EP0416011A1 - Composition chimiotherapeutique pour le traitement du sida - Google Patents

Composition chimiotherapeutique pour le traitement du sida

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Publication number
EP0416011A1
EP0416011A1 EP89906537A EP89906537A EP0416011A1 EP 0416011 A1 EP0416011 A1 EP 0416011A1 EP 89906537 A EP89906537 A EP 89906537A EP 89906537 A EP89906537 A EP 89906537A EP 0416011 A1 EP0416011 A1 EP 0416011A1
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EP
European Patent Office
Prior art keywords
dpm
azt
cells
composition
hiv
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Ceased
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EP89906537A
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German (de)
English (en)
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EP0416011A4 (en
Inventor
John N. Weinstein
Janos Szebeni
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US Department of Commerce
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US Department of Commerce
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Publication of EP0416011A1 publication Critical patent/EP0416011A1/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
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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 present invention is related to treating or preventing AIDS or other similar viral diseases. More particularly, the present invention is related to a combination of an antiviral agent and a potentiating agent, the combination being more effective against virus than either agent used alone.
  • HIV human immunodeficiency virus
  • chain-terminator compounds such as azidothymidine (AZT), other dideoxynucleosides, dideoxynucleotides, and their analogs have a degree of efficacy against the AIDS virus (Mitsuya, et al. 1985, PNAS, USA. 82:7096; Yarchoan, et al, 1988, Lancet(i) 76-80; Dahlberg et al, 1987, PNAS, USA., 84: 2469). They appear to be phosphorylated within cells and. in the triphosphate form, to ⁇ terminate DNA chains being formed in the presence of viral reverse transcriptase. These drugs may act by additional mechanisms as well, for example, by competing for the enzymes active in phosphorylation of physiological nucleosides and nucleotides.
  • Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that modifies glycosylation by inhibiting ⁇ -glycosldase I.
  • Other compounds of the same group referred to as trimming glycosidase inhibitors because they inhibit the normal removal of glucose residues during the processing of glycoproteins
  • DMDP dihydroxymethyldihydroxypyrrolidine
  • DNJ or DNM deoxynojirimycin
  • Dipyridamole also called Persa ⁇ tine
  • DPM Dipyridamole
  • Its best studied mechanism of action is inhibition of nucleoside transport. Through this mechanism it affects principally the "salvage" pathways for nucleotide production.
  • DPM has also been reported to 'have activity against some viruses, but activity either against the AIDS virus or against retroviruses has not been reported (Tonew et al, 1977. Acta Virol.21:146-150).
  • an object of the present invention to provide a drug combination with synergistic advantage as inhibitor of HIV and other retrovirus activity compared to each drug used alone. It is a further object of the present invention to provide a method of arresting HIV proliferation, comprising contacting HIV infected cells by the drug-combination of the present invention in an amount sufficient to prevent HIV replication. It is yet another object of the present invention to provide a method of treating or preventing AIDS and other
  • HIV-related syndromes comprising administering to a host infected or uninfected with the AIDS virus sufficient amount of the drug-combination of the present invention to inhibit AIDS-virus activity.
  • Figure 1 demonstrates the effect of dipyridamole (DPM), alone and in combination with chain-terminating dideoxynucleosides, on HIV-1 replication in human monocyte/macrophage (M/M) cultures.
  • DPM dipyridamole
  • A Adherence-purified, cryopreserved monocyte/macrophageis treated with AZT alone, DPM alone, or AZT plus DPM. The cells were treated on day 1 and re-fed on days 6, 11, and 14 with medium containing the appropriate drug concentrations. 100- ⁇ l samples of supernatant were analyzed for HIV-1 p24. The figure shows the results on day 14.
  • AZT 0 denotes DPM treatment alone. The data show that DPM potentiates the antiviral action of AZT.
  • B Experiment similar to that in A. except that elutriated monocytes were used. The cells were treated with the drugs on day 1 of culturing and were re-fed by replacing 100 ⁇ l of medium (without drugs) at 3-4 day intervals. The data show p24 levels on day 17 and demonstrate DPM's antiviral activity and potentiation of antiviral activity of AZT. The pattern of effects was similar on day 14.
  • C and D From the same experiment as in B but using 2',3'-dideoxycytldine (ddC) and 2',3'-dideoxycytidine triphosphate (ddCTP), respectively.
  • ddC 2',3'-dideoxycytldine
  • ddCTP 2',3'-dideoxycytidine triphosphate
  • Figure 2 shows the results of time course study of HIV-1 infection in elutriated M/M treated with DPM alone.
  • Figure 3 shows the interaction between AZT and DPM in their cytotoxic effects on human bone marrow granulocyte-monocyte precursors.
  • the CFU GM assay, calculation of the combination index (C.I.), and other experimental details are described herein infra.
  • Panels A and B show the C.I. values calculated on the basis of the mutually exclusive model (identical site/mechanism of action) and the mutually nonexclusive model (non-identical sites/mechanisms of action), respectively.
  • the different symbols indicate cells from different healthy donors.
  • the results show no synergy between AZT and DPM in bone marrow toxicity.
  • FIG. 4 shows the effect of combination chemotherapy with DPM-AZT (panel A) and DPM-castanospermine (panel B) in T-lymphoblastoid cells (CEM-SS) infected with HIV-1.
  • Panel A shows the results when the cells were infected by cocultivation with H9 cells carrying HIV-1(III B ). Treatment was begun on day 1.
  • Panel B shows the result of an analogous study for DPM and castanospermine. At high concentrations, DPM potentiated the antiviral effect of castanospermine. This result emphasizes that a wide range of agents may interact favorably with DPM against HIV and other viruses.
  • FIGS. 5A and 5B give HPLC profiles showing the effect of DPM on intracellular accumulation of phosphorylated derivatives of 3 H-thymidine and 3 H-AZT during 6-hour incubations at 37oC with elutriated human monocyte/macrophages.
  • DPM greatly decreased the appearance of phosphorylated thymidine (THY) but only slightly reduced that of phosphorylated AZT.
