WO2017096161A1

WO2017096161A1 - Compositions and methods for reactivating latent immunodeficiency virus using an akt activator

Info

Publication number: WO2017096161A1
Application number: PCT/US2016/064614
Authority: WO
Inventors: Andrea Gramatica; Warner C. Greene
Original assignee: The J. David Gladstone Institutes
Priority date: 2015-12-04
Filing date: 2016-12-02
Publication date: 2017-06-08

Abstract

The present disclosure provides compositions and methods for reactivating latent immunodeficiency virus using a protein kinase B (Akt) activator, e.g., SC-79 (2-amino-6-chloro-α -cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acid ethyl ester), or a pharmaceutically acceptable salt or derivative thereof.

Description

COMPOSITIONS AND METHODS FOR REACTIVATING LATENT IMMUNODEFICIENCY VIRUS

USING AN AKT ACTIVATOR

CROSS -REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/263,081, filed December 4, 2015, which application is incorporated herein by reference in its entirety.

INTRODUCTION

[0002] HIV latency is established early during acute infection and is primarily found within memory CD4+ T cell subsets. This reservoir, although almost transcriptionally silent, is fully capable of generating infectious virus when the host cell is reactivated by antigen or cytokine stimulation or when antiretroviral therapy is interrupted. The latent HIV reservoir is principally found in lymphoid tissues where 98% of the CD4+ T cells reside. Although highly active antiretroviral therapy is capable of suppressing viral replication, it fails to eradicate latent reservoirs.

[0003] Efforts to purge latent HIV have initially focused on reactivating latent proviruses with cytokines or T cell receptor activating agents. However, these strategies often result in severe side effects and generally have low efficacy. The so-called "Shock and Kill" strategy, instead, involves reactivation of transcriptionally silent proviruses through administration of latency reversal agents (LRAs), which are chemical compounds able to induce HIV transcription without fully activating the cells of the immune system. However, LRAs used so far in clinical and pre-clinical studies have largely been inadequately active and/or associated with some toxicity. Accordingly, there is a need in the art for new LRAs.

SUMMARY

[0004] The present disclosure provides compositions and methods for reactivating latent

immunodeficiency virus using a protein kinase B (Akt) activator, e.g., SC-79 (2-amino-6- chloro-a-cyano-3-(ethoxycarbonyl)-4H-l-benzopyran-4-acetic acid ethyl ester), or a pharmaceutically acceptable salt or derivative thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 depicts the effects of various agents on the reversal of HIV latency and cell

viability in tonsillar HLAC cells (Panel A), PBMCs (Panel B), and splenic HLAC cells (Panel C). Treatments from left to right for each panel are as follows: uninfected, DMSO, CD3/CD28, Prostratin 200nM, Prostratin 400nM, JQ1 ΙΟΟηΜ, JQ1 ΙμΜ, Panibinostat 50nM, Panobinostat 5μΜ, HMBA ImM, HMBA 5mM, Bryostatin-1 5nM, Bryostatin-1 ΙΟηΜ, SC-79 500nm, and SC-79 ΙμΜ. Abbreviations: Pr=Prostratin; Pa=Panobinostat; J=JQ1; B=Bryostatin-l; H=HMBA; S=SC-79. Bars in upper graphs for each panel represent the percentage of viable cells, normalized to the uninfected control (uninfected=100% viability).

[0006] Figure 2 depicts the effects of various combinations of agents on the reversal of HIV latency and cell viability in tonsillar HLAC cells (Panel A), PBMCs (Panel B), and splenic HLAC cells (Panel C). Treatments from left to right for each panel are as follows: uninfected, DMSO, CD3/CD28, Pa+Pr, Pr+J, Pr+H, Pr+B, Pr+S, Pa+J, Pa+H, Pa+B, Pa+S, J+H, J+B, J+S, H+B, H+S, and B+S. Abbreviations: Pr=Prostratin; Pa=Panobinostat; J=JQ1;

B=Bryostatin-l; H=HMBA; S=SC-79. Bars in upper graphs for each panel represent the percentage of viable cells, normalized to the uninfected control (uninfected=100% viability).

[0007] Figure 3 provides a graph showing the effect of various LRA treatments, including SC-79, on virion release in the supernatant of patient-derived CD4⁺ T cells.

[0008] Figure 4 provides a graph showing the expression of three different activation markers

(CD69, CD25 and PD-1) on the surface of CD4⁺ T cells, upon treatment with SC-79. Bry-1= Bryostatin-1; Pan= Panobinostat.

[0009] Figure 5 provides a graph showing CD4⁺ T cell viability for cells treated with SC-79. Bry- 1= Bryostatin-1; Pan= Panobinostat.

DEFINITIONS

[0010] The term "immunodeficiency virus" includes human immunodeficiency virus (HIV), feline immunodeficiency virus, and simian immunodeficiency virus. The term "human

immunodeficiency virus" as used herein, refers to human immunodeficiency virus-1 (HIV- 1); human immunodeficiency virus-2 (HIV -2); and any of a variety of HIV subtypes and quasispecies.

[0011] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease.

[0012] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.

[0013] A "therapeutically effective amount" or "efficacious amount" refers to the amount of a

compound that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound or the cell, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0014] The terms "co-administration" and "in combination with" include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.

[0015] As used herein, a "pharmaceutical composition" is meant to encompass a composition

suitable for administration to a subject, such as a mammal, especially a human. In general a "pharmaceutical composition" is sterile, and is free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the

pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal and the like. In some embodiments the composition is suitable for

administration by a transdermal route, using a penetration enhancer other than

dimethylsulfoxide (DMSO). In other embodiments, the pharmaceutical compositions are suitable for administration by a route other than transdermal administration. A

pharmaceutical composition will in some embodiments include a subject compound and a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutically acceptable excipient is other than DMSO.

[0016] As used herein, "pharmaceutically acceptable derivatives" of a compound of the invention include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof. Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization. The compounds produced may be administered to animals or humans without substantial toxic effects and are either pharmaceutically active or are prodrugs.

[0017] A "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2- ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4- chlorobenzene sulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,

camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(34iydroxy-2-ene-l-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

[0018] The term "solvate" as used herein refers to a complex or aggregate formed by one or more molecules of a solute, e.g. a prodrug or a pharmaceutically-acceptable salt thereof, and one or more molecules of a solvent. Such solvates are typically crystalline solids having a substantially fixed molar ratio of solute and solvent. Representative solvents include by way of example, water, methanol, ethanol, isopropanol, acetic acid, and the like. When the solvent is water, the solvate formed is a hydrate.

[0019] "Prodrug" refers to a derivative of an active agent that requires a transformation within the body to release the active agent. In certain embodiments, the transformation is an enzymatic transformation. Prodrugs are frequently, although not necessarily, pharmacologically inactive until converted to the active agent.

DEFINITION OF SELECT CHEMICAL TERMINOLOGY

[0020] The nomenclature of certain compounds or substituents are used in their conventional sense, such as described in chemistry literature including but not limited to Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002, pp. 360-361, 1084- 1085; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001.

[0021] As used herein, the term "alkyl" by itself or as part of another substituent refers to a saturated branched or straight-chain monovalent hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane. Typical alkyl groups include, but are not limited to, methyl; ethyl, propyls such as propan-l-yl or propan-2-yl; and butyls such as butan-l-yl, butan-2-yl, 2-methyl-propan-l-yl or 2-methyl-propan-2-yl. In some embodiments, an alkyl group comprises from 1 to 20 carbon atoms. In other embodiments, an alkyl group comprises from 1 to 10 carbon atoms. In still other embodiments, an alkyl group comprises from 1 to 6 carbon atoms, such as from 1 to 4 carbon atoms.

[0022] "Alkanyl" by itself or as part of another substituent refers to a saturated branched, straight- chain or cyclic alkyl radical derived by the removal of one hydrogen atom from a single carbon atom of an alkane. Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-l-yl, propan-2-yl (isopropyl), cyclopropan-l-yl, etc.; butanyls such as butan-l-yl, butan-2-yl (sec-butyl), 2 -methyl -propan-l-yl (isobutyl), 2- methyl-propan-2-yl (t-butyl), cyclobutan-l-yl, etc.; and the like.

[0023] "Alkylene" refers to a branched or unbranched saturated hydrocarbon chain, usually having from 1 to 40 carbon atoms, more usually 1 to 10 carbon atoms and even more usually 1 to 6 carbon atoms. This term is exemplified by groups such as methylene (-CH₂-), ethylene (-CH₂CH₂-), the propylene isomers (e.g., -CH₂CH₂CH₂- and -CH(CH₃)CH₂-) and the like.

[0024] "Alkenyl" by itself or as part of another substituent refers to an unsaturated branched,

straight-chain or cyclic alkyl radical having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of an alkene. The group may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-l-en-l-yl, prop-l-en- 2-yl, prop-2-en-l-yl (allyl), prop-2-en-2-yl, cycloprop-l-en-l-yl; cycloprop-2-en-l-yl;

butenyls such as but-l-en-l-yl, but-l-en-2-yl, 2-methyl-prop-l-en-l-yl, but-2-en-l-yl, but-2- en-l-yl, but-2-en-2-yl, buta-l,3-dien-l-yl, buta-l,3-dien-2-yl, cyclobut-l-en-l-yl, cyclobut-1- en-3-yl, cyclobuta-l,3-dien-l-yl, etc.; and the like.

[0025] "Alkynyl" by itself or as part of another substituent refers to an unsaturated branched,

straight-chain or cyclic alkyl radical having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of an alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-l-yn-l-yl, prop-2-yn- 1-yl, etc.; butynyls such as but-l-yn-l-yl, but-l-yn-3-yl, but-3-yn-l-yl, etc.; and the like.

[0026] "Acyl" by itself or as part of another substituent refers to a radical -C(0)R³⁰, where R³⁰ is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein and substituted versions thereof. Representative examples include, but are not limited to formyl, acetyl, cyclohexylcarbonyl,

cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, piperonyl, succinyl, and malonyl, and the like.

[0027] The term "aminoacyl" refers to the group -C(0)NR²¹R²², wherein R²¹ and R²² independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R²¹ and R²² are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0028] "Alkoxy" by itself or as part of another substituent refers to a radical -OR³¹ where R³¹

represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy and the like.

[0029] "Alkoxycarbonyl" by itself or as part of another substituent refers to a radical -C(0)OR³¹ where R³¹ represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, cyclohexyloxycarbonyl and the like.

[0030] "Aryl" by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of an aromatic ring system. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s- indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4- diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like. In certain embodiments, an aryl group comprises from 6 to 20 carbon atoms. In certain embodiments, an aryl group comprises from 6 to 12 carbon atoms. Examples of an aryl group are phenyl and naphthyl.

