TREATMENT OF HUMAN VIRAL INFECTIONS

Abstract: Treatment of cells or humans carrying or infected with a virus capable of causing an immunodeficiency disease with compounds of formula (I), wherein R, R1 and R2 are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R1 and R2 are other than hydrogen.

Description: TREATMENT OF HUMAN VIRAL INFECTIONS BACKGROUND OF THE INVENTION The present application is a continuation-in-part of U.S. Serial No. 07/933,460, filed on August 21, 1992. 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 (1984); Levy, et al. , Science. Ill'-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, F. , AIDS. 1:135-140. This disease is characterized by a long asymptomatic period followed by the 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 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 function genomic organization, having regulatory genes such as tat, rev, nef. in addition to structural genes such as env. gag and pol. 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 the AIDS virus seem to live many years with little or no symptoms, but appear to have persistent infections. Another group of humans suffers 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 the HIV are believed to be persistently infective to others. The activation of the latent HIV provirus from the asymptomatic period has been reported to be governed by long terminal repeats (LTR) in the viral DNA. See Ranki, A., et al. , Lancet ii: 589-593 (1987); Fauci, A.S., et al., Science. 23£:617-622 (1988); Zagury, D., et al., Science. 231:850-853 (1985); Mosca, J.D., 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, B.R. , et al., Cell. 5_8:423-426 (1989). 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, A.S., Science. 239:617-622 (1988); Griffin, G.E., et al. , Nature (London), 3£:70-73 (1989); Nabel, G., et al., Science. 219:1299-1302 (1988). Specifically, phorbol 12-myristate-13-acetate (PMA) and Tumor Necrosis Factor-α (TNFα) are believed to be potent activators. In particular, TNFα is present in markedly enhanced levels in HIV infected individuals, suggesting that the cytokine plays an important role in the pathogenesis of AIDS. Lahdevirta, J., Am. J. Med.. 85:289-291 (1988). Most known methods for treating individuals infected by HIV have focused on preventing integration of the virus into the host cell's chromosome or on stages other than 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, ddl have not been reported to have inhibitory effect on chronic infections. Ro3-3335 was reported to be effective on chronic infection. See Hsu, M-C, Science. 25^:1799-1800 (1992). It thus would be desirable to have a new compound that can inhibit expression of provirus of HIV in HIV infected cells and inhibit chronic infections. 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 now discovered that compounds of the following formula I are inhibitory on expression of an immunodeficiency provirus, such as HIV, preferably HIV-1 and thus are useful for treating cells that have been acutely or chronically infected by immunodeficiency viruses such as HIV: wherein R, R*-_ and R are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R-, and Ro are other than hydrogen. These compounds have at least one chiral center. The present invention includes use of racemic mixtures of stereoisomers of the above compounds. Preferably the compounds are optically enriched, i.e., substantially more of one enantiomer (or diastereomer) than the other stereoisomer(s) . The biological activity of the optically active stereoisomer can be determined empirically as disclosed herein. Typically the compounds where the carbon at the 4 position has the (R) configuration are more preferred. Preferred compounds of formula I include those where R is alkyl, R-. is hydroxyl and/or R is heteroalkyl. Especially preferred compounds include those where R is hydrogen, Ri is hydroxyl and R2 is heteroalkyl, particularly alkyleneamino including monoalkyl- or dialkyl(alkylene)amino. Specifically preferred compounds include Topotecan, i.e., the compound of formula I where R is hydrogen, R**_ is 9-hydroxyl, and R2 is 10-N-N-dimethyl(methylene)amino (i.e., (CH^^NC^-) , and the compound of formula I where R is ethyl, R^ is hydrogen and R is 9-hydroxyl, wherein said 9- and 10- prefixes refer to positions of ring members as indicated in formula I above. The compounds of formula I can inhibit activity of an LTR of an immunodeficiency virus such as the HIV LTR and reduce or inhibit expression of genes operably linked to the HIV LTR. In one embodiment, the compounds of formula I 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 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) . Using such drugs in combination with the present compounds is anticipated to result in a synergistic result. Similarly, the pressent compounds should be effective in cells that are resistant to such compounds. For example, compounds of tne present invention can be used to block HIV-1 LTR directed expression in AZT resistant cell lines. The invention also provides pharmaceutical compositions comprising a compound of formula I and a suitable carrier therefor for use in the conditions referred to above. