WO1997032587A1 - Methods for treating viral infections - Google Patents

Abstract

The present invention includes, inter alia, methods of treating cells infected with a virus capable of causing an immunodeficiency disease and related pharmaceutical compositions. Such methods comprise using a combination of compounds comprising a tat inhibitor and an NF-λB inhibitor.

Description

METHODS FOR TREATING VIRAL INFECTIONS

BACKGROUND OF THE INVENTION

The human immunodeficiency virus type 1 (HIV- 1 , also referred to as HTLV-III, LAV or HTLV-III/LAV) and, to a lesser extent, human immunodeficiency virus type 2 (HIV-2) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and related disorders. (Barre-Sinoussi, et al., Science,

220:868-871 (1983); Gallo, et al., Science, 224:500-503 ( 1 984); Levy, et al., Science, 225:840-842 (1984); Popovic, et al., Science, 224:497-500 (1984); Sarngadharan, et al., Science, 224:506-508 (1984); Siegal, et al., N. Engl. J. Med., 305: 1439-1444 (1981 ); Clavel., AIDS, 1 : 135-140 (1987)).

This disease is characterized by a long asymptomatic period followed by progressive degeneration of the immune system and the central nervous system. Studies of the virus indicate that replication is highly regulated, and both latent and lytic infection of the CD4 positive helper subset of T-lymphocytes occur in the tissue culture. (Zagury, et al., Science, 231:850-853 ( 1986)). The expression of the virus in infected patients also appears to be regulated as the titer of infectious virus remains low throughout the course of the disease. Both HIV-1 and 2 share a similar structural and functional genomic organization, having regulatory genes such as tat, rev, nef, in addition to structural genes such as env, gag and pot.

While AIDS, itself, does not necessarily cause death, in many individuals the immune system is so severely depressed that various other diseases (secondary infections or unusual tumors) such as herpes, cytomegalovirus, Kaposi's sarcoma and Epstein-Barr virus related lymphomas among others occur, which ultimately results in death. These secondary infections may be treated using other medications. However, such treatment can be adversely affected by the weakened immune system. Some humans infected with AIDS virus seem to live many years with little or no symptoms, but appear to have persistent infections. Another group of humans suffer mild immune system depression with various symptoms such as weight loss, malaise, fever and swollen lymph nodes. These syndromes have been called persistent generalized lymphadenopathy syndrome (PGL) and AIDS related complex (ARC) and may or may not develop into AIDS. In all cases, those infected with HIV are believed to be persistently infective to others.

The activation of the latent HIV provirus from asymptomatic period has been reported to be governed by long terminal repeats (LTR) in the viral DNA. See (Ranki, et al., Lancet ii:589-593 ( 1 987); Fauci, et al., Science, 235:61 7-622

( 1 988); Zaguary, et al., Science 237:850-853 ( 1985); Mosca, Nature (London) 325:67-70 (1987)). The activity of HIV-1 is determined by the complex interaction of positive and negative transcriptional regulators that bind to specific sequences within the LTR. (Cullen, et al.. Cell 55:423-426 ( 1 989) . Changes in the quantity or quality of these factors may underlie the activation of transcription of HIV-1 and HIV-2 latent provirus by a myriad of stimuli. See (Fauci, Science 235:61 7-622 ( 1 988); Griffin, et al., Nature (London) 339:70-73 ( 1 989); Nabel, et al., Science 235: 1299-1 302 (1 988)) . Phorbol 1 2-myristate-1 3- acetate (PMA) and Tumor Necrosis Factor-σ (TNF-σ) may be potent activators. TNF-σ is present in enhanced levels in HIV infected individuals, suggesting that the cytokine plays an important role in the pathogenesis of AIDS. (Lahdevirta, Am. J. Med. 35:289-291 (1988)).

Most known methods for treating individuals infected with HIV have focused on preventing integration of the virus into the host cells' chromosome or on stages other than dealing with the provirus. Thus, one area of interest has been drugs that affect reverse transcriptase. Many of the proposed therapeutic methods, however, have not proven clinically effective. Indeed, even treatments that have resulted in clinical utility such as AZT (zidovudine) have not been reported to prevent the breakdown of the immune system in many patients after a number of years of treatment. Few methods have been reported to inhibit both expression of integrated provirus and chronic infection of HIV- 1 . Reverse transcriptase inhibitors, e.g., AZT, ddC, and ddl, have not been reported to have an inhibitory effect on chronic infections.

Another method of treatment that has been proposed is to look for drugs that affect regulatory functions such as tat. The viral switch from latency to active replication requires regulatory proteins including tat. The tat protein transactivates regulatory elements in the HIV-LTR and amplifies viral replication many thousand fold. Thus, it has been proposed that by inhibiting tat function, the virus will be stopped or greatly inhibited at the latent stage of viral infection with subsequent replication of the provirus substantially impeded. See, for example, Hsu, et al.. U.S. Patent No.5,036,101, U.S. Patent No.5,041,438, (Hsu, Science 254:1799-1800 (1992)) and (Hsu, et al., Proc. Natl. Acad. Sci. USA 50:6395-6399 (1993)).

The viral protein tat plays an essential role in upregulating the HIV-1 promoter and virus replication. Viruses with a defective tat do not proliferate nor show cytopathic effects. The tat protein stimulates the rates of provirus transcription and elongation, and plays an unique role in emergence of the virus from the latent state. For example, tat interacts with nascent

RNA sequence synthesized by the viral DNA, known as the trans-activator response {tar) element extending from -17 to + 42 bp, to activate the viral promoter (Dayton, et al. Cell 44, 941-947 (1986); Rosen, C.A., et al., Cell 47:813-823 (1985); Fisher, A.G., et al., Nature 320:361-371 (1986); Sim, I.S., et al., Acad. Sci. USA 616:64-72 (1990); Jeang, K-T, et al., AIDS 5:S3-S14 (1991)).

The tat protein up regulates HIV-1 promoter by two separate mechanisms. One mode of action is mediated via the far-dependent pathway. However tat up regulates a far-less TNF (tumor necrosis factor) promoter, supporting a tat action independent of the far element (Taylor, J.P., et al., J. Virol. 53:3971-3981 (1994); Buonaguro, L., et al., J. Virol. 55:7159- 7167 (1992)). In this mechanism, it is postulated that the chain of reactions, following virus infection, starts with overexpression of genes encoding cytokines including tumor necrosis factor alpha (TNF-σ). However, the reported clinical attempts at targeting tat have not been successful. For example, 7-chloro-5-(2-pyrryl)- 3H- 1 ,4-benzodiazepin-2( 1 H)-(one) (Ro 5-3335), a faf-inhibitor, was considered a promising compound but animal studies revealed some toxicity. An evaluation of 400 analogues resulted in finding 7-chloro-N-methyl-5-(1 H-pyrrol-2-yl)-3H- 1 ,4- benzodiaezepin-2 amine (Ro 24-7429) and in the clinical testing of Ro 24-7429. While that drug was clinically tested, the clinical tests were stopped because the drug was not considered clinically effective.

We discovered that the use of combinations of drugs that target tat function and also NF-κB function can result in obtaining a synergistic result. See U.S.S.N. 08/1 59,509. Our work demonstrates that tat induces overexpression of genes encoding cytokines (Biswas, D.K., et al., J. Virol. In press ( 1 995); Buonaguro, L., et al., J. Virol. 66:supra; Rautonen, J., et al., AIDS Res. Hum. Retro Viruses 70:781 -785 ( 1 994); Puri, R.K., et al., Cancer Res. 52:3787-3790 ( 1 992); Biswas, D.K., et al., Proc. Natl. Acad. Sci. USA 50: 1 1044-1 1048 ( 1 993)) and this can be observed in the absence of any externally administered activators of NF-κB. Popik and Pitha (Popik, W., et al., J. Virol. 57: 1094- 1099 ( 1 993) reported that HIV- 1 provirus with defective tat can replicate in the presence of TNF, suggesting that at least in part tat action could be mediated via this cytokine.

