EP0655919A1 - Traitement d'infections virales chez l'homme - Google Patents

Traitement d'infections virales chez l'homme

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Publication number
EP0655919A1
EP0655919A1 EP93920283A EP93920283A EP0655919A1 EP 0655919 A1 EP0655919 A1 EP 0655919A1 EP 93920283 A EP93920283 A EP 93920283A EP 93920283 A EP93920283 A EP 93920283A EP 0655919 A1 EP0655919 A1 EP 0655919A1
Authority
EP
European Patent Office
Prior art keywords
hiv
cells
hydrogen
substituted
ltr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93920283A
Other languages
German (de)
English (en)
Other versions
EP0655919A4 (fr
Inventor
Arthur B. Pardee
Jia-Qiang Li
Clyde Crumpacker
Lin Zhang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dana Farber Cancer Institute Inc
Beth Israel Deaconess Medical Center Inc
Original Assignee
Dana Farber Cancer Institute Inc
Beth Israel Hospital Association
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Publication date
Application filed by Dana Farber Cancer Institute Inc, Beth Israel Hospital Association filed Critical Dana Farber Cancer Institute Inc
Publication of EP0655919A1 publication Critical patent/EP0655919A1/fr
Publication of EP0655919A4 publication Critical patent/EP0655919A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • 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.
  • AIDS acquired immune deficiency syndrome
  • AIDS 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.
  • PDL persistent generalized lymphadenopathy syndrome
  • ARC AIDS related complex
  • LTR long terminal repeats
  • TNF ⁇ Tumor Necrosis Factor- ⁇
  • 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).
  • 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.
  • 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.
  • Preferred compounds of formula I include those where R is alkyl, R-. is hydroxyl and/or R is heteroalkyl.
  • Particular 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.
  • 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.
  • immunodeficiency viruses for example, HIV, preferably HIV-1
  • 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) .
  • reverse transcriptase inhibitors such as zidovudine (AZT), 2' ,3' -dideoxyinosine (ddl) and 2' ,3' -dideoxycytidine (ddC) .
  • AZT zidovudine
  • ddl 2' ,3' -dideoxyinosine
  • ddC 2' ,3' -dideoxycytidine
  • the pressent compounds should be effective in cells that are resistant to such compounds.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, ary
  • 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.
  • the optically active compounds where the chiral carbon of the lactone moiety i.e., the ring carbon of position 4 as depicted above
  • 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).
  • 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.
  • a gene operably linked to the HIV LTR preferably the gene is operably linked to an HIV-1 LTR.
  • operably linked means that the gene is under the control of the HIV LTR and positioned in a nucleotide sequence to accomplish this.
  • the gene is downstream of the
  • the present invention can be used in treating a serum positive but asymptomatic patient.
  • a preventative it can also be used prophylactically as a preventative for high risk individuals.
  • 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.
  • 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.
  • a marker gene e.g. CAT, Lac Z, etc.
  • 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 (TNF ⁇ ) or phorbol-12-myristate-13-acetate (PMA) .
  • TNF ⁇ tumor necrosis factor
  • PMA phorbol-12-myristate-13-acetate
  • One product of this expression, i.e. tat can further augment HIV-1 gene expression.
  • 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.
  • 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 block activation or suppress activity of HIV-1 LTR and thus expression of genes under its control in both chronically and acutely infected cells.
  • compounds of formula (I) in a dose dependent fashion inhibit HIV LTR directed TNF ⁇ 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.
  • 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.
  • 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.
  • a suitable effective dose of one or more compounds of formula I will be in the range 0.4 to 10,000 ⁇ g per kilogram body weight of recipient per day, preferably in the range of 1 to 1,000 ⁇ g per kilogram, still more preferably in the range of 5 ⁇ g to 500 ⁇ g 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 ⁇ g, preferably 5 to 250 ⁇ g.
  • 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),
  • reverse transcriptase inhibitors such as dideoxynucleosides, e.g. zidovudine (AZT)
  • 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.
  • interferon e.g., alpha-interferon, interleukin II, and phosphonoformate (Foscarnet)
  • 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.
  • the present compounds may be effective when the above-described drugs are not or are no longer effective.
  • compounds of the present invention can be used in cells that are resistant to reverse transcriptase inhibitors such as AZT, ddl, and ddC.
  • 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.
  • the present compounds can block HIV-1 LTR directed expression in an AZT resistant strain of HIV-1.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HIV-1 was obtained from the culture supernatant of HTLV-III B -producing H9 (H9/HTLV-III ⁇ ) 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 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 TNF ⁇ .
  • PNAZ Dulbecco's modified Eagle's medium
  • PBMC Human peripheral blood mononuclear cells
  • 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.
  • PBMC Peripheral blood mononuclear cells
  • RTU reverse transcriptase unit
  • 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.
  • 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).
  • the HIV LTR activity of 293.27.2 cells were simulated with TNF ⁇ 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 ⁇ M.
  • 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.
  • 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) .
  • 293.27.2 cells were plated as described in Example 1 above.
  • TNF ⁇ 40 ⁇ /ml
  • Topotecan 0.5 ⁇ M
  • Topotecan was added to the cells 0.5, 2, and 5.5 hours after addition of TNF ⁇ , i.e., Topotecan was added to the cells after HIV LTR activation.
  • the cells were harvested 6 hours after the addition of TNF ⁇ .
  • ⁇ 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 TNF ⁇ in the absence of Topotecan.
  • TNF ⁇ stimulated cells with Topotecan at 2.0 and 5.5 hours as described rapidly and thoroughly stopped accumulation.
  • Topotecan Treatment of the TNF ⁇ stimulated cells with Topotecan at 2.0 and 5.5 hours as described rapidly and thoroughly stopped accumulation.
  • TNF ⁇ 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 TNF ⁇ (See Figure 3) .
  • 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.
  • 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).
  • 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.
  • 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.
  • 293.27.2 cells were plated at 1 X 10 6 cells/plate in 175 mm plates. After 48 hours, cells were treated with TNF ⁇ (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 TNF ⁇ .
  • Northern blot analysis was carried out as described in the General Comments and the results depicted in Figure 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 TNF ⁇ .
  • 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.
  • 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 ⁇ Ci/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.
  • TPT Topotecan
  • ICCQ Topotecan
  • the promyelocytic cell line OMlO.l shows activation of HIV-1 P 24 antigen following treatments with 20 units of TNF ⁇ and this is effectively blocked by treatment with TPT.
  • TPT inhibits HIV-1 LTR directed expression and viral P 24 antigen and HIV RNA production in 60
  • TPT also prevents TNF a mediated activation of HIV P 24 and OMlO.l cells.
  • Topotecan was added to cultures of OMlO.l cells (10 5 cells/ml) at the same time as TNF ⁇ (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 ⁇ M TPT was applied to the cell culture in duplicate.
  • OMlO.l cells were stimulated with TNF- ⁇ (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.