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows inhibition of cytokine stimulated HIV LTR directed gene expression by Topotecan (varying concentrations) . Figure 2 shows the effect of Topotecan on cell survival. Figure 3 shows inhibitory effects of Topotecan on activated HIV LTR. Figure 4 shows inhibition of HIV-1 replication by Topotecan (varying concentrations) in acutely infected human peripheral blood mononuclear cells. Figure 5 shows inhibition of HIV-1 replication by Topotecan, as determined by levels of mRNA of reverse transciptase in acutely infected human peripheral blood mononuclear cells. Figure 6 shows inhibition of HIV-1 expression by Topotecan (varying concentrations) in chronically infected human T cells. Figure 7 shows selective effects of Topotecan on the accumulation of HIV LTR directed mRNA and cellular 3-actin mRNA. Figure 8 shows dose dependent inhibition by Topotecan (varying concentrations) on the accumulation of HIV-1 LTR directed mRNA. Figure 9 shows the effect of Topotecan (varying concentrations) on total RNA synthesis of cell. DETAILED DESCRIPTION OF THE INVENTION We have discovered that compounds of the following formula I can be used to inhibit expression of an immunodeficiency provirus, for example, to treat cells infected with an immunodeficiency virus such as HIV and thus can be used for treatment in HIV infected individuals: wherein R, R-, and R2 are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R-, and R2 are other than hydrogen. The alkyl groups preferably have from 1 to about 12 carbon atoms, more preferably from about 1 to 6 carbon atoms. As used herein, the term alkyl unless otherwise modified refers to both cyclic and noncyclic groups, although of course cyclic groups will comprise at least three carbon ring members. Straight or branched chain noncyclic groups are generally more preferred than cyclic groups. Straight chain groups are still more preferred. The alkenyl substituents preferably have from 2 to about 12 carbon atoms, more preferably from 2 to about 6 carbon atoms. Heteroalkyl groups include those noncyclic groups that comprise one or more hetero atoms and each hetero atom has one or more alkyl linkages such as alkyl linkages having from 1 to 8 carbon atoms or 1 to 4 carbon atoms. Thus suitable heteroalkyl groups include those where a hetero atom is directly bonded to the general ring system of formula I as well as those groups where a hetero atoms is spaced from the ring system by an alkylene linkage of, e.g., one to four carbon atoms. Particularly preferred heteroalkyl groups include aminoalkyl groups including primary, secondary and tertiary alkylamines, and especially preferred are N-N-dialkyl(alkylene)amino groups, e.g., groups of the formula (alkyl)2 N ( CH 2- ) n" w ^ ere n - ~ an integer of 1 to 4. A particularly perferred gruop is N-N-dimethyImethyleneamino. The term "noncylic" heteroalkyl, is intended to include straight and branched chain moieties, but not groups that comprise a closed ring structure including those groups that form a ring comprising two or more aromatic carbons at positions 7 to 10 as depicted above in formula I. Said substituted R, Ri and R2 groups may be substituted at one or more available position by one or more suitable groups such as, for example, halogen such as fluoro, chloro or bromo, alkyl such as alkyl having from 1 to 12 carbon atoms or from 1 to 6 carbon atoms, alkenyl such as alkenyl having from 2 to 10 carbon atoms or 2 to 6 carbon atoms, aryl having from 6 to 10 carbon atoms, and N, 0, S, including heteroalkyl, e.g., heteroalkyl alkyl having one or more of said hetero atoms and from 1 to 10 carbon atoms or from 1 to 6 carbon atoms. The present invention includes use of both racemic mixtures and optically active stereoisomers of compounds of formula I. The optically active stereoisomers of formula I are preferred. Typically still more preferred are the optically active compounds where the chiral carbon of the lactone moiety (i.e., the ring carbon of position 4 as depicted above) is of the (R) configuration according to the Cahn-Ingold-Prelog nomenclature system. See Carey, F.A. , Advanced Organic Chemistry. Part A, p. 65-66 (2d ed. , Plenum Press 1984). It is believed that the compounds of formula I provide effective therapy of chronically infected cells (i.e. cells infected by a virus which is an immunodeficiency virus such as FIV, SIV, HIV, etc.) as evidenced by a reduction in, preferably a complete repression of, e.g. HIV LTR directed gene expression. Thus, in an HIV infected cell addition of an effective amount of a compound of formula I will reduce the expression of a gene operably linked to the HIV LTR. 