WO2019246427A1 - Crystalline forms of pyridinone-containing tricyclic compounds and methods of preparing and using same - Google Patents

Abstract

The present invention includes crystalline forms of certain substituted pyridinone-containing tricyclic compounds, and compositions comprising the same, that can be used to treat or prevent hepatitis B virus (HBV) 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 Pyridinone-Containing Tricyclic 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. 62/688,659, filed June 22, 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, 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 on being infected with, HBV and/or HDV. There is thus a need to identify scalable synthetic routes for those anti -HBV (or anti -HBV - HDV) antiviral agents. The present invention addresses this need.

BRIEF SUMMARY OF THE INVENTION

The invention provides certain (S)-6-isopropyl-2-methoxy-3-(3-methoxypropoxy)-l0- oxo-5, lO-dihydro-6H-pyrido[l,2-h][l,7]naphthyridine-9-carboxylic acid (I) free acid crystalline solids, each of which is characterized by a certain X-ray diffraction pattern (XRPD). The invention further provides pharmaceutical compositions comprising at least one pharmaceutically acceptable carrier and at least one free acid crystalline solid of the invention. The invention further provides a method of treating or preventing hepatitis B virus infection in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one free acid crystalline solid of the invention and/or a pharmaceutical composition comprising the same. The invention further 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 a therapeutically effective amount of at least one free acid crystalline solid of the invention and/or a pharmaceutical composition comprising the same.

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 is a polarized light microscopy (PLM) image of fSj-6-isopropyl-2-metho\y-3- (3-methoxypropoxy)-l0-oxo-5,l0-dihydro-6H-pyrido[l,2-h][l,7]naphthyridine-9-carboxylic acid (I) crystalline pattern 1.

FIG. 2 is an XRPD spectrum of crystalline pattern 1 of (I).

FIG. 3 is a TGA analysis of crystalline pattern 1 of (I).

FIG. 4 is a DSC analysis of crystalline pattern 1 of (I).

FIG. 5 is an overlaid XRPD spectrum of (a) crystalline pattern 1 reference and (b) the wet solid collected from an ethyl acetate slurry (crystalline patern 2).

FIG. 6 is an overlaid XRPD spectrum of (a) crystalline patern 1 reference and crystalline patern 3 derived from: (b) dried solid collected from acetone evaporation (c) wet solid collected from ethanol slurry (d) wet solid collected from 2-methyl-THF (2-methyl- tetrahydrofuran) slurry and (e) wet solid collected from methanol slurry.

FIG. 7 is an overlaid XRPD spectrum of (a) crystalline patern 1 reference and (b) dried solid collected from dichloromethane evaporation (crystalline patern 4).

FIG. 8 is an overlaid XRPD spectrum of (a) crystalline patern 1 reference and (b) dried solid collected from water/acetonitrile evaporation (crystalline patern 5).

FIG. 9 is an overlaid XRPD spectrum of various crystalline patern 1 materials: (a) starting material reference, (b) wet solid collected from 2-propanol slurry, (c) wet solid collected from water slurry, (d) wet solid collected from water/methanol slurry, and (e) wet solid collected from water/ethanol slurry.

FIG. 10 is an overlaid XRPD spectrum of various crystalline patern 1 materials: (a) starting material reference, (b) wet solid collected from CPME slurry, (c) wet solid collected from toluene slurry, (d) wet solid collected from MTBE slurry, and (e) wet solid collected from isopropyl acetate slurry.

FIG. 11 is an XRPD analysis of solid collected from ethyl acetate slurry (crystalline patern 2).

FIG. 12 is a TGA analysis of solid collected from ethyl acetate slurry (crystalline patern 2).

FIG. 13 is a DSC analysis of solid collected from ethyl acetate slurry (crystalline patern 2).

FIG. 14 is an XRPD analysis of solid collected from 2-methyl-THF (crystalline patern 3).

FIG. 15 is a TGA analysis of solid collected from 2-methyl-THF slurry (crystalline patern 3).

FIG. 16 is a DSC analysis of solid collected from 2-methyl-THF slurry (crystalline pattern 3).

FIG. 17 is an XRPD analysis of solid collected from dichloromethane (crystalline pattern 4).