Mutational studies indicate that the TNF-σ- induced upregulation of HIV-1 promoter is mediated via NF-κB activation (Nabel, G.J., et al., Nature 325:71 1 -71 3 ( 1 987); Vlach, J., et al., Virology 187:63-72 ( 1 992)). This is substantiated by blocking extracellular TNF-σ antibody, which decreases active NF-κB and inhibits HIV- 1 promoter activity (Biswas, D.K. et al., J. Virol, supra). NF-κB in turn activates genes encoding cytokines. Thus, over-production of cytokines via the far- independent tat pathway establishes an up regulatory loop (Biswas, D.K. et al., J. Virol, supra)..

The faf-induced super-activation of HIV- 1 promoter is apparently dependent on both over production of cytokines and on its far-mediated action. Synergistic interaction between these two pathways induces super-activation of HIV-1 promoter (Biswas, D.K., et al., Proc. Natl. Acad. Sci. USA 30: 1 1 044- 1 1048 ( 1 993) . In U.S.S.N. 08/1 59,509 we showed that combined inhibitors of specific steps of these two pathways of tat action influence HIV-1 transcription synergistically and should also influence virus replication.

We disclosed a number of classes of drugs that can be used, specifically exemplifying the use of certain groups. However, it is important in treating a virus that has the ability to mutate as rapidly as HIV to be able to find even more effective combinations that can be used.

Thus, it would be desirable to have new compounds or a combination of compounds that more effectively inhibit expression or replication of the HIV provirus in HIV infected cells and chronic infections. It would also be desirable to be able to effectively administer combinations of drugs at lower cumulative doses than currently possible. It would be particularly desirable to have a new therapy that can be used to treat already infected cells by means of inhibiting expression of provirus, or a means to keep the provirus dormant within infected cells.

SUMMARY OF THE INVENTION

We have discovered that combinations of drugs that inhibit tat function, i.e. tat inhibitors, and drugs that inhibit NF- B function wherein the latter drug is a diterpene such as a cembrane-type diterpene of the formula

wherein R R₄ ^are selected from the group consisting of: hydrogen, lower straight-chain or branched alkyls (C, - C₆), lower alkoxy groups (OR, where R is C, - C₆), lower alkenyls (C₂ - C₆), halogens (F, Cl, Br and I), carboxylic acids (RCOOH, where R is hydrogen or lower alkyls (C, - C₆), and amino groups (NRR' where R and R' are hydrogen or lower alkyls (C, - C₆₎₎.

It is preferred that either R, and R₂ or R₃ and R₄ is identical. It is most preferred that R,-R₄ are identical, and more preferable that all are CH₃.

These compounds include sarcophytol A or its derivatives such as 3, 7, 1 1 -trimethylcyclodeca 3E, 7E, 1 1 E-triene-1 -l and 2, 8, 1 2-trimethyldeca- 1 , 5Z, 7E, 1 1 -tetraene-4-ol or analogs of the formula

where R,-R₄ ^are defined as above, X is a substituted or unsubstituted alkylene of 1 to about 10 carbon atoms, or a substituted or unsubstituted alkyenylene of 2 to about 10 carbon atoms, or a substituted or unsubstituted alkyenylene of 2 to about 10 carbon atoms, n is 1 or 2 , Y is a substituted methylene (CH₂), N, O, or S; more preferably X has about 3-6 carbon atoms. Substituents include N, O, S and halogens. Preferably the compound has a cyclohexene such as

OH

For example, 2-isopropyl-4-isopropylidenecyclohex2-ene-1 -ol (Canventol).

A preferred Sarcophytol A derivative is [(dl)-trans-2- isopropyl-4-phenyleyleonex-2-enc-1 -01 ]

The use of such compounds with a tat inhibitor work together in a synergistic manner. For example, the combined use of faf inhibitors such as benzodiazepine compounds with the above-described class of NF-/cB inhibiting compounds inhibits expression and replication of the HIV provirus. The diterpene such as those having a cembrane-type structure and derivatives are effective inhibitors of TNF-σ. Analogs wherein the cembrane ring has been replaced by a simpler structure (X-Y) such as cyclohexene (e.g. canventol) appear to work on multiple targets (e.g. inhibiting protein isoprenylation as well as inhibiting TNF- a) . One preferred combination includes Ro 24-7429 and Canventol.

In one embodiment, the combination of compounds can treat cells infected by immunodeficiency viruses, for example,

HIV, preferably HIV-1 , and thus can be used to treat humans infected by HIV. For example, treatment of those diagnosed as having AIDS as well as those having ARC, PGL and those not yet exhibiting such conditions. These combinations of compounds can be used against a different target than the conventional drugs being used to treat humans infected by HIV, e.g., reverse transcriptase inhibitors such as zidovudine (AZT), 2', 3'-dideoxyinosine (ddl) and 2',3'- dideoxycytidine (ddC). Furthermore, using this combination yields a synergistic result. Thus, lower dosages may be used.

Similarly, the present compounds should be effective in cells that are resistant to reverse transcription inhibitors.

The invention also provides pharmaceutical compositions comprising a combination of the compounds and a suitable carrier therefor for use in the conditions referred to above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and B show the influence of a compound coming within this invention, canventol, on HIV-1 , replication.

Figure 1 A. 2X10⁶ Jurkat cells were plated in 2 ml medium in each well of a 6 well plate and infected with HIV-1 strain MN. Cell growth and infection conditions were as described in Examples. Virus replication in control and treated cells was measured by the level of virus antigen protein p24 in the supernatant of cells infected and treated with the drug for 7 days. Cells were refed with fresh medium and drugs after 3 days of growth in the same medium. Viability of infected cells was measured by MTT procedure. No effect on cell viability was observed in infected Jurkat cells treated with indicated concentrations of Canventol.

Figure 1 B. A laboratory strain of chronically infected U 1 cells were treated with indicated concentrations of canventol or Ro24-7429 or with two drugs in combinations for 2 hours followed by activation of the virus from the latent state with TNF-σ (0.1 ng/ml) . After 3 days of growth under these conditions p24 levels in the supernatant were measured as described above.

Jurkat cells were transiently transfected with the indicated expression plasmid constructs using the DEAE dextran procedure. Cell extracts were prepared seventy two hours after transfection, and CAT activity in 10 μg of protein was measured. The numbers under each panel indicate the μg of input plasmids used to transfect the cells.

Figures 2 A-D show the multiple pathways of tat action.

Figure 2A. Cells were transfected with fusion gene construct of HIV-1 LTR carrying wild type (wt) or mutated (mut) NF-KB motifs and wild type far element fused to the reporter gene chloramphenicol acetyl transf erase (CAT). Figure 2B. Cells were transfected with the mutated plasmid construct, and also with the indicated amounts of the Taf-expressing plasmid pSV-7af.

Figure 2C. Cells were transfected with a fusion gene construct of HIV-1 LTR lacking the far element (Δ tar) cotransfected with 7af-expressing plasmid psV- 7af.

Figure 2D. Cells were cotransfected with a fusion gene construct with wild type LTR and with pSV- Tat.