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 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 tat gene, HIV rev gene, etc. Hence, in one preferred embodiment the present invention can be used in treating a serum positive but asymptomatic patient. For example, as a preventative, it can also be used prophylactically as a preventative for high risk individuals. This, method of protection comprised administering an effective amount of the compound of formula I is by means as set forth below. Compounds of formula I can be used to treat cells, especially mammalian cells and in particular human cells, infected by an immunodeficiency virus such as HIV infected cells. As a result of treatment with compounds of formula I viral expression is significantly reduced. For example, viral expression of HIV can be studied by a number of methods such as looking at the expression of a marker gene, e.g. CAT, Lac Z, etc., operably linked to the HIV LTR, which acts as the promoter. Use of the present compounds such as Topotecan can significantly reduce expression of such a marker. HIV-1 viral expression is turned on and enhanced by HIV LTR stimulators such as tumor necrosis factor (TNFα) or phorbol-12-myristate-13-acetate (PMA) . One product of this expression, i.e. tat can further augment HIV-1 gene expression. Using a marker gene such as Lac Z operably-linked to the HIV LTR in HIV infected cells, the addition of an effective amount of compounds of formula I significantly inhibits expression of lac Z gene, thereby indicating that HIV expression under the control of the HIV LTR such as HIV envelope glycoprotein expression has been inhibited if not completely stopped. P 24 , a major structural protein (product of gag), has been widely used for monitoring HIV-1 replication in cells and vireamia in individuals. Use of present compounds such as Topotecan, at concentrations that do not significantly adversely affect cells, can dramatically reduce HIV-1 replication, as determined by P 24 levels, i.e., preferably a reduction of more than 25% as determined by P 24 levels, more preferably a reduction of more than 50% , and still more preferably a reduction of HIV-1 replication of more than 80% as determined by P 24 levels. The effective amount used to obtain such a result is at micromolar and even nanomolar concentrations. Furthermore, the administration of the compounds of the present invention at effective concentrations, which inhibit for example, HIV expression, do not adversely affect the cell. 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 virus of the present invention can be used prophylactically for such individuals to minimize their risk. Compounds of formula I can readily be made or obtained. See, generally, U.S. Patent No. 5,004,758. Other compounds of formula I can be prepared by procedures well known to those skilled in the synthesis art. As demonstrated in the examples which follow, compounds of formula I block activation or suppress activity of HIV-1 LTR and thus expression of genes under its control in both chronically and acutely infected cells. In particular, it has been found that compounds of formula (I) in a dose dependent fashion inhibit HIV LTR directed TNFα and PMA stimulated gene expression. Moreover, such inhibition is provided with essentially no adverse effects on cell survival or cellular mRNA or total cellular RNA synthesis. Thus, it is believed compounds of formula I 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. While not wishing to be bound by theory, the absence of cytotoxicity of the compounds of formula I indicates that these compounds affect positive or negative regulators of HIV LTR, preferably HIV-1 LTR, that are more critical to the retrovirus than the host cell. In general for the immunodeficiency infections, for example an HIV infection, a suitable effective dose of one or more compounds of formula I will be in the range 0.4 to 10,000 μg per kilogram body weight of recipient per day, preferably in the range of 1 to 1,000 μg per kilogram, still more preferably in the range of 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 0.1 to 250 μg, preferably 5 to 250 μg. 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) , TAT antagonists such as Ro 3-3335, and Ro 24-7429, protease inhibitors and other agents such as 9- (2-hydroxyethoxymethyl)guanine (acyclovir) , interferon, e.g., alpha-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 a synergistic result 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, 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 compounds of formula (I), can be used to block HIV-1 LTR directed LTR expression in such resistant cell lines and for treatment of such resistant strains. For example, the present compounds can 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, R03-3335, 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 one or more compounds of formula I may be administered alone, they also may be present as part of a pharmaceutical composition. The compositions of the invention comprise at least one compound of formula I 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 to be administered ingredients 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; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion or packed in liposomes and 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 agent. 