FIG. 18 is a TGA analysis of solid collected from dichloromethane evaporation (crystalline pattern 4).

FIG. 19 is a DSC analysis of solid collected from dichloromethane evaporation (crystalline pattern 4).

FIG. 20 is an XRPD analysis of solid collected from water/acetonitrile evaporation (crystalline pattern 5).

FIG. 21 is a TGA analysis of solid collected from water/acetonitrile evaporation (crystalline pattern 5).

FIG. 22 is a DSC analysis of solid collected from water/acetonitrile evaporation (crystalline pattern 5).

FIG. 23 is an XRPD analysis of solid collected from THF slurry (crystalline pattern

6).

FIG. 24 is a TGA analysis of solid collected from THF slurry (crystalline pattern 6).

FIG. 25 is a DSC analysis of solid collected from THF slurry (crystalline pattern 6).

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 HBV-HDV) infection and related conditions in a subject. In certain embodiments, the compounds were originally described in WO 2018/085619, published on May 11, 2018, which is incorporated herein in its entirety by reference.

In certain embodiments, the present invention provides developable forms of [(<S)-6- isopropy l-2-methoxy-3 -(3 -methoxy propoxy )- 10-oxo-5 , 10-dihy dro-6H-pyrido[ 1 ,2- h][l,7]naphthyridine-9-carboxylic acid], (I), or a salt or solvate thereof:

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

As described herein, six crystalline solvated forms of (I) free acid were identified during a crystallization screen from solvents such as ethyl acetate, 2-methyl-THF, THF, dichloromethane, water/acetonitrile, acetone, ethanol, methanol, water/ethanol, water, isopropyl acetate, cyclopentylmethane (CPME), toluene, and methyl tert-butyl ether (MTBE). Two solvates comprising ethyl acetate or dichloromethane (crystalline patterns 2 and 4, respectively) and four anhydrous forms (crystalline patterns 1, 3, 5 and 6) were identified in such screens.

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 ²H, 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 are synthesized using techniques and materials described herein 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 infections include: (a) reverse transcriptase inhibitors; (b) capsid inhibitors; (c) cccDNA formation inhibitors; (d) sAg secretion inhibitors; (e) oligomeric nucleotides targeted to the Hepatitis B genome; and (f) immunostimulators.

(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, (lR,2R,3R,5R)-3-(6-amino-9H-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 (lR,2R,3R,5R)-3-(6-amino-9H-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 ((((lR,3R,4R,5R)-3-(6-amino-9H-purin-9-yl)-4-fluoro-5-hydroxy-2-methylenecyclopentyl) methoxyXphenoxy) phosphoryl)-(D or L)-alaninate and methyl ((((lR,2R,3R,4R)-3-fluoro-2- hydroxy-5-methylene-4-(6-oxo-l,6-dihydro-9H-purin-9-yl)cyclopentyl)methoxy)(phenoxy) phosphoryl)-(D or L)-alaninate. Also included are the individual diastereomers thereof, which include, for example, methyl ((R)-(((lR,3R,4R,5R)-3-(6-amino-9H-purin-9-yl)-4- fluoro-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-(D or L)- alaninate and methyl ((S)-(((lR,3R,4R,5R)-3-(6-amino-9H-purin-9-yl)-4-fluoro-5-hydroxy-2- methylenecyclopentyl) methoxyXphenoxy )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) sAg Secretion Inhibitors

As described herein, the term“sAg secretion inhibitor” includes compounds that are capable of inhibiting, either directly or indirectly, the secretion of sAg (S, M and/or L surface antigens) bearing subviral particles and/or DNA containing viral particles from HBV-infected cells. For example, in certain embodiments, the inhibitor detectably inhibits the secretion of sAg as measured, e.g., using assays known in the art or described herein, e.g., ELISA assay or by Western Blot. In certain embodiments, the inhibitor inhibits the secretion of sAg by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%. In certain embodiments, the inhibitor reduces serum levels of sAg in a patient by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or at least 90%.

Reported sAg secretion inhibitors 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 sAg secretion inhibitors 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 and are incorporated herein in their entirety by reference.

(e) Immunostimulators

The term“immunostimulator” includes compounds that are capable of modulating an immune response (e.g., stimulate an immune response (e.g, an adjuvant)).