Figures 3A-D show the influence of canventol on superactivation, faf-induced, far-independent and far- dependent activation of HIV-1 LTR.

Jurkat cells were transfected with indicated amounts of the specific fusion gene constructs as described about with respect to Figure 2.

Figure 3A shows the influence of Canventol on 7af- induced superactivation of HIV-1 LTR. Cells were transfected with pHIV-LTR-CAT (5 μg) and pSV- Tat (5 μg) . As NF-κB- sites and the tar element in the LTR sequence are wild type in this fusion gene construct both NF- B- and 7af-induced activation pathways of HIV- 1 promoter were operational.

Figure 3B shows the influence of different concentrations of Canventol on superactivation of HIV-1 promoter in the presence of indicated concentration of TNF. Canventol treatment was for forty eight hours and TNF treatment was for the last 18 hours of the transfection period. Figure 3C shows the influence of Canventol on the far- independent mode of action of Tat. Cells were transfected with 10 μg of the fusion gene construct with HIV-1 LTR lacking the tar element (pHIV-1 LTR-Δ-CAT) cotransfected with the indicated amounts of pSV-Taf. Twenty four hours later

Canventol was added at the indicated concentrations to the transfected cells and remained for forty eight hours. TNF-σ (10ng/ml) treatment was for the last 18 hours of the transfection period. Cell extracts were prepared seventy two hours after transfection.

Figure 3D shows the effect of Canventol on far-dependent action of 7af. Cells were transfected with 10 μg of pHIV-1 LTR- NF-mut-CAT and indicated amounts of pSV- Tat. NF-κB- mediated trans-activation of HIV-1 promoter is nonfunctional in this assay because of mutations in the two interaction sites in the LTR. Canventol and Ro24-7429 treatments, at the indicated concentrations, preparation of cell extracts, measurement and quantitation of CAT activity were as described above.

Figure 4 shows the influence of canventol on faf-induced activation of NF-AB.

Jurkat cells were transfected with 1 0 μg of pSV- Tat. Twenty four hours later cells were treated with Canventol at the indicated concentrations and continued for forty eight hours. TNF-treatment at the indicated concentration was for the last eighteen hours of the transfection period. Seventy two hours after transfection nuclear extracts were prepared. 10 μg of nuclear extract proteins were then incubated with ³²P-labeled synthetic oligonucleotide carrying the NF-/cB motif²¹ and subjected to 6% poiyacrylamide gel electrophoresis under non denaturing conditions.

Lane 1 shows the basal level of active nuclear NF-κB (indicated by the arrow).

Lane 2 shows NF- fB in Jurkat cells transfected with 10 μg pSV-7af.

Lanes 3-4, 5-6 and 7-8 show NF-κB in nuclear extracts of cells treated with 10,50 and 100 /vM Canventol respectively.

Lane 9 shows the influence of TNF-σ (20 ng/ml) on Tat expressing Jurkat cells.

Lane 10 shows the influence of TNF-σ (20 ng/ml) on Canventol inhibition.

Figures 5A-D show the effect of a combination of an NF- zrB inhibitor, canventol, and a faf-inhibitor, Ro 24-7429 on faf-induced superactivation of the HIV-1 promoter.

Jurkat cells were transfected with 5 μg of pHIV- 1 LTR- CAT and pSV-7af. Transfected cells were then treated with Ro24-7429 and Canventol in combination (Figure 5A) or with

Canventol alone (Figure 5B) or Ro24-7429 alone (Figure 5C) at the indicated concentrations. Seventy two hours after transfection and after forty eight hours of drug treatment, CAT activity in the cell extracts were measured and quantitated. Figure 5D shows cell viability. DETAILED DESCRIPTION OF THE INVENTION

We have now discovered certain combinations of compounds that can both inhibit different aspects of LTR mediated gene expression work together in synergistic manners. The effect of LTRs on gene expression in a wide range of immunodeficiency viruses is well-known. For example, in the primate lentiviruses, such as SIV, HIV-1 , and HIV-2. This is also true with respect to other immunodeficiency virus, such as feline immunodeficiency virus (FIV), among others. While the primate lentiviruses have varying degrees of sequence homology, they share a strong degree of organizational and functional homology. For example, regulation of immunodeficiency type virus type- 1 (HIV-1 ) and type 2 (HIV-2) gene expression is mediated via interplay of multiple cellular and viral positive (frar?s-activator) and negative (repressor) factors interacting within small segments of DNA with the long terminal repeat (LTR) sequence (Lu, Y., et al., Genetic Structure and Regulation of HIV, Raven Press, NY:41 5-435 (1991 ); Gaynor, P., et al., Genetic Structure and Regulation of HIV, Raven Press, NY: 107-1 34 (1991 )). These DNA/protein interactions confer specialized gene regulatory properties to the c/s-acting elements in the LTR sequence which are essential to effective viral replication. For example, deletions and mutations of specific sites can decrease LTR-driven activation of genes.

Mapping of the LTR by in vitro transcription competition and linker-scanning mutagenesis suggests that specific sequences in the U3-R region (-453 to + 80) are necessary for gene regulation (Zeichner, S.L., et al., J. Virol. 55:2436-2444 (1991 )) . For example, the tat protein plays a major role in frar/s-activating (stimulating) viral gene expression by interaction with the tar element which is present in this region. Similarly, the nuclear factor NF-/cB can also stimulate viral gene expression through its interaction with sequences present in the LTR (Sen, R., et al., Cell 47:921 -928

( 1 986); Cullen, B.R., et al., Microbiol. Rev. 55:375-394 (1 992)). For example, mutations in the NF-/cB response element reduce HIV- 1 LTR driven gene expression (Nabel, G.J., et al., Nature 325:71 1 -71 3 ( 1 987); Nabel, G.J., et al., Science 233: 1299- 1 302 (1 988); Biswas, D.K., et al., J. Acquir. Immun. Defic.

Syndr. 5:7778-786 ( 1 993)) but do not prevent viral replication (Leonard, J., et al., J. Virol. 53:491 9-4924 ( 1 989)) . Deletions of this site, together with mutations in the tar element, do render viral replication incompetent. This suggests that concerted action of these two frarjs-activators plays a role in viral gene regulation and viral replication (Leonard, J . , et al., J. Virol. 63, supra; Liu, J., et al., J. Virol. 55:3883-3887 ( 1 992); Kammine, J., et al., J. Virol. 55:3932-3936 (1992); Kadonaga, J.T., et al., Science 242: 1 566- 1 570 (1 988); Doppler, C , et al., AIDS Res. Hum. Retro. Viruses 3:245-252 ( 1 992)).

This has lead to various attempts at looking for agents that can affect either of these functions, i.e. tat inhibitors or NF¬ KB inhibitors. However, to date, the clinical results reported have not been encouraging.

Canventol (2-isopropyl-4-isopropylidenecyclohex-2-ene- 1 - ol) qualifies as an anti-NF- B agent because it inhibits the release of cytokines (Atsumasa, K., et al., Cancer Res. 53:3462-3464 (1993) and thereby NF- fB activation. The site of action of Canventol is different from that of other NF-κB inhibitors, such as PKC inhibitors, PTX and Go 6976 (Qatsha, K.A., et al., Proc. Natl. Acad. Sci. USA 50:4674-4678 ( 1 993). The net result of treating cells with any of these compounds is to decrease active NF-xB, thereby stalling the faf-induced upregulatory loop. The inhibitory influence of Canventol on HIV-1 replication is thus due to the inhibition of release and reduction of the level of TNF-σ available for its downstream upregulatory functions, a mechanism similar to anti-TNF-σ antibody. We have found that canventol, even when used individually, provides surprisingly effective results in cells infected with HIV.