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 basis, 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 comprising one or more compounds of formula I and 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 tablets of the kind previously described. It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention ma Y 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 herein 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 COMMENTS The following reagents and procedures were employed as specified in the examples . Virus. HIV-1 was obtained from the culture supernatant of HTLV-III B -producing H9 (H9/HTLV-III β ) cells. During the exponential phase of growth, cell free supernatant was harvested, standardized for reverse transcriptase (RT) activity, and frozen in aliquots at -70°C. Clinical isolates of HIV-1 were prepared from patients testing positive for the human immunodeficiency virus, and standardized for RT activity. Cells. Cell clone 293.27.2, obtained from L.A. Herzenberg (Stanford University) , was derived from human embronic kidney epithelial cells, which were cultured in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% Fetal Calf Serum (FCS, Sigma) plus L-glutamine. See Roederer, M. , et al., Proc. Natl. Acad. Sci. USA. 87:4884-4888 (1990). This cell clone had been stably transfected with PNAZ, which is an expression construct of lacZ gene driven by HIV-1 LTR. Expression of S-galactosidase can be greatly induced by PMA or TNFα. Human peripheral blood mononuclear cells (PBMC) were prepared by Ficoll-Hypaque gradient centrifugation of blood from HIV-seronegative individuals, and cultured in RPMI 1640 supplemented with 20% FCS (Sigma), penicillin, streptomycin, and L-glutamine in the presence of PHA (3 μg/ml) . RPMI 8402 cell line, received from Toshiwo Ando (Aichi Cancer Research Institute, Nagoya, Japan), is a human T cell line. It was grown in RPMI 1640 medium supplemented with 15% FCS and L-glutamine. Stock Solution. A stock solution was prepared in Topotecan in water at concentration of 20 mM. Aliquots of the stock solution were stored frozen at -20°C. Ouantitation of HIV-1 LTR directed gene expression. Exponentially growing 293.27.2 (L.A. Herzenberg of Stanford University) cells were plated in 6 well plates at 2 x 10 s cells per well in 2 ml of growth medium. After 48 hours, cells were stimulated with 40 u/ml of TNFα (Genzyme, Cambridge, MA) or 2 ng/ml of PMA (Sigma) . Various concentrations of the specified compound of formula I were added to the medium at designated times after stimulation. Controls were run with final concentrations of ethanol, less than 0.1% (vol/vol) . After 6-8 hours incubation at 37°C, medium was aspirated, cells were harvested, washed 4 times with PBS, and lysed in lacZ buffer (60 mM Na 2 HP0 4 , 40 M NaH 2 P0 4 , 10 mM KC1, 1 mM MgSO^) . / 3-Galactosidase activities of cell lysates were quantitated by using ONPG as substrate. See Herbomel, et al. , Cell. 19:653-662 (1984). Protein concentration was measured. Cell visibility was determined by the colony formation assay after cells were treated as above. Total cellular RNA synthesis. 293.27.2 cells were plated at I X 10 6 cells per P150 plate in 20 ml of growth medium. After 48 hours incubation, cells were treated with different concentrations of Topotecan for 2 hours. Cells were then incubated for another 1 hour in the presence of 3 H-uridine at 1 uCi/ml. Total cellular RNA was prepared by the guanidium-CsCl step gradient method as described in Ausubel, F. , et al. , Current Protocols in Molecular Biology. I (Wiley Interscience 1990) . Newly synthesized RNA was quantitated by scintillation counting, which was adjusted against RNA concentrations measured at 260 nm. Northern blot analysis. 293.27.2 cells in log phase growth were stimulated with TNFα at 40 u/ml in the presence or absence of drugs. After 2 to 6 hours incubation, cells were harvested. Total cellular RNA was prepared by the guanidium-CsCl step gradient method by procedures described supra. RNA was equally loaded, fractionated and Northern transferred, and then hybridized with a lacZ gene fragment (Hindlll/EcoR I from PHSV lac 19) or a -actin probe. Treatment in acute HIV-1 infection. Peripheral blood mononuclear cells (PBMC) , after 72 hours stimulation with 3 μg/ml PHA, were infected with either HTLV-IIIg or a clinical isolate of HIV-1 at 1 reverse transcriptase unit (RTU) per 10 cells. Infection was carried out at 37°C for 2 hours. Then PBMC were washed with PBS to remove free virus and replated at 4.5 X 10 6 cells per well in 2 ml medium in the absence or presence of different concentrations of drugs. The cells were then continuously exposed to drugs for 6 days. On day 3, 1 ml of medium was removed from each well and replaced with 1 ml of fresh media containing drug at the previous concentration. On day 6, cells and media were harvested. Cell viability was determined by the trypan blue exclusion method and MTT metabolic assays. See Mosman, T. , J. Immunological Methods. 