or” is used to refer to a nonexclusive“or” unless otherwise indicated. The statement“at least one of A and B” or“at least one of A or B” has the same meaning as“A, B, or A and B.”

As used herein, the term“about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein,“about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. In one aspect, the terms“co-administered” and“co-administration” as relating to a subject refer to administering to the subject a compound and/or composition of the invention along with a compound and/or composition that may also treat or prevent a disease or disorder contemplated herein. In certain embodiments, the co-administered compounds and/or compositions are administered separately, or in any kind of combination as part of a single therapeutic approach. The co-administered compound and/or composition may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.

For the purposes of the present invention the terms“compound,”“analog,” and “composition of matter” stand equally well for the prodrug agent described herein, including all enantiomeric forms, diastereoisomeric forms, salts, and the like, and the terms “compound,”“analog,” and“composition of matter” are used interchangeably throughout the present specification.

As used herein, a“disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate.

As used herein, a“disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.

As used herein, an“effective amount,”“therapeutically effective amount” or “pharmaceutically effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.

“Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the composition and/or compound of the invention in a kit. The instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container that contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression

communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.

As used herein, the term“pharmaceutical composition” or“composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject.

As used herein, the term“pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the invention, and is relatively non-toxic, /. e.. the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term“pharmaceutically acceptable carrier” means a

pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil;

glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

As used herein,“pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The“pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention.

Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

As used herein, the language“pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.

As used herein, a“pharmaceutically effective amount,”“therapeutically effective amount,” or“effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.

The term“prevent,”“preventing” or“prevention,” as used herein, means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences. Disease, condition and disorder are used interchangeably herein.

As used herein, a“patient” or“subject” may be a human or non-human mammal or a bird. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human.

The term“treat,”“treating” or“treatment,” as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.

Certain abbreviations used herein follow: cccDNA, covalently closed circular DNA; CPME, cyclopentylmethane; DMSO, dimethylsulfoxide; DSC, Differential scanning calorimetry; DVS, Dynamic Vapor Sorption; GC, gas chromatography; GPC, gel- permeation chromatography; HBV, hepatitis B virus; HDV, hepatitis D virus; HPLC, high performance liquid chromatography; MTBE, methyl tert-butyl ether; NMR, Nuclear Magnetic Resonance; PLM, polarized light microscopy; rh, relative humidity; sAg, surface antigen; TGA, thermogravimetric analysis; TG-FTIR, Thermogravimetric Fourier transform infrared; THF, tetrahydrofuran; TLC, thin layer chromatography; XRPD, X-ray powder diffraction.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

It is to be understood that, wherever values and ranges are provided herein, the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, all values and ranges encompassed by these values and ranges are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. The description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. For example, a range of“about 0.1% to about 5%” or“about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement“about X to Y” has the same meaning as“about X to about Y,” unless indicated otherwise. Likewise, the statement“about X, Y, or about Z” has the same meaning as“about X, about Y, or about Z,” unless indicated otherwise. This applies regardless of the breadth of the range.

The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein. EXAMPLES

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

Methods

X-Ray Powder Diffraction (XPRD)

Powder X-ray diffraction was carried out with a Stoe Stadi P diffractometer equipped with a MythenlK detector operating with Cu-K_aΐ radiation. The measurements were performed in transmission mode at a tube voltage of 40 kV and a tube power of 40 mA. A curved Ge monochromator enabled testing with Cu-K_aΐ radiation. The following parameters were used: 0.02° 20 step size, 12 s step time, 1.5-50.5° 20 scanning range, and l°20 detector step (detector modein step scan). For a typical sample preparation, about 20 mg of sample was placed between two acetate foils and mounted into a Stoe transmission sample holder. The sample was rotated during the measurement. All sample preparation and measurement was carried out under an ambient air atmosphere.

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry was carried out with a TA Instruments Q2000 instrument in a hermetically sealed aluminum sample pan at a heating rate of 10 K/min.

Nuclear Magnetic Resonance (NMR)

A Bruker DPX300 spectrometer was used for NMR measurements. Proton frequency of 300.13 MHz; 30° excitation pulse; recycle delay of 1 s; accumulation of 16 scans;

deuterated DMSO as the solvent; solvent peak used for referencing; chemical shifts reported on the TMS scale.

