WO2008103346A1 - Compositions and methods for treating viral infections - Google Patents

Abstract

The present invention relates to a combination of an agent that inhibits NF-KB with an antiviral agent. The invention also relates to methods for using the inventive combination.

Description

COMPOSITIONS AND METHODS FOR TREATING VIRAL INFECTIONS

BACKGROUND OF THE INVENTION

Human immunodeficiency virus (HIV) is a retrovirus that causes Acquired Immunodeficiency Syndrome (AIDS), a condition in humans in which the immune system begins to fail, leading to life-threatening opportunistic infections. HIV is classified as a member of the genus lentivirus. Two species of HIV infect humans: HIV-I and HIV-2. HIV-I is more virulent, is easily transmitted, and is the cause of the majority of HIV infections globally. HIV-2 is less transmittable and is largely confined to West Africa. HIV is transmitted as single-stranded, positive-sense, enveloped RNA virus.

HIV enters host macrophages and CD4⁺ T cells by the interaction of glycoproteins on the viral surface with host receptors (both CD4 receptors and chemokine receptors), followed by fusion of the viral envelope with the cell membrane. After the fusion step, the HIV core, containing the HIV RNA and various viral enzymes, including virally encoded reverse transcriptase, integrase, ribonuclease, and protease, is released into the cellular cytoplasm. The viral RNA genome is then converted to double-stranded DNA by the virally encoded reverse transcriptase, followed by integration into the host cellular DNA by the virally encoded integrase. The integrated virus can then enter into a latent or quiescent state, or can become active and generate a large number of virus particles that can then infect other target cells. To actively produce the virus, certain cellular transcription factors need to be present, the most important of which appears to be NF-κB, which is upregulated when T cells become activated.

Antiretroviral drugs can be broadly classified by the phase of the retrovirus life-cycle that the drug inhibits. This method generates five broad classifications of antiretroviral drugs with only the first three classes currently having licensed examples: (1) Entry inhibitors block HIV-I from gaining entry to the host cell, for example by binding to CCR5, a molecule on the host cell membrane (termed a co- receptor) that HIV-I normally uses for entry into the cell, or by blocking HIV from fusing with a cell's membrane to enter and infect it. (2) Reverse transcriptase inhibitors (RTIs) target construction of viral DNA by inhibiting activity of reverse transcriptase. There are two subtypes of RTIs with different mechanisms of action: nucleoside and nucleotide analogue RTIs (NRTIs and NtRTIs, respectively) are incorporated into the viral DNA leading to chain termination, while non-nucleoside- analogue RTIs (NNRTIs) act as competitive inhibitors of the reverse transcriptase enzyme. (3) Integrase inhibitors inhibit the enzyme integrase, which is responsible for integration of viral DNA into the DNA of the infected cell. (4) Protease inhibitors (PIs) target viral assembly by inhibiting the activity of protease, an enzyme used by HIV to cleave nascent proteins for final assembly of new virons. (5) Maturation inhbitors inhibit the final assembly of the viral particle. Currently, commercially available drugs are in the entry inhbitors, RTIs, and protease inhibitors classes with new drugs in each class under development. There are several integrase and maturation inhibitors currently under clinical trial but none are commercially available at this time.

In light of rapid rates of viral replication, the highly error-prone HIV-I reverse transcriptase, and the inability of currently available antiretro viral agents to completely inhibit HIV-I replication, the development of resistance to antiretroviral drugs has been an inevitable consequence of drug exposure. Retroviral therapy, especially for HIV, is now thought to be a life-long process. Therefore, it is crucial to develop effective treatments that can be successfully administered for long periods of time to suppress retroviral replication, and in particular, to prevent and/or inhibit HIV. Further, it would be desirable to eliminate, or at least minimize, the clinical toxicity associated with the administration of antiviral agents otherwise determined to be effective. It is generally recognized that the toxicity of an antiviral agent may be avoided or at least minimized by administration of a reduced dose of the antiviral agent; however, it is also recognized that the effectiveness of an antiviral agent generally decreases as the dose is reduced.

With rare exceptions, no individual antiretroviral drug has been demonstrated to suppress an HIV infection for long; these agents must be taken in combinations in order to have a lasting effect. As a result the standard of care is to use combinations of antiretroviral drugs. Combinations of antiretrovirals are subject to positive and negative synergies, which limit the number of useful combinations. For example, ddl and AZT inhibit each other, so taking them together is less effective than taking either one separately. Other issues further limit treatment options from antiretroviral drug combinations, including their complicated dosing schedules, often severe side effects, and the risk of developing drug resistance. Current treatment for HIV consists of highly active antiretroviral therapy, or HAART. Current HAART options are combinations (or "cocktails") comprising at least three drugs belonging to at least two classes of antiretroviral agents. Typically, these combinations comprise two nucleoside analogue reverse transcriptase inhibitors (NRTIs) plus either a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor (NNRTI).

NF-κB, or Nuclear Factor kappa B, is a primary transcription factor found in all cell types. NF-κB promotes the expression of over 150 target genes in response to inflammatory stimulators. These genes include interleukin-1, -2, -6 and TNF-R, as well as genes encoding immunoreceptors, cell adhesion molecules, and enzymes such as cyclooxygenase-II and iNOS (Karin, 2006; Tergaonkar, 2006). NF-κB also plays a key role in the progression of diseases associated with viral infections such as HIV- 1. Thus, NF-κB is considered a promising target for antiretroviral therapy.

Members of the NF-κB family include RelA/p65, ReIB, c-Rel, p5O/plO5 (NF-KB I), and p52/ pi 00 (NF-κB2) (Hayden and Ghosh, 2004; Hayden et al, 2006a; Hayden et al, 2006b). The ReI family members function as either homodimers or heterodimers with distinct specificity for czs-binding elements located within the promoter domains of NF-κB-regulated genes (Bosisio et al,

2006; Natoli et al, 2005; Saccani et al, 2004). Classical NF-κB, composed of the RelA/p65 and p50 heterodimer, is the best-studied form of NF-κB (Burstein and Duckett, 2003; Hayden and Ghosh, 2004; and references therein). Prior to cellular stimulation, classical NF-κB resides in the cytoplasm as an inactive complex bound to the IκBα inhibitor proteins. Inducers of NF-κB such as bacterial lipopolysaccharides, inflammatory cytokines, or HFV-I Vpr protein release active NF-κB from the cytoplasmic complex by activating the IκB-kinase complex (IKK), which phosphorylates IκBα (Greten and Karin, 2004; Hacker and Karin, 2006; Israel, 2000; Karin, 1999; Scheidereit, 2006). Phosphorylation of IKB marks it for subsequent ubiquitinylation and degradation by the 26S proteosome. Free NF-κB dimers translocate into the nucleus where they stimulate the transcription of their target genes. The infection and life-cycle of HIV-I appears to be tightly coupled to the NF-κB pathway in human mononuclear cells. The form of the virus that is the primary infectious agent requires both the CD4 receptor and the chemokine (C-C motif) receptor-5 (CCR-5) co-receptor to gain access to its host cells in the human body. Deletion analysis of the CCR-5 promoter has demonstrated that loss of the 3'- distal NF-κB/AP-1 site drops transcription by >95% (Liu et al, 1998). Once in the cell, the virus is uncoated and the RNA genome is transcribed into DNA. This DNA pro-viral genome is integrated into the host DNA where it is transcribed into mRNA that can be translated into new viral proteins. Numerous studies have shown that activation of NF-κB is required for transcription of the integrated DNA-pro-virus (Baba, 2006; Iordanskiy et al, 2002; Mukerjee et al, 2006; Palmieri et al, 2004; Rizzi et al, 2004; Sui et al, 2006). In fact, lack of NF-κB activation leads to the generation of a population of cells harboring latent virus which is a major block to eliminating the virus from infected patients (Williams et al, 2006). Once the viral pre-protein is generated it is cleaved into the constituent proteins necessary to generate new viral particles.

Recently a new connection between HIV-I infection and NF-κB activation has begun to be appreciated. The over stimulation of T-cells through the activation of NF-κB, as a result of viral infection, leads to the depletion of these critical cells and is the hallmark of AIDS (reviewed in Argyropoulos and Mouzaki, 2006).

SUMMARY OF THE INVENTION

The present invention relates to a combination of an agent that inhibits NF- KB with an antiviral agent. The invention also relates to methods for using the inventive combination. Surprisingly, the present inventors have found that the combinations provide unexpectedly potent antiviral activity, particularly antiretroviral activity.

In one embodiment of the present invention, a method of treating or preventing a viral infection, comprising administering to a subject in need thereof a therapeutically effective amount of a combination of an NF-κB inhibitor and an antiviral agent, is provided. In a preferred embodiment, the viral infection is a retroviral infection, particularly an HIV-I infection.

In another preferred embodiment, the subject is treatment experienced and the viral infection has resistance to antiviral therapy. hi an additional preferred embodiment, the NF-κB inhibitor is parthenolide,

JSH-23, SM-7368, resveratrol, curcumin, or derivatives thereof, and the antiviral agent is an entry inhibitor, an NRTI, an NtRTI, or an integrase inhibitor.

A further aspect of the present invention is a composition comprising an NF- KB inhibitor and an antiviral agent.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the concentration dependent inhibition of NF-κB by parthenolide.

FIG. 2 shows the concentration dependent inhibition of HIV- IBaI and HIV- 1 IIIB by parthenolide.

FIG. 3 shows the cytotoxicity of parthenolide in activated hPBMCs. FIG. 4 shows the cell cycle controlling activity of parthenolide. FIG. 5 shows the concentration dependent inhibition of CCR-5 by parthenolide. FIG. 6 shows synergy plots of HAART drugs and parthenolide.

FIG. 7 shows the reduction Of IC₅₀ values of HAART drugs co-administered with parthenolide.

FIG. 8 shows the concentration dependent inhibition of NF-κB by JSH-23. FIG. 9 shows the concentration dependent inhibition of HIV- IBaI and HIV- HIIB by JSH-23.

FIG. 10 shows the cytotoxicity of JSH-23 in activated hPBMCs. FIG. 11 shows the cell cycle controlling activity of JSH-23. FIG. 12 shows the concentration dependent inhibition of CCR-5 by JSH-23. FIG. 13 shows synergy plots of HAART drugs and JSH-23. FIG. 14 shows the reduction of IC₅₀ values of HAART drugs coadministered with JSH-23. FIG. 15 shows the concentration dependent inhibition of NF-κB by SM- 7368.

FIG. 16 shows the concentration dependent inhibition of HIV- IBaI and HIV-IIIIB by SM-7368. FIG. 17 shows the cytotoxicity of SM-7368 in activated hPBMCs.

FIG. 18 shows the cell cycle controlling activity of SM-7368.

FIG. 19 shows the concentration dependent inhibition of CCR- 5 by SM- 7368.

FIG. 20 shows synergy plots of HAART drugs and SM-7368. FIG. 21 shows the reduction of IC₅₀ values of HAART drugs coadministered with SM-7368.

FIG. 22 shows the concentration dependent inhibition of NF-κB by resveratrol.

FIG. 23 shows the concentration dependent inhibition of HIV- IBaI and HIV-IIIIB by resveratrol.

FIG. 24 shows the cytotoxicity of resveratrol in activated hPBMCs.

FIG. 25 shows the cell cycle controlling activity of resveratrol.

FIG. 26 shows the concentration dependent inhibition of CCR- 5 by resveratrol. FIG. 27 shows synergy plots of HAART drugs and resveratrol.

FIG. 28 shows the reduction of IC₅₀ values of HAART drugs coadministered with resveratrol.

FIG. 29 shows the concentration dependent inhibition of NF-κB by curcumin. FIG. 30 shows the concentration dependent inhibition of HIV-IBaI and

HIV-IIIIB by curcumin.

FIG. 31 shows the cytotoxicity of curcumin in activated hPBMCs.

FIG. 32 shows the cell cycle controlling activity of curcumin.

FIG. 33 shows the concentration dependent inhibition of CCR- 5 by curcumin.

FIG. 34 shows synergy plots of HAART drugs and curcumin. FIG. 35 shows the reduction Of IC₅₀ values of HAART drugs coadministered with curcumin.

FIG. 36 shows the structure of various NF-κB inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compositions comprising a combination of an NF-κB inhibitor and an antiviral agent. The present invention also provides a method of treating a viral infection comprising administering to a subject in need thereof an effective amount of a combination of an NF-κB inhibitor and an antiviral agent.

NF-κB inhibitor. As used herein, an NF-κB inhibitor is any compound that inhibits the activity NF-κB. An NF-κB inhibitor includes a compound that blocks the activation of the p65/RelA subunit in the cytoplasm, prevents translocation of the p65/RelA subunit to the nucleus, prevents binding of the p65/RelA subunit to its' cognate DNA binding site, prevents p65/RelA subunit recruitment of chromatin remodeling complexes and/or transcription machinery, and/or prevents derepression of the chromatin locus associated with the cognate DNA-binding site. See Hoberg et al., 2006.

Examples of known NF-κB inhibitors are set forth in the following table.

NF-kB inhibitors

NF-κB inhibitors suitable for the present invention also include analogues and derivatives of the NF-κB inhibitors disclosed herein, and/or pharmaceutically acceptable conjugates, precursors, metabolites or salts thereof, that maintain NF-κB inhibitory function, hi a preferred embodiment of the invention, the NF-κB inhibitor is parthenonolide, JSH-23, SM-7368, resveratrol, curcumin, or analogues or derivatives thereof.

Parthenolide. Parthenolide, a sesquiterpene lactone derived from Tanacetum parthenum, is the active ingredient of the herbal remedy feverfew and has been used to prevent migraines and treat arthritis among other inflammatory ailments (Brown et al, 1997; Kwok et al, 2001; Reuter et al, 2002). The majority of parthenolide's biological activity is due to inhibition of NF-κB signaling (Garcia- Pineres et al, 2001; Lopez-Franco et al, 2006; Nam, 2006). This inhibition, as described below, is due to the blockade of the p65/RelA subunit of NF-κB from binding to its cognate DNA-binding site. Parthenolide has also been shown to induce apoptosis in human cancer cell lines by other mechanisms including lowering levels of thiols with resultant caspase activation and by promoting GADDl 53 activity (Nozaki et al., 2001; Wen et al., 2002). Recently, parthenolide has also been shown to decrease in vitro cancer cell growth and enhance taxane-induced cytotoxicity (Bellarosa et al, 2005; Dziadyk et al, 2004; Miglietta et al, 2004; Patel et al, 2000).

Sesquiterpene lactones are a group of compounds produced in a variety of plants, mostly from the Asteraceae family, as secondary metabolites (Wagner et al, 2006). These compounds comprise a structural class for which more than 4000 structures are known. Numerous species of the Asteraceae family are used in traditional medicine for the treatment of inflammation, fever, arthritis, migraine, skin disorders, urogenital complaints, and for relief from morning sickness (Groenewegen et al, 1992; Knight, 1995). In cases where the active ingredients have been isolated from these herbal remedies the majority have been shown to be members of the sesquiterpene lactone family. All of the conditions treated with these herbal remedies are characterized by the induction of an inflammatory response. The anti-inflammatory activity of sesquiterpene lactones has been corroborated using various assays, and several studies have established that they exert their activity by inhibiting the transcription factor NF-κB (Garcia-Pineres et al, 2001; Hilmi et al, 2003; Lopez-Franco et al, 2006; Nam, 2006; Wagner et al, 2006). Wagner et al, using two unique sesquiterpene lactones as models, have shown that DNA binding of NF-κB is prevented by alkylation of cysteine-38 in the p65/RelA subunit of NF-κB (Garcia-Pineres et al, 2001; Garcia-Pineres et al, 2004; Rungeler et al, 1999; Wagner et al, 2006). According to Wagner et al, this may be a general mechanism for sesquiterpene lactones, which possess α,β-unsaturated carbonyl structures such as α-methylene-γ-lactones or α,β-unsaturated cyclopentenones. These functional groups are known to react with nucleophiles, especially with the sulfhydryl group of cysteine, in a Michael-type addition (Rungeler et al, 1999; Schmidt et al, 1999).