We have further found that when using these drugs (even where clinically ineffective, individually) in combination, one can obtain a synergistic effect. As used herein, the term synergistic means that the effect of the two drugs when used together is greater than their additive effect would be based upon their use alone.

Where the NF- fB inhibitor is selected from the group wherein of diterpene such disclosed below, the combination is particularly effective. Preferably, it is as a cembrane-type diterpene of the formula

wherein R,-R₄ are selected from the group consisting of: hydrogen, lower straight-chain or branched alkyls (C, - C₆), lower alkoxy groups (OR, where R is C, - C₆), lower alkenyls (C₂ - C₆), halogens (F, Cl, Br and I), carboxylic acids (RCOOH, where R is hydrogen or lower alkyls (C, - C₆), and amino groups (NRR' where R and R' are hydrogen or lower alkyls (C, - C₆)) .

It is preferred that either R, and R₂ or R₃ and R₄ is identical. It is most preferred that R,-R₄ are identical, and more preferable that all are CH₃.

These compounds include sarcophytol A or its derivatives such as 3, 7, 1 1 -trimethylcyclodeca 3E, 7E, 1 1 E-triene-1 -l and 2, 8, 1 2-trimethyldeca-1 , 5Z, 7E, 1 1 -tetraene-4-ol or analogs. A particularly preferred compound is [(dl)-trans-2-isopropyl-r- phyenylcyclohex-2-ene-1 -0l] (sometimes referred to as 59a) . Also included are analogs of the formula

OH

where R,-R₄ are defined as above, X is a substituted or unsubstituted alkylene of 1 to about 10 carbon atoms, or a substituted or unsubstituted alkyenylene of 2 to about 10 carbon atoms, or a substituted or unsubstituted alkyenylene of 2 to about 10 carbon atoms, n is 1 or 2 , Y is a substituted methylene (CH₂), N, O, or S; more preferably X has about 3-6 carbon atoms. Substituents include N, O, S and halogens.

Preferably the compound has a cyclohexene such as

For example, 2-isopropyl-4-iospropylidenecyclohex2-ene-1 -ol (Canventol) . For example, cembrane-type diterpenes such as

Sarcophytol A [Fujiki, et al., J. Cancer Res. Clin. Oncol. , 1 1 5:25-28 ( 1 989)], its derivatives such as 3, 7, 1 1 - trimethylcyclodeca-3E, 7E, 1 1 E-triene-1 -ol and 2, 8, 1 2- trimethyldeca- 1 , 5Z, 7E, 1 1 -Tetraene-4-ol [Fujiki, H., et al., in Phenolic Compounds in Food and Their Effects on Health II. T.

Huang, et al., Eds, pp. 380-387 Washington, DC, American Chemical Society ( 1 992)], and analogs as described above wherein the cembrane ring has been replaced by a X-Y such as a cycloalkene, preferably a cyclohexene such as 2-isopropyl-4- iospropylidenecyclohex-2-ene-1 -ol (canventol) [Komori, et al.,

Cancer Res. 53:3462-3464 ( 1993)] are typically surprisingly more effective.

This means that one can obtain a more potent effect using these combinations than using either drug alone. It also means that one can use lower dose levels of these drugs together than one could cumulatively. This is significant as many drugs have been shown to have deleterious effects at high levels.

The tat inhibitors include those compounds that seek to affect the interaction of a tat protein with the tar element. For example, attempts have been made to use oligonucleotides targeted for inhibition of tatltar interaction and thereby, HIV-1 LTR-driven gene expression [Vickert, et al., Nuc. Acids. Res. 75:3359-3368 (1991 )]. More preferably, one would use anti-faf drugs. One series of anti-faf drugs have been described by Hsu, et al. These compounds include aryl-(2-pyrryl) ketone compounds such as 2-glycinamido-5-chlorophenyl (2-pyrryl) ketone and compounds such as benzodiazepine compounds, preferably bezodiazepines, such as 7-chloro-5-(2-pyrryl)-3H- 1 ,4- benzodiazepin-2(1 H)-one (Ro 5-3335) and 7-chloro-N-methyl-5- (1 H-pyrrol-2-yl)-3H-1 ,4-benzodiazepin-2-amine.

The synthesis of these compounds is disclosed in U.S. Patent Nos. 5,041 ,438; 5,036, 1 01 ; 3,405, 1 22; 3,398, 1 59;

3,407,21 1 ; and 3,400, 1 28, all which are hereby incorporated by reference. Ro 24-7429 was synthesized from Ro 5-3335 by reaction with methylamine in the presence of titanium tetrachloride.

Other groupings of compounds that can be used in place of, or in addition to, the tat inhibitors disclosed above include those described in PCT/US93/07878; PCT/US93/07879; and PCT/US93/07934 of which three drugs were reported by Li, et al. (Li, C.J., et al., Proc. Natl. Acad. Sci. USA 50: 1 839- 1 842) to inhibit LTR function and viral replication. The synthesis of these compounds are described in the above references, all of which are hereby incorporated by reference. Specifically preferred compounds include topotecan, Mapachone, and allyl-3- lapachone, particularly, 3-allyl- ?-lapachone and curcumin.

In cells of non-B-lineage, cytoplasmic NF-/ B is detected as a complex with the inhibitory protein, \κB. Thus, phosphorylation of \κB releases NF- B, which is then translocated into the nucleus, where it interacts with its response element and stimulates HIV-1 LTR-regulated gene expression. Thus, inhibitors of NF-κB may either block the cytoplasmic activation or interfere with the interaction of NF-KB with response elements in the LTR sequence.

These diterpenes such as cembrane-type diterpenes, derivatives and analogs are effective inhibitors of the release of TNF-σ. Preferred compounds include natural compounds such as Sarcophytol A, isolated from the soft coral, Sarcophyton glaucum, derivatives such as 3, 7, 1 1 -trimethylcyclodeca-3E,

7E, 1 1 E-triene- 1 -ol, 2, 8, 1 2-trimethyldeca 1 , 5Z, 7E, 1 1 - tetraene-4-ol and analogs of the formula

OH

wherein R,-R₄, X, Y and n are as defined above. Preferably X-Y form a cyclohexane such as canventol. For example, a compound such as Sarcophytol A is a more potent TNF-σ inhibitor than canventol. However, analogs where the cembrane ring have been replaced with an alkylene can mimic other compounds and display secondary effects. For example, using a cyclohexene such as in canventol can also result in inhibition of protein isoprenylation. Compounds exhibiting such a dual mode of action are preferred in certain embodiments. Compounds such as canventol exhibiting a dual mode of action can in certain embodiments be used individually.

For example, canventol and derivatives are novel specific inhibitor of far-independent LTR activation by tat. This mechanism in part involves TNF-σ and NF-/cB trans-activation. These compounds inhibit tat mediated activation of HIV- 1 promoter via the far-independent pathway and influence viral - 24 - gene expression due to inhibition of TNF-σ release, restricting down-stream events that upregulate HIV- 1 promoter. These derivatives can be made by techniques known in the art. For example, canventol can be synthesized by the method described by [Komori, et al., in Cancer Research 53: 3462-3464 (Aug. 1 ,

1 993)), incorporated herein by reference] .