5:55-63 (1983). P 24 levels in the culture supernatant were quantitated by ELISA assay with HIV-1 P 24 Antigen Kinetics Assay Kit (Coulter, Hialeah, FL) according to the manufacturer's protocol. RNA of HIV-1 reverse transcriptase was assayed with HIV-1 RNA Detection Kit (GeneTrak, Framingham, MA) according to manufacturer's instructions. Briefly, total cellular RNA was prepared, dot blotted onto a nitrocellulose membrane, and hybridized with 32 P labeled probe for RT RNA. Treatment in chronic HIV-1 infection. RPMI 8402, cells have been chronically infected with HTLV-IIIg, was cultured at 4 X 10 s cells per well in 2 ml of medium in the presence or absence of Topotecan. On day 3 and 6, P 24 levels in the culture supernatant were measured with the procedure described above. Cell viability was determined by trypan blue exclusion and MTT metabolic assay as described by Mosman, T. , J . Immunological Methods. 61:55-63 (1983). EXAMPLE 1 The HIV LTR activity of 293.27.2 cells were simulated with TNFα and PMA to greater than a 20 fold increase in a period of 6 hours. The cells were treated with varying concentrations of Topotecan as indictaed in Figure 1. The Topotecan effectively inhibited the LTR stimulations at a Topotecan dose of 0.5 μM. In Figure 1, enzymatic activity is expressed as percentage of maximum expression in drug untreated samples (taken as 100). Basal 3-galactosidase activity is defined as in samples without cytokine stimulation. Each data point is the average of triplicate culture wells . Experiments were repeated on at least three independent occasions. The IC was approximately 50 nM. A similar inhibitory effect was observed upon similar treatment of RPMI 8402 (a CD4 + T-cell line transiently transfected with PNAZ) with Topotecan. Specifically, an inhibition of gene expression 62% relative to the untreated control was observed at a Topotecan concentration of 125 nM (data not depicted in Figures). EXAMPLE 2 293.27.2 cells were treated with Topotecan as described in Example 1 above. After treatment with varying concentrations of Topotecan for 6 hours, cells were trypsinized and replaced in triplicate at 100, 1,000, 10,000 cells per 60 mm plate. After 7 day incubation, cell colonies (more than 50 cells) were counted. Plating efficiency was around 20%. The results are depicted in Figure 2 where each data point is expressed as percent of control survival, i.e. from drug-untreated plates (taken as 100) . EXAMPLE 3 293.27.2 cells were plated as described in Example 1 above. TNFα (40 μ/ml) was added to culture media after 48 hours. Topotecan (0.5 μM) was added to the cells 0.5, 2, and 5.5 hours after addition of TNFα, i.e., Topotecan was added to the cells after HIV LTR activation. The cells were harvested 6 hours after the addition of TNFα. ^9-Galactosidase was assayed as depicted in Figure 3 where each data point represents an average of triplicate culture wells. Experiments were repeated twice independently. As shown in Figure 3, -galactosidase accumulated time-dependently after stimulation with TNFα in the absence of Topotecan. Treatment of the TNFα stimulated cells with Topotecan at 2.0 and 5.5 hours as described rapidly and thoroughly stopped accumulation. When cells were treated with Topotecan at a concentration of 0.5 uM for 2.0 hours before TNFα stimulation and washing (3X) with fresh medium showed 60% inhibition of 3-galactosidase accumulation. Substantially no /3-galactosidase accumulation was observed upon simultaneous treatment of the cells with Topotecan and TNFα (See Figure 3) . EXAMPLE 4 The inhibition of HIV-1 replication in acutely infected human PBMC by compounds of formula I was investigated. The effect of Topotecan on viral replication was measured by P 24 release onto the culture supernatant. Human PBMC were infected with HIV-1 (HTLV-IIIg or clinical isolate) at 37°C for 2 hours. Free virus was removed by washing with PBS. Infected PBMC were then aliquoted at 4.5 X 10 s cells/well. Various concentrations of Topotecan were added to culture media. After 6 days, cell viability was determined by trypan blue exclusion. P 24 levels were determined and RT mRNA analyzed as described in the General Comments above. P 24 levels were expressed as percent of P 24 concentration of drug treated samples versus untreated samples (taken as 100) and are depicted in Figure 4 where results are representative of two independent studies where each data point was obtained from duplicate samples. As