As used herein, reference will be made to parthenolide, with the understanding that whatever is disclosed in connection with parthenolide is expected by the present inventors to apply at least to all sesquiterpene lactones, salts thereof, stereoisomers, enantiomers, regioisomers, diasteromers, derivatives thereof, and to plant or herbaceous extracts containing such sesquiterpene lactones. Sesquiterpene lactones identified in Wagner et al as compounds 9, 12, 14, 21, 48, 51, 53, 65, and 71 {see Table 6 at page 2246) are of particular interest.

JSH-23. Shin et al. describe a novel aromatic diamine (4-Methyl-Nl-(3- phenyl -prop yl)-benzene- 1 ,2-diamine) compound designated JSH-23 (Shin et al., 2004). This compound was generated in a series of compounds looking for molecules with anti-inflammatory properties (Min et al, 2004). These authors were interested in the compounds' ability to inhibit nitric oxide production and NF-κB transcriptional activity in lipopolysaccharide (LPS)-stimulated macrophage cell line (RAW 264.7). In the manuscript by Shin et al, the authors describe the molecular mechanism by which JSH-23 inhibits NF-κB transcription induction. In the described experiments the authors demonstrate that the JSH-23 compound can inhibit LPS-induced DNA binding activity and nuclear translocation of the NF-κB p65 subunit. However, the suppression of nuclear transport was not due to inhibition of IκBα degradation. JSH-23 holds great promise as the scaffold upon which to build a powerful NF-κB inhibitor. As used herein, reference will be made to JSH-23, with the understanding that whatever is disclosed in connection with JSH-23 is expected by the present inventors to apply at least to salts thereof, cis- and trans-isomers thereof, stereoisomers, enantiomers, regioisomers, diasteromers, oligomers thereof, polymers thereof, derivatives thereof, and to plant or herbaceous extracts containing such compounds.

SM-7368. SM-7368 is a novel benzamide compound (3-Chloro-4-nitro-N- (5-nitro-b2-thiazolyl)-benzamide) identified from a chemical library by Lee et al. for its ability to block TNF-α-induced matrix metalloproteinase-9 (MMP-9) production (Lee et al, 2005). The expression of the MMP-9 gene has been reported to require the activations of NF-κB and AP-I in some cell types (Bond et al, 2001; Bond et al, 1998; Chase et al, 2002; Woo et al, 2004; Woo et al, 2005). In the study by Lee et al, the authors demonstrate that SM-7368 blocks the expression of an NF-κB reporter gene but has no effect on the expression of a reporter gene under the control of AP-I in a human fibrosarcoma cell line (HT1080). The authors further show that TNF-α-induced NF-κB activity was completely inhibited by SM-7368 compound. These results demonstrate that SM-7368 has the properties desired in a specific NF- KB inhibitor making it a good lead candidate for further development.

As used herein, reference will be made to SM-7368, with the understanding that whatever is disclosed in connection with SM-7368 is expected by the present inventors to apply at least to salts thereof, cis- and trans-isomers thereof, stereoisomers, enantiomers, regioisomers, diasteromers, oligomers thereof, polymers thereof, derivatives thereof, and to plant or herbaceous extracts containing such compounds. Resveratrol. Resveratrol, 3, 4', 5-trihydroxyl-trα«5-stilbene, is a naturally occurring phytoalexin that was first isolated from the roots of white hellebore (yeratrum grandiflorum O.Loes), but has subsequently been found in the skin of grapes, red wine, peanuts, olive oil, cranberries, and other foods (reviewed in Aggarwal et al, 2004). Resveratrol is produced by a variety of plants in response to stress, injury, ultraviolet irradiation, and fungal infection. Transfer of the biosynthetic genes involved in the synthesis of resveratrol conveys greater resistance to fungal infection to the recipient (Beekwilder et al, 2006; Hain et al, 1993; Jeandet et al, 2002; Richter et al, 2006). Resveratrol has shown a wide range of biological activities, such as antioxidative (Frankel et al, 1993), antiplatelet (Aburjai, 2000; Olas et al, 2005), antifungal (Jung et al, 2005; Schouten et al, 2002; Seppanen et al, 2004), phytoestrogen^ (Klinge et al, 2005; Klinge et al, 2003; Lu and Serrero, 1999), cardioprotective (Bradamante et al, 2004; Das and Maulik, 2006; Wu et al, 2001), and anti-viral activities (Heredia et al, 2000). Resveratrol has also been shown to function as an anti-neoplastic (Jang et al, 1997; Jang and Pezzuto, 1999). This data has pushed resveratrol into a number of clinical trials for colon and prostate cancer as well as a chemopreventative agent (Aggarwal et al, 2004; Bemis et al, 2006). A plethora of recent reports have demonstrated that resveratrol could extend the lifespan of many organisms from yeast to mice, implicating the potential of resveratrol as an anti-aging agent (Bauer et al, 2004; Baur et al, 2006; Collins et al, 2006; Howitz et al, 2003; Morris, 2005; Valenzano and Cellerino, 2006; Valenzano et al, 2006). Plants containing resveratrol have been used in traditional Asian medicine for hundreds of years for the treatment of a number of the ailments listed above.

Resveratrol appears to exert its effects on cells through multiple mechanisms. Resveratrol acts as an antioxidant (Olas et al, 2005), inhibits cyclooxygenases (COX; (Kundu et al, 2006a; Szewczuk et al, 2004; Torres-Lopez et al, 2002)) and protein kinase C (PKC; (Li et al, 2005; Woo et al, 2004)), prevents cytokine release (Baolin et al, 2004; Boscolo et al, 2003; Culpitt et al, 2003; Donnelly et al, 2004), and NF-κB and AP-I transcription factor induction (reviewed in Aggarwal et al, 2004; Kundu et al, 2006b; Nam, 2006). Other effects of resveratrol include increasing high-density lipoprotein, cholesterol, and lipid peroxidation (Rimando et al, 2005). Resveratrol also inhibits mammalian ribonucleotide reductase (reviewed in Aggarwal et al, 2004) and DNA polymerases (Sun et al, 1998). Many of these effects directly or indirectly affect the expression of genes involved in the inflammatory response and are controlled by the NF-κB signal transduction pathway. It has been suggested that resveratrol inhibits NF-κB by blocking the activity of IKK, the stress-induced kinase that facilitates the degradation of NF-κB's homeostatic inhibitor, IKB (Aggarwal and Shishodia, 2006; Kundu et al, 2006a). It has also been demonstrated that resveratrol induces the activity of SIRTl, an NAD+ dependent protein deacetylase. Activation of SIRTl by resveratrol inhibits NF-κB signaling by promoting deacetylation of Lys310 of RelA/p65 (Chen et al, 2005; Chen et al, 2001; Yeung et al, 2004). Acetylation of Lys310 allows recruitment of the enhansosome complex which is responsible for recruitment of chromatin modifying and transcriptional machinery required for transcription of NF-κB responsive genes (Chen et al, 2001; Hoberg et al, 2006).

Heredia et al, explored resveratrol in combination with the nucleoside analogue ddl against HIV-I as an anti-viral treatment in part due to the fact that it was a widely used natural product, with the potential of being nontoxic and less costly. The authors reasoned that resveratrol' s ability to effect the cell cycle (prolongation of the S phase), its activity against protein kinase C, and particularly inhibit ribonucleotide reductase would deplete the deoxyadenosine triphosphate (dATP) pools. As a consequence of the depleted dATP pools, more ddATP generated from ddl may be available to be incorporated into the HIV-I DNA.

Although resveratrol worked well in culture and synergized with ddl to inhibit HIV- 1 replication in cultured cells it was not pursued due to metabolism issues. Resveratrol is rapidly metabolized and cleared through glucuronidation and sulfation mainly in the small intestine and to a lesser extent in the liver (Brill et al, 2006; De Santi et al, 2000; Kuhnle et al, 2000; Marier et al, 2002; Sabolovic et al, 2006; Urpi-Sarda et al, 2007; Wenzel et al, 2005). Wang et al, tested the major metabolites of resveratrol for their ability to inhibit HVI-I replication and found no activity (Wang et al, 2004).

As noted above, resveratrol is present in a number of plants used in traditional Asian medicine. In addition, resveratrol is an ingredient in several over- the-counter vitamin supplements. Therefore, resveratrol or derivatives thereof are available as extracts or powders of natural products, mainly extracted from Vitaceae species and particularly from the skin, grapes, grape-seeds, grape-stalks, and leaves of grapevines. Its concentration is greater in grape plants affected by typical diseases of the vine. Further sources of resveratrol or derivatives thereof may include extracts of the root, rhizome, stalk, leaf, fruit, cotyledon, or seed of sources such as Vitaceae, Umbellifereae, Myrtaceae, Dipterocarpaceae, Cyperaceae, Gnetaceae, Leguminosae, cereals, Sericeae, Haemodoraceae, Musaceae, Polygonacea, Pinaceae, Cupressaceae, Cesalpiniaceae, Poaceae, or Solanaceae, for example. Resveratrol is commercially available from SIGMA (St. Louis, Mo.) and from PHARMASCIENCE as RESVERIN™ (Montreal, Quebec, Canada). As used herein, reference will be made to resveratrol, with the understanding that whatever is disclosed in connection with resveratrol is expected by the present inventors to apply at least to dihydroxystilbenes, trihydroxystilbenes, tetrahydroxystilbenes, salts thereof, cis- and trans-isomers thereof, stereoisomers, enantiomers, regioisomers, diasteromers, oligomers thereof, polymers thereof, derivatives thereof, and to plant or herbaceous extracts containing such compounds.

Particularly preferred derivatives of resveratrol include alkyl, alkoxy derivatives such as pterostilbene (trans-3,5-dimethoxy-4'-hydroxystilbene), or carbohydrate derivatives such as the glycoside derivative, 3,4',5-trihydroxystilbene- 3-β-mono-D-glucoside (trans-polydatin, piceid). Further trihydroxystilbenes contemplated for methods and compositions of the present invention include trans- 3,3',5-trihydroxystilbene, trans-3,4,4'-trihydroxystilbene, 3,3',5-trihydroxy-4'- methoxystilbene-3-O-β-D-glucoside (rhapontin, SIGMA, St. Louis Mo.) or the like.

Tetrahydroxystilbenes contemplated for methods and compositions of the present invention include trans-3,3',4,5'-tetrahydroxystilbene (piceatannol, SIGMA), trans-3,3', 5,5'-tetrahydroxystilbene, or the like. A dihydroxystilbene is 3,5- dihydroxystilbene (pinosylvin). Heynekamp et al, 2006, describe substituted trans- stilbenes, including analogues of resveratrol, that inhibit NF-κB. Substituted trans- stilbenes numbered 6cc, 6r, 4cc, 4k, and 6p are of particular interest (see Table 1, page 7186). PCT publication WO 99/03816 is incorporated by reference herein for disclosure of resveratrol compositions and preparation thereof.

Curcumin. Curcumin [l,7-bis(4-hydroxy-3-methoxyphenyl)-l,6- heptadiene-3,5-dione; diferuloylmethane], is a non-nutritive, non-toxic, crystalline compound. It is the major constituent of the yellow spice turmeric derived from the rhizome of the plant Curcuma longa (L. Zingiber aceae). Turmeric has been used for centuries in India and elsewhere in cooking and as an herbal medicinal treatment of wounds, jaundice, and rheumatoid arthritis and many of the therapeutic effects have been confirmed by contemporary scientific research. In preclinical studies, curcumin has been shown to have chemopreventive potential in several different animal tumor models, including colon (Chen et ah, 2006; Perkins et ah, 2002), duodenal (Huang et ah, 1994), stomach (Singh et ah, 1998), prostate (Dorai et ah, 2001), and breast (Choudhuri et ah, 2002) carcinogenesis. Curcumin has also been shown to prevent metastasis in a number of bioassay systems (Aggarwal et ah, 2005; Hong et ah, 2006; Skommer et ah, 2007; Wang et ah, 2006). In human clinical trials curcumin has been administered in doses up to 8 g/day with no dose- limiting toxicity suggesting that it could be used safely in the prevention and treatment of cancer and other diseases (Aggarwal et ah, 2003; Cheng et ah, 2001). Mechanisms by which curcumin prevents cancer are thought to involve up- regulation of carcinogen detoxifying enzymes such as glutathione S-transferases (GST) (Piper et ah, 1998; Susan and Rao, 1992)), antioxidation , and suppression of expression of the isoenzyme cyclooxygenase-2 (COX-2) (Plummer et ah, 2001; Plummer et ah, 1999)). The regulation of these enzymes and others are probably related to the regulation of the NF-kB signaling pathway by curcumin. Curcumin has been shown to inhibit cytokine-mediated I-κB phosphorylation, degradation and I-κB kinase activity which in turn inhibit nuclear factor kappa-B (NF-κB) activation (Li et ah, 2004; Youn et ah, 2006). Recently, curcumin has been shown to inhibit NF-κB nuclear translocation in cell lines which lack the cytoplasmic NF-κB inhibitory protein IκBα (Thomas et ah, 2005).

Interestingly, curcumin has been explored as an anti-viral drug used to inhibit the protease and integrase enzymes of HIV-I (Gupta and Nagappa, 2003; Mazumder et ah, 1996; Sui et ah, 1993; Vlietinck et ah, 1998). Its integrase inhibitory activity has been reported with IC_5O values equal to 150 μM for 3' processing and 140 μM for strand transfer (Mazumder et ah, 1995). This activity may be related to the aryl β-diketo moiety contained in curcumin. The inhibition of HIV-I and HIV-2 protease by curcumin has been reported to be 100 and 250 μM, respectively (Sui et ah, 1993). With these properties in mind curcumin was evaluated in an early clinical trial against HIV-I infected patients (James, 1996; Liu et ah, 2005). The two trials had mixed results with one reporting modest effects and the other reporting no effects of curcumin on viral load in the treated patients. These results can be explained in light of recent papers demonstrating the rapid biotransformation and clearance of curcumin by glucuronidation and sulfation in the small intestine (Asai and Miyazawa, 2000; Basu et al, 2004; Pan et al, 1999). These results make it clear that curcumin will have to be altered or formulated to avoid these problems. Curcumin has not been pursued for these applications as much more effective compounds have been developed. Curcumin and its derivatives have much more potential as modulators of NF-κB as they inhibit NF- KB activation at a concentration (8 μM for curcumin (Weber et al. , 2006)) that is greater than 10-fold lower that those reported for its activity against HIV-I protease and integrase.

As used herein, reference will be made to curcumin, with the understanding that whatever is disclosed in connection with curcumin is expected by the present inventors to apply at least to salts thereof, cis- and trans-isomers thereof, stereoisomers, enantiomers, regioisomers, diasteromers, oligomers thereof, polymers thereof, derivatives thereof, and to plant or herbaceous extracts containing such agents. Weber et al, 2006 describe curcumin analogues that inhibit NF-κB. Curcumin analogues identified as analogue numbers 21 , 24, 26, 27, and 63 are of particular interest.

Antiviral Agents. Antiviral agents of the present invention can be entry inhibitors, reverse transcriptase inhibitors classified as NRTIs (nucleoside analogue reverse transcriptase inhibitors) or NtRTIs (nucleotide analogue reverse transcriptase inhibitors), and integrase inhibitors. Antiviral agents are well known in the art (see, e.g., Bean, 2005; Barbara et al., 2005; and Pereira et al., 2004 for review).