The combination of compounds described provide more effective therapy of chronically infected cells as evidenced by a reduction in, and preferably a complete repression of, HIV LTR directed gene expression. Thus, in an HIV infected cell, addition of an effective amount of a compound of the combination will reduce the expression of a gene operably linked to the HIV LTR by use of lower amounts of the two drugs together than can be achieved by either drug alone. Preferably, the gene is operably linked to an HIV- 1 LTR. As used herein, the term operably linked means that the gene is under the control of the HIV LTR and positioned in a nucleotide sequence in a way to accomplish this. Typically, the gene is downstream of the LTR, which acts as a promoter. Preferably, the gene corresponds to a viral gene such as the HIV env gene, HIV rev gene, etc.

Hence, in one preferred embodiment, the present invention can be used in treating those diagnosed as having AIDs as well as those having ARC, PGL, and those seropositive but asymptomatic patient. For example, for high risk individuals it can also be used prophylactically as a preventative. Thus, a method of protection comprises administering an effective amount of the combination by means as set forth below. The dual action derivatives such as canventol and the combinations disclosed herein can be used to treat cells, especially mammalian cells and in particular human cells, infected by an immunodeficiency virus such as HIV infected cells. Preferably, combinations are used. As a result of treatment with the combination, viral expression is significantly reduced. Furthermore, one can use lower dosages. Thus, side effects seen with high levels of administration can be avoided. Though a compound such as canventol has a relatively low toxicity to cells (Komori, Can. Res. 53:3462 (Aug. 1 , 1 993)), it is preferable to use compounds in as low a dose range as practical and compounds such as Ro 5-3335 demonstrated unacceptable toxicity when used alone in large doses. However, used in lower dosages in combination with, for example, Canventol, such drugs may be acceptable. Further, compounds such as Ro 24-7429, which by itself was not considered clinically effective, can be used successfully in combination.

For example, viral expression of HIV can be studied by a number of methods such as looking at the expression of a reporter gene, e.g., CAT lacZ, etc., operably linked to the HIV LTR, which acts as the promoter. Use of the present combination of compounds, Canventol and Ro 24-7429, can significantly reduce expression as demonstrated by looking at expression of such reporters. HIV-1 viral expression can be "turned on" and enhanced by HIV LTR stimulators such as tumor necrosis factor-σ (TNF-σ) or phorbol-1 2-myristate-1 3- acetate (PMA). The use of faf, can further augment HIV-1 gene expression. Using a reporter gene such as chloramphenicol - 26 - acetyl transf erase (CA T) operably linked to the HIV LTR in HIV infected cells, one can monitor that HIV expression under the control of the HIV LTR such as HIV envelope glycoprotein expression. The results show that the addition of an effective amount of the presently described combination of compounds significantly inhibits expression of the gene, and that viral protein expression would be inhibited if not completely stopped.

P²⁴, a major structural protein (product of gag), has been widely used for monitoring HIV-1 replication in cells and viraemia in individuals. Use of the present combination of compounds, at concentrations that do not significantly adversely affect cells, can dramatically reduce HIV- 1 replication, as determined by P²⁴ levels. For example, use of diterpenes such as canventol alone reduces P²⁴ levels by more than 20%, more preferably more than 40%, and most preferably by more than 50% . Therefore, in combination with a faf-inhibitor, a reduction of preferably more than 25% as determined by P²⁴ levels should occur, more preferably a reduction of more than 50%, and still more preferably a reduction of HIV- 1 replication of more than

60% as determined by P²⁴ levels, and even more preferably a reduction of 75% or more.

The effective amount used to obtain such a result will be at lower levels than those cumulatively used, preferably at micromolar and even more preferably at nanomolar concentrations. For example, it is believed that one can reduce the cumulative dosage level of constituents by at least 20%, more preferably at least 25%, even more preferably at least 50%, still more preferably by at least 70%, and most preferably at least 90% . Furthermore, the administration of the combination of compounds of the present invention at effective concentrations, which inhibit, for example, HIV expression, do not adversely affect the cell.

Further, an alternative mechanism of action of derivatives such as Canventol on virus replication activity in addition to its inhibitory influence of NF- B activation is virus replication inhibition. The later possibility may explain the results that the combined effect of the faf-inhibitor, Ro24-7429 and Canventol on virus replication in chronically infected cells is at the most additive although these two drugs in combination showed a synergistic effect on faf/NF-κB-induced superactivation of HIV- 1 promoter.

The compounds of the present invention can be administered to HIV infected individuals or to individuals at high risk for HIV infection. For example, those having sexual relations with an HIV infected partner, intravenous drug users, etc. Because of its inhibitory effect, the combination of compounds of the present invention can be used prophylactically as a method of prevention for such individuals to minimize their risk. One would administer an effective amount of the combination as set forth below by the methodology described herein.

As demonstrated in the Examples which follow, the combinations of compounds described block activation or suppress activity of HIV-1 LTR at a synergistic level when compared to either component by itself, and, thus, expression of genes under its control in both chronically and acutely infected cells. In particular, it has been found that combinations in a dose dependent fashion inhibit HIV LTR directed TNF-σ stimulated gene expression. Moreover, such inhibition is i provided with essentially no adverse affects on cell survival.

Thus, it is believed that the combinations will have utility in inhibiting the progression of an HIV infection and other retroviral infections in cells and in a human, including utility in extending the latency of an HIV infection in a human.

In general, for the treatment of immunodeficiency infections, for example an HIV or FIV infection, more preferably, an HIV infection, a suitable effective dose of one or more compounds of the combination will be in the range of 0.01 to

100,000 μg per kilogram body weight of recipient per day, preferably in the range of 0.1 to 1 ,000 /^yg, still more preferably in the range of 0.5 μg to 500 μg per kilogram body weight per day. The desired dose is suitably administered once or several more sub-doses administered at appropriate intervals throughout the day, or other appropriate schedule. These sub-doses may be administered as unit dosage forms, for example, containing 0.01 to 100 μg, preferably 0.5 to 100 μg. These dosages will be lower than that of either compound by itself. For example, if canventol was being used by itself the dose range would increase by more than 20% over the cumulative dose, typically about 50% or more than the cumulative dose. Preferably, the combined amount will be less than 400 mg thrice daily, still more preferably, less than about 350 mg, even more preferably less than about 250 mg, and most preferably less than about 200 mg.

Administration of the compounds of the invention may be by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral

(including subcutaneous, intramuscular, intravenous and intradermal) with oral or parenteral being preferred. It will be appreciated that the preferred route may vary with, for example, the condition and age of the recipient.

The administered ingredients may be used in therapy in conjunction with other medicaments such as reverse transcriptase inhibitors such as dideoxynucleosides, e.g. , zidovudine (AZT), 2', 3'-dideoxyinosine (ddl) and 2', 3'- dideoxycytidine (ddC), protease inhibitors and other agents such as 9-(2-hydroxyethoxymethyl)guanine (acyclovir), interferon, e.g., σ-interferon, interleukin II, and phosphonoformate (Foscarnet) or in conjunction with other immune modulation agents including bone marrow or lymphocyte transplants or other medications such as levamisol or thymosin which would increase lymphocyte numbers and/or function as is appropriate. Because many of these drugs are directed to different targets, e.g., reverse transcription, it is anticipated that further advantages will be obtained by this combination.

Similarly, the present compounds may be effective when the above-described drugs are not or are no longer effective. For example, the combination of compounds of the present invention can be used in cells that are resistant to reverse transcriptase inhibitors such as AZT, ddl and ddC. For instance, the combination of compounds, can be used to block HIV- 1 LTR directed expression in an AZT resistant strain of HIV-1 . Accordingly, the present invention can be used therapeutically in an individual as that individual develops resistance to drugs that act on different targets such as AZT, ddl, ddC, etc. It is expected that the present invention can be used for treatment of HIV-1 infected individuals who develop resistance to any drug that targets a different state in the viral life cycle than the present compounds.