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  • Epidemiology (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Traitement de cellules ou de sujets humains infectés par ou porteur d'un virus capable de provoquer une maladie à immunodéficience au moyen de composés de la formule (I), dans laquelle R, R1 et R2 sont chacun indépendamment choisis dans le groupe constitué par hydrogène, alkyle substitué et non substitué, hétéroalkyle non cyclique, et alcényle substitué et non substitué, à condition qu'au moins deux des substituants de R, R1 et R2 soient différents de l'hydrogène.
EP93920283A 1992-08-21 1993-08-23 Traitement d'infections virales chez l'homme. Withdrawn EP0655919A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US93346092A 1992-08-21 1992-08-21
US933460 1992-08-21
US632093A 1993-01-19 1993-01-19
US6320 1993-01-19
PCT/US1993/007934 WO1994004160A1 (fr) 1992-08-21 1993-08-23 Traitement d'infections virales chez l'homme

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EP0655919A1 true EP0655919A1 (fr) 1995-06-07
EP0655919A4 EP0655919A4 (fr) 1995-10-18

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EP (1) EP0655919A4 (fr)
JP (1) JPH08502044A (fr)
CA (1) CA2142831A1 (fr)
WO (1) WO1994004160A1 (fr)

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Publication number Priority date Publication date Assignee Title
US5491237A (en) * 1994-05-03 1996-02-13 Glaxo Wellcome Inc. Intermediates in pharmaceutical camptothecin preparation
WO1997032587A1 (fr) * 1996-03-04 1997-09-12 Dana-Farber Cancer Institute Methodes de traitement d'infections virales
US6395541B1 (en) 1996-05-23 2002-05-28 The Rockefeller University Methods for the identification of compounds capable of inhibiting HIV-1 viral replication employing murine cell lines expressing human topoisomerase I
US6559309B2 (en) 1996-11-01 2003-05-06 Osi Pharmaceuticals, Inc. Preparation of a camptothecin derivative by intramolecular cyclisation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0220601A1 (fr) * 1985-10-21 1987-05-06 Daiichi Seiyaku Co., Ltd. Dérivés de la pyranoindolizine et procédé de préparation

Family Cites Families (2)

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Publication number Priority date Publication date Assignee Title
US5004758A (en) * 1987-12-01 1991-04-02 Smithkline Beecham Corporation Water soluble camptothecin analogs useful for inhibiting the growth of animal tumor cells
US5422344A (en) * 1990-05-08 1995-06-06 The United States Of America As Represented By The Secretary Of The Department Of Health & Human Services Method of treating retroviral infections in mammals

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0220601A1 (fr) * 1985-10-21 1987-05-06 Daiichi Seiyaku Co., Ltd. Dérivés de la pyranoindolizine et procédé de préparation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AIDS RES. HUM. RETROVIRUSES, vol.7, no.1, 1991 pages 65 - 72 E. PRIEL ET AL. 'Inhibition of Human Immunodeficiency Virus (HIV-1) replication in vitro by noncytotoxic doses of camptothecin, a topoisomerase I inhibitor' *
IXTH INTERNATIONAL CONFERENCE ON AIDS AND THE IVTH STD WORLD CONGRESS. MEETING, BERLIN, GERMANY, JUNE 6-11, 1993.; PAGE 33. CRUMPACKER C et al 'TOPOTECAN A TOPOISOMERASE I INHIBITOR INHIBITS HIV-1 REPLICATION IN ACUTELY AND CHRONICALLY INFECTED CELLS' *
PROC NATL ACAD SCI U S A (UNITED STATES), MAR 1 1993, VOL. 90, NO. 5, PAGE(S) 1839-42, Li CJ et al 'Three inhibitors of type 1 human immunodeficiency virus long terminal repeat-directed gene expression and virus replication.' *
See also references of WO9404160A1 *

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EP0655919A4 (fr) 1995-10-18
JPH08502044A (ja) 1996-03-05
WO1994004160A1 (fr) 1994-03-03
CA2142831A1 (fr) 1994-03-03

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