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WO1996028162A1 - Nouvelle composition de medicaments pour le traitement d'affections virales - Google Patents

Nouvelle composition de medicaments pour le traitement d'affections virales Download PDF

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Publication number
WO1996028162A1
WO1996028162A1 PCT/US1996/003502 US9603502W WO9628162A1 WO 1996028162 A1 WO1996028162 A1 WO 1996028162A1 US 9603502 W US9603502 W US 9603502W WO 9628162 A1 WO9628162 A1 WO 9628162A1
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Prior art keywords
azt
cells
hiv
die
infection
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PCT/US1996/003502
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English (en)
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Roger Strair
Daniel Medina
Peter Tung
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University Of Medicine & Dentistry Of New Jersey
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Publication of WO1996028162A1 publication Critical patent/WO1996028162A1/fr

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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof

Definitions

  • the present invention relates to a method for treating a human with human immunodeficiency virus infection.
  • the method comprises administering to the human a therapeutically effective amount of a thymidine analog, which analog acts as an inhibitor of viral reverse transcriptase necessary for viral replication of human immunodeficiency virus, and a thymidylate synthase inhibitor.
  • the method further comprises admimstering to the human a therapeutically effective amount of a folate antagonist or hydroxyurea, or both.
  • AIDS Acquired immunodeficiency syndrome
  • human immunodeficiency virus Human immunodeficiency virus
  • Human immunodeficiency virus is a retrovirus which replicates in a human host cell.
  • the human immunodeficiency virus appears to preferentially attack helper T-cells (T-lymphocytes or OKT4-bearing T-cells).
  • helper T-cells T-lymphocytes or OKT4-bearing T-cells.
  • helper T-cells When the helper T-cells are invaded by the virus, the T-cells become a human immunodeficiency virus producer.
  • the helper T-cells are quickly destroyed causing the B-cells and other T-cells, normally stimulated by helper T-cells, to no longer function normally or produce sufficient lymphokines and antibodies to destroy the invading virus or other invading microbes.
  • Human immunodeficiency virus is an extremely heterogeneous virus. The clinical significance of this heterogeneity is evidenced by the ability of the virus to evade immunological pressure, survive drug selective pressure, and adapt to a variety of cell types and growth conditions. A comparison of isolates among infected patients has revealed significant diversity, and within a given patient, changes in the predominant isolate over time have been noted and characterized. In fact, each patient infected with human immunodeficiency virus harbors a "quasispecies" of virus with a multitude of undetected viral variants present and capable of responding to a broad range of selective pressures, such as those imposed by the immune system or antiviral drug therapy.
  • Human immunodeficiency virus infection has multiple mechanisms to maximize its potential for genetic heterogeneity. These mechanisms result in an extremely diverse population of virus capable of responding to a broad range of selective pressures, including the immune system and antiretroviral therapy, with the outgrowth of genetically altered virus.
  • the prevalence of viral variants containing mutations encoding resistance to TIBO R82150 was reflected by the prevalence of recombinant viruses capable of infecting HeLa-T4 cells in the presence of TIBO R82150.
  • the presence of reporter genes in the recombinant viruses allowed for a quantitative analysis of a single cycle of infection on a single cell basis.
  • United States patent no. 4,724,232 discloses a method for treating a human having acquired immunodeficiency syndrome which comprises administering to the human 3'-azido-3'-deoxythymidine.
  • Figure 1 is a schematic representation of the production of recombinant HIV-gpt by COS cell transfection or rescue from the H9/HIV-gpt cell line.
  • Figure 2 is a schematic representation of the analysis of colonies arising after COS cell derived HIV-gpt infection of HeLa-T4 cells in the presence of 10 ⁇ M AZT.
  • Figure 3 is a graph showing the infection of a clone of HeLa-T4 cells "persistently resistant" to the antiviral effects of AZT (clone R116) and a control clone (SI) with replication-competent HIV-IIIIB in the presence of O.l ⁇ M AZT.
  • Figures 4A and 4B are graphs illustrating thymidine metabolism-
  • Figure 5 is a graph showing a comparison of thymidine kinase mRNA levels (A) and enzyme activity (B) in cell lines sensitive and persistently resistant to the antiretroviral effects of AZT.
  • Figure 6 is a graph showing cellular toxicity of AZT.
  • Figure 7A and Figure 7B are graphs showing the suppression of viral breakthrough in cells sensitive and refractory to the antiviral effects of AZT.
  • Figure 8 is a graph illustrating FUdR cytotoxicity in cells sensitive and refractory to the antiretroviral activity of AZT.