While the combination may be administered alone, it also may be present as part of a pharmaceutical composition. The compositions of the invention comprise at least one combination of compounds together with one or more acceptable carriers, e.g., liposomes, and optionally other therapeutic ingredients, including those therapeutic agents discussed supra. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes and may be prepared by any methods well known in the art of pharmacy.

Such methods include the step of bringing into association the ingredients to be administered with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then, if necessary, shaping the product.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; packed in liposomes; or as a bolus, etc.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing water. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

Compositions suitable for topical administration include lozenges comprising the ingredients in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier.

Compositions suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes using a pharmaceutically acceptable carrier. A suitable topical delivery system is a transdermal patch containing the ingredient to be administered.

Compositions suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Compositions suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. , by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; 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 containers, for example, sealed ampules 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 and tables of the kind previously described.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.

All documents mentioned herein are incorporated by reference.

The present invention is further illustrated by the following Examples. These Examples are provided to aid in the understanding of the invention and are not to be construed as limitations thereof.

GENERAL METHODS

The following reagents and procedures were employed as specified in the examples. Cells: Jurkat cells are CD4 + T lymphocytes obtained from the American Type Culture Collection (ATCC) and are maintained as suspension cultures in RPMI 1 640 (GIBCO/BRL, Gaithersberg, MD) supplemented with 10% fetal bovine serum at 37° C in an atmosphere of 5% CO₂ and 95 % air.

Reagents: Canventol and Ro24-7429 are gifts from Mitsubishi Kasei Corporation, Tokyo, Japan, and Hoffman-La Roche, Nutley, NJ respectively. Tumor necrosis factor alpha (TNF-σ) and anti human-TNF-σ antibody are from Endogen, Cambridge,

MA. DEAE-dextran, acetyl CoA, and 4-B-phorbol-1 2-B-myristate acetate (PMA) are from Sigma Chemical Co. (St. Louis, MO) . '⁴C-Chloramphenicol (CAP) is from DuPont/NEN. The complementary strands of the oligonucleotide (5'- TCGACAGGGACTTTCCGAGAG-3') containing the NF-*B motif

(boldfaced) were custom synthesized in the core facilities of Dana-Farber Cancer Institute.

Canventol can be prepared as follows: Chemicals. Canventol is synthesized as a monocyclic conformationally restricted analogue of sarcophytol A. Its synthesis is summarized as follows:

2-isopropylcyclohexenone is prepared from o- methoxybenzoic acid according to Taber's procedure, J. Org.

Chem. 41 :2649-2650 ( 1 976). Formation of the thermodynamic enolate and trapping with trimethylchlorosilane produced the trimethylsilyl enol ether which is exposed to 2,2- dimethoxypropane and titanium tetrachloride in dichloromethane at -78°C. The enone is isolated in 49% overall yield Elimination of methanol from the enone takes place at 35-40°C in 2,2,2- trifluoroethanol in the presence of perchloric acid to produce a dienone in 51 % yield. Carbonyl reduction with sodium borohydride and cerous chloride in methanol at 25 °C (Gemal, et al., J. Am. Chem. Soc. 1 03, 5454-5459 (1981 )) produces a

96% yield of crystalline (d, l)-canventol (melting point), 45- 47°C) . Canventol to be used for the experiments is 99.9% pure.

Plasmids: The expression plasmids pHIV- 1 LTR-CAT and pHIV-

1 LTR-mut-NF-/ B-CAT¹⁴ are gifts from Dr. G. Nabel of Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Ml pSV-faf²⁰ and pHIV-LTR-Δfar-CAT² are gifts from Dr. J. Sodroski of Dana-Farber Cancer Institute.

Transfections: Transient transfections of Jurkat cells with the specified fusion plasmids was performed by the DEAE/dextran procedure as described in Biswas, D.K., et al., Proc. Natl. Acad. Sci. USA 90, supra) . CAT activities was measured and quantitated by scanning the TLC paper with a Betascope 603 blot analyzer (Betagen, Waltham, MA) . CAT activity was expressed as percentage [¹⁴C]-chloramphenicol (CAP) derivatives produced per hour. The procedures for nuclear and whole cell extract preparations, determination of protein content, CAT activity and ?-galactosidase activity were as described previously (Biswas, D.K., et al., Proc. Natl. Acad. Sci. USA 90, supra; Biswas, D.K, et al., J. Acquir. Immune Defic. Syndr. 5:778-786 (1 993); Biswas, D.K., et al., Mol. Med. 7, 31 -34 (1993)). The transfection efficiency in these assays was determined by cotransfection with psVlacZ (Molnar, G., et al. , Mol. Cell. Biol. 74:5242-5248 (1 994)) plasmid and measuring the level of SV40 promoter-driven 3-galactosidase activity in cell extracts. As SV40 promoter was not affected by any of these external modulators of HIV- 1 LTR the reporter CAT activity in control and treated cell extracts were normalized in relation to the level of SV40 promoter-driven /?-galactosidase activity in the same extracts.

Electrophoretic Mobility Shift Assay (EMSA): Nuclear extracts from control and treated cells were prepared as described by

Dignam, et al. (Dignam, J.D., et al., Nucleic Acids Research 7 7 : 1475-1489 (1 983)) . Reaction conditions with ³²P-labeled double stranded oligonucleotide carrying the NF-κB motif and electrophoretic mobility shift assay (EMSA), quantitation of CAT activity, yff-galactosidase activity and autoradiographic signals of

EMSA were as described. (Biswas, D.K., et al., Proc. Natl. Acad. Sci. USA 90, supra; Biswas, D.K, et al., J. Acquir. Immune Defic. Syndr. 6:supra; Biswas, D.K., et al. , Mol. Med. 7, supra.

Virus replication in Jurkat Cells by Acute Infection with HIV-1 _MN: Jurkat cells were plated in 6 well cloning plates with 2 ml RPMI 1640 supplemented with 10% fetal bovine serum and incubated at 37° in an atmosphere of 5% CO₂ and 95% air. Cells were pretreated with indicated concentrations of Canventol for two hours prior to infection with HIV-1 _MN strain at MOI of 0.1 . After a virus infection period of 1 hour, the cells were washed three times with PBS and replated in the growth medium in the presence of the indicated concentrations of the compound. After 7 days of HIV-1 infection and drug treatment, the level of p24 virus specific antigen was measured in the culture medium of untreated and treated cells by ELISA with an HIV- 1 p24 antigen kinetics assay kit (Abbott) as described. (Marshall, W.L., et al., J. Virol. 55:5492-5499 ( 1 992)). The influence of the compound on viability of infected cells is measured by MTT assay (Mosman, T., et al., J. Immunol. Methods 55:55-64 ( 1 983)). See Fig. 1 A.

The influence of Canventol on virus replication in chronically infected U1 cells was examined by pretreating the cells with the indicated concentrations of the drug for 2 hours followed by activating the cells with either LPS or TNF-σ (0.1 ng/ml) and growth of the cells for 3 days and measurement of p24 level in the supernatant as described above. See Fig. 1 B.