Immunostimulators include, but are not limited to, polyinosinic:polycytidylic acid (poly I:C) and interferons.

Reported immunostimulators include, but are not limited to, agonists of stimulator of IFN genes (STING) and interleukins. Reported immunostimulators further include, but are not limited to, HBsAg release inhibitors, TLR-7 agonists (such as, but not limited to, GS- 9620, RG-7795), T-cell stimulators (such as, but not limited to, GS-4774), RIG-l inhibitors (such as, but not limited to, SB-9200), and SMAC-mimetics (such as, but not limited to, Birinapant).

(†) Oligomeric Nucleotides

Reported oligomeric nucleotides targeted to the Hepatitis B 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 Hepatitis B 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.

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 HBV and hepatitis D virus (HDV) co- 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; sAg secretion inhibitor; oligomeric nucleotide targeted to the Hepatitis B genome; and immunostimulator. 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 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; sAg secretion inhibitor; oligomeric nucleotide targeted to the Hepatitis B genome; and immunostimulator. 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 some combination 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, i.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 combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

The composition may include an antioxidant and a chelating agent which 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 wetting 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 pg to about 6,000 mg, about 100 pg to about 5,500 mg, about 200 pg to about 5,000 mg, about 400 pg to about 4,000 mg, about 800 pg 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 sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline 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.

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.

Definitions

Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present teachings also consist essentially of, or consist of, the recited components, and that the processes of the present teachings also consist essentially of, or consist of, the recited processing steps.

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.

As used herein, unless defined otherwise, all technical and scientific terms 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 organic chemistry, virology, biochemistry and pharmaceutical sciences are those well-known and commonly employed in the art. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. 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 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.

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.

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; DNA, deoxyribonucleic acid;

DSC, differential scanning calorimetry; GC, gas chromatography; GPC, gel-permeation chromatography; HBsAg, HBV surface antigen; HBV, hepatitis B virus; HDV, hepatitis D virus; HPLC, high pressure liquid chromatography; MTBE, methyl tert-butyl ether; NARTI or NRTI, reverse-transcriptase inhibitor; NtARTI or NtRTI, nucleotide analog reverse- transcriptase inhibitor; NMR, Nuclear Magnetic Resonance; PLM, polarized light microscopy; rcDNA, relaxed circular DNA; sAg, surface antigen; SFC, supercritical fluid chromatography; TGA, thermogravimetric analysis; THF, tetrahydrofuran; TLC, thin layer chromatography; XRPD, X-ray powder diffraction.

Ranges: throughout this disclosure, various aspects of the present invention can be presented in a range format. It should be understood that 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 present invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges 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

Polarized Light Microscopy (PLM)

Photomicrographs were taken using an Olympus BX51 polarized light microscope, equipped with a JENOPTIK ProgRes camera and operated by ProgRes Capture Pro 2.8.8 software. Samples were dispersed on a microscope slide with silicon oil and examined under transmitted polarized light.

X-Ray Powder Diffraction (XPRD)

X-ray powder diffraction data were collected under ambient conditions on a Rigaku Miniflex 600 diffractometer using Cu K alpha (1.5406 Angstrom) radiation. Powder patterns were collected on a zero background holder with a 0.1 mm indent at a scan rate of 2 to 40° two theta at 2° per min at 40 kV and 15 mA.

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry was performed with a TA Discovery series DSC using a few milligrams of material in a Tzero aluminum pan sealed with a Tzero hermetic lid containing two pin holes. Samples were scanned at 10 °C per minute under 50 mL per minute of nitrogen flow.

Thermogravimetric Analysis (TGA)

Thermogravimetric analysis data were collected with a TA Discovery series TGA. A few milligrams of material were analyzed in an aluminum sample pan. The data was collected from room temperature to 300 °C with a 10 °C per min scan rate.

Nuclear Magnetic Resonance (NMR)

¹HNMR spectra was recorded on a Varian Inova 300 Hz spectrometer using DMS ti de as the solvent and TMS as the internal standard.