  • Figure 9 is a graph showing AZT-FUdR cytotoxicity in JE6.1 cells sensitive and resistant to the antiviral effects of AZT.
  • Figure 10 is a graph showing that the AZT-FUdR combination inhibits HIV-1 infection of PBMC.
  • This invention pertains to a method for treating a human with human immunodeficiency virus infection (acquired immunodeficiency syndrome) which comprises admimstering to the human a therapeutically effective amount of a thymidine analog, which analog acts as an inhibitor of viral reverse transcriptase necessary for viral replication of human immunodeficiency virus, and a thymidylate synthase inhibitor, or i pharmaceutically acceptable salts thereof.
  • the method further comprises administering to the human a therapeutically effective amount of a folate antagonist or hydroxyurea, or both.
  • Results with these defective 5 HIV indicate that early infection in the presence of AZT often results from the infection of a cell which is refractory to the antiretroviral effects of AZT. Characterization of cell lines derived from such infected cells has demonstrated decreased accumulation of AZT, increased phosphorylation of thymidine to TTP, and increased levels of thymidine kinase activity. In addition, AZT 0 inhibition of replication-competent HIV infection is also significantly impaired in this cell line.
  • the present invention relates to a method for treating a human with human immunodeficiency virus infection.
  • the method comprises 5 administering to the human a therapeutically effective amount of a thymidine analog, which analog acts as an inhibitor of viral reverse transcriptase necessary for viral replication of human immunodeficiency virus, and a thymidylate synthase inhibitor.
  • Thymidine analogs such as 3'-azido-3'- deoxythymidine (AZT) are prodrugs in the treatment of acquired immunodeficiency syndrome.
  • 3 '-Azido-3' -deoxythymidine is converted by cellular enzymes to 3' -azido-3 '-deoxythymidine monophosphate (AZTMP).
  • 3'-azido-3'- deoxythymidine diphosphate (AZTDP) and 3 '-azido-3' -deoxythymidine triphosphate (AZTTP).
  • AZTDP 3'-azido-3'- deoxythymidine diphosphate
  • AZTTP 3 '-azido-3' -deoxythymidine triphosphate
  • 3'-azido-3'- deoxythymidine triphosphate is an inhibitor of the viral reverse transcriptase necessary for viral replication.
  • Some cells do not efficiently metabolize AZT to the triphosphate and may overproduce the natural thymidine triphosphate, which competes with the antiviral activity of AZTTP. Studies have demonstrated that these cells contribute to the early failure of the antiviral activity of AZT.
  • thymidylate synthase inhibitor By coadministering a thymidylate synthase inhibitor with the thymidine analog, applicants have found that that the thymidine analog is a more effective inhibitor of HIV replication.
  • the thymidylate synthase inhibitor may function by resulting in lower levels of thymidine triphosphate to compete with the phosphorylated thymidine analog reverse transcriptase inhibition.
  • the method further comprises administering to the human a therapeutically effective amount of a folate antagonist together with the thymidine analog and the thymidylate synthase inhibitor to modulate the effects of the thymidine analog.
  • the method further comprises administering to the human a therapeutically effective amount of hydroxyurea together with the thymidine analog and the thymidylate synthase inhibitor to modulate the effects of the thymidylate synthase inhibitor.
  • both the folate antagonist and hydroxyurea may be administered with the thymidine analog and the thymidylate synthase inhibitor.
  • replication-defective HIV encoding a selectable marker was used to infect target cells in the presence of 10 ⁇ M AZT.
  • the cells infected with the defective HIV were isolated by expression of the selectable marker.
  • a subset of these infected cells was demonstrated to be readily infected with another HIV in the presence of 10 ⁇ M AZT.
  • These cells were persistently refractory to the antiviral effects of AZT and were demonstrated to have excessive phosphorylation of thymidine to TTP, increased thymidine kinase activity and decreased accumulation of AZTTP.
  • FUdR will be shown to potentiate the antiviral effects of AZT in whole cell populations (including peripheral blood mononuclear cells [PBMC]) as well as in subsets of cells isolated by infection with recombinant HIV in the presence of AZT. Infection of these latter cells will be shown to be extremely sensitive to combined AZT-FUdR therapy .
  • PBMC peripheral blood mononuclear cells
  • prodrug refers to compounds which undergo biotransformation prior to exhibiting their pharmacological effects.
  • drug latentiation is the chemical modification of a biologically active compound to form a new compound which upon in vivo enzymatic attack will liberate the parent compound.