[0031] "Arylalkyl" by itself or as part of another substituent refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, is replaced with an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-l-yl, 2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2- naphthylethen-l-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like. Where specific alkyl moieties are intended, the nomenclature arylalkanyl, arylalkenyl and/or arylalkynyl is used. In certain embodiments, an arylalkyl group is (C₇-C₃₀) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C_I-C_K>) and the aryl moiety is (C₆-C₂o). In certain embodiments, an arylalkyl group is (C₇-C₂o) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (Ci-C₈) and the aryl moiety is (C₆-Ci₂).

[0032] "Arylaryl" by itself or as part of another substituent, refers to a monovalent hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a ring system in which two or more identical or non-identical aromatic ring systems are joined directly together by a single bond, where the number of such direct ring junctions is one less than the number of aromatic ring systems involved. Typical arylaryl groups include, but are not limited to, biphenyl, triphenyl, phenyl-napthyl, binaphthyl, biphenyl-napthyl, and the like. When the number of carbon atoms in an arylaryl group are specified, the numbers refer to the carbon atoms comprising each aromatic ring. For example, (C₅-Ci₄) arylaryl is an arylaryl group in which each aromatic ring comprises from 5 to 14 carbons, e.g., biphenyl, triphenyl, binaphthyl, phenylnapthyl, etc. In certain embodiments, each aromatic ring system of an arylaryl group is independently a (C₅-Ci₄) aromatic. In certain embodiments, each aromatic ring system of an arylaryl group is independently a (C₅-Ci₀) aromatic. In certain embodiments, each aromatic ring system is identical, e.g., biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.

[0033] "Cycloalkyl" by itself or as part of another substituent refers to a saturated or unsaturated cyclic alkyl radical. Where a specific level of saturation is intended, the nomenclature "cycloalkanyl" or "cycloalkenyl" is used. Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane and the like. In certain embodiments, the cycloalkyl group is (C₃-Ci₀) cycloalkyl. In certain embodiments, the cycloalkyl group is (C₃-C₇) cycloalkyl.

[0034] "Cycloheteroalkyl" or "heterocyclyl" by itself or as part of another substituent, refers to a saturated or unsaturated cyclic alkyl radical in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom. Typical heteroatoms to replace the carbon atom(s) include, but are not limited to, N, P, O, S, Si, etc. Where a specific level of saturation is intended, the nomenclature "cycloheteroalkanyl" or "cycloheteroalkenyl" is used. Typical cycloheteroalkyl groups include, but are not limited to, groups derived from epoxides, azirines, thiiranes,

imidazolidine, morpholine, piperazine, piperidine, pyrazolidine, pyrrolidine, quinuclidine and the like.

[0035] "Heteroalkyl, Heteroalkanyl, Heteroalkenyl and Heteroalkynyl" by themselves or as part of another substituent refer to alkyl, alkanyl, alkenyl and alkynyl groups, respectively, in which one or more of the carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatomic groups. Typical heteroatomic groups which can be included in these groups include, but are not limited to, -0-, -S-, -S-S-, -0-S-, - NR ⁷R³⁸-, =N-N= -N=N-, -N=N-NR ⁹R⁴⁰, -PR⁴¹-, -P(0)₂-, -POR⁴²-, -0-P(0)₂-, -S-0-, -S- (O)-, -S0₂-, -SnR⁴³R⁴⁴- and the like, where R³⁷, R³⁸, R³⁹, R⁴⁰, R⁴¹, R⁴², R⁴³ and R⁴⁴ are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl. [0036] "Heteroaryl" by itself or as part of another substituent, refers to a monovalent heteroaromatic radical derived by the removal of one hydrogen atom from a single atom of a heteroaromatic ring system. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, benzodioxole and the like. In certain embodiments, the heteroaryl group is from 5-20 membered heteroaryl. In certain embodiments, the heteroaryl group is from 5-10 membered heteroaryl. In certain embodiments, heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.

[0037] "Heteroarylalkyl" by itself or as part of another substituent, refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylalkenyl and/or heterorylalkynyl is used. In certain embodiments, the heteroarylalkyl group is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-10 membered and the heteroaryl moiety is a 5-20-membered heteroaryl. In certain embodiments, the

heteroarylalkyl group is 6-20 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-8 membered and the heteroaryl moiety is a 5-12-membered heteroaryl.

[0038] "Aromatic Ring System" by itself or as part of another substituent, refers to an unsaturated cyclic or polycyclic ring system having a conjugated π electron system. Specifically included within the definition of "aromatic ring system" are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane, indene, phenalene, etc. Typical aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like.

[0039] "Heteroaromatic Ring System" by itself or as part of another substituent, refers to an

aromatic ring system in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom. Typical heteroatoms to replace the carbon atoms include, but are not limited to, N, P, O, S, Si, etc. Specifically included within the definition of "heteroaromatic ring systems" are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, arsindole, benzodioxan, benzofuran, chromane, chromene, indole, indoline, xanthene, etc. Typical heteroaromatic ring systems include, but are not limited to, arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene and the like.

"Substituted" refers to a group in which one or more hydrogen atoms are independently replaced with the same or different substituent(s). Typical substituents include, but are not limited to, alkylenedioxy (such as methylenedioxy), -M, -R⁶⁰, -O , =0, -OR⁶⁰, -SR⁶⁰, -S^", =S, __nr60r6I _=NR6o _{CN 0CN SCN N0 =N¾ N3} -S(0)₂0 , -S(0)₂OH,

-S(0)₂R⁶⁰, -OS(0)₂0^", -OS(0)₂R⁶⁰, -P(0)(0^")₂, -P(O)(OR⁶⁰)(O ), -OP(O)(OR⁶⁰)(OR⁶¹), -C(0)R⁶⁰, -C(S)R⁶⁰, -C(0)OR⁶⁰, -C(O)NR⁶⁰R⁶¹,-C(O)O^~, -C(S)OR⁶⁰, -NR⁶²C(O)NR⁶⁰R⁶¹, -NR⁶²C(S)NR⁶⁰R⁶¹, -NR⁶²C(NR⁶ )NR⁶⁰R⁶¹ and -C(NR⁶²)NR⁶⁰R⁶¹ where M is halogen; R⁶⁰, R⁶¹, R⁶² and R⁶³ are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R⁶⁰ and R⁶¹ together with the nitrogen atom to which they are bonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring; and R⁶⁴ and R⁶⁵ are independently hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R⁶⁴ and R⁶⁵ together with the nitrogen atom to which they are bonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, substituents include -M, -R⁶⁰, =0, -OR⁶⁰, -SR⁶⁰, -S^", =S, -NR⁶⁰R⁶¹, =NR⁶⁰, -CF₃, -CN, -OCN, -SCN, -NO, -N0₂,=N₂, -N₃, -S(0)₂R⁶⁰, -OS(0)₂0^", -OS(0)₂R⁶⁰, -P(0)(0^")₂, -P(O)(OR⁶⁰)(O^"), -OP(O)(OR⁶⁰)(OR⁶¹), -C(0)R⁶⁰, -C(S)R⁶⁰, -C(0)OR⁶⁰, -C(O)NR⁶⁰R⁶¹,-C(O)O^", -NR⁶²C(O)NR⁶⁰R⁶¹. In certain embodiments, substituents include -M, -R⁶⁰, =0, -OR⁶⁰, -SR⁶⁰, -NR⁶⁰R⁶¹, -CF₃, -CN, -N0₂, -S(0)₂R⁶⁰, -P(O)(OR⁶⁰)(O^"), -OP(O)(OR⁶⁰)(OR⁶¹), -C(0)R⁶⁰, -C(0)OR⁶⁰, -C(O)NR⁶⁰R⁶¹,-C(O)O^". In certain embodiments, substituents include -M, -R⁶⁰,=O, -OR⁶⁰, -SR⁶⁰, -NR⁶⁰R⁶¹, -CF₃, -CN, -N0₂, -S(0)₂R⁶⁰, -OP(O)(OR⁶⁰)(OR⁶¹), -C(0)R⁶⁰, -C(0)OR⁶⁰ ,-C(0)0 , where R⁶⁰, R⁶¹ and R⁶² are as defined above. For example, a substituted group may bear a methylenedioxy substituent or one, two, or three substituents selected from a halogen atom, a (l-4C)alkyl group and a (l-4C)alkoxy group.

[0041] The compounds described herein can contain one or more chiral centers and/or double bonds and therefore, can exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers. Accordingly, all possible enantiomers and stereoisomers of the compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures are included in the description of the compounds herein.

Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan. The compounds can also exist in several tautomeric forms including the enol form, the keto form and mixtures thereof. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds. The compounds described also include isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature.

Examples of isotopes that can be incorporated into the compounds disclosed herein include, but are not limited to, ²H, ¾, ⁿC, ¹ C, ¹⁴C, ¹⁵N, ¹⁸0, ¹⁷0, etc. Compounds can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, compounds can be hydrated or solvated. Certain compounds can exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of the present disclosure.

[0042] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0043] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same

meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0045] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an AKT activator" includes a plurality of such activators and reference to "the HIV-infected cell" includes reference to one or more HIV-infected cells and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0046] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0047] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

[0048] The present disclosure provides methods of reactivating latent HIV integrated into the

genome of an HIV-infected cell. The methods generally involve contacting an HIV-infected cell in which HIV is latent with a protein kinase B (Akt) activator of Formula I, e.g., SC-79 (2-amino-6-chloro-a-cyano-3-(ethoxycarbonyl)-4H-l-benzopyran-4-acetic acid ethyl ester) or a pharmaceutically acceptable salt or derivative thereof. SC-79 binds to the pleckstrin homology domain of Akt and enhances Akt phosphorylation by upstream protein kinases. SC-79 also enables cytosolic activation of Akt. Latently infected cells contain replication- competent integrated HIV-1 genomes that are blocked at the transcriptional level, resulting in the absence of viral protein expression. The present disclosure provides methods for reducing the reservoir of latent immunodeficiency virus in vitro and in vivo.

[0049] The present disclosure further provides detection methods for identifying a cell that has latent HIV. The methods generally involve contacting a cell obtained from an individual with a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof; and detecting expression of an HIV-encoded gene product. If the cell expresses an HIV -encoded gene product when contacted with a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, but does not express detectable levels of the HIV-encoded gene product in the absence of the compound of Formula I, e.g., SC-79 or the pharmaceutically acceptable salt or derivative thereof, the cell is considered to have latent HIV.

[0050] The present disclosure further provides a method of identifying a candidate agent for treating an HIV infection in an individual. The method generally involves contacting a primary cell identified using a subject method with a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, and a test agent; and determining the effect of the test agent on the level of HIV produced in the cell. A test agent that reduces the level of HIV produced in the cell, compared to the level of HIV produced in a control cell contacted with the compound of Formula I, e.g., SC-79 or the pharmaceutically acceptable salt or derivative thereof but not with the test agent, is considered a candidate agent for inhibiting HIV and treating an HIV infection.