Scanning Electron Microscopy (SEM) Procedures

About 1 mg of material was added to a 1/2” slotted head, 1/8” Ted Pella aluminum sample pin with double sided carbon tape and lightly tapped until no loose powder was present on the sample pin. The sample was then sputter coated with gold for 30 seconds prior to imaging. A standard sample stage was used and the sample was imaged at an appropriate magnification.

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

Ethyl 6-isopropyl-2-methoxy-3-(3-methoxypropoxy)-l0-oxo-5H,6H-pyrido[l,2- h] l,7-naphthyridine-9-carboxylate, (230 mg, 0.53 mmol) and lithium hydroxide monohydrate (90 mg, 2.l4mmol) were suspended in a THF/MeOH/H₂0 mixture (3: 1 : 1 (v/v/v), 2 mL) and the reaction was stirred at rt for 1 hour. THF and MeOH were removed under reduced pressure and the crude residue was diluted with water (40 mL), extracted with 2 x 50 ml EtOAc (2 x 50 mL) to get rid of some impurities. The remaining aqueous solution was acidified to pH 2 with aq. 1N HC1 and extracted with EtOAc (3 x 50 mL). The combined organics were dried with sodium sulfate, then concentrated under vacuum to give a crude light brown solid. The solid was further washed with EtO Ac/hexanes mixture (4: 1 (v/v), 10 mL), filtered and dried to give 6-isopropyl-2-methoxy-3-(3-methoxypropoxy)-l0-oxo-5,l0- dihydro-6H-pyrido[l,2-h][l,7]naphthyridine-9-carboxylic acid as a light tan solid (l05mg, 49%, m/z: 403 [M + H]⁺ observed). ^XH NMR (400 MHz, DMSO-d₆) d 16.52 (s, 1H), 8.8 (s, 1H), 7.42 (s, 1H), 7.37 (s, 1H), 4.51 - 4.47 (dd, J= 5.6Hz, J= 8.8 Hz, 1H), 4.11 (m, 2H), 3.96 (s, 3H), 3.48 (t, J=6.4 Hz, 2H), 3.44 -3.4 (m, 1H), 3.34 (s,3H), 3.22 (m, 1H), 2.00 (m, 2H), 1.73 (m, 1H), 0.88 (d, J = 6.4 Hz, 3H), 0.72 (d, J = 6.4 Hz, 3H).

Above racemic mixture was separated by SFC (supercritical fluid chromatography) on a CHIRALCEL OX-H column using liquid C0₂ and Hf CTECN (1 : 1, v/v) and 0.1% diethylamine as modifier. Pure fractions containing (I) were evaporated to dryness to give an off-white solid. The product was crystallized from 2-propanol/water (1:2, v/v) to give anhydrous form 1 of (I).

Example 2: Characterization of (I) Crystalline Form 1

Initial characterization was performed on anhydrous Crystalline Form 1, including polarized light microscopy (PLM), x-ray powder diffraction (XRPD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and nuclear magnetic resonance (¹HNMR). The PLM and XRPD data showed that the material was crystalline, with large rod-shaped particles (FIGs. 1-2). The TGA showed an initial weight loss of 0.1% up to 150 °C, followed by a second weight loss of 0.2% up to 230 °C (FIG. 3). There was a sharp endotherm associated with melting at an onset of 200.5 °C from DSC (FIG. 4). Table 1.

Example 3: Polymorph Screen of (I) Free Form

Crystalline Form 1 of (I) was used as starting material for a solvent-mediated polymorph screen, containing slurry experiments in a number of stressed conditions, with varying temperature, solvent and water activity.

Samples for the slurry experiments were prepared by weighing out 50-60 mg of (I) into 4 mL amber vials. Then 0.5-1 mL of solvent was pipetted in (in 0.5 mL aliquots), followed by a stir bar. The vials were placed onto their respective temperature stir plates to stir at 500 rpm.

After 11 days of stirring, the remaining slurry samples were taken off the stir plates, centrifuged, and their supernatants discarded. The wet solids were analyzed by XRPD. The samples using acetone and acetonitrile remained as solutions after 4 days of stirring at 25°C, and were then cooled to 4°C. After a few days at 4°C, the remaining solutions were allowed to undergo slow evaporation. The evaporated solids were then analyzed by XRPD.