  • Figure 6 shows the inhibition of HIV-1 in human M/M by AZT in combination with nitrobenzylthioinosine (NBTI), like
  • DPM an inhibitor of nucleoside transport.
  • the experiment was performed essentially as for Figure 1A except that the adherence-purified M/M cultures were prepared from freshly drawn donor blood, not cryopreserved cells. At the concentrations tested NBTI inhibited virus replication and potentiated the anti-HIV activity of AZT. Mean ⁇ S.E.M. of quadruplicate wells on day 11.
  • FIG. 7 shows the inhibition of HIV-1 in human M/M by
  • Figure 8 shows the inhibition of HIV-1 in human M/M by AZT in combination with mioflazine.
  • mioflazine is an inhibitor of nucleoside transport. The experiment was performed as was the experiment for Figure 6. At the concentrations tested, mioflazine inhibited virus replication and potentiated the anti-HIV activity of AZT. MF: mioflazine. The pattern of effects was similar on days 13 and 21. Mean ⁇ S.E.M. of quadruplicate wells. DETAILED DESCRIPTION OF THE INVENTION
  • a pharmaceutical composition comprising an effective amount of an antiviral agent and an effective amount of a potentiating agent to inhibit viral replication in infected cells, wherein a combination of the antiviral and the potentiating agent produces greater viral inhibition than expected from the activity of each component alone, and pharmaceutically acceptable carrier.
  • Retroviruses "pr diseases caused thereby, against which the composition and the methods of the present invention can be used include HIV-1, HIV-2, HTLV-I, HTLV-II and .the like.
  • the composition may include such additives as sterilants, adjuvants, non-toxic sterile buffers and the like, as are commonly used in such preparations and which are well known to one of ordinary skill in the art.
  • chain terminating drug refers to a compound or a precursor thereof, which, when inserted into an elongating nucleic acid chain, terminates the growth of the chain.
  • These drugs may act by additional mechanisms as well, for example, by competing for the enzymes active in phosphorylation of physiological nucleosides and nucleotides.
  • Base is selected from the group consisting of adenine, guanine, cytosine, thymine and analogues, derivatives or salts thereof which can be incorporated into growing DNA chains, which are metabolically processed to yield molecules that can be so incorporated or which work as functionally equivalent analogues of such molecules;
  • R is H, azido or another group which does not allow the 3'-attachment of the next nucleotide, thereby preventing chain elongation of retroviral DNA under the influence of reverse transcriptase; and X represents H, mono, di, tri or other phosphates and analogs thereof.
  • the ribose-based dideoxy-sugar is replaced by a different organic moiety in some of the functionally similar chain-terminating compounds, for example in adenylline, cytallene, and PMEA.
  • the compound of the general formula (1) be AZT or another 2',3'-dideoxy nucleoside or nucleotide.
  • chain-terminator nucleosides are as follows: azidothymidine, 2', 3'-dideoxycytidine, 2', 3'-dideoxyadenosine, 2', 3'-dideoxythymidine, 2', 3'-dideoxyguanosine, 2', 3'-dideoxyinosine and the like.
  • the dideoxynucleotides include mono-, di- and triphosphates of the dideoxynucleosides.
  • potentiating agent refers to dipyridamole (DPM) and to similar agents with overlapping mechanisms of action, including nitrobenzylthioinosine (NBTI); dilazep; lidoflazine; hexabendine; 7-bromo-1,5-dihydro-3, 6-dimethylimidazol-(2, 1, 6) quinazolin-2 (3H)-one, mioflazine and the like.
  • NBTI nitrobenzylthioinosine
  • dilazep dilazep
  • lidoflazine hexabendine
  • 7-bromo-1,5-dihydro-3, 6-dimethylimidazol-(2, 1, 6) quinazolin-2 (3H)-one mioflazine and the like.
  • These agents are inhibitors of nucleoside and nucleobase transport.
  • Overlapping in effect on cyclic AMP phosphodiesterase are papaverine and mopidamol. Preferred among these compounds is DPM
  • the term "synergistic” or “potentiated” effect means that a combination of an antiviral agent and a potentiating agent produces greater antiviral effect than expected from the activity of each component alone.
  • Various cell types such as monocytes, T-lymphocytes, T- lymphocyte tumors, macrophages, and the like could be used as the host cell for tests of viral infection or replication.
  • the viral activity, particularly of the HIV can be tested by p24 production, reverse transcriptase activity, trans-activator function, or other marker antigen production. Use of a particular cell type or method of measuring viral activity is not the critical part of the invention and either in vitro or in vivo systems can be used for the testing of viral, activity.
  • the significant part of the invention is the discovery that a potentiated antiviral effect is produced by chain terminators and by the ⁇ -glucosidase inhibitor castanospermine when administered with a nontoxic dosage of DPM or one of the other potentiating agents mentioned above. It is also significant that DPM does not potentiate the activity of AZT against human bone marrow cells in vitro. Bone marrow suppression is known to be the dose-limiting toxicity of AZT in humans. It may further be noted that the chain terminator and/or potentiating components can be administered in combination either as such or carried by liposomes or other delivery vehicles. Methods of preparing liposomes are well known to one of ordinary skill in the art and such techniques are not a critical part of the present invention. The potentiating agent can be administered as such or in a polymeric form.
  • Dipyridamole 2,6-bis-diethanolamino-4,8-di-piperidinopyrimido-(5,4d)-pyrimidine] was obtained from Sigma
  • AZT was prepared by Ash Stevens (Lot #HLR 0221) and obtained through the Developmental Therapeutics Branch, AIDS Program, NIAID.
  • ddC and ddCTP were obtained from Moravek Biochemicals (Brea, CA). 3 H-ddCTP was repurified by HPLC as described below.
  • Nitrobenzylthioinosine (NBTI; also abbreviated NBMPR) was obtained from Aldrich, Milwaukee, Wisconsin, and papaverine was obtained from Sigma. St. Louis, Missouri. Adherence-Purified Monocvte/Macrophages. The cells were prepared and infected with HIV-l NIH/USA/1985/HTLV-III BaL , an HIV-1 isolate recovered from and propagated in M/M, as described by Gartner et al (1986, Science 233, 215-219). In brief, peripheral blood mononuclear leukocytes were obtained from leukapheresed healthy, HIV-1 antibody-negative donors by Ficoll-Hypaque-separation.