Entry Inhibitors. An entry inhibitor includes any compound that blocks the entry of virus into the host cell. HIV entry into its target cell is a multistep process that involves the HIV envelope spike, the CD4 receptor, and either CCR5 or CXCR4. Entry inhibitors can function, for example, by inhibiting the binding of HIV- 1 to host cell receptors , or by inhibiting the fusion of HIV-I with the host cell membrane. More specifically, inhibitors can interfere with entry at any step of the entry process, including (i) interaction of the envelope spike with CD4; (ii) conformational changes within the envelope spike proteins (both gpl20 and gp41) that reveal the coreceptor binding site; (iii) interaction with CCR5 or CXCR4; (iv) additional conformational changes within the gp41 coiled-coil region that drive the formation of a 6-helix bundle; and (v) fusion between the viral and cellular membrane to release the core into the target cell.

Examples of entry inhibitors that target viral envelope spike proteins include peptides such as Enfuvirtide (also called T-20, has the trade name FUZEON®), T- 1249, and CD4M33; small molecules such as BMS-378806, BMS-488043, catechin derivatives, theaflavin derivatives, NBD-556, and NBD-557; monoclonal antibody or monoclonal antibody fragments such as 2F5, 4E10, ZlO, 2G12, M44, M46, M47, M9, M14, Ml 8, and IgGlbl2, PRO542; and lectins derived from plants such as Galanthus nivalis, Hippeastrum hybrid, Narcissus pseudonarcissus, Listera ovata, Cymbidium, Epipactis helleborine, Urtica dioica, and Cyanovirin-N. Entry inhibitors that target the CD4 receptor include monoclonal antibodies such as TNX- 355, Q4120, OKT4a, Leu3a, and Q425. Entry inhibitors that target the chemokine (C-C motif) receptor 5 (CCR5) include small molecules such as TAK-779, TAK- 652, Ancriviroc, Vicriviroc, E913, Maraviroc, GW873140, and UK427857; and monoclonal antibodies or monoclonal antibody fragments such as PRO 140 and ST6. Entry inhibitors that target the chemokine (C-X-C motif) receptor 4 (CXCR4) include small molecules such as AMD3100, AMD070, and KRH- 1636; and peptides such as TN14003 and TC14012. Preferred entry inhibitors are Enfuvirtide. For references describing entry inhibitors see Briz et al, 2006; De Clercq, 2000; Moore and Doms, 2003; Pierson and Doms, 2003; Pohlmann and Reeves, 2006; Sterjovski et al, 2006; Tamamura et al, 2005; and Zhao et al, 2005).

Reverse Transcriptase Inhibitors (RTIs): An NRTI includes any nucleoside analogue that becomes incorporated into the viral DNA, leading to chain termination. Similarly, an NtRTI includes any nucleotide analogue that becomes incorporated into the viral DNA, leading to chain termination. The mode of action of NRTIs and NtRTIs is essentially the same; they are analogues of the naturally occurring deoxynucleotides needed to synthesize the viral DNA and they compete with the natural deoxynucleotides for incorporation into the growing viral DNA chain. However, unlike the natural deoxynucleotides substrates, NRTIs and NtRTIs lack a 3'-hydroxyl group on the deoxyribose moiety. As a result, following incorporation of an NRTI or an NtRTI, the next incoming deoxynucleotide cannot form the next 5'-3' phosphodiester bond needed to extend the DNA chain. Thus, when an NRTI or NtRTI is incorporated, viral DNA synthesis is halted, a process known as chain termination. All NRTIs and NtRTIs are classified as competitive substrate inhibitors. In contrast, NNRTIs have a completely different mode of action. NNRTIs block reverse transcriptase by binding at a different site on the enzyme, compared to NRTIs and NtRTIs. NNRTIs are not incorporated into the viral DNA but instead inhibit the movement of protein domains of reverse transcriptase that are needed to carry out the process of DNA synthesis. NNRTIs are therefore classified as non- competitive inhibitors of reverse transcriptase.

Suitable RTIs include nucleoside analog reverse transcriptase inhibitors (NRTIs) and nucleotide analog reverse transcriptase inhibitors (NtRTIs). Examples of NRTIs include Zidovudine (also called AZT, ZDV, and azido thymidine, has the trade name RETROVIR®), Didanosine (also called ddl, with the trade names VIDEX® and VIDEX EC®), Zalcitabine (also called ddC and dideoxycytidine, has the trade name Hivid®), Stauvidine (also called d4T, has trade names ZERIT® and ZERIT XR®), Lamivudine (also called 3TC, has the trade name EPIVIR®), Abacavir (also called ABC, has the trade name ZIAGEN®, is an analog of guanosine), Emtricitabine (also called FTC, has the trade name EMTRTV A® (formerly Coviracil)), Adefovir (also known as bis-POM PMPA, has the trade name PREVEON®), and Adefovir dipivoxil (has the trade name HEPSERA®). Examples of NtRTIs include Tenofovir (also known as tenofovir disoproxil fumarate, has the trade name VIREAD®). Preferred RTIs and NtRTIs are Tenofovir, Zidovudine, Lamivudine, Stauvidine, Didanosine, Zalcitabine, Emtriciditabine, Abacavir. NRTIs and NtRTIs are described in the art {see, e.g., Bean, 2005).

Integrase Inhibitors: An integrase inhibitor includes any compound that inhibits the activity of viral integrase. Integration is a multistep process that occurs in discrete biochemical stages: (i) assembly of a stable complex with specific DNA sequences at the end of the HIV-I long terminal repeat (LTR) regions, (ii) endonucleolytic processing of the viral DNA to remove the terminal dinucleotide from each 39 end, and (iii) strand transfer in which the viral DNA 39 ends are covalently linked to the cellular (target) DNA. Small molecules, for example, can be used to inhibit one or more of the steps within the integration process.

Examples of integrase inhibitors include diketo acids such as L-731,988 (Antimicrob Agents Chemother. 2002 Oct;46(10):3301-3), L-708,906 (Antimicrob Agents Chemother. 2002 Oct;46(10):3292-7), L-870,810 (Proc Natl Acad Sci U S A. 2004 Aug 3;101(31):l 1233-8), L-870,812 (Science. 2004 JuI 23;305(5683):528- 32) and MK-0518 (AIDS Treat News. 2005 Oct-Nov;(416):3-4) (MERCK), S-1360 (also known as GSK817081) and S-364735 (Curr Opin Investig Drugs. 2003 Feb;4(2):206-9), and 5CITEP (Proc Natl Acad Sci U S A. 1999 Nov 9;96(23): 13040-3) (Shionogi), and Compound 1 (J Med Chem. 2006 Mar

9;49(5): 1506-8) (University of Georgia); benzoic acids such as exophillic acid (J Antibiot (Tokyo). 2003 Dec;56(12): 1018-23) (MERCK); polyketides such as Cytosporic acid (J Nat Prod. 2003 Apr;66(4):551-3) and Integrasone (J Nat Prod. 2004 May;67(5):872-4) (MERCK); complestatins such as Isocomplestatin (J Nat Prod. 2001 Jul;64(7): 874-82) (MERCK); triterpenoids such as Integracide B (J Nat Prod. 2003 Oct;66(10): 1338-44) and Integramycin (Org Lett. 2002 Apr 4;4(7): 1123-6) (MERCK); naphtha-gamma-pyrones such as Isochaetochromin B (Bioorg Med Chem Lett. 2003 Feb 24;13(4):713-7) (MERCK); monoketo acids such as GS-9137 (also known as JTK-303) (JAPAN TABACCO), GS-9160 and GS- 9224 (GILEAD) (J Med Chem. 2006 Jan 26;49(2):445-7); pyrolloquinolines such as Compound 22 (Bioorg Med Chem Lett. 2006 Aug l;16(15):3989-92) (GILIAD); triketo acids such as Compound 19 (Bioorg Med Chem Lett. 2006 Jun l;16(l l):2920-4) (BRISTOL-MEYERS SQUIBB); styrylquinolines such as FZ 41 (BIOALLIANCE) and PL-2500 (PROCYON (now AMBRILLIA)) (MoI Pharmacol. 2004 Jan;65(l):85-98); pyranodipyrimidines such as V-165 (Curr Biol. 2002 JuI 23;12(14):1169-77) (REGA); and oligonucleotides such as ZINTEVIR™ (also known as ARl 77 or T#0177) (Antimicrob Agents Chemother. 1995 Nov;39(l l):2426-35) (Aronex). hi a preferred embodiment of the invention, the integrase inhibitor is L 731988. The present inventors have investigated the ability of NF-κB inhibitors to block HIV-I replication, CCR-5 transcription and progression from Gl to S phase of the cell-cycle in PHA-stimulated human PBMCs. The present inventors have also investigated the ability of NF-κB inhibitors to synergize with the current drugs used in HAART therapy. The data presented in the examples set forth herein shows that NF-κB inhibitors inhibit viral replication, block CCR-5 transcription, and prevent cells from progressing from Gl to S phase of the cell cycle. As also shown in the examples, the present inventors have unexpectedly found that NF-κB inhibitors synergize with entry, NRTI, NtRTI, and integrase inhibitors. In contrast, NF-κB inhibitors do not synergize with NNRTIs or protease inhibitors. Other unexpected benefits of the present invention include a reduction in the amount and/or frequency of antiviral agent dosages required to reach and maintain viral load suppression; slowing or preventing the emergence of viral populations resistant to antiviral therapy, thereby improving the therapeutic utility of antiviral drugs; the recovery of clinical utility of an antiviral drug in subjects who have clinically failed therapy with said drug; and improved compliance to antiviral therapeutic regime.

Subject in need thereof: As used herein, a "subject in need thereof is a mammal, in particular, a human, infected with a virus for which NF-κB inhibitors in combination with entry inhibitors, NRTIs, NtRTIs, and integrase inhibitors, have antiviral activity. The subject, in a preferred embodiment, is treatment-naive and has been recently infected with the virus. Another preferred embodiment is a subject who is a treatment-experienced mammal having resistance to currently employed antiviral agents, or a mammal infected with a virus population that is resistant to antiviral therapy. In another preferred embodiment, the subject is an uninfected mammal at risk for becoming infected due to exposure by known and well-accepted transmission routes. The subject may or may not have seroconverted. A subject in need thereof may be an immune deficient subject. An immune deficient subject has a lower than normal CD4⁺T-cell count, a normal count being about 800- 1000/mm³. A subject having advanced immune deficiency may have a CD4⁺T-cell count of less than about 200/mm . An additional unexpected result of the present combination composition and methods for using the combination composition is that immune reconstitution is made possible, i.e., the CD4⁺T-cell count may increase to a value at or near normal levels.

Virus: A virus includes any virus susceptible to an NF-κB inhibitor, and includes DNA and RNA viruses. In a preferred embodiment, the virus is any virus belonging to the viral family Retroviridae (i.e. a retrovirus) such as Human immunodeficiency virus 1 (HIV-I), Human immunodeficiency virus 2 (HIV-2), Human T-lympho tropic virus type 1 (HTLV-I), Human T-lympho tropic virus type 2 (HTLV-2), Simian immunodeficiency virus (SIV), or Feline immunodeficiency virus (FIV). In a particularly preferred embodiment, the virus is HIV-I. In additional preferred embodiments, the virus is a member of the Hepadnaviridae viral family, such as Hepatitis B virus (HBV) and Hepatitis C virus (HCV); a member of the Herpesviridae viral family, such as Herpes simplex virus 1 (HSV-I), Herpes simplex virus 2 (HSV-2), Cytomegalovirus (CMV), Human herpes virus 6 (HHV6), Epstein Barr virus (EBV), Human herpes virus 8 (HHV8 or Karposi's sarcoma), and Herpesvirus saimiri (HVS 13); a member of the Othomyxoviridae viral family, such as Influenza; a member of the Paramyxoviridae viral family, such as Sendai virus, Measels, and Human respiratory syncytial virus (RSV); or a member of the Picornaviridae viral family, such as Rhino virus. Method of treating a viral infection. A method of treating a viral infection is meant herein to include "prophylactic" treatment or "therapeutic" treatment. A "prophylactic" treatment is a treatment administered to an uninfected subject to prevent the transmission of the viral agent to the uninfected subject via contact with an infected individual. A therapeutic treatment is a treatment administered to a subject who has been exposed to the virus through recognized transmission routes and/or who is infected and does not exhibit signs of a disease and/or who exhibits early signs of the disease. The "therapeutic" treatment is intended, for example, to decrease the risk of developing pathology associated with the viral infection and/or to decrease viral load or the physical amount of virus present in the treated subject. The methods of the present invention are also meant to include a method to slow or prevent the emergence of a virus population that is resistant to antiviral therapy in an infected mammal by administering to a subject in need thereof a therapeutically effective amount of an NF-κB inhibitor in combination with an antiviral agent, a method to recover susceptibility to or therapeutic utility of antiviral therapy in a mammal infected with a virus population that is resistant to antiviral therapy by administering a therapeutically effective amount of an NF-κB inhibitor in combination with an antiviral agent, a method to reduce viral replication (viral load) in an infected mammal by administering a therapeutically effective amount of an NF-κB inhibitor in combination with an antiviral agent, a method to reduce the effective dose of an antiviral agent by administering a therapeutically effective amount of an NF-κB inhibitor in combination with an antiviral agent, a method to reduce the adverse events and/or toxic effects related to the administration of an antiviral agent in an infected mammal by administering a therapeutically effective amount of an NF-κB inhibitor in combination with an antiviral agent, and a method to modulate an inflammatory response associated with a viral infection and reduce viral replication in an infected mammal by administration of a therapeutic amount of an NF-κB inhibitor in combination with an antiviral agent.

The methods of the present invention are also meant to include a method of reducing the risk of transmitting a viral agent by administering a therapeutically effective amount of an NF-κB inhibitor in combination with an antiviral agent to an infected mammal at risk of transmitting the viral agent, and a method of reducing the risk of infection by a viral agent by administering a therapeutically effective amount of an NF-κB inhibitor in combination with an antiviral agent to an otherwise uninfected mammal at risk of contracting the viral infection.