  • the chemical alterations of the parent compound are such that the change in physicochemical properties will affect the absorption, distribution and enzymatic metabolism.
  • prodrugs latentiated drugs, and bioreversible derivatives are used interchangeably.
  • latentiation implies a time lag element or time component involved in regenerating the bioactive parent molecule in vivo.
  • prodrug is general in that it includes latentiated drug derivatives as well as those substances which are converted after administration to the actual substance which combines with receptors.
  • prodrug is a generic term for agents which undergo biotransformation prior to exhibiting their pharmacological actions.
  • the thymidine analogs, and prodrugs thereof, which may be employed in the present invention are compounds which act as inhibitors of viral reverse transcriptase necessary for viral replication of human immunodeficiency virus.
  • the thymidine analogs are prodrugs which are converted by cellular enzymes to their respective active monophosphates, diphosphates, and/or triphosphates which are inhibitors of viral reverse transcriptase.
  • Nonlimiting examples of thymidine analogs may be selected from the group consisting of 3 '-azido-3' -deoxythymidine, and D4T.
  • the thymidine analog is 3' -azido-3' -deoxythymidine.
  • the amount of thymidine analog which acts as an inhibitor of viral reverse transcriptase present in the therapeutic compositions of the present invention is a therapeutically effective amount.
  • a therapeutically effective amount of thymidine analog is that amount necessary to inhibit viral reverse transcriptase.
  • All prodrugs or precursors are administered to a human in a therapeutically effective amount sufficient to generate an effective amount of the compound which inhibits viral reverse transcriptase necessary for viral replication of human immunodeficiency virus.
  • a suitable effective dose of the thymidine analog or its pharmaceutically acceptable basic salts will be in the range of about 5mg to 250mg per kilogram body weight of recipient per day, preferably in the range of 7.5mg to lOOmg per kilogram body weight per day, and most preferably in the range lOmg to 40mg per kilogram body weight per day.
  • the thymidylate synthase inhibitors, and prodrugs thereof, which may be employed in the present invention are compounds which are antimetabolites which interfere with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibit the formation of ribonucleic acid (RNA).
  • the thymidylate synthase inhibitors inhibit the synthesis of thymidine triphosphate so that the phosphorylated thymidine analog which acts as an inhibitor of the viral reverse transcriptase can compete more effectively with thymidine triphosphate and will more effectively inhibit viral reverse transcriptase necessary for viral replication of human immunodeficiency virus.
  • Nonlimiting examples of thymidylate synthase inhibitors may be selected from the group consisting of 5-fluorouracil, 5-fluoro-2-pyrimidone (a prodrug of 5- fluorouracil), and floxuridine.
  • the thymidylate synthase inhibitor is floxuridine.
  • Floxuridine is a fluorinated pyrimidine antineoplastic antimetabolite. Chemically, floxuridine is 2'-deoxy-5-fluorouridine. FUdr produces the same toxic and antimetabolic effects as does 5-fluorouracil. The primary effect is to interfere with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibit the formation of ribonucleic acid (RNA). Derivatives of 5 -fluorouracil and floxuridine may also be incorporated into DNA or RNA.
  • O.Olmg to lOmg per kilogram body weight per day and most preferably in the range O.Olmg to 5mg per kilogram body weight per day.
  • the amount of folate antagonist present in the therapeutic compositions of the present invention is a therapeutically effective amount.
  • a therapeutically effective amount of folate antagonist is that amount necessary to modulate the effects of the thymidine analog.
  • a suitable effective dose of folate antagonist or its pharmaceutically acceptable salts will be in the range of about 0.05mg to 25mg per kilogram body weight of recipient per day, N preferably in the range of 0.05mg to lOmg per kilogram body weight per day, and most preferably in the range 0.05mg to 4mg per kilogram body weight per day.
  • the method of the present invention may further comprise administering to a human a therapeutically effective amount of hydroxyurea, and prodrugs thereof, together with the thymidine analog and the thymidylate synthase inhibitor to modulate the effects of the thymidylate synthase inhibitor.
  • Hydroxyurea has the structural formula H2N-CO-NHOH. The precise mechanism by which hydroxyurea produces cytotoxic effects is not known but it is believed that hydroxyurea causes an immediate inhibition of DNA synthesis without interfering with the synthesis of ribonucleic acid or of protein.
  • the amount of hydroxyurea present in the therapeutic compositions of the present invention is a therapeutically effective amount.