  • the purified cells were cryopreserved according to standard procedures in RPMI 1640 containing 20% heat-inactivated fetal calf serum (FCS) and 10% (final concentration) of DMSO. Prior to infection, the cells were thawed, washed to remove DMSO. and suspended in
  • RPMI 1640 supplemented with 10% ppoled human serum, 20% FCS and antibiotics. Infection was done by exposing 10 7 pelleted cells for 45-60 min at 37oC to 1 ml of virus inoculum containing 0.5-1.0 ⁇ 10 6 cpm RT activity. After infection, the cells were washed and seeded in 96-well Costar microtiter plates (8 ⁇ 10 4 cells/well) in RPMI 1640 culture medium supplemented with 20% heat-inactivated FCS and antibiotics. Elutriated Monocvte/Macrophages. Peripheral blood monocytes were obtained by standard counterflow centrifugal elutriation (Wahl et al, 1984, Cell Immunol.
  • ddCTP was incubated with uninfected, elutriated M/M (5 ⁇ 10 6 cells/well) under conditions mimicking those of the viral studies.
  • Samples of the supernatants were centrifuged through Centrifree filters (Amicon Co., Danvers, MA) and analyzed for ddCTP metabolites by HPLC, using a VYDAC 303NT405 nucleotide column (Separations Group, Hesperia, CA). The column was eluted with a 0-50% linear gradient of 0.035 mM ammonium formate pH 4.65 and 0.5 M sodium phosphate pH 2.8 (formed over 10 min).
  • ddCTP Cellular uptake of ddCTP was assessed under similar conditions by incubating 1 ⁇ M a H-labeled ddCTP with uninfected, elutriated M/M cultures (5 ⁇ 10 6 cells/well), followed by thorough washing of the cells (4 times with ice-cold saline), detachment from the plates (with 0.5% Triton X-100), and measurement of cell-associated radioactivity.
  • the supernatants were removed and the wells quickly washed 3 times with iced DMEM containing 20 ⁇ M DPM (to block further transport). 0.9 ml of 0.5% Triton X-100 was then added to each well, and the plates were shaken for 30 minutes at room temperature. The contents were transferred to 1.5 ml polypropylene centrifuge tube (Eppendorf), and 0.1 ml of 50% trichloroacetic acid was added. The tubes were then spun in a Microfuge at maximum speed for 10 minutes. The supernatants were collected, neutralized with 1.2 ml 20% trin-octylamine (in trichlorotrifluoroethane), and vortex-mixed.
  • aqueous phase was transferred to another tube and lyophilized.
  • the sample was then redissolved in 120 ⁇ l of distilled water and vortexmixed.
  • Samples (100 ⁇ l) were assayed by HPLC using the VYDAC column and elution scheme described above. Fifty fractions from each run were counted in scintillation vials. Generally, duplicate wells were harvested and processed separately for each time point.
  • CFU GM granulocyte/monocyte colony formation
  • Cells of the CD4+ CEM-SS and MT-2 T-lyraphoblastoid lines were grown in RPMI-1640 containing 10% FCS. The cells were then infected either with free HIV-1(III b ) stock or with HIV-1 (RF)-infected H9 cells and seeded in 96-well microtiter plates. After 7 days in culture, 50 ml of solution containing the tetrazolium salt "XTT" (1 mg/ml) and the electron acceptor phenazine methosulfate (0.01-0.02 mM) was added to each well. Uninfected cells or cells which are protected by drugs and have continued to proliferate produce a soluble orange formazan whose O.D.
  • PHA-8timulated human T-lymphocytes Mononuclear cells from a healthy volunteer were grown in the presence of phytohemagglutinin (PHA) (5 ⁇ g/ml) for 2 days and then stimulated with IL2 in RPMI 1640 with 15% FCS, 1% L-glutamine, and 0.1% gentamycin. After 3 days the cells were infected with various titers of HIV-1(III B ) for 90 minutes at
  • the 50% inhibitory doses (ID 50 ) for the tested drugs were calculated fay plotting log[f a / (1-f a ) ] versus log D (median-effect plot), where f a is the fraction affected (i.e., percent inhibition /100), and D is the drug dose [Chou et al, 1984, Adv. Enzvme Regul. 22, 27-55; Chou et al, 1987) in New Avenues in Developmental Cancer Chemotherapy, eds. Harrap, K. R. & Connors, T.A. (Bristol Myers Symposium Series, Academic Press, N.Y.), pp. 37-64].
  • AZT effectively inhibited viral replication in cryopreserved, adherence-purified cells. Dipyridamole in the 0.08-10 ⁇ M range had little effect by itself but unexpectedly it greatly potentiated the antiviral efficacy of AZT. In the presence of 2 ⁇ M DPM. for example, the ID 50 and ID 95 levels of AZT (Table 1) were decreased by more than 5-fold and 10-fold, respectively (i.e., to 18 and 8% of the values for AZT alone).
  • Fig. 1B shows that the potentiating influence of DPM, in relative terms, was even more pronounced in elutriated M/M cultures.
  • p24 expression was near baseline levels at each AZT concentration including the lowest, 1.6 nM. This degree of inhibition was achieved only at 1 ⁇ M level when AZT was used alone. Dipyridamole also unexpectedly potentiated the anti-HIV effects of ddC and 2',3'-dideoxycytidine triphosphate (ddCTP). The ID 95 values for these drugs decreased at least 5-fold in the presence of > 2 ⁇ M DPM (Fig. 1C-D). In the elutriated M/M system, DPM by Itself appeared to be Inhibitory, causing a significant and unanticipated decrease in p24 expression (Eig. 2).
  • Toxicity for monocvte/macrophages Cell counting in the culture wells with an inverted microscope showed no consistent differences between control and treatment groups for uninfected cells two weeks after treatment with AZT and/or DPM. Cell viability of infected M/M cells, as evaluated by trypan blue exclusion, was >95% in each of the groups.