Light Microscopy

Light microscopy was performed on a Leitz Orthoplan polarized microscope part #130880, generally a 10x10 magnification was applied. Raman Spectroscopy

FT-Raman spectra were recorded on a Bruker MultiRAM FT-Raman system with a near infrared Nd:YAG laser operating at 1064 nm and a liquid nitrogen-cooled germanium detector. 64 scans with a resolution of 2 cm ¹ were accumulated in the range from 3500 to - 50 cm ¹; however, only data above 100 cm ¹ was evaluated due to filter cutoff effects.

Nominal laser powers were typically 100 or 300 mW.

Thermogravimetric Fourier transform infrared (TG-FTIR) Spectroscopy

Thermogravimetric measurements were carried out with a Netzsch Thermo- Microbalance TG 209 coupled to a Bruker FTIR Spectrometer Vector 22 (sample pans with a pinhole, N₂ atmosphere, heating rate 10 K/min).

Dynamic Vapor Sorption (DVS) Methods

DVS measurements were performed with an SPS1 l-lOOn“Sorptions Priifsystem” from ProUmid (formerly“Projekt Messtechnik“), August- agei-Str. 23, 89079 Ulm

(Germany). About 5 - 20 mg of sample was placed into an aluminum sample pan. Humidity change rates of 5% per hour were used. The applied measurement program is visualized in the figures (thicker line trace). The sample was placed on an aluminum holder on top of a microbalance and allowed to equilibrate at 50% RH before starting the pre-defmed humidity programs:

(1) 2 h at 50% RH

(2) 50 0% RH (5%/h); 5 h at 0% RH

(3) 0 95% RH (5%/h); 5 h at 95% RH

(4) 95 50% RH (5%/h); 2 h at 50% RH

Example 1: Purification and Crystallization of (I) Free Form

A mixture of carbonyldiimidazole (CDI) (97 g, 0.6 mol), (l-methyl-l,2,4-triazol-3- yl)methanol (67.7 g, 0.6 mol), and 2-MeTHF (1150 mL) was stirred at room temperature (20 °C) for 2h. The reaction mixture was added to a stirred mixture of (S)- 1 -amino-N-(3-chloro- 4-fluorophenyl)-7-fluoro-2,3-dihydro-lH-indene-4-carboxamide hydrochloride (143.3 g, 0.4 mol), 2-MeTHF (860 mL), and N,N-diisopropylethylamine (129 g, 1 mol). The mixture was heated to 60 °C and the contents maintained at 60 °C for 6h. The contents were cooled to 20 °C, water (285 mL) was added, and the mixture was stirred for 15 min. The layers were separated and the organic layer was washed with water two times (285 mL each time). The organic layer was filtered through a pad of Celite, and rinsed with 145 mL 2-MeTHF (1 volume). The solution was concentrated to about 700 mL under vacuum. 2-propanol (710 mL) was added and the mixture concentrated to about 700 mL. Additional 2-propanol (710 mL) was added and the mixture concentrated to approximately 1000 mL. The slurry was heated at 45 °C for 2h and cooled to 20 °C, over lh. The slurry was stirred at 20 °C for 2h, and the product filtered. The product was washed with 2-propanol two times (286 mL each wash), and the wet product was dried under vacuum to give (1 -methyl- 1H- 1,2, 4-triazol-3- yl)methyl (S)-(4-((3-chloro-4-fluorophenyl)carbamoyl)-7-fluoro-2,3-dihydro-lH-inden-l- yl)carbamate (I) as solid (144.5 g, 78.4% yield).

Example 2: Characterization of (I) Crystalline Form 1

Powder X-ray Diffraction

Pure (I) was characterized by PXRD and the sample was found to be crystalline in nature. The diffractogram of the transmission mode measurement is presented in FIG. 1. Table 1 reports the XRPD peaks for this solid form. d-Value analysis was performed using EVA software from Bruker (version 14, 0, 0, 0).

Table 1. XRPD peaks for Crystalline Form 1

Solubility Screen

In order to design crystallizations and slurry/evaporation experimental conditions, the approximate solubility of (I) was tested in a variety of solvents and solvent mixtures. (I) was found to be practically insoluble in water, water: methanol (1 : 1), tert-butyl methyl ether (TBME) and toluene; sparingly soluble in ethyl acetate (EtOAc), isopropanol (i-PrOH), 2- methyl tetrahydrofuran (2-Me THF), dichloromethane (DCM), ethanol (EtOH), acetonitrile (MeCN), and water: methanol (1 :9); and soluble in methanol (MeOH), acetone,

tetrahydrofuran (THF), wateracetone (1:9) and THF:water (1 : 1).