New crystalline patterns were observed and labeled Forms 2 to 5. The solids that remained as starting material were labeled Form 1. The list of experimental conditions and results are shown in Table 2. The XRPD data are presented in FIGs. 5-10. The slurry in THF was found to be semi-crystalline from screen and became more crystalline over time showing a new XRPD pattern labeled as Form 6.

Table 2. Polymorph screen of free form (I) with slurry experiments

Example 4: Characterization of (I) Crystalline Form 2 The solid collected from the ethyl acetate slurry (Form 2) was air dried and analyzed by XRPD, TGA and DSC. After air drying, the solid appeared to be a mixture of Form 2 and an unknown pattern (FIG. 11). The TGA showed a weight loss of 6.1% up to l25°C (FIG.

12) and the DSC showed a small endotherm at 75.9°C, followed by a sharp endotherm at 200.4°C (FIG. 13). This solid was further characterized by ¹HNMR, showing the presence of residual ethyl acetate.

Table 3.

Example 5: Characterization of (I) Crystalline Form 3

The solid collected from the 2-methyl-THF slurry (Form 3) was air dried and analyzed by XRPD, TGA and DSC. The solid remained as Form 3 after air drying (FIG. 14). The TGA showed no negligible weight loss up to 200°C (FIG. 15) and the DSC showed a small endotherm, followed by a small exotherm at l90°C, and then a sharp endotherm at 200.6°C (FIG. 16). This solid was further characterized by ¹HNMR.

Table 4.

Example 6: Characterization of (I) Crystalline Form 4

The solid collected from the dichloromethane after evaporation (Form 4) was analyzed by XRPD, TGA and DSC. The XRPD showed a pattern with poor crystallinity (FIG. 17). The TGA showed a weight loss of 0.2% up to 200°C (FIG. 18) and the DSC showed a sharp endotherm at 200.6°C (FIG. 19). This solid was further characterized by ¹HNMR, showing the presence of residual dichloromethane.

Table 5.

Example 7: Characterization of (I) Crystalline Form 5

The solid collected from the water/acetonitrile after evaporation (Form 5) was analyzed by XPRD, TGA and DSC. The XRPD indicated a crystalline solid (FIG. 20) was made. The TGA showed negligible weight loss up to 2lO°C (FIG. 21). The DSC showed a small endotherm at 97.7°C, a small exotherm at l56.3°C and a sharp endotherm at 200.7°C (FIG. 22). This solid was further characterized by ¹HNMR.

Table 6.

Example 8: Characterization of (I) Crystalline Form 6

The solid collected from the THF slurry was initially semi-crystalline. After air drying, the crystallinity increased, which showed a new unknown pattern, Form 6 (FIG. 23). The solid was analyzed by XRPD, TGA and DSC. The TGA showed negligible weight loss up to 200°C (FIG. 24) and the DSC showed a small endotherm at l56.3°C, and then a sharp endotherm at 200.0°C (FIG. 25). This solid was further characterized by ¹HNMR.

Table 7.

Example 9: Competitive slurries of (I) Crystalline Forms 1-6

From screening and characterization results, a series of competitive slurry experiments at various temperatures were conducted to probe the relative stability of new forms to support a robust crystallization process of (I).

Form 1 and Form 3, were used as the starting forms and then seeded with other identified forms. The solid collected from each competitive slurry was analyzed by XRPD. The experimental conditions and results are shown in Table 8.

At temperatures below 50°C, Form 3 was a preferred form from competitive slurry, except in 2-propanol at 25°C. Above 70°C Form 1 was preferred from the slurry. Form 1 and Form 3 may be enantiotropically related, around temperature of 50-70°C.

Using THF as the solvent, Form 6 was preferred at temperatures 25°C to 50°C from the competitive slurry results.