  • Table 2 shows the 0 2 - production by uninfected M/M treated with AZT and/or DPM. The principal significance of these data is that they showed no discernible
  • Fig. 3A the C.I. values obtained from 6 donors over a wide range of inhibition levels clustered near 1 in the "mutually exclusive" model, suggesting essentially additive toxicities.
  • the C.I. values are predominantly >1 in Fig. 3B suggesting an unexpected trend toward antagonism of the toxicities, rather than toward synergy.
  • DPM has an intrinsic effect against HIV and that it significantly potentiates the antiviral action of AZT, ddC and ddCTP in human monocyte/macrophages.
  • DPM did not potentiate the marrow toxicity of AZT. and the in vitro therapeutic index was therefore unexpectedly increased.
  • DPM potentiated the anti-HIV activity of ddC, which enters cells primarily via DPM-sensitive nucleoside transport (Zimmerman et al, 1987, J. Biol. Chem. 262. 5748-5754).
  • DPM-sensitive nucleoside transport Zimmerman et al, 1987, J. Biol. Chem. 262. 5748-5754.
  • ddC is more llpophilic than dC, the physiological nucleoside, because it has one less hydroxyl group. Therefore, a greater inhibitory effect of DPM on the transport of dC might explain the potentiation of ddC activity.
  • Results shown in Figure 4A indicate a beneficial effect of DPM on the AZT treatment of T-lymphoblastoid cell line (CEM-SS); DPM diminished the toxicity of AZT in these cells while increasing or not affecting its activity against HIV-1 (HTLV-III RF ). Consequently, the in vitro therapeutic index increased in an unanticipated manner.
  • MT-2 cells another T-lymphoblastoid cell l ine, were more sens itive to toxic effects of both AZT and DPM.
  • Results presented in Figure 4B also show a surprising potentiation of castanospermine's anti-HIV activity in CEM-SS cells at high concentrations of DPM .
  • FIGS 5A and 5B show results obtained in experiments for a H-thymidine and for a H-AZT. DPM greatly decreased the appearance of phosphorylated thymidine species. whereas it had only a minor effect on the appearance of phosphorylated
  • analogues of DPM were also evaluated for their intrinsic activity against HIV and for their ability to potentiate the activity of the chain-terminating antiviral agent AZT.
  • the nucleoside transport inhibitor NBTI had an intrinsic inhibitory effect on HIV-1 replication in human M/M and also potentiated the inhibitory effect of AZT.
  • papaverine gave similar results.
  • mioflazine also had an intrinsic inhibitory effect and potentiated the inhibitory effect of AZT.
  • Mioflazine like DPM, inhibits nucleoside transport, but it has the possible advantage of crossing the blood-brain barrier (Deckert et al,1988, Life Sciences 42, 1331-1345; Wauquier et al, 1987, Psychopharm. 91, 434-439) more readily than does DPM.
  • DPM blood-brain barrier
  • DPM or its analogues potentiate or act synergistically is most likely due to the well-established activities of DPM in blocking nucleoside transport and/or inhibiting cyclic AMP phosphodiesterase activity.
  • Plasma concentrations of DPM in excess of 10 ⁇ M can be sustained in humans. It is important to note, therefore, that the studies described in Example 1 demonstrated potentiation of dideoxynucleoside-mediated HIV inhibition at DPM concentrations of 2 ⁇ M and in some experiments much less (in 10%-20% fetal calf serum). DPM is largely protein-bound in blood. Hence, the free drug level at a given overall concentration is expected to be higher in tissue culture experiments than in vivo. The other potentiating agents are employed similarly to DPM.
  • the present invention now provides a chemotherapeutic method of treating AIDS or other viral diseases.
  • the method comprises administering to a host afflicted with viral infection, including AIDS-virus infection, an effective amount of the drug-combination of the present invention to inhibit replication of the disease-causing virus.
  • the drug- combination can be administered by any suitable route such as oral, parenteral, intrathecal or the like and administered as often per day as tolerated. Uninfected individuals or those exposed to the virus can be treated in like manner to prevent infection.
  • Antiviral agents such as AZT, ddC and the like, can be administered in combination or in alternating schedule along with DPM and/or its analogues.
  • Table 1 Levels of AZT eliciting 50% and 95% Inhibition of HlV-1 p24 antigen expression in adherence-purifiedhuman WM cultures, in the presence andabsence ofDPM.
  • ID 50 and ID 95 values were calculated from the equations of the medianeffect plots (see Methods for data analysis), r, linear regression coefficients for the plots. The percentages in brackets relate the ID 50 and ID 95 values to those of AZT alone.
  • Table20 The effect of AZT and DPM on superoxide. (O 2 -) production in elutriated human M/M cultures. Superoxide production (on day 14 of culturing) is taken as a functional index of cell health. Experimental details were the same as in Fig.1B-D, exceptthatthe cells were not infected with HIV-1. Values are expressed as nmole O 2 -/weII. Means of quadruplicate wells ⁇ S.E.M. The data were analyzed by the method of Jonckheere (30) for non-parametric testing of trends of ordered alternatives.
  • the present invention is related to treating or preventing AIDS or other similar viral diseases. More particularly, the present invention is related to a combination of an antiviral agent and a potentiating agent, the combination being more effective against virus than either agent used alone.
  • HIV human immunodeficiency virus
  • chain-terminator compounds such as azidothymidine (AZT), other dideoxynucleosides, dideoxynucleotides, and their analogs have a degree of efficacy against the AIDS virus (Mitsuya, et al. 1985, PNAS, USA. 82:7096; Yarchoan, et al, 1988, Lancet(i) 76-80; Dahlberg et al, 1987, PNAS, USA., 84: 2469). They appear to be phosphorylated within cells and, in the triphosphate form, to terminate DNA chains being formed in the presence of viral reverse transcriptase. These drugs may act by additional mechanisms as well, for example, by competing for the enzymes active in phosphorylation of physiological nucleosides and nucleotides.
  • Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that modifies glycosylation by inhibiting ⁇ -glycosidase I.