shown in Figure 4, Topotecan dramatically decreased HIV-1 replication as indicated by P 24 production for both the lab strain (HTLV-IIIg) and clinical isolate of the virus. The median HIV replication inhibition concentration was approximately 20 nM, similar to the effective HIV LTR activity inhibition concentration of Toptecan thus suggesting that HIV LTR is a target of the compounds of formula I. This inhibition was achieved in the absence of significant adverse effect on cell survival. Cell viability of stimulated PBMC was about 90% as determined by trypan blue exclusion. Cell viability was 100% in unstimulated PBMC treated with 500 nM Topotecan. At a concentration of 31 nM, Topotecan reduced RT MRNA to an essentially undetectable level (See results depicted in Figure 5). EXAMPLE 5 RPMI 8402 cells were infected with HTLV-IIIB. Topotecan was added to the culture media of the chronically infected cells (2 X 10 s cell/ml) . Three and six days after addition of Topotecan, P 24 in the culture supernatant was quantitated as described in the General Comments. The thus determined P 24 levels are depicted in Figure 6. In particular, it was found that Topotecan at concentrations of 31 nM reduced HIV-1 replication by more than 80%. Cell viability as shown in Figure 5 was determined by MTT assay as described in the General Comments. EXAMPLE 6 293.27.2 cells were plated at 1 X 10 6 cells/plate in 175 mm plates. After 48 hours, cells were treated with TNFα (40 u/ml) in the presence of different concentrations of Topotecan Total cellular RNA was prepared at 0 hours, 2 hours, or 6 hours after addition of TNFα. Northern blot analysis was carried out as described in the General Comments and the results depicted in Figure 7. EXAMPLE 7 293.27.2 cells were plated and treated with different concentrations of Topotecan, as described in Example 6 above. Total cellular RNA was prepared at 6 hours after addition of TNFα. Northern blot analysis was done as described in the General Comments. The autoradiograms were scanned. HIV LTR directed 3-galactosidase mRNA was determined by densitometry. Results are depicted in Figure 8. EXAMPLE 8 293.27.2 cells were plated as described in Example 6 above. After 48 hours, cells were treated with varying concentrations of Topotecan as shown in Figure 9 for 3 hours. 3 H-uridine was added to the culture media (1 μCi/ml) at the beginning of the third hour. Total cellular RNA was prepared by the end of the third hour. Newly synthesized total cell RNA was quantitated by scintillation counting, and RNA concentrations were measured spectrophotometrically and results depicted in Figure 9 where each data point is an average of triplicate dishes of a representative study of two independent experiments. EXAMPLE 9 The inhibition of HIV-1 replication in HIV-1 infected cells that are resistant to a reverse transcriptase inhibitor was investigated. Topotecan (TPT) at a dose of about 0.006-0.008 μM (ICCQ) was measured in PBMCs acutely infected with an AZT resistant HIV-1 strain. The AZT resistant strain was inhibited by TPT at such doses as measured by the production of HIV P 24 antigen and HIV-1 LTR directed /3-galactosidase gene RNA expression. A chronically infected T-lymphocyte line and the promyelocytic cell line OMlO.l also showed marked inhibition at IC 50 = 0-008 μM following treatment with TPT whereas AZT and ddl had little effect. The promyelocytic cell line OMlO.l shows activation of HIV-1 P 24 antigen following treatments with 20 units of TNF α and this is effectively blocked by treatment with TPT. Thus, TPT inhibits HIV-1 LTR directed expression and viral P 24 antigen and HIV RNA production in 60 ■ 23 - such acutely infected and chronically infected cell lines with minimal cell toxicity. TPT also prevents TNF a mediated activation of HIV P 24 and OMlO.l cells. EXAMPLE 10 Topotecan (TPT) was added to cultures of OMlO.l cells (10 5 cells/ml) at the same time as TNFα (20 units/ml). OMlO.l cells in log growth phase were resuspended in RPMI 1640 medium and aliquoted into a 24-well micro-culture plate at 3 x 10 cells/well. A range of 0.002-0.031 μM TPT was applied to the cell culture in duplicate. OMlO.l cells were stimulated with TNF-α (Endogen Inc., Boston, MA) for 36 hours and P 24 antigen was determined by Coulter P 24 kinetics assay with 1:250 dilution of the specimen. The assay was run in duplicates with results shown in the below Table, where values are the mean of two representative assays. The promyelocytic cell line OMlO.l contains one copy of HIV-1 per cell integrated into its genome and continually produces a low level of HIV-1 proteins. This cell line was kindly supplied by NIH-AIDS Research and Reference Reagent Program. TABLE TOPOTECAN PREVENTS ACTIVATION OF HIV P 24 EXPRESSION BY TNF-α IN OMlO.l CELLS This invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of the disclosure, may make modification and improvements with the spirit and scope of the invention.