  • a therapeutically effective amount of hydroxyurea is that amount necessary to modulate the effects of me thymidylate synthase inhibitor.
  • a suitable effective dose of hydroxyurea or its pharmaceutically acceptable salts will be in the range of about 5mg to 250mg per kilogram body weight of recipient per day, preferably in the range of 7.5mg to lOOmg per kilogram body weight per day, and most preferably in the range lOmg to 40mg per kilogram body weight per day.
  • Administration 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.
  • the preferred route may vary with the condition and age of the recipient.
  • the formulations of the present invention comprise the administered ingredients, as above defined, together with one or more acceptable carriers thereof and optionally other therapeutic ingredients.
  • 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.
  • the formulations 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 may be prepared by any methods well known in the art of pharmacy.
  • Such methods include the step of mixing the ingredients to be administered with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping ⁇ _e product.
  • Formulations 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.
  • Formulations 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.
  • Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the ingredient to be administered and a pharmaceutically acceptable carrier.
  • a preferred topical If? delivery system is a transdermal patch containing the ingredient to be administered.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • Formulations 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.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of die intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and diickening agents.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, or an appropriate fraction thereof, of the administered ingredient. . 7
  • the present invention is further illustrated by the following examples which are presented for purposes of demonstrating, but not limiting, the preparation of the compounds and compositions of this invention.
  • the HIV construct encoding LacZ has been described (26). It contains the LacZ gene driven by an SV40 promoter inserted into a large deletion in the HIV genome extending from the 5' end of the pol gene to the 3' end of the env gene.
  • the HIV-gpt and HXB2env plasmids were kindly provided by Kathleen Page (University of California, San Francisco, CA) (18).
  • the HIV-gpt plasmid contains an HXB2 provirus into which an SV40 promoter gpt (E. coli guanine phosphoribosyl transferase) gene was inserted into the env region.
  • the HXB2 env plasmid contains the HXB2 gpl60 gene driven by an SV40 promoter gpt (E. coli guanine phosphoribosyl transferase) gene driven into the env region.
  • the HXB2 env plasmid contains the HXB
  • the H9/HIV-gpt cell line and HeLa T4/HIV-LacZ cell line were prepared and used as previously described (26).
  • Rescue of defective retroviruses from the H9/H_N-gpt cell line and d e HeLa T4/ HIV-LacZ cell line were performed as previously described (26).
  • the resultant titer of HTV-LacZ or HIV-gpt was determined and die inoculum used to infect HeLa-T4 cells was adjusted depending upon d e number of infectious events to be analyzed.
  • AZT-mediated cytotoxicity was assayed in cells persistently refractory to die antiviral effects of AZT (R116) and in cells sensitive to die antiviral effects of AZT (HT4, S pool and SI) using a standard assay (14).
  • Triplicate wells of 24 - well plates containing 3 x 10 4 cells were cultured in d e absence or presence of various concentrations of AZT.
  • drug cytotoxicity was quantitated widi a standard MTT assay in which die uptake and metabolism of 3-[4, 5-dimethyld ⁇ iazol-2-yl] 2, 5-dephenyltetrazolium bromide (MTT) by cells was measured (14).
  • the amount of formazan produced in 2 hours was determined by dissolving d e product in 100% DMSO and dien measuring the absorbance at 570 nm.
  • RNA from SI and R116 cells were extracted as described previously (4). Equal amounts of total RNAs were electrophoresed on an agarose gel containing 1% formaldehyde and blotted onto a nylon membrane. The RNAs were hybridized widi a 32 P-labled human mymidine kinase probe (3). The labeled bands were visualized using autoradiography and quantitated using a Molecular Dynamics Personal Densitometer.
  • Cell line R116 is a derivative of HeLa-T4 cells that was isolated after infection of HeLa-T4 cells with HIV-gpt in the presence of 10 / xM AZT (31). This cell line was demonstrated to be refractory to die antiviral effects of AZT by virtue of reinfection widi eidier recombinant or replication-competent HIV infection in d e presence of AZT.
  • Cell line SI is a derivative of HeLa-T4 cells that was isolated after infection of HeLa-T4 cells with HIV-gpt in the absence of AZT (31).
  • PBMC Peripheral blood mononuclear cells isolated from healthy HIV-1 seronegative donors were activated widi PHA (lOug/ml) for 72 hours prior to HIV-1 infection.
  • PBMC were maintained in RPMI 1640 supplemented wid 10% interleukin-2 (Advanced Biotechnologies, Columbia, MD), 20% FBS, 2 mM L-glutamine and antibiotics.