  • Other compounds of the same group referred to as trimming glycosidase inhibitors because they inhibit the normal removal of glucose residues during the processing of glycoproteins
  • DMDP dihydroxymethyldihydroxypyrrolidine
  • DNJ or DNM deoxynojirimycin
  • Dipyridamole also called Persantine
  • DPM Dipyridamole
  • cAMP phosphodiesterase to alter prostaglandin production, to alter adenosine levels and CD4 expression, to alter cell surface properties, and to stimulate interferon production.
  • DPM has also been reported to 'have activity against some viruses, but activity either against the AIDS virus or against retroviruses has not been reported (Tonew et al, 1977. Acta Virol .21: 146-150). Moreover, a synergistic combination of DPM or a similar potentiating agent with an antiviral drug has not heretofore been known or described.
  • an object of the present invention to provide a drug combination with synergistic advantage as inhibitor of HIV and other retrovirus activity compared to each drug used alone. It is a further object of the present invention to provide a method of arresting HIV proliferation, comprising contacting HIV infected cells by the drug-combination of the present invention in an amount sufficient to prevent HIV replication. It is yet another object of the present invention to provide a method of treating or preventing AIDS and other
  • HIV-related syndromes comprising administering to a host infected or uninfected with the AIDS virus sufficient amount of the drug-combination of the present invention to inhibit AIDS-virus activity.
  • DPM dideoxynucleosides. on HIV-1 replication in human monocyte/macrophage (M/M) cultures.
  • AZT 0 denotes DPM treatment alone. The data show that DPM potentiates the antiviral action of AZT. Days 6 and 11 showed qualitatively similar results.
  • B Experiment similar to that in A, except that elutriated monocytes were used. The cells were treated with the drugs on day 1 of culturing and were re-fed by replacing 100 ⁇ l of medium (without drugs) at 3-4 day Intervals. The data show p24 levels on day 17 and demonstrate DPM's antiviral activity and potentiation of antiviral activity of AZT. The pattern of effects was similar on day 14.
  • C and D From the same experiment as in B but using 2',3'-dideoxycytidine (ddC) and 2',3'-dideoxycytidine triphosphate (ddCTP), respectively.
  • ddC 2',3'-dideoxycytidine
  • ddCTP 2',3'-dideoxycytidine triphosphate
  • Figure 2 shows the results of time course study of HIV-1 infection In elutriated M/M treated with DPM alone. Experiment similar to that in Fig. 1B-D. The cells were treated on day 1, and samples of culture supernatant were taken for HIV-1 p 24 assay at the time points shown (i.e., at each re-feeding). Samples consisted of 100 ⁇ l out of a total volume of 200 ⁇ l in the well, and cumulative p24 production was calculated by correcting for antigen removed at prior time points. It is noted that DPM alone exhibited antiviral activity. Means of quadruplicate wells ⁇ S.E.M.
  • Figure 3 shows the interaction between AZT and DPM in their cytotoxic effects on human bone marrow granulocyte-monocyte precursors.
  • the CFU GM assay, calculation of the combination index (C.I.), and other experimental details are described herein infra.
  • Panels A and B show the C.I. values calculated on the basis of the mutually exclusive model (identical site/mechanism of action) and the mutually non-exclusive model (non-identical sites/mechanisms of action), respectively.
  • the different symbols indicate cells from different healthy donors.
  • the results show no synergy between AZT and DPM in bone marrow toxicity.
  • FIG. 4 shows the effect of combination chemotherapy with DPM-AZT (panel A) and DPM-castanospermine (panel B) in T-lymphoblastoid cells (CEM-SS) infected with HIV-1.
  • Panel A shows the results when the cells were infected by cocultivation with H9 cells carrying HIV-1(III B ). Treatment was begun on day 1.
  • Panel B shows the result of an analogous study for DPM and castanospermine. At high concentrations, DPM potentiated the antiviral effect of castanospermine. This result emphasizes that a wide range of agents may interact favorably with DPM against HIV and other viruses.
  • FIGS. 5A and 5B give HPLC profiles showing the effect of DPM on intraceilular accumulation of phosphorylated derivatives of a H-thymidine and a H-AZT during 6-hour incubations at 37°C with elutriated human monocyte/macrophages.
  • DPM greatly decreased the appearance of phosphorylated thymidine (THY) but only slightly reduced that of phosphorylated AZT.
  • Figure 6 shows the Inhibition of HIV-1 in human M/M by AZT In combination with nitrobenzylthioinosine (NBTI), like
  • DPM an inhibitor of nucleoside transport.
  • the experiment was performed essentially as for Figure 1A except that the adherence-purified M/M cultures were prepared from freshly drawn donor blood, not cryopreserved cells. At the concentrations tested NBTI inhibited virus replication and potentiated the anti-HIV activity of AZT. Mean ⁇ S.E.M. of quadruplicate wells on day 11.
  • FIG. 7 shows the inhibition of HIV-1 in human M/M by
  • Figure 8 shows the inhibition of HIV-1 in human M/M by AZT in combination with mioflazine: Like DPM. mioflazine is an inhibitor of nucleoside transport. The experiment was performed as was the experiment for Figure 6. At the concentrations tested, mioflazine inhibited virus replication and potentiated the anti-HIV activity of AZT. MF: mioflazine. The pattern of eff-ects was similar on days 13 and 21. Mean ⁇ S.E.M. of quadruplicate wells. DETAILED DESCRIPTION OF THE INVENTION
  • a pharmaceutical composition comprising an effective amount of an antiviral agent and an effective amount of a potentiating agent to inhibit viral replication in infected cells, wherein a combination of the antiviral and the potentiating agent produces greater viral inhibition than expected from the activity of each component alone, and pharmaceutically acceptable carrier.
  • Retroviruses or diseases caused thereby, against which the composition and the methods of the present invention can be used include HIV-1, HIV-2, HTLV-I , HTLV-II and .the like.
  • the composition may include such additives as sterilants, adjuvants, non-toxic sterile buffers and the like, as are commonly used in such preparations and which are well known to one of ordinary skill in the art.
  • chain terminating drug refers to a compound or a precursor thereof, which, when inserted into an elongating nucleic acid chain, terminates the growth of the chain.