Claims: What is claimed is: 1. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective antiviral treatment amount of a compound of formula I wherein R, R-, and R are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R-i and R2 are other than hydrogen, adapted for treating a mammal having an immunodeficiency virus . 2. A method of inhibiting or reducing the expression of genes operably linked to a LTR of an immunodeficiency virus which comprises administering an effective gene expression reduction amount of a compound of the formula I: wherein R, R-*_ and R2 are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R-, and R2 are other than hydrogen. 3. A method for treating cells infected with a virus capable of causing an immunodeficiency disease in a human, comprising administering to the cells an effective antiviral treatment amount of a compound of the following formula I: wherein R, Ri and R2 are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R-, and R2 are other than hydrogen. 4. A method of treating humans having an immunodeficiency disease, wherein the immunodeficiency disease is resistant to a reverse transcriptase inhibitor, comprising administering to said human an effective immunodeficiency disease treatment amount of a compound of the following * formula I: * wherein R, R-, and Ro are each independently selected from the group consisting of hydrogen, substituted and unsubstituted alkyl, noncyclic heteroalkyl, and substituted and unsubstituted alkenyl, provided at least two of the substituents of R, R-, and R2 are other than hydrogen. 5. The method of claim 1 wherein the cell line is a reverse transcriptase inhibitor resistant cell line. 6. The method of claims 1, 2, 4, and 5 wherein the reverse transcriptase inhibitor is selected from the group consisting of zidovudine (AZT), 2' ,3' -dideoxyinosine (ddl), and 2' ,3' -dideoxycytidine (ddC) . 7. The method of claim 6, wherein the reverse transcriptase inhibitor is AZT. 8. The method of claim 1 or 2 wherein the LTR is an HIV LTR. 9. The method of claim 8 wherein the LTR is an HIV-1 LTR. 10. The compound described in claims 1, 2, 3, or 4 where R is ethyl, Ri is hydrogen, and R is 9-hydroxyl. 11. The compound described in claims 1, 2, 3 or 4, wherein the compound of formula I is Topotecan. 12. The method of claim 2 wherein the virus infecting the cells is resistant to a reverse transcriptase inhibitor. 13. The method of claim 2 or 12 where the cells are human cells. 14. The method of claims 1, 2, 4, 5, 12 where the virus is capable of causing in a human, acquired immune deficiency syndrome or an acquired immune deficiency syndrome related complex. 15. The method of claim 14 where the virus is HIV. 16. .The method of claim 15 where the virus is HIV-1.

Inventors: CRUMPACKER CLYDE, LI JIA-QIANG, PARDEE ARTHUR B, ZHANG LIN, CRUMPACKER, CLYDE, LI, JIA-QIANG, PARDEE, ARTHUR, B., ZHANG, LIN
Applicants: BETH ISRAEL HOSPITAL, CRUMPACKER CLYDE, DANA FARBER CANCER INST INC, LI JIA QIANG, PARDEE ARTHUR B, ZHANG LIN, DANA FARBER CANCER INSTITUTE
Application year: 1993
Main classification: A61K31
IPC codes: A61K31, A61P31, A61P37, C07D491