  • HIV-1 IIIB Stock preparations of HIV-1 IIIB were harvested from H9 cells by me "shake off method” (13). An AZT sensitive clinical isolate (HIV-lp re AO8 W was prepared in MT-2 cells. Stock virus infectivity was determined by end-point dilution in MT-2 cells (32). Virus-induced cytopatiiic effect (syncytium formation) was scored 7 days post-infection and die TCID50 was calculated widi the Reed and Muench equation (33).
  • Azidodiymidine (AZT) and Floxuridine (FUdR) were purchased from Sigma Chemical Co. (St. Louis, MO) and were dissolved in phosphate buffered saline, sterile filtered (0.22 um) and stored at -20°C.
  • HIV-1 production in infected cultures was determined by a
  • RT activity was determined by qualification of ⁇ 2 P bound to die DE81 paper by using a Molecular Dynamics phosphorimager. The results are reported as pixel units per microliter of die reaction mixture.
  • HeLa-T4 cells were infected widi a recombinant HIV, HIV-gpt, in d e presence or absence of 10 ⁇ M AZT ( Figure 1). Two separate populations of HIV-gpt were utilized for diese infections. One population of HIV-gpt was produced in COS cells by co-transfection of die HIV-gpt plasmid widi a plasmid encoding die HXB2 env gene. The infectious virions produced by diis co-transfection have little genetic diversity in that tiiey are produced from products encoded by plasmids in COS cells.
  • the second population of HIV-gpt was genetically more diverse, being produced by rescue from the H9/ HIV-gpt cell line widi replication competent HIV-IIIIB that had been propagated in culture (26).
  • die HeLa-T4 cells were placed in gpt selective media and die number of colonies developing by day 10 was used as an indicator of me number of cells initially infected in die absence or presence of lO ⁇ M AZT.
  • die prevalence of colony formation after infection in the presence of AZT was similar (-5 x 10 "4 ) with die two preparations of HIV-gpt.
  • HeLa-T4 cells were infected widi HIV-gpt (prepared in COS cells) in die absence or presence of AZT. Infected cells were selected in gpt selective media and colonies were isolated and expanded into cell lines. Twelve cell lines developing after infection in the presence of AZT were further characterized. To determine if diese cell lines were refractory to the antiretroviral effects of AZT they were infected wid HIV-LacZ in die presence of 10 / ⁇ M AZT. Three days after infection, die cells were stained widi X-gal to detect 13-galactosidase activity. Nine of diese twelve cell lines behaved like wild type HeLa-T4 cells with complete inhibition of infection in die presence of
  • a persistently resistant cell line was compared to a control cell line obtained by HIV-gpt infection in die absence of AZT. Each of these cell lines was incubated widi 3 H-d ⁇ ymidine and diymidine metabolites were assayed by HPLC. As shown in Figures 4A and 4B, die persistently resistant cell line (Rl 16) had a greater phosphorylation of thymidine into TTP compared to d e non-resistant cell line (SI). An identical experiment widi 3 H-AZT indicated a nearly 2 fold reduction in AZTTP in R116 cells compared to SI cells (Table 3).
  • a component of the resistance may be related to a diminished AZTTP/TTP ratio.
  • HIV-gpt a recombinant HIV encoding a selectable marker
  • Infected cells were isolated in gpt selective media and expanded into cell lines.
  • Several such cell lines were refractory to the antiviral effects of AZT as evidenced by d e ability of replication-defective or replication-competent HTV to infect diese cells in die presence of AZT.
  • Several control cell lines were obtained by infection of HeLa-T4 cells with HIV-gpt in die absence of AZT.
  • Cell line R116 is a cell line tiiat was determined to be refractory to the antiviral effects of AZT.
  • Cell line SI is a control cell line.
  • a prior metabolic analysis of diese cell lines indicated tiiat cell line R116 had a reduced accumulation of AZTTP and an increased phosphorylation of diymidine to TTP in comparison to die SI control cell line (31).
  • die addition of a fluoropyrimidine to AZT increased die antiviral efficacy of AZT in the R116 cell line, cells were cultured in the absence or presence of 0.1 ⁇ M AZT or O.Ol ⁇ M FUdR alone or in combination prior to infection widi HIV-1 IIIB at an input multiplicity of infection of 1.