  • These drugs may act by additional mechanisms as well, for example, by competing for the enzymes active in phosphorylation of physiological nucleosides and nucleotides.
  • Base is selected from the group consisting of adenine, guanine, cytosine, thymine and analogues, derivatives or salts thereof which can be incorporated into growing DNA chains, which are metabolically processed to yield molecules that can be so incorporated or which work as functionally equivalent analogues of such molecules;
  • R is H, azido or another group which does not allow the 3'-attachment of the next nucleotide, thereby preventing chain elongation of retroviral DNA under the influence of reverse transcriptase; and X represents H, mono, di, tri or other phosphates and analogs thereof.
  • the ribose-based dideoxy-sugar is replaced by a different organic moiety in some of the functionally similar chain-terminating compounds, for example in adenylline, cytallene, and PMEA.
  • chain-terminator nucleosides are as follows: azidothymidine, 2', 3'-dideoxycytidine, 2', 3'-dideoxyadenosine, 2', 3'-dideoxythymidine, 2', 3'-dideoxyguanosine, 2', 3'-dide ⁇ xyinosine and the like.
  • the dideoxynucleotides include mono-, di- and triphosphates of the dideoxynucleosides.
  • potentiating agent refers to dipyridamole (DPM) and to similar agents with overlapping mechanisms of action, including nitrobenzylthioinosine (NBTI); dilazep; lidoflazine; hexabendine; 7-bromo-1,5-dihydro-3, 6-dimethylimidazol-(2, 1, 6) quinazolin-2 (3H)-one, mioflazine and the like.
  • NBTI nitrobenzylthioinosine
  • dilazep dilazep
  • lidoflazine hexabendine
  • 7-bromo-1,5-dihydro-3, 6-dimethylimidazol-(2, 1, 6) quinazolin-2 (3H)-one mioflazine and the like.
  • These agents are inhibitors of nucleoside and nucleobase transport.
  • Overlapping in effect on cyclic AMP phosphodiesterase are papaverine and mopidamol. Preferred among these compounds is DPM
  • the term "synergistic” or “potentiated” effect means that a combination of an antiviral agent and a potentiating agent produces greater antiviral effect than expected from the activity of each component alone.
  • Various cell types such as monocytes, T-lymphocytes, T-lymphocyte tumors, macrophages, and the like could be used as the host cell for tests of viral infection or replication.
  • the viral activity, particularly of the HIV can be tested by p24 production, reverse transcriptase activity, trans-activator function, or other marker antigen production. Use of a particular cell type or method of measuring viral activity is not the critical part of the invention and either in vitro or in vivo systems can be used for the testing of viral activity.
  • the significant part of the invention is the discovery that a potentiated antiviral effect is produced by chain terminators and by the ⁇ -glucosidase inhibitor castanospermine when administered with a nontoxic dosage of DPM or one of the other potentiating agents mentioned above. It is also significant that DPM does not potentiate the activity of AZT against human bone marrow cells in vitro. Bone marrow suppression is known to be the dose-limiting toxicity of AZT In humans. It may further be noted that the chain terminator and/or potentiating components can be administered in combination either as such or carried by liposomes or other delivery vehicles. Methods of preparing liposomes are well known to one of ordinary skill in the art and such techniques are not a critical part of the present invention. The potentiating agent can be administered as such or in a polymeric form.
  • Dipyridamole 2,6-bis-diethanolamino-4,8-di-piperidinopyrimido-(5,4d)-pyrimidine] was obtained from Sigma
  • AZT was prepared by Ash Stevens (Lot #HLR 0221) and obtained through the Developmental Therapeutics Branch, AIDS Program, NIAID.
  • ddC and ddCTP were obtained from Moravek Biochemicals (Brea, CA).
  • a H-ddCTP was repurified by HPLC as described below.
  • Nitrobenzylthioinosine (NBTI; also abbreviated NBMPR) was obtained from Aldrich, Milwaukee, Wisconsin, and papaverine was obtained from Sigma, St. Louis, Missouri.
  • Adherence-Purified Monocyte/Macrophages The cells were prepared and infected with HIV-1 NIH/USA/1985/HTLV-III BaL , an HIV-1 isolate recovered from and propagated in M/M, as described by Gartner et al (1986, Science 233, 215-219).
  • peripheral blood mononuclear leukocytes were obtained from leukapheresed healthy, HIV-l antibody-negative donors by Ficoll-Hypaque-separation. Purification by overnight adherence yielded M/M populations with >95% non-specific esterase-positive cells.
  • the purified cells were cryopreserved according to standard procedures in RPMI 1640 containing 20% heat-inactivated fetal calf serum (FCS) and 10% (final concentration) of DMSO. Prior to infection, the cells were thawed, washed to remove DMSO, and suspended In RPMI 1640 supplemented with 10% ppoled human serum. 20% FCS and antibiotics. Infection was done by exposing 10 7 pelleted cells for 45-60 min at 37oC to 1 ml of virus inoculum containing 0.5-1.0 ⁇ 10 6 cpm RT activity.
  • FCS heat-inactivated fetal calf serum
  • D-MEM Dulbecco's modified Eagle's medium
  • ddCTP was incubated with uninfected, elutriated M/M (5 ⁇ 10 6 cells/well) under conditions mimicking those of the viral studies.
  • Samples of the supernatants were centrifuged through Centrifree filters (Amicon Co., Danvers. MA) and analyzed for ddCTP metabolites by HPLC, using a VYDAC 303NT405 nucleotide column (Separations Group, Hesperia, CA). The column was eluted with a 0-50% linear gradient of 0.035 mM ammonium formate pH 4.65 and 0.5 M sodium phosphate pH 2.8 (formed over 10 min).
  • ddCTP Cellular uptake of ddCTP was assessed under similar conditions by incubating 1 ⁇ M a H-labeled ddCTP with uninfected, elutriated M/M cultures (5 ⁇ 10 6 cells/well), followed by thorough washing of the cells (4 times with ice-cold saline), detachment from the plates (with 0.5% Triton X-100), and measurement of cell-associated radioactivity.