TREATMENT METHODS

[0051] The present disclosure provides methods for reactivating latent immunodeficiency virus in a cell, the methods generally involving contacting the cell with a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof. The present disclosure provides methods for reducing the reservoir of latent immunodeficiency virus in an individual by administering to the individual an effective amount of a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, that binds Akt in a cell latently infected with HIV. The present disclosure provides methods of treating an immunodeficiency virus infection in an individual, the methods generally involving coadministering to the individual a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof and an anti-HIV agent.

[0052] In some embodiments, an effective amount of a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof that binds Akt in a cell is an amount that reactivates latent HIV and reduces the reservoir of latent HIV in an individual by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. A "reduction in the reservoir of latent HIV" (also referred to as "reservoir of latently infected cells") is a reduction in the number of cells in the individual that harbor a latent HIV infection. Whether the reservoir of latently infected cells is reduced can be determined using any known method, including the method described in Blankson et al. (2000) J. Infect. Disease 182(6): 1636-1642.

[0053] In some embodiments, a subject method of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof; and b) administering to the individual an effective amount of an agent that inhibits an immunodeficiency virus function. The immunodeficiency virus function can be selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity.

[0054] In some embodiments, a compound of Formula I, e.g., SC-79 or a pharmaceutically

acceptable salt or derivative thereof is administered in a combination therapy (i.e., coadministered) with: 1) one or more nucleoside reverse transcriptase inhibitors (e.g., Combivir, Epivir, Hivid, Retrovir, Videx, Zerit, Ziagen, etc.); 2) one or more non-nucleoside reverse transcriptase inhibitors (e.g., Rescriptor, Sustiva, Viramune, etc.); 3) one or more protease inhibitors (e.g., Agenerase, Crixivan, Fortovase, Invirase, Kaletra, Norvir, Viracept, etc.); 4) an anti-HIV agent such as a protease inhibitor and a nucleoside reverse transcriptase inhibitor; 5) an anti-HIV agent such as a protease inhibitor, a nucleoside reverse transcriptase inhibitor, and a non-nucleoside reverse transcriptase inhibitor; 6) an anti-HIV agent such as a protease inhibitor and a non-nucleoside reverse transcriptase inhibitor, and/or 7) an anti-viral (e.g., HIV) agent such as a protein kinase C (PKC) activator (e.g., prostratin). Other combinations of an effective amount of a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, with one or more anti-HIV agents, such as one or more of a protease inhibitor, a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, and a protein kinase C (PKC) activator are contemplated.

[0055] A PKC activator (e.g., prostratin ((lai?, lb5^*,4ai?,7aS^*,7bi?,8i?,9aS)-4a,7b-dihydroxy-3- (hydroxymethyl)-l,l,6,8-tetramethyl-5-oxo-l, la, lb,4,4a,5,7a,7b,8,9-decahydro-9aH- cyclopropa[3,4]benzo[l,2-e]azulen-9a-yl)) can be administered in a separate formulation from a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof. A PKC activator can be co-formulated with a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, and the co- formulation administered to an individual. [0056] Any of a variety of methods can be used to determine whether a treatment method is effective. For example, methods of determining whether the methods of the invention are effective in reducing immunodeficiency virus (e.g., HIV) viral load, and/or treating an immunodeficiency virus (e.g., HIV) infection, are any known test for indicia of

immunodeficiency virus (e.g., HIV) infection, including, but not limited to, measuring viral load, e.g., by measuring the amount of immunodeficiency virus (e.g., HIV) in a biological sample, e.g., using a polymerase chain reaction (PCR) with primers specific for an immunodeficiency virus (e.g., HIV) polynucleotide sequence; detecting and/or measuring a polypeptide encoded by an immunodeficiency virus (e.g., HIV), e.g., p24, gpl20, reverse transcriptase, using, e.g., an immunological assay such as an enzyme-linked immunosorbent assay (ELISA) with an antibody specific for the polypeptide; and measuring the CD4⁺ T cell count in the individual.

SC-79 and Pharmaceutically Acceptable Salts and Derivatives Thereof

[0057] The compositions of the present disclosure include compounds of Formula I, shown below, which formula encompasses SC-79 and pharmaceutically acceptable salts and derivatives thereof. Pharmaceutical compositions and methods of the present disclosure also contemplate compounds of Formula I.

[0058] In one of its composition aspects, the present embodiments provide a compound of Formula

I:

or a pharmaceutically acceptable salt, solvate or prodrug thereof,

wherein each of R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ are independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heterocycloalkyl, substituted heterocycloalkyl, heteroaryl, substituted heteroaryl, hydroxyl, alkoxyl, substituted alkoxyl, substituted acyloxy, alkoxycarbonyl, substituted alkoxycarbonyl, thiol, acyl, amino, substituted amino, aminoacyl, acylamino, azido, carboxyl, carboxylalkyl, cyano, halogen, and nitro.

[0059] In some embodiments, R¹ is hydrogen. In other embodiments, R¹ is alkyl or substituted alkyl.

In other embodiments, R¹ is aryl or substituted aryl. In other embodiments, R¹ is

heterocycloalkyl or substituted heterocycloalkyl. In other embodiments, R¹ is heteroaryl or substituted heteroaryl. In other embodiments, R¹ is hydroxyl, alkoxyl or substituted alkoxyl. In other embodiments, R¹ is acyloxy or substituted acyloxy. In other embodiments, R¹ is alkoxylcarbonyl or substituted alkoxycarbonyl. In other embodiments, R¹ is acyl or substituted acyl. In other embodiments, R¹ is thiol. In other embodiment, R¹ is amino or substituted amino. In other embodiments, R¹ is aminoacyl or substituted aminoacyl. In other embodiments, R¹ is azido. In other embodiments, R¹ is carboxyl, substituted carboxyl, carboxyalkyl or substituted carboxyalkyl. In other embodiments, R¹ is cyano. In other embodiments, R¹ is a halogen, such as -F, -CI, -Br and -I. In other embodiments, R¹ is nitro.

[0060] In some embodiments, R² is hydrogen. In other embodiments, R² is alkyl or substituted alkyl.

In other embodiments, R² is aryl or substituted aryl. In other embodiments, R² is

heterocycloalkyl or substituted heterocycloalkyl. In other embodiments, R² is heteroaryl or substituted heteroaryl. In other embodiments, R² is hydroxyl, alkoxyl or substituted alkoxyl. In other embodiments, R² is acyloxy or substituted acyloxy. In other embodiments, R² is alkoxylcarbonyl or substituted alkoxycarbonyl. In other embodiments, R² is acyl or substituted acyl. In other embodiments, R² is thiol. In other embodiment, R² is amino or substituted amino. In other embodiments, R² is aminoacyl or substituted aminoacyl. In other embodiments, R² is azido. In other embodiments, R² is carboxyl, substituted carboxyl, carboxyalkyl or substituted carboxyalkyl. In other embodiments, R² is cyano. In other embodiments, R² is a halogen, such as -F, -CI, -Br and -I. In other embodiments, R² is nitro.

[0061] In some embodiments, R³ is hydrogen. In other embodiments, R³ is alkyl or substituted alkyl.

In other embodiments, R³ is aryl or substituted aryl. In other embodiments, R³ is

heterocycloalkyl or substituted heterocycloalkyl. In other embodiments, R³ is heteroaryl or substituted heteroaryl. In other embodiments, R³ is hydroxyl, alkoxyl or substituted alkoxyl. In other embodiments, R³ is acyloxy or substituted acyloxy. In other embodiments, R³ is alkoxylcarbonyl or substituted alkoxycarbonyl. In other embodiments, R³ is acyl or substituted acyl. In other embodiments, R³ is thiol. In other embodiment, R³ is amino or substituted amino. In other embodiments, R³ is aminoacyl or substituted aminoacyl. In other embodiments, R³ is azido. In other embodiments, R³ is carboxyl, substituted carboxyl, carboxyalkyl or substituted carboxyalkyl. In other embodiments, R³ is cyano. In other embodiments, R³ is a halogen, such as -F, -CI, -Br and -I. In other embodiments, R³ is chloro.

[0062] In some embodiments, R⁴ is hydrogen. In other embodiments, R⁴ is alkyl or substituted alkyl.

In other embodiments, R⁴ is aryl or substituted aryl. In other embodiments, R⁴ is

heterocycloalkyl or substituted heterocycloalkyl. In other embodiments, R⁴ is heteroaryl or substituted heteroaryl. In other embodiments, R⁴ is hydroxyl, alkoxyl or substituted alkoxyl. In other embodiments, R⁴ is acyloxy or substituted acyloxy. In other embodiments, R⁴ is alkoxylcarbonyl or substituted alkoxycarbonyl. In other embodiments, R⁴ is acyl or substituted acyl. In other embodiments, R⁴ is thiol. In other embodiment, R⁴ is amino or substituted amino. In other embodiments, R⁴ is aminoacyl or substituted aminoacyl. In other embodiments, R⁴ is azido. In other embodiments, R⁴ is carboxyl, substituted carboxyl, carboxyalkyl or substituted carboxyalkyl. In other embodiments, R⁴ is cyano. In other embodiments, R⁴ is a halogen, such as -F, -CI, -Br and -I. In other embodiments, R⁴ is nitro.

[0063] In some embodiments, R⁵ is hydrogen. In other embodiments, R⁵ is alkyl or substituted alkyl.

In other embodiments, R⁵ is aryl or substituted aryl. In other embodiments, R⁵ is

heterocycloalkyl or substituted heterocycloalkyl. In other embodiments, R⁵ is heteroaryl or substituted heteroaryl. In other embodiments, R⁵ is hydroxyl, alkoxyl or substituted alkoxyl. In other embodiments, R⁵ is acyloxy or substituted acyloxy. In other embodiments, R⁵ is alkoxylcarbonyl or substituted alkoxycarbonyl. In other embodiments, R⁵ is acyl or substituted acyl. In other embodiments, R⁵ is thiol. In other embodiment, R⁵ is amino or substituted amino. In other embodiments, R⁵ is aminoacyl or substituted aminoacyl. In other embodiments, R⁵ is azido. In other embodiments, R⁵ is carboxyl, substituted carboxyl, carboxyalkyl or substituted carboxyalkyl. In other embodiments, R⁵ is cyano. In other embodiments, R⁵ is a halogen, such as -F, -CI, -Br and -I. In other embodiments, R⁵ is nitro.

[0064] In some embodiments, R⁶ is hydrogen. In other embodiments, R⁶ is alkyl or substituted alkyl.

In other embodiments, R⁶ is aryl or substituted aryl. In other embodiments, R⁶ is

heterocycloalkyl or substituted heterocycloalkyl. In other embodiments, R⁶ is heteroaryl or substituted heteroaryl. In other embodiments, R⁶ is hydroxyl, alkoxyl or substituted alkoxyl. In other embodiments, R⁶ is acyloxy or substituted acyloxy. In other embodiments, R⁶ is alkoxylcarbonyl or substituted alkoxycarbonyl. In other embodiments, R⁶ is acyl or substituted acyl. In other embodiments, R⁶ is thiol. In other embodiment, R⁶ is amino or substituted amino. In other embodiments, R⁶ is aminoacyl or substituted aminoacyl. In other embodiments, R⁶ is azido. In other embodiments, R⁶ is carboxyl, substituted carboxyl, carboxyalkyl or substituted carboxyalkyl. In other embodiments, R⁶ is cyano. In other embodiments, R⁶ is a halogen, such as -F, -CI, -Br and -I. In other embodiments, R⁶ is nitro. In certain embodiments, R⁶ is alkoxylcarbonyl, such as ethoxycarbonyl.