"Treatment" or "treating," as used herein, refers to any clinically or quantitatively measurable beneficial effect in the condition for which the subject is being treated. A "beneficial effect" includes, for example, rendering the virus incompetent for replication, inhibition of viral replication, inhibition of infection of a further host cell, prevention or slowing of the emergence of a virus population that is resistant to antiviral therapy, recovering susceptibility to or the therapeutic utility of antiviral therapy, increasing CD4⁺T-cell count, and/or preventing viral infection in a subject at risk of infection. The methods of the present invention involve delivering a therapeutically effective amount of a combination of an NF-κB inhibitor and an antiretroviral agent. A "therapeutically effective amount," as used herein, refers to an amount, determined by one skilled in the art, sufficient for treating the condition for which the subject is being treated. Dosages: Doses to be administered are variable according to the NF-κB inhibitor to be used, the antiviral agent to be used, the treatment period, frequency of administration, the host, and the nature and severity of the infection. The dose can be determined by one of skill in the art without an undue amount of experimentation. The compositions of the invention are administered in substantially non-toxic dosage concentrations sufficient to ensure the release of a sufficient dosage unit of the present combination into the patient to provide the desired inhibition of the virus. A substantially non-toxic dosage is minimally anti-proliferative and has an immune reconstitution profile as good as or more promising than current protocols. The actual dosage administered will be determined by physical and physiological factors such as age, body weight, severity of condition, and/or clinical history of the patient. The active ingredients are ideally administered to achieve in vivo plasma concentrations of an NF-κB inhibitor and an antiviral agent of about 0.01 μM to about 100 μM, more preferably about 0.1 to about 10 μM. For example, in the treatment of HIV-positive and AIDS patients, the methods of the present invention may use compositions to provide from about 0.001 to about 1000 mg/kg body weight/day of an NF-κB inhibitor and an antiviral agent. Particular unit dosages of an NF-κB inhibitor and an antiviral agent of the present invention include 50 mg, 100 mg, 200 mg, 500 mg, and 1000 mg amounts, for example, formulated separately, or together as discussed infra. It will be understood, however, that dosage levels that deviate from the ranges provided may also be suitable in the treatment of a given viral infection. In treating a mammal in need thereof, a therapeutically effective amount of the present composition is administered thereto in accordance with the present invention (see above). As used herein, the term "therapeutically effective amount" is an amount of the composition indicated for treatment while not exceeding an amount which may cause significant adverse effects. Methods for evaluating the effectiveness of combinations of the present invention include, for example, PCR- based assays for viral RNA in plasma. Kits including appropriate PCR primer pairs are available commercially, e.g., from CHIRON Corporation, (Emeryville, Calif), ROCHE (BOEHRINGER-MANNHEIM), and ORTHO (Netherlands)). Useful indicators of immunological dysfunction (as defined by excessive levels of immune activation and apoptosis) include: T cell proliferation (e.g. Ki67 expression, Cyclin B expression), apoptosis (e.g. Annexin V, Caspase 3 production, CD95 expression), and activation (e.g. expression of IL-7 receptor, HLA-DR, or CD69), and CCR5 or CCR7 expression. Other markers for immune activation are soluble forms of cellular receptors that are expressed in serum. These include soluble CD27, soluble IL-2R, soluble CD62L, soluble CD50, soluble VCAM-I, soluble TNF-receptor I (p55), soluble TNF-receptor II (p75), neopterin, soluble CD62E, soluble CD30, soluble CD25, and soluble CD54. Overexpression of these soluble receptors tracks exceptionally well with disease progression. The CD4+T cell count can be used along with HIV-I RNA in plasma as a predictor of the rate of disease progression. Markers for immunological disfunction are discussed in the art {see, e.g., Messele et al, 2001; Rizzardi et al, 1996; Lawn et al, 2000; Kourtis et al, 2000; Hober et al, 1996a; Hober et al, 1996b; Zangerle et al, 1994; Lang et al, 1988; Atlas et al, 2004; De Milto et al, 2003; Nkengasong et al., 2001; Daniel et al, 2001; Fahey et al, 1998; Salazar-Gonzolez et al, 1998; Zangerle et al, 1998; lAu et al, 1997; Rizzardi et al, 1997; Fuchs et al, 1990; and Fahey et al, 1990).

The dose may be modified according to the patient's hepatic, renal and bone marrow function, functions which are frequently abnormal in patients with viral infections. One may wish to use a higher dose of compositions of the present invention for therapy of certain manifestations of HIV infection, e.g., HIV-related dementia.

The relative amounts of NF-κB inhibitor and antiviral agent required to form a combination composition of the present invention are determined using assays conventional in the art. For example, cells infected with a virus, against which a given NF-κB inhibitor is active, are subjected to varying concentrations of that NF- KB inhibitor in the presence of varying concentrations of an antiviral agent. In this way, the various combinations of concentrations of NF-κB inhibitor and antiviral agent that form combination compositions can be determined by observing subsequent viral replication and comparing results thereof. Thus, for example, the relative amounts of the NF-κB inhibitor and the antiviral agent required to form a combination composition capable of inhibiting HIV-I are determined by incubating cells infected with HIV-I in the presence of varying concentrations of NF-κB inhibitor and antiviral agent. From these analyses, dosages effective to treat a virally-infected mammal can be determined in an established manner. Formulations and Administration: Combination compositions of the present invention may be in any form suitable for co-administration separately or as an admixture. Such administrable forms include tablets, buffered tablets, pills, capsules, enteric-coated capsules, dragees, cachets, powders, granules, aerosols, liposomes, suppositories, creams, lotions, ointments, skin patches, parenterals, lozenges, oral liquids such as suspensions, solutions and emulsions (oil-in-water or water-in-oil), ophthalmic liquids and injectable liquids, or sustained-release forms thereof. The desired dose may be provided in several increments at regular intervals throughout the day, by continuous infusion, or by sustained release formulations, or may be presented as a bolus, electuary or paste.

Combination compositions of the present invention may be administered alone in solution, hi one embodiment, compositions of the present invention are prepared by admixture of an NF-κB inhibitor and an antiviral agent of the present invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" means acceptable for use in the pharmaceutical and veterinary arts, compatible with other ingredients of the formulation, and not being toxic or otherwise unacceptable.

The selection of carrier depends on the intended mode of administration of the combination composition. Thus, compositions to be administered orally are prepared using substances that are suitably combined with an NF-κB inhibitor and/or the antiviral agent for oral ingestion. Such substances include, without limitation, sugars, such as lactose (hydrous, fast flow), glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, including microcrystalline cellulose, sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragancanth; colloidal silicon dioxide; croscarmellose sodium; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and corn oil; polyols such as propylene glycol, glycerine, sorbitol, mannitol and polyethylene glycol; agar; alginic acids; antacids such as aluminum hydroxide or magnesium hydroxide; buffer such as sodium citrate, acetate, or bicarbonate; water; isotonic saline and phosphate buffer solutions. Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers, tabletting agents, anti-oxidants, preservatives, coloring agents and flavoring agents may also be present.

Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats or solutes which render the formulation isotonic with blood; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi- dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules or tablets, or the like. Likewise, compositions for ophthalmic administration are prepared in suitable liquid carriers such as buffered or physiological saline, liposomes or basic amino acids. Creams, lotions and ointments may be prepared for topical application using an appropriate base such as triglyceride base, liposomes, or basic amino acids. Such creams, lotions and ointments may also contain a surface active agent.

A pharmaceutical composition of the invention can take the form of a lyophilized powder of the active substance, to be dissolved immediately before use in a physiological solution for the purpose of injection.

Compositions of the present invention may be administered parenterally, intravenously, intraperitoneally, intraosseously, in the cerebrospinal fluid, or the like. Further modes of administration include rectal, nasal, buccal, sublingual, vaginal, subcutaneous, intramuscular, or intradermal administration. In an embodiment of the present invention, the NF-κB inhibitor and antiviral agent are administered in separate compositions rather than administered admixed in a single composition. This is particularly preferred when the desired mode of administration of the NF-κB inhibitor and antiviral agent differ. Thus, a composition comprising an NF-κB inhibitor is prepared by admixture of the NF -KB inhibitor with at least one suitable pharmaceutically acceptable carrier to achieve an NF-κB inhibitor composition in the desired administrable form. Likewise, a composition comprising an antiviral agent is prepared by admixture with at least one suitable pharmaceutically acceptable carrier to achieve a composition in the desired administrable form. The NF-κB inhibitor and antiviral agent compositions may be administered together as an admixture, administered separately but concurrently, or separately but substantially concurrently, at appropriate dosage levels. For a perinatal subject, the drug combination of the present invention may be, for example, administered orally after 36 weeks of pregnancy and continued through delivery. Interventions around the time of late gestation and delivery (when the majority of transmissions are thought to occur) are most efficacious.

A pharmaceutical combination in kit form may be provided which includes in packaged combination a carrier means adapted to receive a container means in close confinement therewith and a first container means including a pharmaceutical NF-κB inhibitor composition and a pharmaceutical antiviral agent composition, hi such a kit, the NF-κB inhibitor and antiviral agent compositions may be in different administrable forms. For example, the NF-κB inhibitor may be in an orally administrable form such as tablet, pill, capsule or powder form, while the antiviral agent composition may be in a form suitable for administration by injection, i.e., in solution form. Such kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Printed instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.

The present invention also includes use of combination compositions as presented herein further in combination with other medical compositions intended for the treatment of those viral infections set forth herein.

Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples. The examples are not to be construed in any way as limiting the scope of this invention. Those of skill in the art should, in light of the present disclosure, appreciate that changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLES Materials and Methods

Cell culture. Normal human donor leukopheresis products were obtained from Interstate Blood Bank (Memphis, TN), processed by Ficoll-Hypaque separation to isolate peripheral blood mononuclear cells (hPBMCs), and frozen. Cryogenically stored hPBMCs were thawed, washed, and resuspended in RPMI 1640 (MEDIATECH, Herndon, VA) containing 20% FBS (MEDLATECH), 100 U/ml penicillin (MEDIATECH), 100 μg/ml streptomycin (MEDIATECH), and 2 rnM L-glutamine (MEDIATECH). hPBMCs were activated with 5 μg/ml

Phytohemagglutinin PHA-P (SIGMA- ALDRICH, St. Louis, MO) in the presence of 100 IU/ml recombinant human IL-2 (rhIL-2) (CHIRON, Emeryville, CA) for 3 days at 37° C in a humidified 5% CO₂ atmosphere.

NF-κB reporter assay. Cryogenically stored 293/NFκB-luc cells (PANOMICS, Fremont, CA) were thawed, washed, and resuspended in DMEM

(MEDIATECH) containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. Cells were plated in 96-well opaque-wall tissue culture plates at a concentration of 5x10⁵ cells/ml (0.1 ml per well) and incubated overnight at 37° C in a humidified 5% CO₂ atmosphere. Known and unknown NF-κB inhibitor compounds were added to each well at the appropriate concentration and incubated with the cells for 2 hours. After incubation period, TNF-α (R&D SYSTEMS, Minneapolis, MN) was added to each well at a final concentration of 50 ng/ml. Cells were incubated for 24 hours. Luciferase activity of 293/NF-κB-luc cells was detected by adding lOOμl BRIGHT-GLO Reagent (PROMEGA, Madison, WI) to each well, mixing with pipette, and incubating at room temperature for 5 minutes. Luminescence was read with a DTX 880 MULTLMODE DETECTOR (BECKMAN COULTER, Fullerton, CA). Data was analyzed with EXEL 2003 (MICROSOFT, Redmond, WA) software.

HIV-I replication assay. Activated hPBMCs were washed and resuspended to 2xlO⁶ cells/ml in RPMI 1640 containing 20% FBS, lOOU/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 200 IU/ml rhIL-2. 1x10⁵ (50 μl) hPBMCs were transferred to each well of a 96-well plate. Cell cycle inhibitor compounds were added to each well at the appropriate concentration. HIV-I Ba-L (R5) or IIIB (X4) strains were diluted to 2x10⁴ TCID₅₀/ml in RPMI 1640 containing 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine and sonicated for 10 seconds. hPBMCs were infected at a MOI of 0.01 by transferring 1x10³ (50 μl) TdD₅₀ of HIV-I to each well, mixing with pipette, and incubating plates for 3 days at 37° C in a humidified 5% CO₂ atmosphere. After infection, cells were removed by centrifugation. HIV-I was lysed by adding 0.5% (v/v) Triton X- 100 and 0.02% (w/v) NaN₃ to supernatant and incubating at room temperature for 30 minutes. Lysed HIV-I containing supernatant was diluted 1 :100 and HIV-I p24 antigen levels detected by ELISA (IHV μQUANT Facility, Baltimore, MD).

Absorbance was read at 450nm on an AD 200 (BECKMAN COULTER). Data was analyzed with EXEL 2003 (MICROSOFT) software.

Cell cycle analysis: Activated hPBMCs were washed and resuspended to IxIO⁶ cells/ml in RPMI 1640 containing 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 100 IU/ml rhIL-2. hPBMCs were transferred to a 96-well plate (250 μl per well). Cell cycle inhibitor compounds were added to each well at the appropriate concentration and incubated with the hPBMCs for 3 days. hPBMCs were fixed by washing with ice cold IX PBS, resuspending in 100 μl ice cold 80% EtOH, and incubating overnight at -30° C. Fixed hPBMCs were rehydrated by resuspending in 100 μl IX PBS and incubating at 37° C for 30 minutes. Rehydrated hPBMCs were stained by resuspending in 100 μl IX PBS containing 0.1% Triton X-100, 0.1 mM EDTA, 50 μg/ml RNase A (INVITROGEN, Carlsbad, CA), and 50 μg/ml propidium iodide (KPL, Gaithersburg, MD) and incubating at room temperature for 30 minutes. Stained hPBMCs were washed with and resuspended in IX PBS containing 1% FBS and 0.1% NaN₃. Propidium iodide staining was detected with a FACSCALIBUR (BD, Franklin Lakes, NJ) flow cytometer. Cell cycle data was analyzed with FLOWJO version 7.1.3 (TREE STAR, Ashland, OR) software.

Synergy with HAART assay: Activated hPBMCs were washed and resuspended to 2x10⁶ cells/ml in RPMI 1640 containing 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 200 IU/ml rhIL-2. IxIO⁵ (50 μl) hPBMCs were transferred to each well of a 96-well plate. HAART drugs along with cell cycle inhibitor compounds were added to each well at the appropriate concentration. HIV-I Ba-L (R5) or IIIB (X4) strains were diluted to 2x10⁴ TdD₅o/ml in RPMI 1640 containing 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 niM L-glutamine and sonicated for 10 seconds. hPBMCs were infected at a MOI of 0.01 by transferring IxIO³ (50 μl) TCID_5O of HIV-I to each well, mixing with pipette, and incubating plates for 3 days at 37° C in a humidified 5% CO₂ atmosphere. After infection, cells were removed by centrifugation. HIV-I was lysed by adding 0.5% (v/v) Triton X-100 and 0.02% (w/v) NaN₃ to supernatant and incubating at room temperature for 30 minutes. Lysed HIV-I containing supernatant was diluted 1:100 and HIV-I p24 antigen levels detected by ELISA (IHV μQUANT Facility, Baltimore, MD). ELISA absorbance was read at 450 nm on an AD 200 (BECKMAN COULTER). ELISA data was analyzed for synergy with SIGMAPLOT (SYSTAT Software, Richmond, CA) software. Example 1. Parthenolide inhibits NF-κB in cells.