  • cytotoxicity to various concentrations of FUdR were determined. As shown in Figure 8, the R116 cell line had an ED50 of 0.7 ⁇ M FUdR whereas die SI cell line, parental HeLa-T4 cells and a pool of control cell lines all had an ED50 of 7 ⁇ M. These results further substantiate die presence of metabolic differences in cells refractory to die antiviral effects of AZT as opposed to cells sensitive to die antiviral effects of AZT. Efficacy of AZT in Combination with FUdR in Inhibiting HTV-1 Infection of Lymphoid cells
  • die JE6.1AZTR cells represent a mixture of cells, some of which require an increased concentration of AZT to inhibit HIV infection. This is reflected by a 2 fold increase AZT IC50 when analyzing die entire population. Strikingly, die combination of FUdR wid AZT dramatically suppresses HIV infection of mis population. A greater tiian 600 fold reduction of AZT IC50 is seen during infection of diese cells in die presence of AZT and FUdR. In fact, diese cells, which were initially isolated as cells infected in die presence of AZT, were more sensitive to die antiviral effects of the AZT-FUdR combination tiian were control or parental cells.
  • die mechanisms responsible for cellular resistance because reversal of diis resistance may greatly reduce viral burden and delay die outgrowth of virus with genetic resistance. It is important to emphasize that die cells tiiat were detected as refractory to d e antiviral effects of AZT were only exposed to AZT for a short period of time. There was no preselection of cells prior to infection widi the recombinant viruses.
  • cellular resistance is likely to contribute to viral breaktiirough during an in vivo infection and multiple mechanisms may contribute to cellular resistance.
  • the prevalence of resistant cells detected in single cell lines derived during infection in these studies raises interesting speculation concerning the prevalence of similar resistant cells during an in vivo infection involving multiple cell types.
  • Figure 1 is a schematic representation of the production of recombinant HIV-gpt by COS cell transfection or rescue from the H9/ HIV-gpt cell line.
  • Figure 2 is a schematic representation of the analysis of colonies arising after COS cell derived HIV-gpt infection of HeLa-T4 cells in the presence of 10 ⁇ M AZT. Twelve such colonies were expanded and infected widi HIV-LacZ in die presence and absence of 10 ⁇ M AZT. Ten control colonies derived from HTVgpt infection of HeLa-T4 cells in the absence of AZT were studied in parallel. "Persistent" cellular resistance was defined by a high level infection widi HIVLacZ in die presence of AZT, as shown for colony number 2. HIV-LacZ contains the LacZ gene driven by an SV40 promoter inserted into a large deletion in die HIV genome extending from the pol gene to the 3' end of die env gene. HIVLacZ virus production has been previously described (16).
  • Figure 3 is a graph showing the infection of a clone of HeLa-T4 cells "persistently resistant" to the antiviral effects of AZT (clone R116) and a control clone (SI) with replicationcompetent HIV-IIIIB in the presence of 0. l ⁇ M AZT.
  • P24 was assayed, compared to a control infection in the absence of AZT and plotted as a function of time.
  • P24 values in the absence of AZT were 1857 + 104 ng/ml for SI and 1717+ 113ng/ml for R116.
  • Figures 4A and 4B are graphs illustrating diymidine metabolism- HPLC analysis of clones obtained after infection of HeLa-T4 cells with
  • Table 3 shows the concentration of phosphorylated AZT metabolites in the "persistently resistant" (R116) and sensitive (SI) cell lines. Pool sizes were determined by incubation of cells widi 3 H-AZT for 4 hours followed by cellular extraction and HPLC. The numbers are expressed as pmoles/10" cells. The numbers in parentheses represent die percentage of total radioactive species in tiiat pool.
  • HIV in the presence of AZT in vitro was previously undertaken (31).
  • applicants Based upon a prior study demonstrating increased phosphorylation of diymidine to TTP and decreased AZTTP in a subset of cells infected widi drug-sensitive HIV in die presence of AZT, applicants have attempted to modulate die antiviral efficacy of AZT by combining AZT therapy with floxuridine.
  • die combination was much more effective tiian AZT alone at inhibiting HIV infection of an unfractionated lymphoid cell line and PBMC.
  • the enhanced antiviral activity of the combination therapy is not restricted to cell lines, recombinant viruses, or laboratory strains of virus and may therefore have clinical utility.
  • AZT-FUdR The increased efficacy of AZT-FUdR in suppressing HIV infection of cells readily infected widi HIV in the presence of AZT is particularly striking. Since this population of cells is a mixture of cells with and without persistent refractoriness to the antiviral effects of AZT (i.e. , infection of a subset of mis population is repeatedly refractory to the antiviral effects of AZT), the AZT IC50 for this population is only minimally elevated. Nevertheless, infection of this entire population is extremely sensitive to inhibition by the AZT-FUdR combination. The supersensitivity of infection of 3 -3- this population of cells to combination therapy was unanticipated and is likely to be explained by metabolic features tiiat are responsible for the efficacy of the combination.