Table 8 .Conditions and results of competition slurry experiments

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 a fS')-6-isopropyl-2-metho\y-3-(3-metho\ypropo\y)- 1 ()-oxo- 5,lO-dihydro-6H-pyrido[l,2-h][l,7]naphthyridine-9-carboxylic acid (I) free acid crystalline solid, which is characterized by an X-ray diffraction pattern (XRPD) selected from the group consisting of:

(a) Crystalline Form 1, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 4.09, 8.28, 8.72, 9.24, 9.52, 10.56, 11.90, 12.32, 13.18, 15.23, 15.93, 16.73, 17.16, 17.65, 18.68, 19.25, 19.54, 19.97, 20.65, 21.05, 22.14, 22.77, 24.07, 25.51, 26.54, 28.27, 29.69, 34.13, 34.89 and 36.24;

(b) Crystalline Form 2, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 5.73, 6.54, 9.41, 9.99, 14.60, 15.69, 19.01, 19.31, 21.54, 24.09, 24.79, 25.90, 26.36, 27.39, 28.73, 33.84, 34.41, 35.21, and 38.83;

(c) Crystalline Form 3, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 2.09, 4.27, 6.75, 8.62, 9.27, 10.44, 11.10, 12.80, 14.33, 17.35, 17.68, 18.80, 20.53, 20.96, 21.45, 22.53, 23.59, 24.10, 26.20, 27.55 and 29.41;

(d) Crystalline Form 4, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 2.35, 6.13, 7.84, 8.55, 9.91, 12.13, 14.99, 16.54, 17.25, 20.55, 22.15, 24.05, 24.65 and 26.10;

(e) Crystalline Form 5, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 2.44, 8.42, 9.46, 10.34, 13.93, 15.39, 16.37, 18.07, 20.85, 22.55, 24.25, 24.70, 26.25, 27.26, 28.19, 28.58, 31.48, 32.38 and 34.69;

(f) Crystalline Form 6, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 6.07, 8.78, 11.04, 11.79, 12.19, 17.71, 18.85, 20.93, 24.08, 24.70, 25.65 and 36.56;

wherein the XRDP is measured with a Copper X-ray source.

Embodiment 2 provides the solid of Embodiment 1(a), wherein the solid is obtained by crystallizing (I) free acid in at least one solvent selected from the group consisting of 2- propanol, water, water/methanol mixture, water/ethanol mixture, ethanol,

cyclopentylmethane, toluene, isopropyl acetate, and methyl tert-butyl ether.

Embodiment 3 provides the solid of Embodiment 2, wherein the water/methanol mixture is about 1 : 3 (v/v).

Embodiment 4 provides the solid of Embodiment 2, wherein the water/ethanol mixture is about 1 : 3 (v/v).

Embodiment 5 provides the solid of Embodiment 1(b), comprising ethyl acetate.

Embodiment 6 provides the solid of Embodiment 5, wherein the solid is obtained by crystallizing (I) free acid in ethyl acetate.

Embodiment 7 provides the solid of Embodiment 1(c), wherein the solid is obtained by crystallizing (I) free acid in at least one solvent selected from the group consisting of acetone, 2-methyl tetrahydrofuran, ethanol, and methanol.

Embodiment 8 provides the solid of Embodiment 1(d), comprising dichloromethane.

Embodiment 9 provides the solid of Embodiment 8, wherein the solid is obtained by crystallizing (I) free acid in dichloromethane.

Embodiment 10 provides the solid of Embodiment 1(e), wherein the solid is obtained by crystallizing (I) free acid in at least one solvent selected from the group consisting of acetonitrile and water/acetonitrile mixture.

Embodiment 11 provides the solid of Embodiment 10, wherein the water/acetonitrile mixture is about 1 : 3 (v/v).

Embodiment 12 provides the solid of Embodiment 1(f), wherein the solid is obtained by crystallizing (I) free acid in tetrahydrofuran.

Embodiment 13 provides the solid of any of Embodiments 1-12, wherein at least one applies: the solid in (a) is characterized by a Differential Scanning Calorimetry (DSC) thermogram having a single maximum value at about 200.5 °C; the solid in (b) is characterized by a DSC thermogram having endotherms at about 75.9 °C and about 200.4 °C; the solid in (c) is characterized by a DSC thermogram having an exotherm and an endotherm at about 190 °C and an endotherm at about 200.5 °C; the solid in (d) is characterized by a DSC thermogram having a single maximum value at about 200.6 °C; the solid in (e) is characterized by a DSC thermogram having an endotherm at about 97.7°C, an exotherm at about l56.3°C, and an endotherm at about 200.7°C; the solid in (f) is characterized by a DSC thermogram having endotherms at about l56.3°C and about 200.0°C.