Parthenolide is reported to be a potent inhibitor of NF-κB at low-μM concentrations (5±10 μM, HeLa cells; (Hehner et ah, 1998)). Wagner et al., reported that it can completely abrogate p65/RelA binding to DNA at 20 μM. To verify that the parthenolide purchased (SIGMA) was functioning properly its ability to inhibit NF-κB in a 293 -Luc reporter cell was assessed. Cells were treated with 0, 0.1, 1, 5, 10, 20 and 50 μM parthenolide for 2 hours then induced with 50ng/ml TNF-α for 18 hours. Following the induction luminescence was quantified using a BECKMAN-COULTER 2300 plate reader. Figure 1 shows the dose response curve generated from the luminescence data. Parthenolide clearly inhibited the expression of the luciferase gene in a dose dependent manner with a median IC₅₀ (Inhibitory Concentration of 50%) of 6.2±0.1 μM. This value is the median value generated from three independent experiments ± the standard error. As a control 0.5% DMSO treated and untreated cells were compared to verify that the parthenolide carrier had no effect on the expression of luciferase or in the readout of the assay. There was a slight decrease in the output from the assay in the DMSO treated population although it was not statistically significant. As a result of the controls the decrease in activity in the drug treated samples were compared to the DMSO control sample. The data also confirms the observation that 20 μM parthenolide gives 100% inhibition of NF-κB activity. This observation along with the fact that the determined IC₅₀ value is within the reported range, gives confidence that this compound is functioning as reported in the literature. The experimental IC₅₀ value can also be used to assess the efficacy of the compound against HTV-I replication. Example 2. Parthenolide inhibits CCR-5 and CXCR-4 tropic viruses

Given the critical role NF-κB plays in the progression of the HIV life cycle and the onset of AIDS we assessed parthenolide's ability to inhibit HIV replication in human peripheral blood mononuclear cells (hPBMCs). Human PBMCs from a single donor were thawed and grown in the presence of 100U/ml IL-2 and activated with 5 μg/ml phytohemagglutinin (PHA) for three days. Activated hPBMCs were washed and resuspended in media containing 100U/ml IL-2 at a concentration of 1x10⁶ cells/ml and 100 μl transferred to each well of a 96 well plate. The cells were treated with 0, 0.1, 1, 5, 10, 20 and 50 μM parthenolide in triplicate for each experiment. One set of the triplicate drug-treated cells was infected with HIV-I IIIb (X4-trophic) and another was infected with HIV-I BaL (R5-trophic) at an MOI of 0.01 for both viruses. Viral replication was assayed by p24 elisa after 72 hours. Parthenolide inhibited the growth of both viruses in a concentration dependent manor. The carrier for parthenolide (0.5% DMSO) had no effect on viral replication, with p24 values in these treatment groups equal or greater than untreated cells. Figure 2 shows the dose response curves generated from the p24 elisa. Figure 2 A is one of the data sets for inhibition of HIV-I BaL (R5 -trophic), and Figure 2 B is one of the data sets for inhibition of HIV-I IIIb (X4-trophic). The median IC₅₀ values ± the standard error generated from the depicted data and two other experiments was determined to be 1.8±0.4 μM for BaL and 0.3±0.1 μM for IIIb. The IC₅₀ for the IIIb strain was consistently 3 to 10-fold lower than that for BaL. This could be explained by the observations of Cicala et al., who treated Jurkat cells with either X4 or R5 pi 20 envelope and determined the differences in the gene expression profiles for the two treatment groups. Interestingly, these authors found that the p65/RelA subunit of NF-κB was down regulated in the X4 envelope treated cells. Example 3. The cytotoxicity profile of parthenolide

The cytotoxicity of parthenolide was determined for the same pool of hPBMCs used in the infectivity assay described above. As with the infectivity assay the hPBMCs were stimulated with PHA for three days and then triplicate samples were treated with 0, 0.2, 2, 10, 20, 40 and 100 μM parthenolide for an additional 72 hours. The number of viable cells was determined using MTS reagents (CALBIOCHEM) and one representative experiment is shown in Figure 3. As the graph shows there was no detectable toxicity to these cells up to the 100 μM tested. Cells treated with 0.5% DMSO showed no detectable cytotoxic effects as compared to the same cells untreated. Parthenolide has been reported to have a good cytotoxicity profile with reports of IC_5O values between 60 and 200 μM depending on the cell line (Nakagawa et al, 2005; O'Neill et al, 1987; Shin et al, 2004). The potential usefulness of a compound is defined, at least in part by, the therapeutic or selectivity index (the ratio of cytotoxicity IC₅₀ to inhibitory IC₅₀). For parthenolide, using the reported values for cytotoxicity this would yield a therapeutic index of 30 to 100 with our data suggesting a number closer to the higher value. Example 4. Parthenolide inhibits progression from Gl to S phase of the cell- cycle.

NF-κB inhibitors including parthenolide have been reported to inhibit cell cycle progression from Gl to S phase of many different cell types (Ralstin et al, 2006). This effect is most likely due to the block of cyclin D expression which is regulated by NF-κB. This could have beneficial effects for the treatment of HIV-I as this virus requires progression through this point of the cell cycle to replicate (Nekhai et al, 2000; Tobiume et al, 1998). To verify parthenolide's effect on cell cycle progression hPBMCs were thawed and activated for three days as described above. The activated cells were treated in duplicate with 0, 1, 10 and 50 μM parthenolide for three days and the cell cycle distribution was determine by quantifying the DNA content using flow cytometry. Control cells, both untreated and those treated with 0.5% DMSO, had approximately 78% of cells in the Gl phase of the cell cycle. Figure 4 shows that upon addition of parthenolide the number of cells in Gl increases to approximately 85% at 1 μM and approximately 90% at 10 μM. At 50 μM the analysis was not possible as it was not possible to identify activated T-cells. These results verify that parthenolide can block progression of from Gl-S phase of the cell cycle.

Example 5. Parthenolide down regulates CCR-5 expression.

Analysis of the CCR-5 promoter indicates that NF-κB is the major inducer of transcription on the context of the full promoter (Liu et al, 1998). It has been shown that even modest reductions in the surface density of CCR-5 can have a dramatic effect on HIV-I infectivity (Heredia et al, 2003). To investigate whether parthenolide' s inhibition of NF-κB has an effect on CCR-5 expression hPBMCs were thawed and activated for three days as described above. The activated cells were treated in duplicate with 50 μM parthenolide for three days. Total RNA was isolated from the cells. One microgram of total RNA was used in each reaction to amplify the CCR-5 and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) gene by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) reaction. It is desirable when designing RT-PCR primers to have them span an intron exon boundary at the 3' end of the gene so that amplification from genomic DNA can be distinguished from amplification from the mRNA. However, in the case of CCR-5 exon 4 contains the open reading frame, the complete 3 -prime UTR, and 11 nucleotides of the 5-prime UTR (Mummidi et al, 1997). Therefore, primers were designed as described in Lai et al., who used these primers for QRT-PCR. In addition the total RNA was subjected to DNase digestion prior to the amplification step as described in the onestep RT-PCR kit. The primers used were, CCR5-up, 5'- CAAAAAGAAGGTCTTCATTACACC-S' (position, 512 to 535 of the mRNA); CCR5-down, 5'-CCTGTGCCTCTTCTTCTCATTTCG-S' (position, 677 to 700 of the mRNA). The CCR-5 primer pair generated a 189 bp PCR product. To control for the integrity and amount of RNA used for RT-PCR, the mRNA of a housekeeping gene, GAPDH, was amplified. The primers for GAPDH were 5'- GGTGGTCTCCTCTGACTTC AAC A-3' (sense) and 5'- GTTGCTGTAGCC AAATTCGTTGT-3' (antisense). The GAPDH primer pair generated a 126 bp PCR product. RT-PCR products were resolved on a 2% agarose gel and visualized by staining with ethidium bromide. In order to avoid saturation of the PCR signal a range of cycles were compared to determine where CCR-5 amplification was still expanding linearly in total RNA from untreated hPBMCs (Figure 5 A). From the results in Figure 5 A, 25 cycles was chosen to examine CCR-5 message levels and 20 cycles for GAPDH. Figure 5 B shows the results of amplification of CCR-5 from two independent pools of parthenolide treated hPBMCs. The average reduction in CCR-5 message was 72.5%. These data show for the first time that an NF- KB inhibitor can influence the level of CCR-5 mRNA. Example 6. Parthenolide synergizes with HAART drugs

Having demonstrated that parthenolide can inhibit viral replication with IC_5O values ofl.8±0.4 μM for BaL and 0.3±0.1 μM for HIb we next determined whether it could synergize with any of the drugs currently approved for the treatment of HIV-I by the US Food and Drug Administration (FDA). One drug from each class (entry, NRTI, NNRTI, integrase, and protease inhibitor) was initially evaluated to determine if there was any effect of the combination treatment. As above, hPBMCs were stimulated with PHA for three days and the stimulated cells were plated in a 96-well tissue culture plate. The stimulated cells were treated with parthenolide in a range up to 5μM, the HAART drug in a range up to their IC_5O values or in all combination of the two treatment series (see Treatment Grids: Tables 1 to 5 below). The treated cells were infected with BaL, as described above, and viral replication was assayed by p24 elisa after 72 hours. Synergy of the combination was calculated using the formula described in Verrier et al., (118 Tony). Significant synergy was considered to be an SI value greater than 100 that was not a single random point, i.e. the surface area on the resultant 3D mesh plot contained peaks with reasonable slopes. Synergy was seen with the entry inhibitor (T-20), the NRTI (tenofovir), and the integrase inhibitor (L-731,988) being developed by MERCK (Hazuda et al., 2000) (Figure 6 Panel A, B, D). No synergy was seen with the NNRTI (Efavirenz), or the protease inhibitor (Nelfinavir) (Figure 6 Panel C, E). Table 1. T-20 and partbenolide plating grid.

Table 2. Tenofovir and parthenolide plating grid.

Table 3. Efavirenz and parthenolide plating grid.

Table 5. Nelfinovir and parthenolide plating grid.

The reduction in IC₅₀ values (mean ±SD of three independent experiments) for the HAART drugs was determined for each concentration of parthenolide (Figure 7 Panel A-E). The IC_5O for T-20 as single treatment ranged from 2 to 10 nM for BaL and significantly fell to 0.58 μM (on average) when used in combination with Parthenolide. This decrease is a 3.4-fold reduction in IC₅₀ value for T-20. For the NRTI (tenofovir), there was a 4-fold reduction of the IC_5O values when used in combination with parthenolide, dropping the IC₅₀ value from 150 nM to 35 nM. However, there was no reduction in IC₅₀ when parthenolide was used in combination with the NNRTI (Efavirenz). The integrase inhibitor showed the greatest drop in IC₅₀ value in combination with parthenolide. The IC₅₀ value for this compound dropped from 1.5 μM to 0.05 μM (a decrease of 30-fold). Lastly, the protease inhibitor (Nelfinavir) in combination with parthenolide showed no reduction in IC₅₀ value.

These finding are new and unpredicted from the previous literature on NF- KB and HIV-I infection. The data presented demonstrates for the first time that certain classes of currently used HAART drugs, entry, reverse transcriptase, and integrase inhibitors, synergize with parthenolide. The reduction in the IC₅₀ of entry, reverse transcriptase, and integrase inhibitors by parthenolide by, 3.4-, 4-, and 30- fold respectively, has profound implication for the treatment of HIV-I infected individuals. Co-administration of parthenolide with any/or all of the HAART drugs has the potential to increase patient compliance due to the reduction in adverse effects of the drugs given at lower doses. The synergy of parthenolide with the specific classes of HAART drugs provides the opportunity for co-formulation of a single pill which again has the benefit of lowering pill burden and improving patient compliance. Example 7. JSH-23 inhibits NF-κB in cells. JSH-23 is reported to be a good inhibitor of NF- KB at low-μM concentrations (IC_5O of 14.4 μM, (Min et al, 2004)). Shin et al, reported that it can completely prevent nuclear transport of the p65/RelA and/or abrogate its binding to DNA at 30 μM (Shin et al, 2004). To verify that the JSH-23 purchased (EMD Biosciences) was functioning properly its ability to inhibit NF-kB in a 293-Luc reporter cell was assessed. Cells were treated with 0, 0.1, 1, 5, 10, 20 and 50 μM JSH-23 for 2 hours then induce with 50ng/ml TNF-α for 18 hours. Following the induction luminescence was quantified using a DTX 880 multimode plate reader (Beckman Coulter). Figure 8 shows the dose response curve generated from the luminescence data. JSH-23 clearly inhibited the expression of the luciferase gene in a dose dependent manor with a median IC₅₀ (Inhibitory Concentration of 50%) of 16.6±1.2 μM. This value is the median value generated from three independent experiments ± the standard error. As a control 0.5% DMSO treated and untreated cells were compared to verify that the JSH-23 carrier had no effect on the expression of luciferase or in the readout of the assay. There was a slight decrease in the output from the assay in the DMSO treated population although it was not statistically significant. As a result of the controls the decrease in activity in the drug treated samples were compared to the DMSO control sample. The data also confirms the observation that 30 μM JSH-23 gives 100% inhibition of NF-κB activity. This observation along with the fact that the determined IC₅₀ value is within the reported range, gives confidence that this compound is functioning as reported in the literature and that it is indeed a good lead for developing new NF-κB inhibitors. The experimental IC₅₀ value can also be used to assess the efficacy of the compound against HIV-I replication.

Example 8. JSH-23 inhibits CCR-5 (R5-tropic) and CXCR-4 (X4-tropic) viruses. Given the critical role NF-κB plays in the progression of the HIV life cycle and the onset of AIDS we assessed JSH-23's ability to inhibit HIV replication in human peripheral blood mononuclear cells (hPBMCs). Human PBMCs from a single donor were thawed and grown in the presence of 100U/ml IL-2 and activated with 5 μg/ml phytohemagglutinin (PHA) for three days. Activated hPBMCs were washed and resuspended in media containing 100U/ml IL-2 at a concentration of 1x10⁶ cells/ml and lOOμl transferred to each well of a 96 well plate. The cells were treated with 0, 0.1, 1, 5, 10, 20 and 50 μM JSH-23 in triplicate for each experiment. One set of the triplicate drug-treated cells was infected with HIV-I HIb (X4-trophic) and another was infected with HIV-I BaL (R5-trophic) at an MOI of 0.01 for both viruses. Viral replication was assayed by p24 elisa after 72 hours. JSH-23 inhibited the growth of both viruses in a concentration dependent manor. The carrier for JSH- 23 (0.5% DMSO) had no effect on viral replication, with p24 values in these treatment groups equal or greater than untreated cells. Figure 9 shows the dose response curves generated from the p24 elisas for hPBMCs infected with each virus. Figure 9 A is one of the data sets for inhibition of HIV-I BaL (R5-trophic), and Figure 9 B is one of the data sets for inhibition of HIV-I HIb (X4-trophic). The median IC₅₀ values ± the standard error generated from the depicted data and two other experiments was determined to be 6.9±1.1 μM for BaL and 0.39±0.08 μM for HIb. The IC₅₀ for the IHb strain was consistently 3 to 10-fold lower than that for BaL with all of the NF-κB inhibitors tested. This could be explained by the observations of Cicala et al., who treated Jurkat cells with either X4 or R5 pi 20 envelope and determined the differences in the gene expression profiles for the two treatment groups (Cicala et al, 2006). Interestingly, these authors found that the p65/RelA subunit of NF-κB was down regulated in the X4 envelope treated cells. Furthermore, it has been reported that NF-κB plays a role in CXCR-4 receptor expression (Helbig et al, 2003) which would further effect the sensitivity of PBMCs harboring X4-tropic viruses to these compounds. Example 9. The cytotoxicity proΩle of JSH-23.

The cytotoxicity of JSH-23 was determined for the same pool of hPBMCs used in the infectivity assay described above. As with the infectivity assay the hPBMCs were stimulated with PHA for three days and then triplicate samples were treated with 0, 0.2, 2, 10, 20, 40 and 100 μM JSH-23 for an additional 72 hours. The number of viable cells was determined using MTS reagents (CalBioChem) and one representative experiment is shown in Figure 10. As the graph shows there was no detectable toxicity to these cells up to the 100 μM tested. Cells treated with 0.5% DMSO showed no detectable cytotoxic effects as compared to the same cells untreated. JSH-23 has been reported to have a good cytotoxicity profile with no toxicity being reported at concintrations up to 100 μM in macrophages RAW 264.7 (Min et al, 2004). The potential usefulness of a compound is defined, at least in part by, the therapeutic or selectivity index (the ratio of cytotoxicity IC₅₀ to inhibitory IC₅₀). For JSH-23, using the reported values for cytotoxicity this would yield a therapeutic index of 15 to 250 depending on the viral tropism.

Example 10. JSH-23 inhibits progression from Gl to S phase of the cell-cycle.