Canventol Inhibits HIV-1 Replication:

Canventol inhibited HIV-1 replication in acutely and chronically infected cells pretreated with the compound prior to infection with HIV-1 . The inhibition of virus replication by Canventol as judged by the level of viral antigen p24 in infected calls, was observed at concentrations as low as 1 nM and was maximal at 50-60% after seven days in acutely infected Jurkat cells and after three days in TNF-σ or LPS-activated chronically infected U 1 cells. These results were repeated in five separate experiments. Simultaneous treatment of the cells with virus and

Canventol was not as effective as pretreatment of the cells prior to infection. A comparatively slightly higher concentration of Canventol was required to obtain a similar degree of inhibition in chronically infected U1 cells. This may be due to the higher concentration of the drug necessary to overcome the effect of agents such as TNF-σ or LPS initially used to activate the virus from the latent state in chronically infected cells.

Mechanism of Anti-HIV-1 Action of Canventol: Two pathways of 7af-Action. Investigation of the basis for action of Canventol on virus replication depended upon separating various steps in the action of tat on HIV- 1 LTR. The level of transactivation in the absence of tat and in the absence of any externally added NF-κB modulators was determined by transient transfection of Jurkat cells with a pHIV- 1 LTR-CAT¹⁴ fusion plasmid and measuring reporter CAT activity (Fig.2) . This activity depends upon the basal level of active NF-κB because it was completely eliminated by mutations in the NF-κB motifs (Fig. 2A) . These results also suggest that contributions from other cellular transactivators, such as SP1 , are insignificant in

Jurkat cells under these assay conditions. The far-dependent mode of action of tat in the absence of any contribution from the cellular factor NF-κB was demonstrated by transfection of Jurkat cells with pHIV-1 LTR-mutNF-κB-CAT (Nabel, G.J., et al., Nature 325:71 1 -713 (1 987)) cotransfected with faf-expression vector pSV-faf (Caputo, A., et al., J. Acquir. Immune. Defic. Syndr. 3:372-379 (1990)) m as shown in Figure 2. The second mode of action of faf that is independent of tar was established by cotransfection of pHIV-1 LTR-NF-κB-Δfar-CAT with pSV-faf, and demonstrating reporter gene activity (Figure 2C). The low level transactivation of HIV- 1 promoter (reproduced in three separate experiments) by tat via the individual pathways, was elevated 60-100 fold when both pathways of faf action are operational (Figure 2D). These results demonstrate that NF-KB and tat act in synergy to induce super-activation of the HIV- 1 promoter.

Canventol Inhibits 7^*af/NF-/r-Mediated Superactivation of the HIV- 1 Promoter. See Fig. 3. Super activation of HIV-1 promoter was assayed by transient transfection of Jurkat cells with fusion plasmid pHIV-1 LTR-CAT (Nabel, G.J. , et al., Nature 326, supra)) cotransfected with the tat expression plasmid pSV-tat (Caputo, A., et al., J. Acquir. Immune. Defic. Syndr. 3, supra)) and then measuring the LTR-driven reporter CAT activity in cell extracts (Biswas, D.K., et al., Proc. Natl. Acad. Sci. USA 30: 1 1 044-1 1048 (1993)). In this assay system both modes of action of tat are operative. Canventol blocked this activation in a concentration dependent fashion, by about 60% at 100 μM (Fig. 3A). Because Canventol blocks release of TNF-σ,

(Atsumasa, K., et al., Cancer Res. 53:3462-3464 (1 993)) these results suggest that this inhibition of HIV- 1 promoter activation is due to the reduced level of TNF-σ. This conclusion is substantiated by the result that externally administered TNF-σ overcame this inhibitory influence of Canventol (Fig. l B) .

Canventol Inhibits the far-Independent Pathway of Action of tat. Fig. 3C. To establish the role of cytokines and relevance of the far-independent pathway of action of tat we examined the influence of Canventol upon each pathway. The far- independent upregulation of HIV-1 promoter by tat was assayed by transient transfection of Jurkat cells with a fusion gene construct with HIV-1 LTR lacking the far element (pHIV-1 LTR- Δ-far-CAT²) cotransfected with tat expression plasmid pSV-faf. Canventol inhibited the far-independent activation in a concentration dependent fashion. Again, this inhibitory influence of the drug was overcome by externally administered TNF-σ (Fig. 3C).

Canventol Down Regulates faf-lnduced Activation of NF- B in Jurkat cells. The downstream consequence of Canventol's blocking the release of TNF-σ was investigated by its influence on the level of nuclear active NF-κB as determined by electrophoretic mobility shift assay (Biswas, D.K., et al. , Mol. Med. 1 :31 -43 ( 1 994)) . Nuclear active NF-κB was elevated in the presence of tat expressed from the transfected pSV-faf plasmid (Fig. 4, lane 2). Canventol blocked this binding in a concentration dependent fashion (lanes 3-8) . This inhibition of NF- B activation by Canventol was overcome by external administration of TNF-σ (lanes 9,10). These results demonstrated that Canventol blocked a step in the far- independent pathway of action of faf upstream of the TNF-σ induced NF- B activation step. The sequential events of far- independent tat activation are thus overexpression and secretion of cytokines, followed by activation of NF-/cB and up regulation of HIV-1 promoter.

These experiments show that virus replication is inhibited by Canventol at nM concentrations, qualifying Canventol and its derivatives as potential anti-HIV-1 agents.

The combination of Canventol with Ro24-7429 Synergistically Inhibits HIV-1 LTR Activation. In contract to Canventol a TNF-σ inhibitor, the drug Ro24-7429 inhibits far-dependent but not far- independent action of Tat. By acting at different sites in the two separate pathways of tat action, these compounds in combination synergistically inhibited HIV- 1 promoter activity (Fig. 5A) . The concentrations of these compounds used in combination that were required to achieve the same level of inhibition were at least one log lower than those when used singly (Fig. 5B&C). For example, 100/vM Canventol inhibited

CAT expression by 60% vs 10μM Canventol when used in combination with 0.2//M Ro24-7429. Canventol and Ro24- 7429 applied separately at these concentrations did not significantly affect the reporter gene activity (Fig. 5B&C) . These results again demonstrate that the two separate modes of action of tat cooperate, leading to super-activation of the HIV-1 promoter. In addition to our previous report (Biswas, D.K., et al., Proc. Natl. Acad. Sci. USA 90, supra) they suggest that inhibitors with different activation mechanisms can synergistically down regulate HIV- 1 promoter.

Antiviral drugs that target the HIV- 1 LTR are attractive because the LTR harbors sites of multifactorial virus specific interplay between cellular and viral factors in regulating the expression of viral genes. Agents that interfere with LTR- mediated gene regulatory function by limiting the availability or activity of frar/s-activators should adversely affect HIV-1 replication. We have found that concerted interaction of two frar?s-activators, the cellular factor NF-/cB and the viral factor tat, lead to the super-activation of HIV- 1 LTR in Jurkat cells.

Concerted interplay between cellular and viral factors has been demonstrated in other cell systems [Liu, J., et al., J. Virol. 55:3883-3887 ( 1 992); Kammine, J., et al., J. Virol. 55:3932- 3936 ( 1992); Kadonaga, J.T., et al., Science 242: 1 566-1 570 (1 988); Doppler, C, et al., AIDS Res. Hum. Retro. Viruses 3:245-252 (1992); Somapayrac, L.M., et al., supra). The cellular and viral effects of TNF-σ, an activator of NF-κB, are increased in a synergistic manner in the presence of faf, which may be explained again on the basis of multifactorial interplay between cellular and viral frarjs-activators [Somapayrac, L.M., et al., supra] . The molecular mechanism of the NF-κB and tat induced concerted activation of HIV-1 LTR is not clear. The basal level of active NF-KB in unstimulated cells was sufficient to display super-activation of HIV-1 LTR in the presence of tat in cells co-transfected with a plasmid that provides the sites of interaction for simultaneous action of both frarjs-activators. In PMA-stimulated cells, as expected, the super-activation was exaggerated in the presence of a higher level of active NF-κB.