Embodiment 14 provides the solid of Embodiment 13, wherein the DSC measurement is performed by heating the solid from 25 °C at 10 °C/min to at least 20 °C above the solid’s melting point.

Embodiment 15 provides the solid of any of Embodiments 13-14, wherein the DSC measurement is performed with standard aluminum DSC sample pans and covers, with a nitrogen gas purge rate at about 50 ml/min.

Embodiment 16 provides a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and the solid of any one of Embodiments 1-15.

Embodiment 17 provides the pharmaceutical composition of Embodiment 16, which is in solid dosage form for oral administration.

Embodiment 18 provides the pharmaceutical composition of any of Embodiments 16-

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

Embodiment 19 provides the pharmaceutical composition of any of Embodiments 16-

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

Embodiment 20 provides the pharmaceutical composition of Embodiment 19, 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; sAg secretion inhibitor; oligomeric nucleotide targeted to the Hepatitis B genome; and immunostimulator.

Embodiment 21 provides a method of treating or preventing hepatitis B virus (HBV) infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the solid of any of Embodiments 1-15 and/or the pharmaceutical composition of any of Embodiments 16-20.

Embodiment 22 provides the method of Embodiment 21, wherein the subject is further administered at least one additional agent useful for treating HBV infection.

Embodiment 23 provides the method of Embodiment 22, wherein the solid or pharmaceutical composition, and the at least one additional agent, are coformulated.

Embodiment 24 provides the method of any of Embodiments 21-23, wherein the subject is further infected with hepatitis D virus (HDV).

Embodiment 25 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 a therapeutically effective amount of the solid of any of

Embodiments 1-15 and/or the pharmaceutical composition of any of Embodiments 16-20.

Embodiment 26 provides the method of Embodiment 25, wherein the subject is further administered at least one additional agent useful for treating HBV infection. Embodiment 27 provides the method of Embodiment 26, wherein the solid or pharmaceutical composition, and the at least one additional agent, are coformulated.

Embodiment 28 provides the method of any of Embodiments 25-27, wherein the subject is further infected with hepatitis D virus (HDV).

Embodiment 29 provides the method of any of Embodiments 21-28, wherein the subject is a mammal.

Embodiment 30 provides the method of any of Embodiments 21-29, 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

CLAIMS What is claimed is:

1. A CV)-6-isopropyl-2-methoxy-3-(3-methoxypropoxy)- 10-oxo-5.10-dihydro-6H- pyrido[l,2-h][l,7]naphthyridine-9-carboxylic acid (I) free acid crystalline solid, which is characterized by an X-ray diffraction pattern (XRPD) selected from the group consisting of:

(a) Crystalline Form 1, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 4.09, 8.28, 8.72, 9.24, 9.52, 10.56, 11.90, 12.32, 13.18, 15.23, 15.93, 16.73, 17.16, 17.65, 18.68, 19.25, 19.54, 19.97, 20.65, 21.05, 22.14, 22.77, 24.07, 25.51, 26.54, 28.27, 29.69, 34.13, 34.89 and 36.24;

(b) Crystalline Form 2, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 5.73, 6.54, 9.41, 9.99, 14.60, 15.69, 19.01, 19.31, 21.54, 24.09, 24.79, 25.90, 26.36, 27.39, 28.73, 33.84, 34.41, 35.21, and 38.83;

(c) Crystalline Form 3, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 2.09, 4.27, 6.75, 8.62, 9.27, 10.44, 11.10, 12.80, 14.33, 17.35, 17.68, 18.80, 20.53, 20.96, 21.45, 22.53, 23.59, 24.10, 26.20, 27.55 and 29.41;

(d) Crystalline Form 4, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 2.35, 6.13, 7.84, 8.55, 9.91, 12.13, 14.99, 16.54, 17.25, 20.55, 22.15, 24.05, 24.65 and 26.10;

(e) Crystalline Form 5, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 2.44, 8.42, 9.46, 10.34, 13.93, 15.39, 16.37, 18.07, 20.85, 22.55, 24.25, 24.70, 26.25, 27.26, 28.19, 28.58, 31.48, 32.38 and 34.69;

(f) Crystalline Form 6, with a X-ray powder diffraction spectrum comprising 2Q values (in degrees) of about: 6.07, 8.78, 11.04, 11.79, 12.19, 17.71, 18.85, 20.93, 24.08,

24.70, 25.65 and 36.56;

wherein the XRDP is measured with a Copper X-ray source.