NF-κB inhibitors have been reported to inhibit cell cycle progression from Gl to S phase of many different cell types (Ralstin et al, 2006). This effect is most likely due to the block of cyclin D expression which is regulated by NF-κB. This could have beneficial effects for the treatment of HIV-I as this virus requires progression through this point of the cell cycle to replicate (Nekhai et al, 2000; Tobiume et al, 1998). To determine if JSH-23 can affect the progression of the cell cycle from Gl phase to S phase hPBMCs were thawed and activated for three days as described above. The activated cells were treated in duplicate with 0, 1, 10 and 50 μM JSH-23 for three days and the cell cycle distribution was determine by quantifying the DNA content using flow cytometry. Control cells, both untreated and those treated with 0.5% DMSO, had approximately 78% of cells in the Gl phase of the cell cycle. Figure 11 shows that upon addition of JSH-23 the number of cells in Gl increases to approximately 80% at 1 μM and approximately 94% at 10 and 50 μM. These results verify that JSH-23 can block progression of from Gl-S phase of the cell cycle. Example 11. JSH-23 down regulates CCR-5 expression.

Analysis of the CCR-5 promoter indicates that NF-kB is the major inducer of transcription on the context of the full promoter (Liu et al., 1998). It has been shown that even modest reductions in the surface density of CCR-5 can have a dramatic effect on HIV-I infectivity (Heredia et al, 2003). To investigate whether JSH-23's inhibition of NF-κB has an effect on CCR-5 expression hPBMCs were thawed and activated for three days as described above. The activated cells were treated in duplicate with 50 μM JSH-23 for three days. Total RNA was isolated from the cells. One microgram of total RNA was used in each reaction to amplify the CCR-5 and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) gene by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) reaction. It is desirable when designing RT-PCR primers to have them span an intron exon boundary at the 3' end of the gene so that amplification from genomic DNA can be distinguished from amplification from the mRNA. However, in the case of CCR-5 exon 4 contains the open reading frame, the complete 3 -prime UTR, and 11 nucleotides of the 5-prime UTR (Mummidi et al., 1997). Therefore, primers were designed as described in Lai et al., who used these primers for QRT-PCR. In addition the total RNA was subjected to DNase digestion prior to the amplification step as described in the onestep RT-PCR kit. The primers used were, CCR5-up, 5'- CAAAAAGAAGGTCTTCATTACACC-S¹ (position, 512 to 535 of the mRNA); CCR5-down, 5'-CCTGTGCCTCTTCTTCTCATTTCG-3¹ (position, 677 to 700 of the mRNA). The CCR-5 primer pair generated a 189 bp PCR product. To control for the integrity and amount of RNA used for RT-PCR, the mRNA of a housekeeping gene, GAPDH, was amplified. The primers for GAPDH were 5'- GGTGGTCTCCTCTGACTTCAACA-3 ' (sense) and 5 '-

GTTGCTGT AGCCAAATTCGTTGT-3' (antisense). The GAPDH primer pair generated a 126 bp PCR product. RT-PCR products were resolved on a 2% agarose gel and visualized by staining with ethidium bromide. In order to avoid saturation of the PCR signal a range of cycles were compared to determine where CCR-5 amplification was still expanding linearly in total RNA from untreated hPBMCs (Figure 12 A). From the results in Figure 12 A, 25 cycles was chosen to examine CCR-5 message levels and 20 cycles for GAPDH. Figure 12 B shows the results of amplification of CCR- 5 from two independent pools of JSH-23 treated hPBMCs. The average reduction in CCR-5 message was 60.5%. These data show for the first time that an NF-κB inhibitor can influence the level of CCR-5 mRNA. Example 12. JSH-23 synergizes with HAART drugs. Having demonstrated that JSH-23 can inhibit viral replication with IC₅₀ values of 6.9±1.1 μM for BaL and 0.39±0.08 μM for HIb we next determined whether it could synergize with any of the drugs currently approved for the treatment of HIV-I by the US Food and Drug Administration (FDA). One drug from each class (entry, NRTI, NNRTI, integrase, and protease inhibitor) was initially evaluated to determine if there was any effect of the combination treatment. As above, hPBMCs were stimulated with PHA for three days and the stimulated cells were plated in a 96-well tissue culture plate. The stimulated cells were treated with JSH-23 in a range up to 10 μM, the HAART drug in a range up to their IC₅₀ values or in all combination of the two treatment series (see Treatment Grids: Tables 6 to 10 below). The treated cells were infected with BaL, as described above, and viral replication was assayed by p24 elisa after 72 hours. Synergy of the combination was calculated using the formula described in (Hess et al, 1997; Verrier et al, 2001). Significant synergy was considered to be an SI value greater than 100 that was not a single random point, i.e. the surface area on the resultant 3D mesh plot contained peaks with reasonable slopes. Synergy was seen with the entry inhibitor (T-20), the NRTI (tenofovir), and the integrase inhibitor (L-731,988) being developed by MERCK (Hazuda et al, 2000) (Figure 13 Panel A, B, D). No synergy was seen with the NNRTI (Efavirenz), or the protease inhibitor (Nelfmavir) (Figure 13 Panel C, E). Synergy was seen with the anti- viral drugs that require some level of cellular activation and/or cell cycle progression for their activity which is consistent with the block of the cell cycle by this class of drugs.

The reduction in IC₅₀ values (mean ±SD of three independent experiments) for the HAART drugs was determined for each concentration of JSH-23 (Figure 14 Panel A-E). The IC₅₀ for T-20 as single treatment ranged from 2 to 10 nM for BaL and significantly fell to 0.24 μM (on average) when used in combination with JSH- 23. This decrease is a 8.2-fold reduction in IC₅₀ value for T-20. For the NRTI (tenofovir), there was an 8.3 -fold reduction of the IC₅₀ values when used in combination with JSH-23, dropping the IC₅O value from 250 nM to 30 nM. However, there was no reduction in IC₅₀ when JSH-23 was used in combination with the NNRTI (Efavirenz). The integrase inhibitor showed the greatest drop in IC₅₀ value in combination with JSH-23. The IC₅₀ value for this compound dropped from 1.0 μM to 0.025 μM a decrease of 40-fold. Lastly, the protease inhibitor (Nelfinavir) in combination with JSH-23 showed no reduction in IC₅₀ value.

These finding are new and unpredicted from the previous literature on NF- KB and HIV-I infection. The data presented demonstrates for the first time that certain classes of currently used HAART drugs, entry, reverse transcriptase, and integrase inhibitors, synergize with JSH-23. The reduction in the IC₅₀ of entry, reverse transcriptase, and integrase inhibitors by JSH-23 by, 8.2-, 8.3-, and 40-fold respectively, has profound implication for the treatment of HIV-I infected individuals. Co-administration of JSH-23 with any/or all of the HAART drugs has the potential to increase patient compliance due to the reduction in adverse effects of the drugs given at lower doses. The synergy of JSH-23 with the specific classes of HAART drugs provides the opportunity for co-formulation of a single pill which again has the benefit of lowering pill burden and improving patient compliance.

Table 6. T-20 and JSH-23 plating grid.

Table 8. Efavirenz and JSH-23 plating grid.

Example 13. SM-7368 inhibits NF-κB in cells.

SM-7368 is reported to be a good inhibitor of NF-κB at low-μM concentrations (IC₅Q of 4.4 μM,(Lee et al, 2005)). Lee et al., reported that 10 μM SM-7368 can reduce NF-κB activation to less than 10% of untreated cells (Lee et al, 2005). To verify that the SM-7368 purchased (EMD Biosciences) was functioning properly its ability to inhibit NF-κB in a 293-Luc reporter cell was assessed. Cells were treated with 0, 0.1, 1, 5, 10, 20 and 50 μM SM-7368 for 2 hours then induce with 50ng/ml TNF-α for 18 hours. Following the induction luminescence was quantified using a DTX 880 multimode plate reader (Beckman Coulter). Figure 15 shows the dose response curve generated from the luminescence data. SM-7368 clearly inhibited the expression of the luciferase gene in a dose dependent manor with a median IC₅₀ (Inhibitory Concentration of 50%) of 2.9±0.6 μM. This value is the median value generated from three independent experiments ± the standard error. As a control 0.5% DMSO treated and untreated cells were compared to verify that the SM-7368 carrier had no effect on the expression of luciferase or in the readout of the assay. There was a slight decrease in the output from the assay in the DMSO treated population although it was not statistically significant. As a result of the controls the decrease in activity in the drug treated samples were compared to the DMSO control sample. The data also confirms the observation that 10 to 20 μM SM-7368 gives 100% inhibition of NF-κB activity. This observation along with the fact that the determined IC₅₀ value is within the reported range, gives confidence that this compound is functioning as reported in the literature and that it is indeed a good lead for developing new NF-κB inhibitors.

The experimental IC_5O value can also be used to assess the efficacy of the compound against HIV-I replication.

Example 14. SM-7368 inhibits CCR-5 (R5-tropic) and CXCR-4 (X4-tropic) viruses. Given the critical role NF-κB plays in the progression of the HIV life cycle and the onset of AIDS we assessed SM-7368's ability to inhibit HIV replication in human peripheral blood mononuclear cells (hPBMCs). Human PBMCs from a single donor were thawed and grown in the presence of 100U/ml IL-2 and activated with 5 μg/ml phytohemagglutinin (PHA) for three days. Activated hPBMCs were washed and resuspended in media containing 100U/ml IL-2 at a concentration of 1x10⁶ cells/ml and lOOμl transferred to each well of a 96 well plate. The cells were treated with 0, 0.1, 1, 5, 10, 20 and 50 μM SM-7368 in triplicate for each experiment. One set of the triplicate drug-treated cells was infected with HIV-I HIb (X4-trophic) and another was infected with HIV-I BaL (R5 -trophic) at an MOI of 0.01 for both viruses. Viral replication was assayed by p24 elisa after 72 hours. SM-7368 inhibited the growth of both viruses in a concentration dependent manor. The carrier for SM-7368 (0.5% DMSO) had no effect on viral replication, with p24 values in these treatment groups equal or greater than untreated cells. Figure 16 shows the dose response curves generated from the p24 elisas for hPBMCs infected with each virus. Figure 16 A is one of the data sets for inhibition of HIV-I BaL (R5-trophic), and Figure 16 B is one of the data sets for inhibition of HIV-I HIb (X4-trophic). The median IC₅₀ values ± the standard error generated from the depicted data and two other experiments was determined to be 2.6±0.8 μM for BaL and 0.53±0.11 μM for HIb. The IC₅₀ for the HIb strain was consistently 3 to 10-fold lower than that for BaL with all of the NF-κB inhibitors tested. This could be explained by the observations of Cicala et ah, who treated Jurkat cells with either X4 or R5 pi 20 envelope and determined the differences in the gene expression profiles for the two treatment groups (Cicala et al., 2006). Interestingly, these authors found that the p65/RelA subunit of NF-κB was down regulated in the X4 envelope treated cells. Furthermore, it has been reported that NF-κB plays a role in CXCR-4 receptor expression (Helbig et al., 2003) which would further effect the sensitivity of PBMCs harboring X4-tropic viruses to these compounds. Example 15. The cytotoxicity profile of SM-7368.

The cytotoxicity of SM-7368 was determined for the same pool of hPBMCs used in the infectivity assay described above. As with the infectivity assay the hPBMCs were stimulated with PHA for three days and then triplicate samples were treated with 0, 0.2, 2, 10, 20, 40 and 100 μM SM-7368 for an additional 72 hours. The number of viable cells was determined using MTS reagents (CalBioChem) and one representative experiment is shown in Figure 17. As the graph shows there was no detectable toxicity to these cells up to the 100 μM tested. Cells treated with 0.5% DMSO showed no detectable cytotoxic effects as compared to the same cells untreated. Although the cytotoxicity of SM-7368 has not been reported it was identified in a cell based screen indicating that it was not toxic to the cells in which it was identified. The potential usefulness of a compound is defined, at least in part by, the therapeutic or selectivity index (the ratio of cytotoxicity IC₅₀ to inhibitory IC₅₀). For SM-7368, using the upper limit of our cytotoxicity assay would yield a therapeutic index of 40 to 200 depending on the viral tropism. Example 16. SM-7368 inhibits progression from Gl to S phase of the cell- cycle.

NF-κB inhibitors have been reported to inhibit cell cycle progression from Gl to S phase of many different cell types (Ralstin et al., 2006). This effect is most likely due to the block of cyclin D expression which is regulated by NF-κB. This could have beneficial effects for the treatment of HIV-I as this virus requires progression through this point of the cell cycle to replicate (Nekhai et al., 2000; Tobiume et al., 1998). To determine if SM-7368 can affect the progression of the cell cycle from Gl phase to S phase hPBMCs were thawed and activated for three days as described above. The activated cells were treated in duplicate with 0, 1, 10 and 50 μM SM-7368 for three days and the cell cycle distribution was determine by quantifying the DNA content using flow cytometry. Control cells, both untreated and those treated with 0.5% DMSO, had approximately 78% of cells in the Gl phase of the cell cycle. Figure 18 shows that upon addition of SM-7368 the number of cells in Gl increases to approximately 83% at 1 μM and approximately 92% at 10 and 50 μM. These results verify that SM-7368 can block progression of from Gl-S phase of the cell cycle.

Example 17. SM-7368 down regulates CCR-5 expression.

Analysis of the CCR-5 promoter indicates that NF-kB is the major inducer of transcription on the context of the full promoter (Liu et al, 1998). It has been shown that even modest reductions in the surface density of CCR-5 can have a dramatic effect on HIV-I infectivity (Heredia et al., 2003). To investigate whether SM-7368's inhibition of NF-κB has an effect on CCR-5 expression hPBMCs were thawed and activated for three days as described above. The activated cells were treated in duplicate with 50 μM SM-7368 for three days. Total RNA was isolated from the cells. One microgram of total RNA was used in each reaction to amplify the CCR-5 and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) gene by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) reaction. It is desirable when designing RT-PCR primers to have them span an intron exon boundary at the 3 ' end of the gene so that amplification from genomic DNA can be distinguished from amplification from the mRNA. However, in the case of CCR- 5 exon 4 contains the open reading frame, the complete 3 -prime UTR, and 11 nucleotides of the 5-prime UTR (Mummidi et al, 1997). Therefore, primers were designed as described in Lai et al, who used these primers for QRT-PCR. In addition the total RNA was subjected to DNase digestion prior to the amplification step as described in the onestep RT-PCR kit. The primers used were, CCR5-up, 5'- CAAAAAGAAGGTCTTCATTACACC-3' (position, 512 to 535 of the mRNA); CCR5-down, 5'-CCTGTGCCTCTTCTTCTCATTTCG-S' (position, 677 to 700 of the mRNA). The CCR-5 primer pair generated a 189 bp PCR product. To control for the integrity and amount of RNA used for RT-PCR, the mRNA of a housekeeping gene, GAPDH, was amplified. The primers for GAPDH were 5'- GGTGGTCTCCTCTGACTTC AACA-3' (sense) and 5'- GTTGCTGT AGCCAAATTCGTTGT-3' (antisense). The GAPDH primer pair generated a 126 bp PCR product. RT-PCR products were resolved on a 2% agarose gel and visualized by staining with ethidium bromide. In order to avoid saturation of the PCR signal a range of cycles were compared to determine where CCR-5 amplification was still expanding linearly in total RNA from untreated hPBMCs (Figure 19 A). From the results in Figure 19 A, 25 cycles was chosen to examine CCR-5 message levels and 20 cycles for GAPDH. Figure 19 B shows the results of amplification of CCR-5 from two independent pools of SM-7368 treated hPBMCs. The average reduction in CCR-5 message was 61.5%. These data show for the first time that an NF-κB inhibitor can influence the level of CCR-5 mRNA. Example 18. SM-7368 synergizes with HAART drugs. Having demonstrated that SM-7368 can inhibit viral replication with IC_5O values of 2.6±0.8 μM for BaL and 0.53±0.11 μM for HIb we next determined whether it could synergize with any of the drugs currently approved for the treatment of HIV-I by the US Food and Drug Administration (FDA). One drug from each class (entry, NRTI, NNRTI, integrase, and protease inhibitor) was initially evaluated to determine if there was any effect of the combination treatment. As above, hPBMCs were stimulated with PHA for three days and the stimulated cells were plated in a 96-well tissue culture plate. The stimulated cells were treated with SM-7368 in a range up to 2.5 μM, the HAART drug in a range up to their IC₅0 values or in all combination of the two treatment series (see Treatment Grids: Tables 11 to 15 below). The treated cells were infected with BaL, as described above, and viral replication was assayed by p24 elisa after 72 hours. Synergy of the combination was calculated using the formula described in (Hess et al, 1997; Verrier et al, 2001). Significant synergy was considered to be an SI value greater than 100 that was not a single random point, i.e. the surface area on the resultant 3D mesh plot contained peaks with reasonable slopes. Synergy was seen with the entry inhibitor (T-20), the NRTI (tenofovir), and the integrase inhibitor (L-731,988) being developed by MERCK (Hazuda et al, 2000) (Figure 20 Panel A, B, D). No synergy was seen with the NNRTI (Efavirenz), or the protease inhibitor (Nelfmavir) (Figure 20 Panel C, E). Synergy was seen with the anti-viral drugs that require some level of cellular activation and/or cell cycle progression for their activity which is consistent with the block of the cell cycle by this class of drugs.