  • the supernatants were removed and the wells quickly washed 3 times with iced DMEM containing 20 ⁇ M DPM (to block further transport). 0.9 ml of 0.5% Triton X-100 was then added to each well, and the plates were shaken for 30 minutes at room temperature. The contents were transferred to 1.5 ml polypropylene centrifuge tube (Eppendorf), and 0.1 ml of 50% trichloroacetic acid was added. The tubes were then spun in a Microfuge at maximum speed for 10 minutes. The supernatants were collected, neutralized with 1.2 ml 20% tri-n-octylamine (in trichlorotrifluoroethane), and vortex-mixed.
  • aqueous phase was transferred to another tube and lyophilized.
  • the sample was then redissolved in 120 ⁇ l of distilled water and vortex-mixed.
  • Samples (100 ⁇ l) were assayed by HPLC using the VYDAC column and elution scheme described above. Fifty fractions from each run were counted in scintillation vials. Generally, duplicate wells were harvested and processed separately for each time point.
  • CFU GM granulocyte/monocyte colony formation
  • Cells of the CD4+ CEM-SS and MT-2 T-lymphoblastoid lines were grown in RPMI-1640 containing 10% FCS. The cells were then infected either with free HIV-1(III b ) stock or with HIV-1 (RF ) -infected H9 cells and seeded in 96-well microtiter plates. After 7 days in culture, 50 ml of solution containing the tetrazolium salt "XTT" (1 mg/ml) and the electron acceptor phenazine methosulfate (0.01-0.02 mM) was added to each well. Uninfected cells or cells which are protected by drugs and have continued to proliferate p.roduce a soluble orange formazan whose O.D.
  • PHA-stiouIated human T-lymphocytes Mononuclear cells from a healthy volunteer were grown in the presence of phytohemagglutinin (PHA) (5 ⁇ g/ml) for 2 days and then stimulated with IL2 In RPMI 1640 with 15% FCS, 1% L-glutamine, and 0.1% gentamycin. After 3 days the cells were
  • PHA phytohemagglutinin
  • AZT effectively inhibited viral replication in cryopreserved, adherence-purified cells. Dipyridamole in the 0.08-10 ⁇ M range had little effect by itself but unexpectedly it greatly potentiated the antiviral efficacy of AZT. In the presence of 2 ⁇ M DPM, for example, the IDoo and ID 95 levels of AZT (Table 1) were decreased by more than 5-fold and 10-fold, respectively (i.e.. to 18 and 8% of the values for AZT alone).
  • Fig. 1B shows that the potentiating influence of DPM, in relative terms, was even more pronounced in elutriated M/M cultures.
  • p24 expression was near baseline levels at each AZT concentration including the lowest, 1.6 nM. This degree of inhibition was achieved only at 1 ⁇ M level when AZT was used alone. Dipyridamole also unexpectedly potentiated the anti- HIV effects of ddC and 2',3'-dideoxycytidine triphosphate (ddCTP). The ID 95 values for these drugs decreased at least 5-fold in the presence of ⁇ 2 ⁇ M DPM (Fig. 1C-D). In the elutriated M/M system, DPM by itself appeared to be inhibitory, causing a significant and unanticipated decrease In p24 expression (Eig. 2).
  • Toxicity for monocvte/macrophages Cell counting in the culture wells with an inverted microscope showed no consistent differences between control and treatment groups for uninfected cells two weeks after treatment with AZT and/or DPM. Cell viability of infected M/M cells, as evaluated by trypan blue exclusion, was >95% in each of the groups.
  • Table 2 shows the 0 2 - production by uninfected M/M treated with AZT and/or DPM. The principal significance of these data is that they showed no discernible
  • Fig. 3A the C.I. values obtained from 6 donors over a wide range of inhibition levels clustered near 1 in the "mutually exclusive" model, suggesting essentially additive toxicities.
  • the C.I. values are predominantly > 1 in Fig. 3B suggesting an unexpected trend toward antagonism of the toxicities, rather than toward synergy.
  • DPM has an intrinsic effect against HIV and that it significantly potentiates the antiviral action of AZT, ddC and ddCTP in human monocyte/macrophages.
  • DPM did not potentiate the marrow toxicity of AZT, and the in vitro therapeutic index was thereJfore unexpectedly increased.
  • DPM potentiated the anti-HIV activity of ddC, which enters cells primarily via DPM-sensitive nucleoside transport (Zimmerman et al, 1987, J. Biol. Chem. 262, 5748-5754).
  • ddC is more lipophilic than dC, the physiological nucleoside, because it has one less hydroxyl group. Therefore, a greater inhibitory effect of DPM on the transport of dC might explain the potentiation of ddC activity.
  • FIGS. 5A and 5B show results obtained in experiments for 3 H-thymidine and for a H-AZT.
  • DPM greatly decreased the appearance of phosphorylated thymidine species, whereas it had only a minor effect on the appearance of phosphorylated AZT.
  • Two independent experiments showed qualitatively similar inhibitory effects of DPM on the intracellular appearance of phosphorylated a H-thymidine .
  • DPM increased or did not change the amount of phosphorylated a H-AZT in the cells.
  • analogues of DPM were also evaluated for their intrinsic activity against HIV and for their ability to potentiate the activity, of the chain-terminating antiviral agent AZT.
  • the nucleoside transport Inhibitor NBTI had an intrinsic inhibitory effect on HIV-1 replication in human M/M and also potentiated the inhibitory effect of AZT.
  • papaverine gave similar results.
  • mioflazine also had an intrinsic inhibitory effect and potentiated the inhibitory effect of AZT.
  • Mioflazine like DPM, Inhibits nucleoside transport, but it has the possible advantage of crossing the blood-brain barrier (Deckert et al,1988.
  • DPM or its analogues potentiate or act synergistically is most likely due to the well-established activities of DPM in blocking nucleoside transport and/or inhibiting cyclic AMP phosphodiesterase activity.