[0065] In some embodiments, R⁷ is hydrogen. In other embodiments, R⁷ is alkyl or substituted alkyl.

In other embodiments, R⁷ is aryl or substituted aryl. In other embodiments, R⁷ is

heterocycloalkyl or substituted heterocycloalkyl. In other embodiments, R⁷ is heteroaryl or substituted heteroaryl. In other embodiments, R⁷ is hydroxyl, alkoxyl or substituted alkoxyl. In other embodiments, R⁷ is acyloxy or substituted acyloxy. In other embodiments, R⁷ is alkoxylcarbonyl or substituted alkoxycarbonyl. In other embodiments, R⁷ is acyl or substituted acyl. In other embodiments, R⁷ is thiol. In other embodiment, R⁷ is amino or substituted amino. In other embodiments, R⁷ is aminoacyl or substituted aminoacyl. In other embodiments, R⁷ is azido. In other embodiments, R⁷ is carboxyl, substituted carboxyl, carboxyalkyl or substituted carboxyalkyl. In other embodiments, R⁷ is cyano. In other embodiments, R⁷ is a halogen, such as -F, -CI, -Br and -I. In other embodiments, R⁷ is nitro. In certain embodiments, R⁷ is amino. [0066] In some embodiments, R¹, R² and R⁴ are independently hydrogen. In these embodiments, R³ is halogen, such as -CI. In some embodiments, R⁵ is substituted alkoxycarbonyl such as an ot-cyano substituted alkoxylcarbonyl. For instance, R⁵ may be -CCNC(0)OCH₂CH₃. In these embodiments, R⁶ is alkoxycarbonyl such as ethoxycarbonyl, -C(0)OCH₂CH₃. In certain embodiments, R⁷ is amino.

[0067] In certain embodiments, a compound of interest and salts or solvates or stereoisomers

thereof, include:

(2-amino-6-chloro-a-cyano-3(ethoxycarbonyl)-4H-l-benzopyran-4-acetic acid ethyl ester) ("SC- 79").

[0068] Additional compounds for reactivating latent immunodeficiency virus according to

embodiments of the present disclosure are found in, e.g., WO2012/1 16170;

WO2012/069525; WO2012/055880; WO2012/055879; WO201 1/143651 ; WO201 1/054848; WO201 1/054846; WO201 1/54845; WO201 1/054843; and WO2011/054553, the disclosures of each of which are incorporated herein by reference.

FORMULATIONS, DOSAGES, AND ROUTES OF ADMINISTRATION

[0069] In general, an active agent (e.g., a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof) is prepared in a pharmaceutically acceptable composition(s) for delivery to a host.

[0070] Pharmaceutically acceptable carriers preferred for use with active agents (and optionally one or more additional therapeutic agent) may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, and microparticles, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. A composition comprising an active agent (and optionally one or more additional therapeutic agent) may also be lyophilized using means well known in the art, for subsequent reconstitution and use according to the invention. Formulations

[0071] An active agent is administered to an individual in need thereof in a formulation with a pharmaceutically acceptable excipient(s). A wide variety of pharmaceutically acceptable excipients is known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7^th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3^rd ed. Amer.

Pharmaceutical Assoc. For the purposes of the following description of formulations, "active agent" includes an active agent as described above, and optionally one or more additional therapeutic agent.

[0072] In a subject method, an active agent may be administered to the host using any convenient means capable of resulting in the desired degree of reduction of immunodeficiency virus transcription. Thus, an active agent can be incorporated into a variety of formulations for therapeutic administration. For example, an active agent can be formulated into

pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi -solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. In an exemplary embodiment, an active agent is formulated as a gel, as a solution, or in some other form suitable for intravaginal administration. In a further exemplary embodiment, an active agent is formulated as a gel, as a solution, or in some other form suitable for rectal (e.g., intrarectal) administration.

[0073] In pharmaceutical dosage forms, an active agent may be administered in the form of its pharmaceutically acceptable salts, or it may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

[0074] In some embodiments, an active agent is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strengths from about 5 mM to about 100 mM. In some embodiments, the aqueous buffer includes reagents that provide for an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars e.g., mannitol, dextrose, sucrose, and the like. In some embodiments, the aqueous buffer further includes a non-ionic surfactant such as polysorbate 20 or 80. Optionally the formulations may further include a preservative.

Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol,

chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at about 4°C. Formulations may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like.

Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures.

[0075] For oral preparations, an active agent can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0076] An active agent can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0077] An active agent can be utilized in aerosol formulation to be administered via inhalation. An active agent can be formulated into pressurized acceptable propellants such as

dichlorodifluoromethane, propane, nitrogen and the like.

[0078] Furthermore, an active agent can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. An active agent can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

[0079] Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more active agents. Similarly, unit dosage forms for injection or intravenous administration may comprise the active agent(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[0080] Unit dosage forms for intravaginal or intrarectal administration such as syrups, elixirs, gels, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet, unit gel volume, or suppository, contains a predetermined amount of the composition containing one or more active agents.

[0081] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of an active agent, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for a given active agent will depend in part on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0082] Other modes of administration will also find use with the subject invention. For instance, an active agent can be formulated in suppositories and, in some cases, aerosol and intranasal compositions. For suppositories, the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), e.g. about 1% to about 2%.

[0083] An active agent can be administered as an injectable. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.

[0084] An active agent will in some embodiments be formulated for vaginal delivery. A subject formulation for intravaginal administration comprises an active agent formulated as an intravaginal bioadhesive tablet, intravaginal bioadhesive microparticle, intravaginal cream, intravaginal lotion, intravaginal foam, intravaginal ointment, intravaginal paste, intravaginal solution, or intravaginal gel.

[0085] An active agent will in some embodiments be formulated for rectal delivery. A subject

formulation for intrarectal administration comprises an active agent formulated as an intrarectal bioadhesive tablet, intrarectal bioadhesive microparticle, intrarectal cream, intrarectal lotion, intrarectal foam, intrarectal ointment, intrarectal paste, intrarectal solution, or intrarectal gel.

[0086] A subject formulation comprising an active agent includes one or more of an excipient (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate), a binder (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, poly(ethylene glycol), sucrose or starch), a disintegrator (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate or calcium citrate), a lubricant (e.g., magnesium stearate, light anhydrous silicic acid, talc or sodium lauryl sulfate), a flavoring agent (e.g., citric acid, menthol, glycine or orange powder), a preservative (e.g., sodium benzoate, sodium bisulfite, methylparaben or propylparaben), a stabilizer (e.g., citric acid, sodium citrate or acetic acid), a suspending agent (e.g., methylcellulose, polyvinylpyrrolidone or aluminum stearate), a dispersing agent (e.g., hydroxypropylmethylcellulose), a diluent (e.g., water), and base wax (e.g., cocoa butter, white petrolatum or polyethylene glycol).

[0087] Tablets comprising an active agent may be coated with a suitable film-forming agent, e.g., hydroxypropylmethyl cellulose, hydroxypropyl cellulose or ethyl cellulose, to which a suitable excipient may optionally be added, e.g., a softener such as glycerol, propylene glycol, diethylphthalate, or glycerol triacetate; a filler such as sucrose, sorbitol, xylitol, glucose, or lactose; a colorant such as titanium hydroxide; and the like.

[0088] Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985. The composition or formulation to be

administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.

[0089] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Dosages

[0090] Although the dosage used will vary depending on the clinical goals to be achieved, a suitable dosage range of an active agent is one which provides up to about 1 mg to about 5000 mg, e.g., from about 1 mg to about 25 mg, from about 25 mg to about 50 mg, from about 50 mg to about 100 mg, from about 100 mg to about 200 mg, from about 200 mg to about 250 mg, from about 250 mg to about 500 mg, from about 500 mg to about 1000 mg, or from about 1000 mg to about 5000 mg of an active agent, which can be administered in a single dose.

[0091] Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0092] In some embodiments, a suitable dose of a compound of Formula I, e.g., SC-79 or a

pharmaceutically acceptable salt or derivative thereof, is in the range of from about 1 mg/kg body weight to about 500 mg/kg body weight, e.g., from about 5 mg/kg body weight to about 500 mg/kg body weight, from about 10 mg/kg body weight to about 500 mg/kg body weight, from about 20 mg/kg body weight to about 500 mg/kg body weight, from about 30 mg/kg body weight to about 500 mg/kg body weight, from about 40 mg/kg body weight to about 500 mg/kg body weight, from about 50 mg/kg body weight to about 500 mg/kg body weight, from about 60 mg/kg body weight to about 500 mg/kg body weight, from about 70 mg/kg body weight to about 500 mg/kg body weight, from about 80 mg/kg body weight to about 500 mg/kg body weight, from about 90 mg/kg body weight to about 500 mg/kg body weight, from about 100 mg/kg body weight to about 500 mg/kg body weight, from about 200 mg/kg body weight to about 500mg/kg body weight, from about 300 mg/kg body weight to about 500mg/kg body weight, or from about 400 mg/kg body weight to about 500mg/kg body weight.

[0093] In some embodiments, a suitable dose of a compound of Formula I, e.g., SC-79 or a

pharmaceutically acceptable salt or derivative thereof, is in the range of from about 1 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kg body weight to about 10 mg/kg body weight, from about 10 mg/kg body weight to about 20 mg/kg body weight, from about 20 mg/kg body weight to about 30 mg/kg body weight, from about 30 mg/kg body weight to about 40 mg/kg body weight, from about 40 mg/kg body weight to about 50 mg/kg body weight, from about 50 mg/kg body weight to about 100 mg/kg body weight, or from about 100 mg/kg body weight to about 500 mg/kg body weight.

[0094] In some embodiments, a single dose of an active agent is administered. In other

embodiments, multiple doses of an active agent are administered. Where multiple doses are administered over a period of time, an active agent is administered, e.g., twice daily (qid), daily (qd), every other day (qod), every third day, three times per week (tiw), or twice per week (biw) over a period of time. For example, an active agent is administered qid, qd, qod, tiw, or biw over a period of from one day to about 2 years or more. For example, an active agent is administered at any of the aforementioned frequencies for one week, two weeks, one month, two months, six months, one year, or two years, or more, depending on various factors.

[0095] Where two different active agents are administered, a first active agent and a second active agent can be administered in separate formulations. A first active agent and a second active agent can be administered substantially simultaneously, or within about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 16 hours, about 24 hours, about 36 hours, about 72 hours, about 4 days, about 7 days, or about 2 weeks of one another.

Routes of Administration

[0096] An active agent is administered to an individual using any available method and route

suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.