Table 11. T-20 and SM-7368 plating grid.

Table 13. Efavirenz and SM-7368 plating grid.

Table 14. The integrase inhibitor (L-731,988) and SM-7368 plating grid.

The reduction in ICs₀ values (mean ±SD of three independent experiments) for the HAART drugs was determined for each concentration of SM-7368 (Figure 21 Panel A-E). The IC₅₀ for T-20 as single treatment ranged from 2 to 10 nM for BaL and significantly fell to 0.29 μM (on average) when used in combination with SM-7368. This decrease is a 7.2-fold reduction in IC₅₀ value for T-20. For the NRTI (tenofovir), there was a 5.2-fold reduction of the ICs₀ values when used in combination with SM-7368, dropping the IC₅₀ value from 200 nM to 39 nM. However, there was no reduction in IC₅₀ when SM-7368 was used in combination with the NNRTI (Efavirenz). The integrase inhibitor showed the greatest drop in IC₅₀ value in combination with SM-7368. The IC₅₀ value for this compound dropped from 1.5 μM to 0.058 μM a decrease of 30-fold. Lastly, the protease inhibitor (Nelfinavir) in combination with SM-7368 showed no reduction in IC₅₀ value.

These finding are new and unpredicted from the previous literature on NF- KB and HTV-I infection. The data presented demonstrates for the first time that certain classes of currently used HAART drugs, entry, reverse transcriptase, and integrase inhibitors, synergize with SM-7368. The reduction in the IC₅₀ of entry, reverse transcriptase, and integrase inhibitors by SM-7368 by, 7.2-, 5.2-, and 30-fold respectively, has profound implication for the treatment of HIV-I infected individuals. Co-administration of SM-7368 with any/or all of the HAART drugs has the potential to increase patient compliance due to the reduction in adverse effects of the drugs given at lower doses. The synergy of SM-7368 with the specific classes of HAART drugs provides the opportunity for co-formulation of a single pill which again has the benefit of lowering pill burden and improving patient compliance. Example 19. Resveratrol inhibits NF-κB in cells.

Resveratrol is reported to be a good inhibitor of NF-κB at low-μM concentrations (IC₅₀ of 10-20 μM, (Csiszar et al, 2006; Heynekamp et al, 2006)). To verify that the resveratrol purchased (SIGMA) was functioning properly its ability to inhibit NF-κB in a 293-Luc reporter cell was assessed. Cells were treated with 0, 0.1, 1, 5, 10, 20 and 50 μM resveratrol for 2 hours then induce with 50ng/ml TNF-α for 18 hours. Following the induction luminescence was quantified using a DTX 880 multimode plate reader (BECKMAN COULTER). Figure 22 shows the dose response curve generated from the luminescence data of two independent assays. Resveratrol clearly inhibited the expression of the luciferase gene in a dose dependent manor with a median IC₅₀ (Inhibitory Concentration of 50%) of 16.6±1.2 μM. This value is the median value generated from three independent experiments ± the standard error. As a control 0.5% DMSO treated and untreated cells were compared to verify that the resveratrol carrier had no effect on the expression of luciferase or in the readout of the assay. There was a slight decrease in the output from the assay in the DMSO treated population although it was not statistically significant. As a result of the controls the decrease in activity in the drug treated samples were compared to the DMSO control sample. The fact that the determined IC₅₀ value is within the reported range, gives confidence that this compound is functioning as reported in the literature and that it is indeed a good lead for developing new NF-κB inhibitors. The experimental IC_5O value can also be used to assess the efficacy of the compound against HIV-I replication. Example 20. Resveratrol inhibits CCR-5 (R5-tropic) and CXCR-4 (X4-tropic) viruses.

Given the critical role NF- KB plays in the progression of the HIV life cycle and the onset of AIDS we assessed resveratrol's ability to inhibit HIV replication in human peripheral blood mononuclear cells (hPBMCs). Human PBMCs from a single donor were thawed and grown in the presence of 100 U/ml IL-2 and activated with 5 μg/ml phytohemagglutinin (PHA) for three days. Activated hPBMCs were washed and resuspended in media containing 100U/ml IL-2 at a concentration of 1x10 cells/ml and 100 μl transferred to each well of a 96 well plate. The cells were treated with 0, 0.1, 1, 5, 10, 20 and 50 μM resveratrol in triplicate for each experiment. One set of the triplicate drug- treated cells was infected with HIV-I HIb (X4-trophic) and another was infected with HIV-I BaL (R5-trophic) at an MOI of 0.01 for both viruses. Viral replication was assayed by p24 elisa after 72 hours. Resveratrol inhibited the growth of both viruses in a concentration dependent manor. The carrier for resveratrol (0.5% DMSO) had no effect on viral replication, with p24 values in these treatment groups equal or greater than untreated cells. Figure 23 shows the dose response curves generated from the p24 elisas for hPBMCs infected with each virus. Figure 23 A is one of the data sets for inhibition of HIV-I BaL (R5-trophic), and Figure 23 B is one of the data sets for inhibition of HIV-I HIb (X4-trophic). The median IC₅₀ values ± the standard error generated from the depicted data and two other experiments was determined to be 10.1±2.2 μM for BaL and 3.1±0.04 μM for HIb. The IC₅₀ for the HIb strain was consistently 3 to 10-fold lower than that for BaL with all of the NF-κB inhibitors tested. This could be explained by the observations of Cicala et al., who treated Jurkat cells with either X4 or R5 p 120 envelope and determined the differences in the gene expression profiles for the two treatment groups (Cicala et al, 2006). Interestingly, these authors found that the p65/RelA subunit of NF-κB was down regulated in the X4 envelope treated cells. Furthermore, it has been reported that NF-κB plays a role in CXCR-4 receptor expression (Helbig et al, 2003) which would further effect the sensitivity of PBMCs harboring X4-tropic viruses to these compounds. Example 21. The cytotoxicity profile of resveratrol.

The cytotoxicity of resveratrol was determined for the same pool of hPBMCs used in the infectivity assay described above. As with the infectivity assay the hPBMCs were stimulated with PHA for three days and then triplicate samples were treated with 0, 0.2, 2, 10, 20, 40 and 100 μM resveratrol for an additional 72 hours. The number of viable cells was determined using MTS reagents (CalBioChem) and one representative experiment is shown in Figure 24. As the graph shows there was only slight toxicity at the two highest doses of 40 and 100 μM. This dose response curve gave an approximated IC₅₀ of 70 μM. Cells treated with 0.5% DMSO showed no detectable cytotoxic effects as compared to the same cells untreated. Resveratrol has been reported to have a reasonable cytotoxicity profile with toxicity being reported at concentrations between 100 to 400 μM depending on the cell type (Babich et al., 2000). The potential usefulness of a compound is defined, at least in part by, the therapeutic or selectivity index (the ratio of cytotoxicity IC₅₀ to inhibitory ICs₀). For resveratrol, using the cytotoxicity values generated in our assay would yield a therapeutic index of 7 to 25 depending on the viral tropism.

Example 22. Resveratrol inhibits progression from Gl to S phase of the cell- cycle.

NF-κB inhibitors have been reported to inhibit cell cycle progression from Gl to S phase of many different cell types (Ralstin et al., 2006). This effect is most likely due to the block of cyclin D expression which is regulated by NF-κB. This could have beneficial effects for the treatment of HIV-I as this virus requires progression through this point of the cell cycle to replicate (Nekhai et al, 2000; Tobiume et al., 1998). To determine if resveratrol can affect the progression of the cell cycle from Gl phase to S phase hPBMCs were thawed and activated for three days as described above. The activated cells were treated in duplicate with 0, 1, 10 and 50 μM resveratrol for three days and the cell cycle distribution was determine by quantifying the DNA content using flow cytometry. Control cells, both untreated and those treated with 0.5% DMSO, had approximately 78% of cells in the Gl phase of the cell cycle. Figure 25 shows that upon addition of resveratrol the number of cells in Gl increases to approximately 77% at 1 μM and approximately 90% and 92% at 10 and 50 μM respectively. These results verify that resveratrol can block progression of from Gl-S phase of the cell cycle.

Example 23. Resveratrol down regulates CCR-5 expression.

Analysis of the CCR-5 promoter indicates that NF-κB is the major inducer of transcription on the context of the full promoter (Liu et al., 1998). It has been shown that even modest reductions in the surface density of CCR-5 can have a dramatic effect on HIV-I infectivity (Heredia et al., 2003). To investigate whether resveratrol' s inhibition of NF-κB has an effect on CCR-5 expression hPBMCs were thawed and activated for three days as described above. The activated cells were treated in duplicate with 50 μM resveratrol for three days. Total RNA was isolated from the cells. One microgram of total RNA was used in each reaction to amplify the CCR-5 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) reaction. It is desirable when designing RT-PCR primers to have them span an intron exon boundary at the 3' end of the gene so that amplification from genomic DNA can be distinguished from amplification from the mRNA. However, in the case of CCR-5 exon 4 contains the open reading frame, the complete 3 -prime UTR, and 11 nucleotides of the 5-prime UTR (Mummidi et al., 1997). Therefore, primers were designed as described in Lai et al., who used these primers for QRT-PCR. In addition the total RNA was subjected to DNase digestion prior to the amplification step as described in the onestep RT-PCR kit. The primers used were, CCR5-up, 5'- CAAAAAGAAGGTCTTCATTACACC-S¹ (position, 512 to 535 of the mRNA); CCR5-down, 5'-CCTGTGCCTCTTCTTCTCATTTCG-3' (position, 677 to 700 of the mRNA). The CCR-5 primer pair generated a 189 bp PCR product. To control for the integrity and amount of RNA used for RT-PCR, the mRNA of a housekeeping gene, GAPDH, was amplified. The primers for GAPDH were 5'- GGTGGTCTCCTCTGACTTC AACA-3' (sense) and 5'- GTTGCTGT AGCC AAATTCGTTGT-3' (antisense). The GAPDH primer pair generated a 126 bp PCR product. RT-PCR products were resolved on a 2% agarose gel and visualized by staining with ethidium bromide. In order to avoid saturation of the PCR signal a range of cycles were compared to determine where CCR-5 amplification was still expanding linearly in total RNA from untreated hPBMCs (Figure 26 A). From the results in Figure 26 A, 25 cycles was chosen to examine CCR-5 message levels and 20 cycles for GAPDH. Figure 26 B shows the results of amplification of CCR-5 from two independent pools of resveratrol treated hPBMCs. The average reduction in CCR-5 message was 55%. These data show for the first time that an NF-κB inhibitor can influence the level of CCR-5 mRNA. Example 24. Resveratrol synergizes with HAART drugs. Having demonstrated that resveratrol can inhibit viral replication with IC_5O values of 10.1±2.21 μM for BaL and 3.1±0.04 μM for UIb we next determined whether it could synergize with any of the drugs currently approved for the treatment of HIV-I by the US Food and Drug Administration (FDA). Heradia et al., have already shown that resveratrol can synergize with ddl an NRTI but there is no data on other classes of compounds used for the treatment of the disease. One drug from each class (entry, NRTI, NNRTI, integrase, and protease inhibitor) was initially evaluated to determine if there was any effect of the combination treatment. As above, hPBMCs were stimulated with PHA for three days and the stimulated cells were plated in a 96-well tissue culture plate. The stimulated cells were treated with resveratrol in a range up to 10 μM, the HAART drug in a range up to their IC₅₀ values or in all combination of the two treatment series (see Treatment Grids: Tables 16 to 20 below). The treated cells were infected with BaL, as described above, and viral replication was assayed by p24 elisa after 72 hours. Synergy of the combination was calculated using the formula described in (Hess et al, 1997; Verrier et al, 2001). Significant synergy was considered to be an SI value greater than 100 that was not a single random point, i.e. the surface area on the resultant 3D mesh plot contained peaks with reasonable slopes. Synergy was seen with the entry inhibitor (T-20), the NRTI (tenofovir), and the integrase inhibitor (L-731,988) being developed by MERCK (Hazuda et al, 2000) (Figure 27, Panel A, B, D). No synergy was seen with the NNRTI (Efavirenz), or the protease inhibitor (Nelfinavir) (Figure 27, Panel C, E). Synergy was seen with the anti-viral drugs that require some level of cellular activation and/or cell cycle progression for their activity which is consistent with the block of the cell cycle by this class of drugs.

Table 18. Efavirenz and Resveratrol plating grid.

The reduction in IC₅₀ values (mean ±SD of three independent experiments) for the HAART drugs was determined for each concentration of resveratrol (Figure 28, Panel A-E). The IC₅₀ for T-20 as single treatment ranged from 2 to 10 nM for BaL and significantly fell to 0.69 μM (on average) when used in combination with resveratrol. This decrease is a 5.1 -fold reduction in IC₅₀ value for T-20. For the NRTI (tenofovir), there was an 7.7-fold reduction of the IC₅₀ values when used in combination with resveratrol, dropping the IC₅₀ value from 160 nM to 21 nM. However, there was no reduction in IC₅₀ when resveratrol was used in combination with the NNRTI (Efavirenz). The integrase inhibitor showed the greatest drop in IC₅₀ value in combination with resveratrol. The IC_5O value for this compound dropped from 4.8 μM to 0.27 μM a decrease of 17.8-fold. Lastly, the protease inhibitor (Nelfmavir) in combination with resveratrol showed no reduction in IC₅₀ value.

These findings are new and unpredicted from the previous literature on NF- KB and HIV-I infection. The data presented demonstrates for the first time that certain classes of currently used HAART drugs, entry, reverse transcriptase, and integrase inhibitors, synergize with resveratrol. The reduction in the IC_5O of entry, reverse transcriptase, and integrase inhibitors by resveratrol by, 5.1-, 7.7-, and 17.8- fold respectively, has profound implication for the treatment of HIV-I infected individuals. Co-administration of resveratrol with any/or all of the HAART drugs has the potential to increase patient compliance due to the reduction in adverse effects of the drugs given at lower doses. The synergy of resveratrol with the specific classes of HAART drugs provides the opportunity for co-formulation of a single pill which again has the benefit of lowering pill burden and improving patient compliance. Example 25. Curcumin inhibits NF-κB in cells.