Approximate solubilities were determined by incremental addition of solvent to about 10 mg of the compound. If the substance was not dissolved by addition of a total of at least

10 mL solvent, the solubility is indicated as <1 mg/mL. Due to the experimental error inherent in this method, the solubility values were intended to be regarded as rough estimates and were used solely for the design of crystallization experiments. Table 2. Approximate solubility of (I), Crystalline Form 1

TG-FTIR analysis

TG-FTIR analysis indicated that a small amount of i-PrOH was present in the Crystalline Form 1 sample of (I) studied. The i-PrOH was released at ~l80°C, close to the melting point of (I) (l82.9°C). It was not clear whether the solvent was trapped in the particles and released when the compound melted, or if solvent was incorporated within the crystalline structure and released as part of the decomposition process. 'H NMR suggested (see elsewhere herein) that the solvent was present even at RT in similar amounts, therefore it is possible that the solvent was trapped in the particles during the crystallization process and was not incorporated into the crystal lattice of Crystalline Form 1. The thermogram is depicted in FIG. 2.

DSC Analysis

DSC showed a sharp endotherm event starting at l8l.0°C (onset T) centered at l82.9°C which is associated with an enthalpy of -0.95 J/g, assigned to melting. Glass transition was not observed in the DSC, therefore it is unlikely that amorphous material is present in the sample (FIG. 3).

NMR spectroscopy

The 'H NMR spectrum of Crystalline Form 1 was recorded in DMSO d₆ and the observed spectrum agreed with the structure of (I). As suggested from the TG-FTIR, isopropanol peaks were observed at d = 1.04, with a doublet assigned to the -CH₃ groups (J = 6 Hz) and a multiplet partially overlapped with a water peak, centered at 3.78 ppm. The i- PrOH ratio measured in the 'H-NMR spectrum was in near agreement with the value observed in the TG-FTIR spectrum (~ 0.4 %wt).

DVS Studies

A maximum mass uptake of 0.15% wt at 95% of r.h. and less than 0.1% at 80% r.h. were observed. Based on these measurements, the material was considered non-hygroscopic (FIGs. 4A-4B).

Light microscopy

Light microscopy imaging confirmed the crystalline nature of the compound showing birefringent particles and agglomerates (FIG. 5).

Raman Spectroscopy

The Raman spectrum of Crystalline Form 1 showed sharp bands further confirming the crystalline nature of the compound. The complete spectrum is displayed in FIG. 6A and a zoomed-in region of the fingerprint region is depicted in FIG. 6B.

Example 3: Polymorph Screen of (I) Free Base

Twenty suspension equilibration experiments were performed by suspending (I) free base in a variety of solvents at three different temperatures (RT, 5 °C and 50 °C). The suspensions were filtered by centrifugation. Three more experiments were carried out using a slow evaporation technique and seven were carried out using a lyophilized substance (high energy phase) in order to try to obtain a different crystalline form. Three additional cooling crystallization experiments were also carried out. All the resulting solid materials were characterized by powder X-ray diffraction (PXRD). Only one of set of conditions (Exp. 29) gave a mixture of a new form with the starting material. Some of the diffractograms suggested partial or total loss of crystallinity (see for example Exp. 35). Some of the relative intensities in the PXRD spectra of certain experiments were different from the previously recorded spectrum of Crystalline Form 1, especially at high angle; however, all of the reflections of these samples were observed at the same 2 theta positions, suggesting the presence of preferred orientations. The summary of the results is presented in Table 3 and FIGs. 7A-7H.

Table 3. Polymorph Screen Conditions Summary

* Note: all solvent mixtures are reported as (v/v) ratios.

Example 4: Isolation and Characterization of Crystalline Form 2 A new form, labeled as Crystalline Form 2, was obtained in a minor amount along with Crystalline Form 1 in polymorph screen Exp. 29. This form was a metastable form which formed on the walls of the crystallization vial during the slow evaporation process of THF (FIG. 7G). The diffractogram shares all the reflections with the starting material, in addition to a number of new peaks (2Q = 2.5, 3, 5.6, 12.6, 23.2°).

1H NMR was carried out to exclude degradation of (I) and the chemical shifts and J- coupling values were found to be the same between the starting material and the Exp. 29 material (FIG. 8A). Some extra resonances were found in the Exp. 29 material (FIG. 8B), attributable to remaining THF and trace butylated hydroxy toluene (a common additive in commercial THF to prevent oxidation). The isopropanol resonances disappeared during crystallization, likely due to evaporation alongside the THF solvent.