  • FUdR has moderate antiviral activity when used by itself.
  • the mechanisms by which this inhibition occurs are also currently unknown and may also be related to perturbations of normal thymidine metabolite pools, direct inhibition of viral or cellular processes or by incorporation into the viral DNA during reverse transcription.
  • AZT-resistance and diese mutations emerge over several montiis-years. Suppression of early HIV replication with AZTsensitive virus in the presence of AZT could delay, or even prevent die emergence of AZT resistant virus by diminishing the substrate for subsequent genetic changes. Therefore, studies that define die mechanisms of early viral breakthrough infection have potential long term therapeutic implications.
  • Figure 7 is a graph showing die suppression of viral breakthrough in cells sensitive and refractory to the antiviral effects of AZT.
  • Figure 8 is a graph illustrating FUdR cytotoxicity in cells sensitive and refractory to die antiretroviral activity of AZT.
  • Cells sensitive, parental HT4 (open circle), SI (solid square), Spool (solid circle) and refractory, R116 (open square) were grown in the presence of various concentrations of FUdR. Three days latter, cell viability was determined by die MTT reduction method.
  • Spool cells are a population of control cells obtained by infection with HIV-gpt in the absence of AZT (7).
  • Figure 9 is a graph showing AZT-FUdR cytotoxicity in JE6.1 cells sensitive and resistant to die antiviral effects of AZT. Cytotoxicity of 10 ⁇ M AZT in combination widi 0.025 ⁇ M FUdR was determined in JE6.1 cells sensitive (solid circle), JE ⁇ .lcon (open circle) and resistant, JE6,1AZTR (open triangle) to die antiviral effects of AZT as described in Materials and Metiiods.
  • Figure 10 is a graph showing that die AZT-FUdR combination inhibits HIV-1 infection of PBMC.
  • PBMC were infected with HIV-1 in the absence of drug (cross) widi AZT alone (x), with various concentrations of FUdR alone, [0.005 ⁇ M FUdR (open circle), O.Ol ⁇ M FUdR (open square), 0.025 ⁇ M FUdR (open triangle)], or with combinations of FUdR and AZT [AZT + 0.005 ⁇ M FUdR (closed circle), AZT + 0.01 ⁇ M FUdR (solid square) AZT + 0.025 ⁇ M FUdR (solid triangle).
  • HIV-1 IIIB in the presence of 0.001 ⁇ M AZT, 0.01 ⁇ M AZT, 0.1 ⁇ M AZT, 1 ⁇ M AZT or 10 ⁇ M AZT in the presence of 0.005 ⁇ M FUdR, 0.01 ⁇ M FUdR or 0.025 ⁇ M FUdR.
  • IC50 represents die concentration of AZT required for 50% inhibition of reverse transcriptase activity at day 6 of infection.

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Abstract

Cette invention concerne un procédé permettant de traiter une personne atteinte d'une infection par virus d'immunodéficience chez l'homme, lequel procédé consiste à administrer à la personne, dans une quantité suffisante pour être efficace sur le plan thérapeutique, d'une part, un analogue de thymidine, cet analogue jouant le rôle d'un inhibiteur de transcriptase virale inverse nécessaire à la réplication virale du virus d'immunodéficience chez l'homme, et, d'autre part, un inhibiteur de synthase de thymidylate ou un de ses sels acceptables sur le plan pharmaceutique.