[0097] Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, transdermal, subcutaneous, intradermal, topical application, intravenous, vaginal, nasal, and other parenteral routes of administration. In some embodiments, an active agent is administered via an intravaginal route of administration. In other embodiments, an active agent is administered via an intrarectal route of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. The composition can be administered in a single dose or in multiple doses. [0098] An active agent can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.

[0099] Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, vaginal, transdermal, subcutaneous, intramuscular, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration can be carried to effect systemic or local delivery of the agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.

[00100] An active agent can also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.

[00101] By treatment is meant at least an amelioration of the symptoms associated with the pathological condition afflicting the host, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the pathological condition being treated, such as the number of viral particles per unit blood. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition.

[00102] A variety of hosts (wherein the term "host" is used interchangeably herein with the terms "subject" and "patient") are treatable according to the subject methods. Generally such hosts are "mammals" or "mammalian," where these terms are used broadly to describe organisms which are within the class mammalia, and primates (e.g., humans, chimpanzees, and monkeys), that are susceptible to immunodeficiency virus (e.g., HIV) infection. In many embodiments, the hosts will be humans.

Kits, Containers, Devices, Delivery Systems

[00103] Kits with unit doses of the active agent, e.g. in oral, vaginal, rectal, transdermal, or injectable doses (e.g., for intramuscular, intravenous, or subcutaneous injection), are provided. In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the drugs in treating an immunodeficiency virus (e.g., an HIV) infection. Suitable active agents and unit doses are those described herein above.

[00104] In many embodiments, a subject kit will further include instructions for practicing the subject methods or means for obtaining the same (e.g., a website URL directing the user to a webpage which provides the instructions), where these instructions are typically printed on a substrate, which substrate may be one or more of: a package insert, the packaging, formulation containers, and the like.

[00105] In some embodiments, a subject kit includes one or more components or features that increase patient compliance, e.g., a component or system to aid the patient in remembering to take the active agent at the appropriate time or interval. Such components include, but are not limited to, a calendaring system to aid the patient in remembering to take the active agent at the appropriate time or interval.

[00106] The present invention provides a delivery system comprising a compound of

Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof. In some embodiments, the delivery system is a delivery system that provides for injection of a formulation comprising an active agent subcutaneously, intravenously, or intramuscularly. In other embodiments, the delivery system is a vaginal or rectal delivery system.

[00107] In some embodiments, an active agent is packaged for oral administration. The

present invention provides a packaging unit comprising daily dosage units of an active agent. For example, the packaging unit is in some embodiments a conventional blister pack or any other form that includes tablets, pills, and the like. The blister pack will contain the appropriate number of unit dosage forms, in a sealed blister pack with a cardboard, paperboard, foil, or plastic backing, and enclosed in a suitable cover. Each blister container may be numbered or otherwise labeled, e.g., starting with day 1.

[00108] In some embodiments, a subject delivery system comprises an injection device.

Exemplary, non-limiting drug delivery devices include injections devices, such as pen injectors, and needle/syringe devices. In some embodiments, the invention provides an injection delivery device that is pre-loaded with a formulation comprising an effective amount of a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof. For example, a subject delivery device comprises an injection device preloaded with a single dose of a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof. A subject injection device can be re-usable or disposable.

[00109] Pen injectors are well known in the art. Exemplary devices which can be adapted for use in the present methods are any of a variety of pen injectors from Becton Dickinson, e.g., BD™ Pen, BD™ Pen II, BD™ Auto-Injector; a pen injector from Innoject, Inc.; any of the medication delivery pen devices discussed in U.S. Pat. Nos. 5,728,074, 6,096,010, 6, 146,361, 6,248,095, 6,277,099, and 6,221,053; and the like. The medication delivery pen can be disposable, or reusable and refillable.

[00110] The present invention provides a delivery system for vaginal or rectal delivery of an active agent to the vagina or rectum of an individual. The delivery system comprises a device for insertion into the vagina or rectum. In some embodiments, the delivery system comprises an applicator for delivery of a formulation into the vagina or rectum; and a container that contains a formulation comprising an active agent. In these embodiments, the container (e.g., a tube) is adapted for delivering a formulation into the applicator. In other

embodiments, the delivery system comprises a device that is inserted into the vagina or rectum, which device includes an active agent. For example, the device is coated with, impregnated with, or otherwise contains a formulation comprising the active agent.

[00111] In some embodiments, the vaginal or rectal delivery system is a tampon or tamponlike device that comprises a subject formulation. Drug delivery tampons are known in the art, and any such tampon can be used in conjunction with a subject drug delivery system. Drug delivery tampons are described in, e.g., U.S. Pat. No. 6,086,909. If a tampon or tampon-like device is used, there are numerous methods by which an active agent can be incorporated into the device. For example, the drug can be incorporated into a gel-like bioadhesive reservoir in the tip of the device. Alternatively, the drug can be in the form of a powdered material positioned at the tip of the tampon. The drug can also be absorbed into fibers at the tip of the tampon, for example, by dissolving the drug in a pharmaceutically acceptable carrier and absorbing the drug solution into the tampon fibers. The drug can also be dissolved in a coating material which is applied to the tip of the tampon. Alternatively, the drug can be incorporated into an insertable suppository which is placed in association with the tip of the tampon.

[00112] In other embodiments, the drug delivery device is a vaginal or rectal ring. Vaginal or rectal rings usually consist of an inert elastomer ring coated by another layer of elastomer containing an active agent to be delivered. The rings can be easily inserted, left in place for the desired period of time (e.g., up to 7 days), then removed by the user. The ring can optionally include a third, outer, rate-controlling elastomer layer which contains no drug. Optionally, the third ring can contain a second drug for a dual release ring. The drug can be incorporated into polyethylene glycol throughout the silicone elastomer ring to act as a reservoir for drug to be delivered.

[00113] In other embodiments, a subject vaginal or rectal delivery system is a vaginal or rectal sponge. The active agent is incorporated into a silicone matrix which is coated onto a cylindrical drug-free polyurethane sponge, as described in the literature.

[00114] Pessaries, tablets, and suppositories are other examples of drug delivery systems which can be used, e.g., in carrying out a method of the present disclosure. These systems have been described extensively in the literature.

[00115] Bioadhesive microparticles constitute still another drug delivery system suitable for use in the present invention. This system is a multi-phase liquid or semi-solid preparation which does not seep from the vagina or rectum as do many suppository formulations. The substances cling to the wall of the vagina or rectum and release the drug over a period of time. Many of these systems were designed for nasal use but can be used in the vagina or rectum as well (e.g. U.S. Pat. No. 4,756,907). The system may comprise microspheres with an active agent; and a surfactant for enhancing uptake of the drug. The microparticles have a diameter of 10-100 μιη and can be prepared from starch, gelatin, albumin, collagen, or dextran.

[00116] Another system is a container comprising a subject formulation (e.g., a tube) that is adapted for use with an applicator. The active agent is incorporated into creams, lotions, foams, paste, ointments, and gels which can be applied to the vagina or rectum using an applicator. Processes for preparing pharmaceuticals in cream, lotion, foam, paste, ointment and gel formats can be found throughout the literature. An example of a suitable system is a standard fragrance free lotion formulation containing glycerol, ceramides, mineral oil, petrolatum, parabens, fragrance and water such as the product sold under the trademark JEPvGENS™ (Andrew Jergens Co., Cincinnati, Ohio). Suitable nontoxic pharmaceutically acceptable systems for use in the compositions of the present invention will be apparent to those skilled in the art of pharmaceutical formulations and examples are described in Remington's Pharmaceutical Sciences, 19th Edition, A. R. Gennaro, ed., 1995. The choice of suitable carriers will depend on the exact nature of the particular vaginal or rectal dosage form desired, e.g., whether the active ingredient(s) is/are to be formulated into a cream, lotion, foam, ointment, paste, solution, or gel, as well as on the identity of the active ingredient(s). Other suitable delivery devices are those described in U.S. Pat. No. 6,476,079. Combination therapy

[00117] In some embodiments, a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, is administered in combination therapy with one or more additional therapeutic agents. Suitable additional therapeutic agents include agents that inhibit one or more functions of an immunodeficiency virus; agents that treat or ameliorate a symptom of an immunodeficiency virus infection; agents that treat an infection that occurs secondary to an immunodeficiency virus infection; and the like.

[00118] Therapeutic agents include, e.g., beta-lactam antibiotics, tetracyclines,

chloramphenicol, neomycin, gramicidin, bacitracin, sulfonamides, nitrofurazone, nalidixic acid, cortisone, hydrocortisone, betamethasone, dexamethasone, fluocortolone, prednisolone, triamcinolone, indomethacin, sulindac, acyclovir, amantadine, rimantadine, recombinant soluble CD4 (rsCD4), anti-receptor antibodies (e.g., for rhinoviruses), nevirapine, cidofovir (Vistide™), trisodium phosphonoformate (Foscarnet™), famcyclovir, pencyclovir, valacyclovir, nucleic acid/replication inhibitors, interferon, zidovudine (AZT, Retrovir™), didanosine (dideoxyinosine, ddl, Videx™), stavudine (d4T, Zerit™), zalcitabine

(dideoxycytosine, ddC, Hivid™), nevirapine (Viramune™), lamivudine (Epivir™, 3TC), protease inhibitors, saquinavir (Invirase™, Fortovase™), ritonavir (Norvir™), nelfinavir (Viracept™), efavirenz (Sustiva™), abacavir (Ziagen™), amprenavir (Agenerase™) indinavir (Crixivan™), ganciclovir, AzDU, delavirdine (Rescriptor™), kaletra, trizivir, rifampin, clathiromycin, erythropoietin, colony stimulating factors (G-CSF and GM-CSF), non-nucleoside reverse transcriptase inhibitors, nucleoside inhibitors, adriamycin, fluorouracil, methotrexate, asparaginase and combinations thereof. Anti-HIV agents are those in the preceding list that specifically target a function of one or more HIV proteins.

[00119] In some embodiments, a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, is administered in combination therapy with two or more anti-HIV agents. For example, a compound of Formula I, e.g., SC-79 or a

pharmaceutically acceptable salt or derivative thereof, can be administered in combination therapy with one, two, or three nucleoside reverse transcriptase inhibitors (e.g., Combivir, Epivir, Hivid, Retrovir, Videx, Zerit, Ziagen, etc.). A compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, can be administered in combination therapy with one or two non-nucleoside reverse transcriptase inhibitors (e.g., Rescriptor, Sustiva, Viramune, etc.). A compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, can be administered in combination therapy with one or two protease inhibitors (e.g., Agenerase, Crixivan, Fortovase, Invirase, Kaletra, Norvir, Viracept, etc.). A compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, can be administered in combination therapy with a protease inhibitor and a nucleoside reverse transcriptase inhibitor. A compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, can be administered in combination therapy with a protease inhibitor, a nucleoside reverse transcriptase inhibitor, and a non- nucleoside reverse transcriptase inhibitor. A compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, can be administered in combination therapy with a protease inhibitor and a non-nucleoside reverse transcriptase inhibitor. Other combinations of a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, with one or more of a protease inhibitor, a nucleoside reverse transcriptase inhibitor, and a non-nucleoside reverse transcriptase inhibitor are contemplated.