The thermodynamic stability of Crystalline Form 2 was tested by suspending the compound at RT in DCM (Exp. 30). The additional PXRD peaks disappeared after 5 days of stirring, suggesting that Crystalline Form 1 is the most stable form. The diffractogram of the competitive slurry experiment is shown in FIG. 9.

Enumerated Embodiments

The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance.

Embodiment 1 provides (l-methyl-lH-l,2,4-triazol-3-yl)methyl (S)-(4-((3-chloro-4- fluorophenyl)carbamoyl)-7-fluoro-2,3-dihydro-lH-inden-l-yl)carbamate, (I) free base crystalline solid, which is characterized by an X-ray diffraction pattern (XRPD) with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 2.61, 4.67, 8.06, 9.14, 9.49, 9.95, 10.53, 14.06, 15.94, 16.66, 17.05, 18.37, 19.08, 19.82, 20.29, 21.06, 21.82, 21.98, 22.98, 23.56, 24.18, 24.35, 24.67, 24.96, 25.27, 25.89, 26.19, 26.42, 26.76, 27.21,

27.71, 27.87, 28.13, 28.59, 28.79, 29.86, 30.53, 30.72, 31.06, 31.37, 31.67, 32.21, 32.87,

33.29, 33.64, 34.63, 35.17, 36.56, 36.94, 37.25, 37.57, 38.10, 38.28, 38.89, 39.33, 40.22, and

40.47.

Embodiment 2 provides the solid of Embodiment 1, wherein the solid is obtained by crystallizing (I) free base in at least one solvent selected from the group consisting of acetonitrile, tetrahydrofuran (THF), 2-methyl tetrahydrofuran, acetone, isopropanol, methanol, ethanol, toluene, ethyl acetate, t-butyl methyl ether, dichloromethane, THF/water mixture, acetone/water mixture, and methanol/water mixture. Embodiment 3 provides the solid of Embodiment 2, wherein the THF/water mixture is about 1: 1 (v/v).

Embodiment 4 provides the solid of Embodiment 2, wherein the acetone/water mixture is about 9: 1 (v/v).

Embodiment 5 provides the solid of Embodiment 2, wherein the methanol/water mixture is about 1: 1 (v/v).

Embodiment 6 provides the solid of Embodiment 2, wherein the solid comprises isopropanol.

Embodiment 7 provides the solid of any of Embodiments 1-6, further characterized by a DSC thermogram having an exotherm at about l82.9°C.

Embodiment 8 provides the solid of Embodiment 7, wherein the DSC measurement is performed by heating the solid starting from at most 25 °C at 10 °C/min to at least 20 °C above the solid’s melting point.

Embodiment 9 provides the solid of Embodiment 7, wherein the DSC measurement is performed with a standard aluminum DSC sample pan and cover, with a nitrogen gas purge rate at about 50 mL/min.

Embodiment 10 provides a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and the solid of any one of Embodiments 1-9.

Embodiment 11 provides the pharmaceutical composition of Embodiment 10, wherein the composition is in solid dosage form for oral administration.

Embodiment 12 provides the pharmaceutical composition of any of Embodiments 10-

11, which is part of a tablet, dragee, drop, suppository, capsule, caplet, and/or gel cap.

Embodiment 13 provides the pharmaceutical composition of any of Embodiments 10-

12, further comprising at least one additional agent useful for treating hepatitis virus infection.

Embodiment 14 provides the pharmaceutical composition of Embodiment 13, wherein the hepatitis virus comprises hepatitis B virus (HBV) and/or hepatitis D virus (HDV).

Embodiment 15 provides the pharmaceutical composition of any of Embodiments 13- 14, wherein the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitor; capsid inhibitor; cccDNA formation inhibitor; RNA destabilizer; oligomeric nucleotide targeted against the HBV genome;

immunostimulator; and GalNAc-siRNA conjugate targeted against an HBV gene transcript.

Embodiment 16 provides a method of treating or preventing hepatitis virus infection in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of the solid of any of Embodiments 1-9 and/or the pharmaceutical composition of any of Embodiments 10-15.

Embodiment 17 provides the method of Embodiment 16, wherein the solid and the at least one additional agent are coformulated.