PCT/US1996/003502 1995-03-14 1996-03-14 Nouvelle composition de medicaments pour le traitement d'affections virales WO1996028162A1 (fr)

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EP0774258A1 (fr) * 1995-11-15 1997-05-21 Bristol-Myers Squibb Company Composition contenant le fluorouracile-5 et le d4T pour le traitement d'infections rétrovirales
US5719132A (en) * 1996-06-27 1998-02-17 Bristol-Myers Squibb Company Compositions and methods of treating HIV with d4T, 5-fluorouracil/tegafur, and uracil
WO1999048504A1 (fr) * 1998-03-26 1999-09-30 Julianna Lisziewicz Compositions inhibitrices du vih, a base d'hydroxyuree et d'inhibiteur de transcriptase inverse
US6593455B2 (en) 2001-08-24 2003-07-15 Tripep Ab Tripeptide amides that block viral infectivity and methods of use thereof
US6680059B2 (en) 2000-08-29 2004-01-20 Tripep Ab Vaccines containing ribavirin and methods of use thereof
US6858590B2 (en) 2000-08-17 2005-02-22 Tripep Ab Vaccines containing ribavirin and methods of use thereof
US6960569B2 (en) 2000-08-17 2005-11-01 Tripep Ab Hepatitis C virus non-structural NS3/4A fusion gene
US7012129B2 (en) 2001-09-19 2006-03-14 Tripep Ab Antiviral composition comprising glycine amide
US7022830B2 (en) 2000-08-17 2006-04-04 Tripep Ab Hepatitis C virus codon optimized non-structural NS3/4A fusion gene
EP1200097B1 (fr) * 1999-05-10 2014-01-08 Vigilent Technologies Methode pour controler la fidelite et la processivite de la reverse transcriptase par incorporation et polymerisation d'analogues de nucleotides acceptes comme substrats de la reaction de reverse transcription sans bloquer son elongation
US8883169B2 (en) 2007-08-16 2014-11-11 Chrontech Pharma Ab Immunogen platform
EP3793561A4 (fr) * 2018-05-15 2022-03-09 National Centre For Cell Science Composés médicamenteux antiviraux et composition associée

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0774258A1 (fr) * 1995-11-15 1997-05-21 Bristol-Myers Squibb Company Composition contenant le fluorouracile-5 et le d4T pour le traitement d'infections rétrovirales
US5719132A (en) * 1996-06-27 1998-02-17 Bristol-Myers Squibb Company Compositions and methods of treating HIV with d4T, 5-fluorouracil/tegafur, and uracil
WO1999048504A1 (fr) * 1998-03-26 1999-09-30 Julianna Lisziewicz Compositions inhibitrices du vih, a base d'hydroxyuree et d'inhibiteur de transcriptase inverse
EP1200097B1 (fr) * 1999-05-10 2014-01-08 Vigilent Technologies Methode pour controler la fidelite et la processivite de la reverse transcriptase par incorporation et polymerisation d'analogues de nucleotides acceptes comme substrats de la reaction de reverse transcription sans bloquer son elongation
US7226912B2 (en) 2000-08-17 2007-06-05 Tripep Ab Hepatitis C virus non-structural NS3/4A fusion gene
US7638499B2 (en) 2000-08-17 2009-12-29 Tripep Ab Hepatitis C virus codon optimized non-structural NS3/4A fusion gene
US6960569B2 (en) 2000-08-17 2005-11-01 Tripep Ab Hepatitis C virus non-structural NS3/4A fusion gene
US6858590B2 (en) 2000-08-17 2005-02-22 Tripep Ab Vaccines containing ribavirin and methods of use thereof
US7022830B2 (en) 2000-08-17 2006-04-04 Tripep Ab Hepatitis C virus codon optimized non-structural NS3/4A fusion gene
US7223743B2 (en) 2000-08-17 2007-05-29 Tripep Ab Hepatitis C virus codon optimized non-structural NS3/4A fusion gene
US7439347B2 (en) 2000-08-17 2008-10-21 Tripep Ab Hepatitis C virus non-structural NS3/4A fusion gene
US7241440B2 (en) 2000-08-17 2007-07-10 Tripep Ab Vaccines containing ribavirin and methods of use thereof
US7244715B2 (en) 2000-08-17 2007-07-17 Tripep Ab Vaccines containing ribavirin and methods of use thereof
US7307066B2 (en) 2000-08-17 2007-12-11 Tripep Ab Hepatitis C virus codon optimized non-structural NS3/4A fusion gene
US7261883B2 (en) 2000-08-29 2007-08-28 Tripep Ab Vaccines containing ribavirin and methods of use thereof
US7244422B2 (en) 2000-08-29 2007-07-17 Tripep Ab Vaccines containing ribavirin and methods of use thereof
US6680059B2 (en) 2000-08-29 2004-01-20 Tripep Ab Vaccines containing ribavirin and methods of use thereof
US6593455B2 (en) 2001-08-24 2003-07-15 Tripep Ab Tripeptide amides that block viral infectivity and methods of use thereof
US7012129B2 (en) 2001-09-19 2006-03-14 Tripep Ab Antiviral composition comprising glycine amide
US8883169B2 (en) 2007-08-16 2014-11-11 Chrontech Pharma Ab Immunogen platform
EP3793561A4 (fr) * 2018-05-15 2022-03-09 National Centre For Cell Science Composés médicamenteux antiviraux et composition associée

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