[00120] In some embodiments, a subject treatment method involves administering: a) a

compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof; and b) an agent that inhibits an immunodeficiency virus function selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity.

[00121] In some embodiments, a subject treatment method involves administering: a) a

compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof; and b) an HIV inhibitor, where suitable HIV inhibitors include, but are not limited to, one or more nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non- nucleoside reverse transcriptase inhibitors (N RTIs), protease inhibitors (Pis), fusion inhibitors, integrase inhibitors, chemokine receptor (e.g., CXCR4, CCR5) inhibitors, and hydroxyurea.

[00122] Nucleoside reverse transcriptase inhibitors include, but are not limited to, abacavir

(ABC; ZIAGEN™), didanosine (dideoxyinosine (ddl); VIDEX™), lamivudine (3TC;

EPIVIR™), stavudine (d4T; ZERIT™, ZERIT XR™), zalcitabine (dideoxycytidine (ddC); HIVID™), zidovudine (ZDV, formerly known as azidothymidine (AZT); RETROVIR™), abacavir, zidovudine, and lamivudine (TRIZIVIR™), zidovudine and lamivudine

(COMBIVIR™), and emtricitabine (EMTRIVA™). Nucleotide reverse transcriptase inhibitors include tenofovir disoproxil fumarate (VIREAD™). Non-nucleoside reverse transcriptase inhibitors for HIV include, but are not limited to, nevirapine (VIRAMUNE™), delavirdine mesylate (RESCRIPTOR™), and efavirenz (SUSTIVA™).

[00123] Protease inhibitors (Pis) for treating HIV infection include amprenavir

(AGENERASE™), saquinavir mesylate (FORTOVASE™, INVIRASE™.), ritonavir (NORVIR™), indinavir sulfate (CRIXIVAN™), nelfmavir mesylate (VIRACEPT™), lopinavir and ritonavir (KALETRA™), atazanavir (REYATAZ™), and fosamprenavir (LEXIVA™).

[00124] Fusion inhibitors prevent fusion between the virus and the cell from occurring, and therefore, prevent HIV infection and multiplication. Fusion inhibitors include, but are not limited to, enfuvirtide (FUZEON™), Lalezari et al., New England J. Med., 348:2175-2185 (2003); and maraviroc (SELZENTRY™, Pfizer).

[00125] An integrase inhibitor blocks the action of integrase, preventing HIV-1 genetic material from integrating into the host DNA, and thereby stopping viral replication. Integrase inhibitors include, but are not limited to, raltegravir (ISENTRESS™, Merck); and elvitegravir (GS 9137, Gilead Sciences).

[00126] Maturation inhibitors include, e.g., bevirimat (3β- (3 -carboxy-3 -methyl - butanoyloxy) lup-20(29)-en-28-oic acid); and Vivecon (MPC9055).

[00127] In some embodiments, a subject treatment method involves administering: a) a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof; and b) one or more of: (1) an HIV protease inhibitor selected from amprenavir, atazanavir, fosamprenavir, indinavir, lopinavir, ritonavir, nelfmavir, saquinavir, tipranavir, brecanavir, darunavir, TMC-126, TMC-114, mozenavir (DMP-450), JE-2147 (AG1776), L- 756423, RO0334649, KNI-272, DPC-681, DPC-684, GW640385X, DG17, PPL-100, DG35, and AG 1859; (2) an HIV non-nucleoside inhibitor of reverse transcriptase selected from capravirine, emivirine, delaviridine, efavirenz, nevirapine, (+) calanolide A, etravirine, GW5634, DPC-083, DPC-961, DPC-963, MIV-150, and TMC-120, TMC-278 (rilpivirene), efavirenz, BILR 355 BS, VRX 840773, UK-453061, and RDEA806; (3) an HIV nucleoside inhibitor of reverse transcriptase selected from zidovudine, emtricitabine, didanosine, stavudine, zalcitabine, lamivudine, abacavir, amdoxovir, elvucitabine, alovudine, MIV-210, racivir, D-d4FC, emtricitabine, phosphazide, fozivudine tidoxil, apricitibine (AVX754), amdoxovir, KP-1461, and fosalvudine tidoxil (formerly HDP 99.0003); (4) an HIV nucleotide inhibitor of reverse transcriptase selected from tenofovir and adefovir; (5) an HIV integrase inhibitor selected from curcumin, derivatives of curcumin, chicoric acid, derivatives of chicoric acid, 3,5-dicaffeoylquinic acid, derivatives of 3,5-dicaffeoylquinic acid, aurintricarboxylic acid, derivatives of aurintricarboxylic acid, caffeic acid phenethyl ester, derivatives of caffeic acid phenethyl ester, tyrphostin, derivatives of tyrphostin, quercetin, derivatives of quercetin, S-1360, zintevir (AR-177), L-870812, and L-870810, MK-0518 (raltegravir), BMS-538158, GSK364735C, BMS-707035, MK-2048, and BA 011; (6) a gp41 inhibitor selected from enfuvirtide, sifuvirtide, FB006M, and TRI-1144; (7) a CXCR4 inhibitor, such as AMD-070; (8) an entry inhibitor, such as SP01A; (9) a gpl20 inhibitor, such as BMS-488043 and/or BlockAide/CR; (10) a G6PD and NADH-oxidase inhibitor, such as immunitin; (11) a CCR5 inhibitors selected from the group consisting of aplaviroc, vicriviroc, maraviroc, PRO-140, INCB15050, PF-232798 (Pfizer), and CCR5 mAb004; (12) another drug for treating HIV selected from BAS-100, SPI-452, REP 9, SP- 01A, TNX-355, DES6, ODN-93, ODN-112, VGV-1, PA-457 (bevirimat), Ampligen, HRG214, Cytolin, VGX-410, KD-247, AMZ 0026, CYT 99007A-221 HIV, DEBIO-025, BAY 50-4798, MDXOIO (ipilimumab), PBS 119, ALG 889, and PA-1050040 (PA-040); (13) any combinations or mixtures of the above.

As further examples, in some embodiments, a subject treatment method involves administering: a) a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof; and b) one or more of: i) amprenavir (Agenerase; (3<S)-oxolan-3-yl N- [(25',3i?)-3-hydroxy-4-[N-(2-methylpropyl)(4-aminobenzene)sulfonamido]-l-phenylbutan-2- yl]carbamate) in an amount of 600 mg or 1200 mg twice daily; ii) tipranavir (Aptivus; N-{3- [(li?)-l-[(2i?)-6-hydroxy-4-oxo-2-(2-phenylethyl)-2-propyl-3,4-dihydro-2H-pyran-5- yl]propyl]phenyl}-5-(trifluoromethyl)pyridine-2 -sulfonamide) in an amount of 500 mg twice daily; iii) idinavir (Crixivan; (25)-l-[(25',4i?)-4-benzyl-2-hydroxy-4-{[(15',2i?)-2-hydroxy- 2,3 -dihydro- lH-inden- 1 -yl] carbamoyl } butyl] -N-fert-butyl-4-(pyridin-3 -ylmethyl)piperazine- 2-carboxamide) in an amount of 800 mg three times daily; iv) saquinavir (Invirase; 2<S)-N- [(25',3i?)-4-[(35)-3-(teri-butylcarbamoyl)-decahydroisoquinolin-2-yl]-3-hydroxy-l- phenylbutan-2-yl]-2-(quinolin-2-ylformamido)butanediamide) in an amount of 1,000 mg twice daily; v) lopinavir and ritonavir (Kaleta; where lopinavir is 2S)-N-[(2S,4S,5S)-5-[2- (2,6-dimethylphenoxy)acetamido]-4-hydroxy- 1 ,6-diphenylhexan-2-yl] -3 -methyl -2-(2-oxo- l,3-diazinan-l-yl)butanamide; and ritonavir is l,3-thiazol-5-ylmethyl N-[(2S,3S,5S)-3- hydroxy-5 -[(2S)-3 -methyl -2-{ [methyl({ [2-(propan-2-yl)- 1 ,3 -thiazol-4- yl]methyl})carbamoyl]amino}butanamido]-l,6-diphenylhexan-2-yl]carbamate) in an amount of 133 mg twice daily; vi) fosamprenavir (Lexiva; {[(2i?,35)-l-[N-(2-methylpropyl)(4- aminobenzene) sulfonamido] -3 -( { [(3<S) -oxolan-3 -yloxy] carbonyl } amino) -4 -phenylbutan-2- yl]oxy}phosphonic acid) in an amount of 700 mg or 1400 mg twice daily); vii) ritonavir (Norvir) in an amount of 600 mg twice daily; viii) nelfinavir (Viracept; (3S,4aS,8aS)-N-tert- butyl-2-[(2i?,3i?)-2-hydroxy-3 -[(3 -hydroxy-2-methylphenyl)formamido] -4- (phenylsulfanyl)butyl]-decahydroisoquinoline-3-carboxamide) in an amount of 750 mg three times daily or in an amount of 1250 mg twice daily; ix) Fuzeon (Acetyl - YTSLIHSLIEESQNQ QEKNEQELLELDKWASLWNWF-amide; SEQ ID NO: 1) in an amount of 90 mg twice daily; x) Combivir in an amount of 150 mg lamivudine (3TC; 2',3'- dideoxy-3'-thiacytidine) and 300 mg zidovudine (AZT; azidothymidine) twice daily; xi) emtricitabine (Emtriva; 4-amino-5 -fluoro- 1 -[(2i?,5<S)-2-(hydroxymethyl)- 1 ,3 -oxathiolan-5 - yl]-l,2-dihydropyrimidin-2-one) in an amount of 200 mg once daily; xii) Epzicom in an amount of 600 mg abacavir (ABV; {(15',4i?)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9- yl]cyclopent-2-en-l-yl} methanol) and 300 mg 3TC once daily; xiii) zidovudine (Retrovir; AZT or azidothymidine) in an amount of 200 mg three times daily; xiv) Trizivir in an amount of 150 mg 3TC and 300 mg ABV and 300 mg AZT twice daily; xv) Truvada in an amount of 200 mg emtricitabine and 300 mg tenofovir (({[(2i?)-l-(6-amino-9H-purin-9- yl)propan-2-yl]oxy}methyl)phosphonic acid) once daily; xvi) didanosine (Videx; 2',3'- dideoxyinosine) in an amount of 400 mg once daily; xvii) tenofovir (Viread) in an amount of 300 mg once daily; xviii) abacavir (Ziagen) in an amount of 300 mg twice daily; xix) atazanavir (Reyataz; methyl N-[(15)-l-{[(25^,,35)-3-hydiOxy-4-[(25)-2- [(methoxycarbonyl)amino]-3,3-dimethyl-N'-{[4-(pyridin-2- yl)phenyl]methyl }butanehydrazido] - 1 -phenylbutan-2-yl] carbamoyl } -2,2- dimethylpropyl] carbamate) in an amount of 300 mg once daily or 400 mg once daily; xx) lamivudine (Epivir) in an amount of 150 mg twice daily; xxi) stavudine (Zerit; 2'-3'- didehydro-2'-3'-dideoxythymidine) in an amount of 40 mg twice daily; xxii) delavirdine (Rescriptor; N-[2-({4-[3-(propan-2-ylamino)pyridin-2-yl]piperazin-l-yl}carbonyl)-lH-indol- 5-yl]methanesulfonamide) in an amount of 400 mg three times daily; xxiii) efavirenz (Sustiva; (45)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-lH-3, 1 - benzoxazin-2-one) in an amount of 600 mg once daily); xxiv) nevirapine (Viramune; 11- cyclopropyl-4-methyl-5,l 1 -dihydro-6H- dipyrido[3,2-6:2',3'-e][l,4]diazepin-6-one) in an amount of 200 mg twice daily); xxv) bevirimat; and xxvi) Vivecon.