Embodiment 18 provides the method of any of Embodiments 16-17, wherein the subject is infected with HBV.

Embodiment 19 provides the method of Embodiment 18, wherein the subject is further infected with HDV.

Embodiment 20 provides a method of inhibiting and/or reducing hepatitis B virus surface antigen (HBsAg) secretion in a virus-infected subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of the solid of any of Embodiments 1-9 and/or the pharmaceutical composition of any of Embodiments 10- 15.

Embodiment 21 provides the method of any of Embodiments 16-20, wherein the subject is a mammal.

Embodiment 22 provides the method of Embodiment 21, wherein the mammal is a human.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

What is claimed is:

1. (l-methyl-lH-l,2,4-triazol-3-yl)methyl (S)-(4-((3-chloro-4-fluorophenyl)carbamoyl)-

7-fluoro-2,3-dihydro-lH-inden-l-yl)carbamate, (I) free base crystalline solid, which is characterized by an X-ray diffraction pattern (XRPD) with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 2.61, 4.67, 8.06, 9.14, 9.49, 9.95, 10.53, 14.06, 15.94, 16.66, 17.05, 18.37, 19.08, 19.82, 20.29, 21.06, 21.82, 21.98, 22.98, 23.56,

24.18, 24.35, 24.67, 24.96, 25.27, 25.89, 26.19, 26.42, 26.76, 27.21, 27.71, 27.87, 28.13,

28.59, 28.79, 29.86, 30.53, 30.72, 31.06, 31.37, 31.67, 32.21, 32.87, 33.29, 33.64, 34.63,

35.17, 36.56, 36.94, 37.25, 37.57, 38.10, 38.28, 38.89, 39.33, 40.22, and 40.47.

2. The solid of claim 1, wherein the solid is obtained by crystallizing (I) free base in at least one solvent selected from the group consisting of acetonitrile, tetrahydrofuran (THF), 2- methyl tetrahydrofuran, acetone, isopropanol, methanol, ethanol, toluene, ethyl acetate, t- butyl methyl ether, dichloromethane, THF/water mixture, acetone/water mixture, and methanol/water mixture.

3. The solid of claim 2, wherein the THF/water mixture is about 1 : 1 (v/v).

4. The solid of claim 2, wherein the acetone/water mixture is about 9: 1 (v/v).

5. The solid of claim 2, wherein the methanol/water mixture is about 1 : 1 (v/v).

6. The solid of claim 2, wherein the solid comprises isopropanol.

7. The solid of claim 1, further characterized by a DSC thermogram having an exotherm at about l82.9°C.

8. The solid of claim 7, wherein the DSC measurement is performed by heating the solid starting from at most 25 °C at 10 °C/min to at least 20 °C above the solid’s melting point.

9. The solid of claim 7, wherein the DSC measurement is performed with a standard aluminum DSC sample pan and cover, with a nitrogen gas purge rate at about 50 mL/min.

10. A pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and the solid of claim 1.

11. The pharmaceutical composition of claim 10, wherein the composition is in solid dosage form for oral administration.

12. The pharmaceutical composition of claim 10, which is part of a tablet, dragee, drop, suppository, capsule, caplet, and/or gelcap.

13. The pharmaceutical composition of claim 10, further comprising at least one additional agent useful for treating hepatitis virus infection.

14. The pharmaceutical composition of claim 13, wherein the hepatitis virus comprises hepatitis B virus (HBV) and/or hepatitis D virus (HDV).

15. The pharmaceutical composition of claim 13, wherein the at least one additional agent comprises at least one selected from the group consisting of reverse transcriptase inhibitor; capsid inhibitor; cccDNA formation inhibitor; RNA destabilizer; oligomeric nucleotide targeted against the HBV genome; immunostimulator; and GalNAc-siRNA conjugate targeted against an HBV gene transcript.

16. A method of treating or preventing hepatitis virus infection in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of the solid of claim 1 and/or the pharmaceutical composition of claim 10.

17. The method of claim 16, wherein the solid and the at least one additional agent are coformulated.

18. The method of claim 16, wherein the subject is infected with HBV.

19. The method of claim 18, wherein the subject is further infected with HDV.

20. A method of inhibiting and/or reducing hepatitis B virus surface antigen (HBsAg) secretion in a virus-infected subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of the solid of claims 1 and/or the pharmaceutical composition of claim 10.

21. The method of claim 16, wherein the subject is a mammal.

22. The method of claim 21, wherein the mammal is a human.