2. The solid of claim 1(a), wherein the solid is obtained by crystallizing (I) free acid in at least one solvent selected from the group consisting of 2-propanol, water, water/methanol mixture, water/ethanol mixture, ethanol, cyclopentylmethane, toluene, isopropyl acetate, and methyl tert-butyl ether.

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

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

5. The solid of claim 1(b), comprising ethyl acetate.

6. The solid of claim 5, wherein the solid is obtained by crystallizing (I) free acid in ethyl acetate.

7. The solid of claim 1(c), wherein the solid is obtained by crystallizing (I) free acid in at least one solvent selected from the group consisting of acetone, 2-methyl tetrahydrofuran, ethanol, and methanol.

8. The solid of claim 1(d), comprising dichloromethane.

9. The solid of claim 8, wherein the solid is obtained by crystallizing (I) free acid in dichloromethane.

10. The solid of claim 1(e), wherein the solid is obtained by crystallizing (I) free acid in at least one solvent selected from the group consisting of acetonitrile and water/acetonitrile mixture.

11. The solid of claim 10, wherein the water/acetonitrile mixture is about 1 : 3 (v/v).

12. The solid of claim 1(f), wherein the solid is obtained by crystallizing (I) free acid in tetrahydrofuran.

13. The solid of claim 1, wherein at least one applies:

the solid in (a) is characterized by a Differential Scanning Calorimetry (DSC) thermogram having a single maximum value at about 200.5 °C;

the solid in (b) is characterized by a DSC thermogram having endotherms at about 75.9 °C and about 200.4 °C;

the solid in (c) is characterized by a DSC thermogram having an exotherm and an endotherm at about 190 °C and an endotherm at about 200.5 °C;

the solid in (d) is characterized by a DSC thermogram having a single maximum value at about 200.6 °C; the solid in (e) is characterized by a DSC thermogram having an endotherm at about 97.7°C, an exotherm at about 156.3°C, and an endotherm at about 200.7°C;

the solid in (f) is characterized by a DSC thermogram having endotherms at about 156.3°C and about 200.0°C.

14. The solid of claim 13, wherein the DSC measurement is performed by heating the solid from 25 °C at 10 °C/min to at least 20 °C above the solid’s melting point.

15. The solid of claim 13, wherein the DSC measurement is performed with standard aluminum DSC sample pans and covers, with a nitrogen gas purge rate at about 50 ml/min.

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

17. The pharmaceutical composition of claim 16, which is in solid dosage form for oral administration.

18. The pharmaceutical composition of claim 16, which is part of a tablet, dragee, drop, suppository, capsule, caplet, and/or gelcap.

19. The pharmaceutical composition of claim 16, further comprising at least one additional agent useful for treating hepatitis virus infection.

20. The pharmaceutical composition of claim 19, 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; sAg secretion inhibitor; oligomeric nucleotide targeted to the Hepatitis B genome; and immunostimulator.

21. A method of treating or preventing hepatitis B virus (HBV) infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the solid of claim 1 or the pharmaceutical composition of claim 16.

22. The method of claim 21, wherein the subject is further administered at least one additional agent useful for treating HBV infection.

23. The method of claim 22, wherein the solid or pharmaceutical composition, and the at least one additional agent, are coformulated.

24. The method of claim 21, wherein the subject is further infected with hepatitis D virus (HDV).

25. 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 a therapeutically effective amount of the solid of claim 1 or the pharmaceutical composition of claim 16.

26. The method of claim 25, wherein the subject is further administered at least one additional agent useful for treating HBV infection.

27. The method of claim 26, wherein the solid or pharmaceutical composition, and the at least one additional agent, are coformulated.

28. The method of claim 25, wherein the subject is further infected with hepatitis D virus (HDV).

29. The method of claim 21 or 25, wherein the subject is a mammal.

30. The method of claim 29, wherein the mammal is a human.