In some embodiments, a subject treatment method involves administering: a) a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof; and b) a PKC activator. An example of a suitable PKC activator is prostratin ((lai?,lb^,4ai? a^,7bi?,8i?,9a^-4a,7b-dihydroxy-3 hydroxymethyl)-14,6,8-tetramethyl-5- oxo-l,la,lb,4,4a,5,7a,7b,8,9-decahydro-9aH-cyclopropa[3,4]benzo[l,2-e]azulen-9a-yl). The PKC activator can be administered in a separate formulation from a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof. A PKC activator can be co-formulated with a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, and the co-formulation administered to an individual. The present disclosure provides a kit comprising a PKC activator in a first container; and a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, in a second container.

SUBJECTS SUITABLE FOR TREATMENT

[00130] The methods of the present disclosure are suitable for treating individuals who have an immunodeficiency virus infection, e.g., who have been diagnosed as having an immunodeficiency virus infection.

[00131] The methods of the present disclosure are suitable for treating individuals who have an HIV infection (e.g., who have been diagnosed as having an HIV infection), and individuals who are at risk of contracting an HIV infection. Such individuals include, but are not limited to, individuals with healthy, intact immune systems, but who are at risk for becoming HIV infected ("at-risk" individuals). At-risk individuals include, but are not limited to, individuals who have a greater likelihood than the general population of becoming HIV infected. Individuals at risk for becoming HIV infected include, but are not limited to, individuals at risk for HIV infection due to sexual activity with HIV-infected individuals. Individuals suitable for treatment include individuals infected with, or at risk of becoming infected with, HIV-1 and/or HIV-2 and/or HIV-3, or any variant thereof.

[00132] The methods of the present disclosure are suitable for treating individuals who have previously been treated with a conventional antiretroviral treatment (ART).

DETECTION METHODS

[00133] The present disclosure provides detection methods for identifying a cell that has latent HIV. The methods generally involve contacting a cell obtained from an individual with a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof; and detecting expression of an FflV-encoded gene product. If the cell expresses an HIV -encoded gene product when contacted with a compound of Formula I, e.g., SC-79 or the pharmaceutically acceptable salt or derivative thereof, but does not express detectable levels of the HIV -encoded gene product in the absence of a compound of Formula I, e.g., SC-79 or the pharmaceutically acceptable salt or derivative thereof, the cell is considered to harbor latent HIV (i.e., to have latent HIV present in the cell genome). Thus, a subject detection method can comprise contacting a cell obtained from an individual with a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof; detecting expression of an HIV-encoded gene product; and comparing the expression, if any, of the HIV-encoded gene product in the cell contacted with the compound of Formula I, e.g., SC-79 or the pharmaceutically acceptable salt or derivative thereof, with expression of the HIV- encoded gene product in a control cell not contacted with the compound of Formula I, e.g., SC-79 or the pharmaceutically acceptable salt or derivative thereof.

[00134] Cells obtained from an individual include cells in a liquid cell suspension sample, and cells in a solid tissue sample. A cell sample obtained from an individual can be from any of a variety of tissues, e.g., brain, blood, saliva, muscle, liver, bronchoalveolar lavage, sputum, etc. The cells can be obtained in any of a variety of forms, e.g., in a buccal swab, in a blood sample, or in any type of tissue biopsy. The cell sample can be obtained from a living individual. The cell sample can be a post-mortem sample. Cells present in the cell sample can be living cells.

[00135] In some embodiments, a cell in a cell sample obtained from an individual is

contacted with a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof; and expression of an HIV-encoded gene product is detected. Gene products include nucleic acids (e.g., mRNA) and protein.

[00136] Methods of detecting nucleic acid gene products are well known in the art; any such method can be used in a subject detection method. For example, a hybridization method can be used, using a suitably labeled nucleic acid probe. Detection can be accomplished by any known method, including, but not limited to, in situ hybridization, PCR, RT-PCR, and "Northern" or RNA blotting, or combinations of such techniques, using a suitably labeled nucleic acid probe.

[00137] In some cases, a polymerase chain reaction (PCR) method (e.g., a reverse

transcription-PCR method; a quantitative PCR method; etc.) is used, employing primers (e.g., pairs of primer oligonucleotides) that amplify an HIV gene. The primer nucleic acids are prepared using any known method, e.g., automated synthesis, and the like. The primer pairs are chosen such that they specifically amplify a cDNA copy of an mRNA encoding an HIV polypeptide.

[00138] Methods using PCR amplification can be performed on the DNA from a single cell, although it is convenient to use at least about 10⁵ cells. A detectable label may be included in the amplification reaction. Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6- carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4' ,5 ' -dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X -rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) ΟΓ Ν,Ν,Ν' ,Ν' -tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels, e.g. ²P, ⁵S, H; etc. The label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high-affinity binding partner, e.g. avidin, specific antibodies, etc. , where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.

[00139] A number of methods are available for determining the expression level of a gene or protein in a particular sample. For example, detection may utilize staining of cells or histological sections with labeled antibodies, performed in accordance with conventional methods. Cells are permeabilized to stain cytoplasmic molecules. The antibodies of interest are added to the cell sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes. The antibody may be labeled with

radioisotopes, enzymes, fluorescers, chemiluminescers, or other labels for direct detection. Alternatively, a second stage antibody or reagent is used to amplify the signal. Such reagents are well known in the art. For example, the primary antibody may be conjugated to biotin, with horseradish peroxidase-conjugated avidin added as a second stage reagent.

Alternatively, the secondary antibody conjugated to a fluorescent compound, e.g.

fluorescein, rhodamine, Texas red, etc. Final detection uses a substrate that undergoes a color change in the presence of the peroxidase. The absence or presence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc.

[00140] Methods of detecting polypeptide gene products are known in the art, and include, e.g., immunological assays such as an enzyme-linked immunosorbent assay (ELISA), a protein blot assay, a radioimmunoassay, and the like, where such assays employ an antibody specific for an HIV -encoded polypeptide.

[00141] A subject detection method can be used to detect the presence, in a cell sample

obtained from an individual, of a cell harboring latent HIV. In some cases, detection in a cell sample obtained from a living individual of a cell harboring latent HIV may indicate that the individual should be treated with an agent that reactivates latent HIV. For example, the individual may be undergoing treatment for an HIV infection at the time the individual is subjected to a subject detection method; in such cases, the individual may be treated with both a treatment regimen for treating the HIV infection, and with an agent that reactivates latent HIV.

[00142] A subject detection method can be used to isolate primary cells harboring latent HIV.

Such cells can be used in a subject screening method, as described below. [00143] In some cases, a subject detection method further comprises isolating a cell that has been identified as harboring latent HIV in its genome.

SCREENING METHODS

[00144] The present disclosure provides a method of identifying a candidate agent for

treating an HIV infection in an individual. The method generally involves contacting a primary cell, identified using a detection method as described herein or using any suitable detection method known in the art, with a compound of Formula I, e.g., SC-79 or a pharmaceutically acceptable salt or derivative thereof, and a test agent; and determining the effect of the test agent on the level of HIV produced in the cell and/or the level of an HIV- encoded gene product in the cell. A test agent that reduces the level of HIV produced in the cell and/or the level of production of an HIV-encoded gene product, compared to the level of HIV produced and/or the level of an HIV-encoded gene product in a control cell contacted with the compound of Formula I, e.g., SC-79 or the pharmaceutically acceptable salt or derivative thereof, but not with the test agent, is considered a candidate agent for inhibiting HIV and treating an HIV infection.

[00145] A test agent that reduces level of HIV produced in the cell and/or the level of

production of an HIV-encoded gene product by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more than 80%, relative to a suitable control, is considered a candidate agent for treating an HIV infection.

[00146] As used herein, the term "determining" refers to both quantitative and qualitative determinations and as such, the term "determining" is used interchangeably herein with "assaying," "measuring," and the like.

[00147] The terms "candidate agent," "test agent," "agent", "substance" and "compound" are used interchangeably herein. Candidate agents encompass numerous chemical classes, typically synthetic, semi -synthetic, or naturally occurring inorganic or organic molecules. Candidate agents include those found in large libraries of synthetic or natural compounds. For example, synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), ComGenex (South San Francisco, CA), and MicroSource (New Milford, CT). A rare chemical library is available from Aldrich

(Milwaukee, Wis.) and can also be used. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from Pan Labs (Bothell, WA) or are readily producible.

[00148] Candidate agents can be small organic or inorganic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents can comprise functional groups necessary for structural interaction with proteins, e.g., hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, and may contain at least two of the functional chemical groups. The candidate agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, and derivatives, structural analogs or combinations thereof.

EXAMPLES

[00149] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p.,

intraperitoneal(ly); s.c, subcutaneous(ly); and the like.

Example 1: Reactivation of latent HIV with the AKT Activator SC-79

MATERIALS AND METHODS

[00150] Isolation of PBMC and HLAC: Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation of buffy coats from HIV- seronegative donors. PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and penicillin/ streptomycin. Human tonsil tissue from routine tonsillectomies was obtained, processed into ex vivo human lymphoid aggregate cultures (HLAC) and cultured as described in Doitsh et al., 2010, Cell, 143:5 789-801.

[00151] Spinoculation: PBMC and HLAC were spinoculated with an NL4-3 virus expressing firefly luciferase under the LTR promoter. Briefly, 50ng of p24Gag per 1.2 million cells were used. Spinoculations were performed in 96-well V-bottom plates with up to 1.2 million of cells per well in RPMI 1640 containing 10% FCS, penicillin/streptomycin and supplemented with 5 μΜ saquinavir. Cells and virus were centrifuged at 1200xg for 1.5-2 hours at 4°C. After spinoculation, cells were moved to 37°C (5%C0₂) and cultured for 72 hours, always in the presence of 5μΜ saquinavir in order to prevent residual spreading infection.