Curcumin is reported to be a good inhibitor of NF-κB at low-μM concentrations (IC₅₀ of 8 μM, (Weber et al, 2006)). To verify that the curcumin purchased (SIGMA) was functioning properly its ability to inhibit NF-kB in a 293- Luc reporter cell was assessed. Cells were treated with 0, 0.1, 1, 5, 10, 20 and 50 μM curcumin for 2 hours then induce with 50ng/ml TNF-α for 18 hours. Following the induction luminescence was quantified using a DTX 880 multimode plate reader (Beckman Coulter). Figure 29 shows the dose response curve generated from the luminescence data for two independent experiments. Curcumin clearly inhibited the expression of the luciferase gene in a dose dependent manor with a median IC₅₀ (Inhibitory Concentration of 50%) of 5.7± 1.3 μM. This value is the median value generated from three independent experiments ± the standard error. As a control 0.5% DMSO treated and untreated cells were compared to verify that the curcumin carrier had no effect on the expression of luciferase or in the readout of the assay. There was a slight decrease in the output from the assay in the DMSO treated population although it was not statistically significant. As a result of the controls the decrease in activity in the drug treated samples were compared to the DMSO control sample. The data also demonstrates that 10 to 20 μM curcumin gives 100% inhibition of NF-κB activity. This observation along with the fact that the determined IC₅₀ value is within the reported range, gives confidence that this compound is functioning as reported in the literature and that it is indeed a good lead for developing new NF-κB inhibitors. The experimental IC_5O value can also be used to assess the efficacy of the compound against HIV-I replication. Example 26. Curcumin inhibits CCR-5 (R5-tropic) and CXCR-4 (X4-tropic) viruses.

Given the critical role NF-κB plays in the progression of the HIV life cycle and the onset of AIDS we assessed curcumin' s ability to inhibit HIV replication in human peripheral blood mononuclear cells (hPBMCs). Human PBMCs from a single donor were thawed and grown in the presence of 100U/ml IL-2 and activated with 5 μg/ml phytohemagglutinin (PHA) for three days. Activated hPBMCs were washed and resuspended in media containing 100U/ml IL-2 at a concentration of 1x10⁶ cells/ml and lOOμl transferred to each well of a 96 well plate. The cells were treated with 0, 0.1, 1, 5, 10, 20 and 50 μM curcumin in triplicate for each experiment. One set of the triplicate drug- treated cells was infected with HIV-I HIb (X4-trophic) and another was infected with HIV-I BaL (R5-trophic) at an MOI of 0.01 for both viruses. Viral replication was assayed by p24 elisa after 72 hours. Curcumin inhibited the growth of both viruses in a concentration dependent manor. The carrier for curcumin (0.5% DMSO) had no effect on viral replication, with p24 values in these treatment groups equal or greater than untreated cells. Figure 30 shows the dose response curves generated from the p24 elisas for hPBMCs infected with each virus. Figure 30 A is one of the data sets for inhibition of HIV-I BaL (R5-trophic), and Figure 30 B is one of the data sets for inhibition of HIV-I HIb (X4-trophic). The median IC₅₀ values ± the standard error generated from the depicted data and two other experiments was determined to be 3.0±1.8 μM for BaL and 1.8±0.7 μM for HIb. The IC₅₀ for the HIb strain was consistently 3 to 10-fold lower than that for BaL with all of the NF-κB inhibitors tested. This could be explained by the observations of Cicala et al, who treated Jurkat cells with either X4 or R5 p 120 envelope and determined the differences in the gene expression profiles for the two treatment groups (Cicala et al, 2006). Interestingly, these authors found that the p65/RelA subunit of NF-κB was down regulated in the X4 envelope treated cells. Furthermore, it has been reported that NF-κB plays a role in CXCR-4 receptor expression (Helbig et al, 2003) which would further effect the sensitivity of PBMCs harboring X4-tropic viruses to these compounds. Example 27. The cytotoxicity profile of curcumin.

The cytotoxicity of curcumin was determined for the same pool of hPBMCs used in the infectivity assay described above. As with the infectivity assay the hPBMCs were stimulated with PHA for three days and then triplicate samples were treated with 0, 0.2, 2, 10, 20, 40 and 100 μM curcumin for an additional 72 hours. The number of viable cells was determined using MTS reagents (CalBioChem) and one representative experiment is shown in Figure 31. As the graph shows there was no detectable toxicity to these cells up to the 100 μM tested. Cells treated with 0.5% DMSO showed no detectable cytotoxic effects as compared to the same cells untreated. Curcumin has been reported to have a good cytotoxicity profile with no toxicity being reported at concentrations up to 8 grams/day for 1 month in humans (Sharma et al, 2005 and references therein). The potential usefulness of a compound is defined, at least in part by, the therapeutic or selectivity index (the ratio of cytotoxicity IC₅₀ to inhibitory IC₅₀). For curcumin, using the reported values for cytotoxicity this would yield a therapeutic index of 30 to 50 depending on the viral tropism.

Example 28. Curcumin inhibits progression from Gl to S phase of the cell- cycle. NF-κB inhibitors have been reported to inhibit cell cycle progression from

Gl to S phase of many different cell types (Ralstin et al, 2006). This effect is most likely due to the block of cyclin D expression which is regulated by NF-κB. This could have beneficial effects for the treatment of HIV-I as this virus requires progression through this point of the cell cycle to replicate (Nekhai et al, 2000; Tobiume et al, 1998). To determine if curcumin can affect the progression of the cell cycle from Gl phase to S phase hPBMCs were thawed and activated for three days as described above. The activated cells were treated in duplicate with 0, 1, 10 and 50 μM curcumin for three days and the cell cycle distribution was determine by quantifying the DNA content using flow cytometry. Control cells, both untreated and those treated with 0.5% DMSO, had approximately 78% of cells in the Gl phase of the cell cycle. Figure 32 shows that upon addition of curcumin the number of cells in Gl increases to approximately 87% at 1 μM and approximately 94% at 10 μM. At 50 μM the analysis was not possible as it was not possible to identify activated T-cells. These results verify that curcumin can block progression of from Gl-S phase of the cell cycle. Example 29. Curcumin down regulates CCR-5 expression. Analysis of the CCR-5 promoter indicates that NF-κB is the major inducer of transcription on the context of the full promoter (Liu et al, 1998). It has been shown that even modest reductions in the surface density of CCR-5 can have a dramatic effect on HTV-I infectivity (Heredia et al, 2003). To investigate whether curcumin's inhibition of NF-κB has an effect on CCR-5 expression hPBMCs were thawed and activated for three days as described above. The activated cells were treated in duplicate with 50 μM curcumin for three days. Total RNA was isolated from the cells. One microgram of total RNA was used in each reaction to amplify the CCR-5 and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) gene by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) reaction. It is desirable when designing RT-PCR primers to have them span an intron exon boundary at the 3' end of the gene so that amplification from genomic DNA can be distinguished from amplification from the mRNA. However, in the case of CCR-5 exon 4 contains the open reading frame, the complete 3 -prime UTR, and 11 nucleotides of the 5-prime UTR (Mummidi et al, 1997). Therefore, primers were designed as described in Lai et al., who used these primers for QRT-PCR. In addition the total RNA was subjected to DNase digestion prior to the amplification step as described in the onestep RT-PCR kit. The primers used were, CCR5-up, 5'- CAAAAAGAAGGTCTTCATTACACC-3' (position, 512 to 535 of the mRNA); CCR5-down, 5'-CCTGTGCCTCTTCTTCTCATTTCG-3^l (position, 677 to 700 of the mRNA). The CCR-5 primer pair generated a 189 bp PCR product. To control for the integrity and amount of RNA used for RT-PCR, the mRNA of a housekeeping gene, GAPDH, was amplified. The primers for GAPDH were 5'- GGTGGTCTCCTCTGACTTC AAC A-3' (sense) and 5'- GTTGCTGT AGCC AAATTCGTTGT-3' (antisense). The GAPDH primer pair generated a 126 bp PCR product. RT-PCR products were resolved on a 2% agarose gel and visualized by staining with ethidium bromide. In order to avoid saturation of the PCR signal a range of cycles were compared to determine where CCR-5 amplification was still expanding linearly in total RNA from untreated hPBMCs (Figure 33 A). From the results in Figure 33 A, 25 cycles was chosen to examine CCR-5 message levels and 20 cycles for GAPDH. Figure 33 B shows the results of amplification of CCR-5 from two independent pools of curcumin treated hPBMCs. The average reduction in CCR-5 message was 85%. These data show for the first time that an NF-κB inhibitor can influence the level of CCR-5 mRNA. Example 30. Curcumin synergizes with HAART drugs.

Having demonstrated that curcumin can inhibit viral replication with IC_5O values of 3.0±1.8 μM for BaL and 1.8±0.7 μM for HIb we next determined whether it could synergize with any of the drugs currently approved for the treatment of

HIV-I by the US Food and Drug Administration (FDA). One drug from each class (entry, NRTI, NNRTI, integrase, and protease inhibitor) was initially evaluated to determine if there was any effect of the combination treatment. As above, hPBMCs were stimulated with PHA for three days and the stimulated cells were plated in a 96-well tissue culture plate. The stimulated cells were treated with curcumin in a range up to 10 μM, the HAART drug in a range up to their IC₅₀ values or in all combination of the two treatment series (see Treatment Grids: Tables 21 to 25 below). The treated cells were infected with BaL, as described above, and viral replication was assayed by p24 elisa after 72 hours. Synergy of the combination was calculated using the formula described in (Hess et ah, 1997; Verrier et ah, 2001). Significant synergy was considered to be an SI value greater than 100 that was not a single random point, i.e. the surface area on the resultant 3D mesh plot contained peaks with reasonable slopes. Synergy was seen with the entry inhibitor (T-20), the NRTI (tenofovir), and the integrase inhibitor (L-731,988) being developed by MERCK (Hazuda et al, 2000) (Figure 34 Panel A, B, D). No synergy was seen with the NNRTI (Efavirenz), or the protease inhibitor (Nelfmavir) (Figure 34 Panel C, E). Synergy was seen with the anti- viral drugs that require some level of cellular activation and/or cell cycle progression for their activity which is consistent with the block of the cell cycle by this class of drugs.

Table 21. T-20 and Curcumin plating grid.

Table 22. Tenofovir and Curcumin plating grid.

The reduction in IC₅₀ values (mean ±SD of three independent experiments) for the HAART drugs was determined for each concentration of curcumin (Figure 35 Panel A-E). The IC₅₀ for T-20 as single treatment ranged from 2 to 10 nM for BaL and significantly fell to 0.13 μM (on average) when used in combination with curcumin. This decrease is a 6.1 -fold reduction in IC₅₀ value for T-20. For the NRTI (tenofovir), there was a 6.7-fold reduction of the IC_5O values when used in combination with curcumin, dropping the IC₅₀ value from 200 nM to 30 nM. However, there was only a slight reduction in IC₅₀ when curcumin was used in combination with the NNRTI (Efavirenz) 2.2-fold. The integrase inhibitor showed the greatest drop in IC₅₀ value in combination with curcumin. The IC₅₀ value for this compound dropped from 1.5 μM to 0.020 μM a decrease of 76-fold. Lastly, the protease inhibitor (Nelfinavir) in combination with curcumin showed no reduction in IC₅₀ value.

These findings are new and unpredicted from the previous literature on NF- KB and HIV-I infection. The data presented demonstrates for the first time that certain classes of currently used HAART drugs, entry, reverse transcriptase, and integrase inhibitors, synergize with curcumin. The reduction in the IC₅₀ of entry, reverse transcriptase, and integrase inhibitors by curcumin by, 6.1-, 6.7-, and 76-fold respectively, has profound implication for the treatment of HIV-I infected individuals. Co-administration of curcumin with any/or all of the HAART drugs has the potential to increase patient compliance due to the reduction in adverse effects of the drugs given at lower doses. The synergy of curcumin with the specific classes of HAART drugs provides the opportunity for co-formulation of a single pill which again has the benefit of lowering pill burden and improving patient compliance. While the present invention has been described with reference to specific embodiments, this application is intended to cover those various changes and substitutions that may be made by those of ordinary skill in the art without departing from the spirit and scope of the appended claims.

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. All publications cited herein are hereby incorporated by reference in their entirety.

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Claims

1. A method of treating or preventing a viral infection, comprising administering to a subject in need thereof a therapeutically effective amount of a combination of (a) an NF-κB inhibitor and (b) an antiviral agent.

2. The method of claim 1, wherein the viral infection is an infection with a member of the Retroviridae virus family.

3. The method of claim 2, wherein the viral infection is an infection with a human immunodeficiency virus (HIV).

4. The method of claim 3, wherein the HIV virus is HIV-I.

5. The method of claim 1, wherein the subject is infected with the virus causing the viral infection.

6. The method of claim 5, wherein the subject is treatment experienced and the viral infection has resistance to antiviral therapy.

7. The method of claim 1, wherein the subject is uninfected but is at risk of contracting the viral infection.

8. The method of claim 1, wherein the NF-κB inhibitor is selected from the group consisting of: a compound that blocks the activation of the p65/RelA subunit in the cytoplasm, a compound that prevents translocation of the p65/RelA subunit to the nucleus, a compound that prevents binding of the p65/RelA subunit to its cognate DNA binding site, a compound that prevents p65/RelA subunit recruitment of chromatin remodeling complexes and/or transcription machinery, and a compound that prevents derepression of the chromatin locus associated with the cognate DNA-binding site

9. The method of claim 1, wherein the NF-κB inhibitor is selected from the group consisting of: parthenolide, JSH-23, SM-7368, resveratrol, curcumin, and analogues derivatives, pharmaceutically acceptable conjugates, precursors, metabolites and salts thereof.

10. The method of claim 1, wherein the antiviral agent is selected from the group consisting of: an entry inhibitor, a nucleoside analogue reverse transcriptase inhibitor (NRTI), a nucleotide analogue reverse transcriptase inhibitor (NtRTI),and an integrase inhibitor.

11. The method of claim 10, wherein the entry inhibitor is T-20.

12. The method of claim 10, wherein the NRTI is tenofovir.

13. The method of claim 10, wherein the integrase inhibitor is L-731988.

14. The method of claim 1, wherein the viral infection is an infection with a member of one of the viral families selected from the group consisting of: the Hepadnaviridae viral family, the Herpesviridae viral family, the Othomyxoviridae viral family, the Paramyxoviridae viral family, and the Picornaviridae viral family.

15. A composition comprising: (a) an NF-κB inhibitor and (b) an antiviral agent.

16. The composition of claim 15, wherein the NF-κB inhibitor is selected from the group consisting of: a compound that blocks the activation of the p65/RelA subunit in the cytoplasm, a compound that prevents translocation of the p65/RelA subunit to the nucleus, a compound that prevents binding of the p65/RelA subunit to its cognate DNA binding site, a compound that prevents p65/RelA subunit recruitment of chromatin remodeling complexes and/or transcription machinery, and a compound that prevents derepression of the chromatin locus associated with the cognate DNA-binding site.

17. The composition of claim 15, wherein the NF-κB inhibitor is selected from the group consisting of: parthenolide, JSH-23, SM-7368, resveratrol, curcumin, and analogues derivatives, pharmaceutically acceptable conjugates, precursors, metabolites and salts thereof.

18. The composition of claim 15, wherein the antiviral agent is selected from the group consisting of: an entry inhibitor, a nucleotide analogue reverse transcriptase inhibitor (NRTI), and an integrase inhibitor.

19. The composition of claim 18, wherein the entry inhibitor is T-20.

20. The composition of claim 18, wherein the NRTI is tenofovir.

21. The composition of claim 18, wherein the integrase inhibitor is L-

731988.

22. The composition of claim 14, further comprising a pharmaceutically acceptable carrier.