Independent of each other, Canventol inhibits NF-κB action and RO24-7429 inhibits tat action in vitro (Vickers, T., et al., Nuc. Acids. Research. 75:3359-3368 ( 1 991 ); Hsu, M-C, et al., Science 254: 1799-1 802 ( 1 991 )), the net result of which is inhibition of HIV- 1 LTR activation. Although the two trans- activators, NF-κB and faf, act in concert, neither drug by itself stops the separate effect. Thus, cooperative interaction of the two factors should continue. The results, however, demonstrate a synergistic inhibition of HIV-1 promoter by Canventol, the inhibitor of NF-κB, in combination with the tat inhibitor RO24-7429. The concentrations of the drugs in combination that effectively inhibited HIV-1 LTR activation were far below the cytotoxic level. Because very little or no inhibition of either NF-κB or tat actions were noticed when drugs at these low concentrations were used individually, it is anticipated that drug combinations that showed cooperative effect on HIV- 1 promoter, will not adversely affect other NF-κB-mediated cellular events.

The results of the three experiments were nearly a log greater inhibition by the combined drugs than by either alone.

It is evident that those skilled in the art given the benefit of the foregoing disclosure may make numerous modifications thereof, and departures from the specific embodiments described herein, without departing from the inventive concepts, and the present invention is to be limited solely to the scope and spirit of the appended claims.

Claims

We claim:

1 . A method of inhibiting or reducing the expression of genes operably linked to an LTR of an immunodeficiency virus, which comprises administering a combination of compounds in an ineffective amount to reduce gene expression, wherein the combination comprises a faf inhibitor and an NF-κB inhibitor of formula I

wherein R,-R are selected from the group consisting of: hydrogen, lower straight-chain or branched alkyls (C, - C6), lower alkoxy groups (OR, where R is C, - C6), lower alkenyls (C₂ - C6), halogens (F, Cl, Br and I), carboxylic acids (RCOOH, where R is hydrogen or lower alkyls (C, - C6)), and amino groups (NRR' where R and R' are hydrogen or lower alkyls (C, - C6) , or formula II

OH

where R,-R₄ are defined as above, X is a substituted or unsubstituted alkylene of 1 to about 10 carbon atoms, or a substituted or unsubstituted alkyenylene of 2 to about 1 0 carbon atoms, or a substituted or unsubstituted alkyenylene of 2 to about 10 carbon atoms, n is 1 or 2 , and Y is a substituted methylene (CH₂), N, O, or S.

2. The method of claim 1 , wherein the NF-κB inhibitor is a compound of formula II.

3. The method of claim 2, wherein the compound has formula III

OH

4. A method of treating a human having an immunodeficiency disease comprising administering a combination of compounds, to said human in a therapeutically effective immunodeficiency disease treatment amount, wherein the combination comprises a faf inhibitor and an NF-κB inhibitor of formula I

wherein R,-R₄ are selected from the group consisting of: hydrogen, lower straight-chain or branched alkyls (C, - C6) , lower alkoxy groups (OR, where R is C, - C₆), lower alkenyls (C₂ - C₆), halogens (F, Cl, Br and I), carboxylic acids (RCOOH, where R is hydrogen or lower alkyls (C, - C₆)), and amino groups (NRR' where R and R' are hydrogen or lower alkyls (C, - C₆)) or formula II

where R,-R₄ ^re defined as above, X is a substituted or unsubstituted alkylene of 1 to about 10 carbon atoms, or a substituted or unsubstituted alkyenylene of 2 to about 10 carbon atoms, or a substituted or unsubstituted alkyenylene of 2 to about 10 carbon atoms, n is 1 or 2 , and Y is a substituted methylene (CH₂), N, O, or S.

5. The method of claim 4, wherein the NF-κB compound is a compound selected from the group consisting of sarcophytol A, 3, 7, 1 1 -trimethylcyclodeca 3E, 7E, 1 1 E-triene-1 -ol, 2, 8, 1 2- trimethyldeca-1 , 5Z, 7E, 1 1 -tetraene-4-ol and 2-isopropyl-4- iospropylidenecyclohex2-ene- 1 -ol.

6. The method of claim 4, wherein the NF-κB compound is a compound of formula II having formula III

7. A pharmaceutical composition comprising a pharmaceutically-acceptable carrier and an effective anti-viral treatment amount of a combination of compounds, wherein the combination comprises a tat inhibitor and an NF-κB inhibitor of formula I

wherein R R₄ are selected from the group consisting of: hydrogen, lower straight-chain or branched alkyls (C, - C6), lower alkoxy groups (OR, where R is C, - C₆), lower alkenyls (C₂ - C₆), halogens (F, Cl, Br and I), carboxylic acids (RCOOH, where R is hydrogen or lower alkyls (C, - C₆)), and amino groups (NRR' where R and R' are hydrogen or lower alkyls (C, - C₆)) or formula II

OH where R,-R₄ are defined as above, X is a substituted or unsubstituted alkylene of 1 to about 10 carbon atoms, or a substituted or unsubstituted alkyenylene of 2 to about 10 carbon atoms, or a substituted or unsubstituted alkyenylene of 2 to about 10 carbon atoms, n is 1 or 2 and Y is a substituted methylene (CH₂), N, O, or S.

8. The method of claim 7, wherein the NF-κB is a compound of formula If having formula III

OH

9. The method of claim 4, wherein the NF-κB compound is selected from the group consisting of sarcophytol A, 3, 7, 1 1 - trimethylcyclodeca 3E, 7E, 1 1 E-triene-1 -ol, 2, 8, 1 2- trimethyldeca-1 , 5Z, 7E, 1 1 -tetraene-4-ol and 2-isopropyl-4- iospropylidenecyclohex2-ene-1 -ol.

10. The NF-κB inhibitor of claims 1 , 3, 4, 5, 7 or 8, wherein R, and R₂ are identical.

1 1 . The NF-κB inhibitor of claims 1 , 3, 4, 5, 7 or 8, wherein R₃ and R₄ are identical.

1 2. The method of claims 1 , 2 or 3, wherein the immunodeficiency virus is the human immunodeficiency virus.

1 3. The method of claims 5 and 9, wherein the human immunodeficiency virus is HIV-1 .

14. The method of claims 1 or 4, wherein the tat inhibitor is selected from the group consisting of aryl-(2-pyrryl) ketones and benzodiazepine.

15. The method of claim 14, wherein the compound is 2- glycinamido-5-chlorophemyl (2-pyrryl) ketone, 7-chloro-5-(2)- pyrryl-3H-1 ,4-benzodiazepin-2( 1 H)-one or 7-chloro-N-methyl-5- (1 H-pyrrol-2-yl)-3H-1 ,4-benzodiazepin-2-amine.

1 6. The method of claim 1 5, wherein the tat inhibitor is 7- chloro-N-methyl-5-(1 H-pyrrol-2-yl)-3(H-1 ,4-benzodiazepin-2- amine.

1 7. The method of the claims 5 or 9, wherein the tat inhibitor is selected from at least one of the group of compounds consisting of 2-glycinamido-5-chlorophemyl (2-pyrryl) ketone, 7- chloro-5-(2)-pyrryl-3H-1 ,4-benzodiazepin-2(1 H)-one or 7-chloro- N-methyl-5-( 1 H-pyrrol-2-yl)-3H-1 ,4-benzodiazepin-2-amine, topotecan, 3-lapachone, arryl- ?-lapachone and curcumin.