[00152] LRA treatment and luciferase assay: 72 hours after spinoculation, cells were treated with the indicated latency reversal agents (LRAs), aCD3/CD28 coated beads, or 0.001% DMSO for 48 hours. All of the different treatments were made in media containing 5μΜ saquinavir and 10μΜ raltegravir. Subsequently, the cells were spun down and lysated for the measure of the luciferase signal. Briefly, cells were harvested 48 hours after LRA treatment, lysed in 30μ1 of Reporter Lysis Buffer (Promega) and immediately subjected to one freeze- and-thaw cycle. ΙΟΟμΙ of substrate (Luciferase Assay System-Promega) was then added to each well and the luciferase activity in cell extracts was quantified with a plate reader.

Relative light units were normalized to the DMSO-treated control (DMSO=l). For the LRA combination experiments (FIG. 2, Panels A-C), the single compounds were used at the highest concentration indicated in FIG. 1, Panels A-C.

[00153] The LRAs were diluted in DMSO (except HMBA, which was diluted in water) and added to the cells at the indicated concentration. The DMSO final concentration, in the presence of the cells, was always equal to or below 0.001% of total media volume. The tested LRAs were: Panobinostat, Bryostatin-1, Prostratin, SC-79 (2-amino-6-chloro-a- cyano-3-(ethoxycarbonyl)-4H-l-benzopyran-4-acetic acid ethyl ester), JQ1 and ΗΜΒΑ.

[00154] Viability assay: Before lysis, 25 μΐ of cells from each well were plated in a new V- bottom plate, treated with cell titer blue and incubated for 4 hours at 37°C, 5% C0₂. Cell viability was measured at a plate reader and shown as a function of viability of untreated/ uninfected cells (untreated=100% viability).

RESULTS

[00155] The efficacy of the Akt activator SC-79 (2-amino-6-chloro-a-cyano-3-

(ethoxycarbonyl)-4H-l-benzopyran-4-acetic acid ethyl ester) on HIV latency reversion was tested. In this study, CD4⁺ T cells extracted from blood or lymphoid tissues (e.g. tonsil, spleen) were latently infected with a fully infectious molecular clone of NL4-3 expressing firefly luciferase from the native LTR promoter. Upon treatment with different LRAs, the amount of cells in which the latent HIV provirus was successfully reactivated was quantified by measuring the resulting luciferase signal.

[00156] The results show a high level of reactivation of latent HIV from CD4⁺ T cells in both blood and lymphoid tissue upon treatment with SC-79 compared to the effects of other LRAs (FIG. 1, Panels A-C). SC-79 displays high stimulation efficiency at low concentration within 48 hours after treatment. While similar levels of reactivation were obtained using a combination of two different LRAs, such reactivation was to the detriment of cell viability (FIG. 2, Panels A-C). [00157] The results show that SC-79 at a concentration of 500 nM can potently reactivate

LTR transcription from latently infected primary CD4⁺ T and other HIV target cells (FIG. 1). Importantly, the overall reactivation achieved is relatively comparable to that achieved with anti-CD3/anti-CD28 antibody stimulation. These antibodies are commonly employed as a highly active positive control in LRA studies.

[00158] Essentially no changes in cell viability were seen when SC-79 is used to induce primary CD4+ T cells harboring latent virus (FIG. 1, Panels A-C).

[00159] These results indicate that SC-79 is a new, highly efficient and non-toxic LRA for the reactivation of HIV latent reservoirs.

Example 2: Reactivation of latent HIV in Patient-Derived CD4⁺ T Cells with the

AKT Activator SC-79

MATERIALS AND METHODS

[00160] Patient Cells: CD4⁺ T cells were extracted from four HIV-1-infected individuals.

The patients were selected by the SCOPE Study (See the website located by placing

"https://" immediately preceding "hiv.ucsf.edu/research/scope.html") on the criteria of suppressive ART (antiretroviral treatment) and undetectable plasma HIV-1 RNA levels (<50 copies per ml) for a minimum of 6 months.

[00161] CD4⁺ T cells were extracted from PBMCs from continuous-flow centrifugation leukapheresis product using density centrifugation on a Ficoll-Hypaque gradient. Resting CD4⁺ lymphocytes were enriched by negative depletion with an EasySepHuman CD4⁺ T Cell Isolation Kit (Stemcell). Cells were cultured in RPMI medium supplemented with 10% fetal bovine serum, penicillin/streptomycin and 5μΜ saquinavir at a concentration of 1 million/ml for all experiments.

[00162] CD4⁺ T cells were stimulated for 48 hours with LRAs in media plus DMSO. The

LRAs were diluted in DMSO and added to the cells at the indicated concentration. The final DMSO percentage was 0.01% (v/v) for all single and combination treatments.

[00163] ddPCR: The release of virions in the supernatant of patient CD4⁺ T cells, following

LRA treatment, was quantified with digital droplet PCR (ddPCR Biorad). Briefly, supernatant of CD4⁺ T cell cultures was clarified via two cycles of centrifugation: first) l,500rpm for 10 minutes to remove cell debris; second) 65,000g for 1 hour to collect the virus. The pelleted virions have been then lysed, the mRNA was extracted with a RNAsy kit (Quiagen) and reverse-transcribed (super Script III, Invitrogen) prior to ddPCR analysis. RESULTS

[00164] The results show reactivation of latent HIV from CD4⁺ T cells extracted from HIV-

1-infected individuals, with as much as about a 7-fold increase in HIV-1 mRNA in the supernatant relative to a DMSO negative control (Figure 3). Example 3: Expression of Activation Markers in Patient-Derived CD4⁺ T Cells

Following Treatment with the AKT Activator SC-79

MATERIALS AND METHODS

Following treatment with LRAs, the CD4⁺ T cells were spun down and stained with antibodies for CD3, CD4, CD69, CD25, PD-1. Briefly, cells were harvested 48 hours after LRA treatment, incubated with the antibodies for 15 minutes at room temperature, than washed and fixed with 1%PFA for 30 minutes; then washed again and analyzed via flow cytometer.

RESULTS

[00165] As shown in Figure 4, activation markers CD69, CD25 and PD-1 (normalized to

100% upon treatment with anti-CD3/CD28 beads) are not up-regulated on the surface of patient derived CD4⁺ T cells following treatment with SC-79, indicating a lack of increased cellular activation, which may be desirable in the context of HIV-1 latency reversion. In contrast, treatment with Bryostatin-1, a known latency-reversing agent, results in an increase in activation marker expression.

Example 4: Viability of Patient-Derived CD4⁺ T Cells Following Treatment with the AKT Activator SC-79

MATERIALS AND METHODS

[00166] Following treatment with LRAs, the CD4⁺ T cells were spun down and stained with the cell-death marker Zombie Violet (BioLegend). Briefly, cells were harvested 48 hours after LRA treatment, incubated with the Zombie dye for 15 minutes at room temperature, than washed and fixed with 1%PFA for 30 minutes; then washed again and analyzed via flow cytometer. Percentage of live (Zombie negative (-)) T Cells (CD3+) was normalized to the DMSO-treated control (DMSO or "untreated" =1).

RESULTS

[00167] The percentage of zombie negative T cells (i.e. viable cells) measured at the flow cytometer for SC-79-treated CD4⁺ T cells was similar to that for the untreated control, indicating that 48h incubation with SC-79 does not reduce cell -viability (Figure 5). In comparison, Panobinostat, a known latency-reversing agent, is highly toxic as shown.

[00168] While the present invention has been described with reference to the specific

embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:

1. A method of reactivating a latent human immunodeficiency virus (HIV) integrated into the genome of a cell infected with HIV, the method comprising contacting the cell with a compound of Formula I, wherein the compound of Formula I reactivates the latent HIV integrated into the genome of the cell.

2. The method of claim 1, wherein the compound of Formula I is SC-79 or pharmaceutically acceptable salt or derivative thereof.

3. A method of reducing the number of cells containing a latent human

immunodeficiency virus in an individual, the method comprising administering to the individual an effective amount of a compound of Formula I, wherein the compound of Formula I reactivates latent HIV integrated into the genome of one or more cells in the individual.

4. The method of claim 3, wherein said administering is effective to reduce the number of cells containing a latent human immunodeficiency virus in the individual by at least 20%.

5. The method of claim 3 or 4, wherein the compound of Formula I is SC-79 or a pharmaceutically acceptable salt or derivative thereof.

6. A method of treating a human immunodeficiency virus (HIV) infection in an individual, the method comprising:

administering to an individual an effective amount of a first active agent, wherein the first active agent is a compound of Formula I, and wherein the compound of Formula I reactivates latent HIV integrated into the genome of a cell in the individual; and

administering to the individual an effective amount of a second active agent, wherein the second active agent inhibits an immunodeficiency virus function selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity.

7. The method of claim 6, wherein the first active agent is SC-79 or a pharmaceutically acceptable salt or derivative thereof.

8. The method of claim 6 or 7, wherein one or both of said administering steps is by a vaginal route of administration, by a rectal route of administration, by an oral route of administration, or by an intravenous route of administration.

9. A drug delivery device comprising:

a) a first container comprising a compound of Formula I, wherein the compound of Formula I reactivates latent immunodeficiency virus transcription; and

b) a second container comprising an agent that inhibits an immunodeficiency virus function selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity.

10. The device of claim 9, wherein the compound of Formula I is SC-79 or

pharmaceutically acceptable salt or derivative thereof.

11. The device of claim 9 or 10, wherein the first and second containers are syringes.

12. A method of identifying a candidate agent for treating a human immunodeficiency virus (HIV) infection in an individual, the method comprising:

contacting a cell comprising latent HIV with a compound of Formula I and a test agent; and

determining the effect of the test agent on the level of HIV produced in the cell and/or the level of an HIV -encoded gene product in the cell,

wherein a test agent that reduces the level of HIV produced in the cell and/or the level of production of an HIV -encoded gene product, compared to the level of HIV produced and/or the level of an HIV -encoded gene product in a control cell contacted with the compound of Formula I in the absence of the test agent, is a candidate agent for treating an HIV infection.

13. The method of claim 12, wherein the compound of Formula I is SC-79 or a pharmaceutically acceptable salt or derivative thereof.

14. A method of detecting in a cell sample a cell comprising latent human

immunodeficiency virus (HIV), the method comprising:

a) contacting a cell sample obtained from an individual with a compound of Formula I, wherein the compound of Formula I reactivates latent immunodeficiency virus transcription; and b) detecting HIV gene expression in the cell, compared to HIV gene expression in a control cell not contacted with the agent,

wherein detection of HIV gene expression in the cell contacted with the agent indicates that the cell comprises latent HIV.

15. The method of claim 14, further comprising isolating the cell from the sample.

16. The method of claim 14 or 15, wherein detection of HIV gene expression comprises detecting an HIV -encoded gene product.

17. The method of any one of claims 14-16, wherein the compound of Formula I is SC- 79 or pharmaceutically acceptable salt or derivative thereof.