  • Plasma concentrations of DPM in excess of 10 ⁇ M can be sustained in humans. It is Important to note, therefore, that the studies described in Example 1 demonstrated potentiation of dideoxynucleoside-mediated HIV inhibition at DPM concentrations of 2 ⁇ M and in some experiments much less (in. 10%-20% fetal calf serum). DPM is largely protein-bound in blood. Hence, the free drug level at a given overall concentration is expected to be higher in tissue culture experiments than in vivo. The other potentiating agents are employed similarly to DPM.
  • the present Invention now provides a chemotherapeutic method of treating AIDS or other viral diseases.
  • the method comprises administering to a host afflicted with viral infection, including AIDS-virus infection, an effective amount of the drug-combination of the present invention to inhibit replication of the disease-causing virus.
  • the drug-combination can be administered by any suitable route such as oral, parenteral, intrathecal or the like and administered as often per day as tolerated. Uninfected individuals or those exposed to the virus can be treated in like manner to prevent infection.
  • Antiviral agents such as AZT, ddC and the like, can be administered in combination or in alternating schedule along with DPM and/or its analogues.
  • Table 1 Levels of AZT eliciting 50% and 95% inhibition of HIV-1 p24 antigen expression in adherence-purifiedhuman M/M cultures, In the presence and absence ofDPM. ID 50 and ID 95 values were calculated from the equations of the medianeffect plots (see Methods for data analysis). r, linear regression coefficients for the plots. Trie percentages in brackets relate the ID 50 and ID 95 values to those of AZT alone.
  • the present invention is related to treating or preventing AIDS or other similar viral diseases. More particularly, the present invention is related to a combination of an antiviral agent and a potentiating agent, the combination being more effective against virus than either agent used alone.
  • HIV human immunodeficiency virus
  • chain-terminator compounds such as azidothymidine (AZT), other dideoxynucleosides, dideoxynucleotides, and their analogs have a degree of efficacy against the AIDS virus (Mitsuya, et al. 1985, PNAS, USA. 82:7096; Yarchoan, et al. 1988. Lancet(i) 76-80; Dahlberg et al. 1987. PNAS. USA., 84: 2469). They appear to be phosphorylated within cells and, in the triphosphate form, to ⁇ terminate DNA chains being formed in the presence of viral reverse transcriptase. These drugs may act by additional mechanisms as well. for example, by competing for the enzymes active in phosphorylation of physiological nucleosides and nucleotides.
  • Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that modifies glycosylation by inhibiting ⁇ -glycosidase I.
  • Other compounds of the same group referred to as tr immi ng glycosidase inhibitors because they inhibit the normal removal of glucose residues during the processing of glycoproteins
  • DMDP dihydroxymethyldihydroxypyrrolidine
  • DNJ or DNM deoxynoj irimycin
  • Dipyridamole also called Persantine.
  • DPM Dipyridamole
  • cAMP phosphodiesterase to alter prostaglandin production, to alter adenosine levels and CD4 expression, to alter cell surface properties, and to stimulate interferon production.
  • DPM has also been reported to 'have activity against some viruses, but activity either against the AIDS virus or against retroviruses has not been reported (Tonew et al, 1977. Acta Virol .21: 146-150). Moreover, a synergistic combination of DPM or a similar potentiating agent with an antiviral drug has not heretofore been known or described.
  • an object of the present invention to provide a drug combination with synergistic advantage as inhibitor of HIV and other retrovlrus activity compared to each drug used alone. It is a further object of the present invention to provide a method of arresting HIV proliferation, comprising contacting HIV infected cells by the drug-combination of the present invention in an amount sufficient to prevent HIV replication. It is yet another object of the present invention to provide a method of treating or preventing AIDS and other
  • HIV-related syndromes comprising administering to a host infected or uninfected with the AIDS virus sufficient amount of the drug-combination of the present invention to inhibit AIDS-vlrus activity.
  • Figure 1 demonstrates the effect of dipyridamole (DPM). alone and in combination with chain-terminating dideoxynucleosides. on HIV-l replication in human monocyte/macrophage (M/M) cultures.
  • DPM dipyridamole
  • A Adherence-purified, cryopreserved monocyte/macrophages treated with AZT alone, DPM alone, or AZT plus DPM. The cells were treated on day 1 and re-fed on days 6, 11, and 14 with medium containing the appropriate drug concentrations. 100- ⁇ l samples of supernatant were analyzed for HIV-1 p24. The figure shows the results on day 14.
  • AZT 0 denotes DPM treatment alone. The data show that DPM potentiates the antiviral action of AZT.
  • Figure 2 shows the results of time course study of HIV-1 infection in elutriated M/M treated with DPM alone.
  • Figure 3 shows the interaction between AZT and DPM in their cytotoxic effects on human bone marrow granulocyte-monocyte precursors.
  • the CFU GM assay, calculation of the combination index (C.I.), and other experimental details are described herein infra.
  • Panels A and B show the C.I. values calculated on the basis of the mutually exclusive model (identical site/mechanism of action) and the mutually non-exclusive model (non-identical sites/mechanisms of action), respectively.
  • the different symbols indicate cells from different healthy donors.
  • the results show no synergy between AZT and DPM in bone marrow toxicity.
  • FIG. 4 shows the effect of combination chemotherapy with DPM-AZT (panel A) and DPM-castanospermine (panel B) in
  • T-lymphoblastoid cells infected with HIV-1.
  • Panel A shows the results when the cells were infected by cocultivation with H9 cells carrying HIV-1(III B ). Treatment was begun on day 1. In this and other similar experiments the effect of DPM on antiviral efficacy of AZT was moderate, but there was striking protection of the cells from AZT toxicity. Hence, the in vitro therapeutic index was greatly increased.
  • Panel B shows the result of an analogous study for DPM and castanospermine. At high concentrations, DPM potentiated the antiviral effect of castanospermine. This result emphasizes that a wide range of agents may interact favorably with DPM against HIV and other viruses.
  • Figure 5 gives HPLC profiles showing the effect of DPM on intracellular accumulation of phosphorylated derivatives of a H-thymidine and a H-AZT during 6-hour incubations at 37°C with elutriated human monocyte/macrophages.
  • DPM greatly decreased the appearance of phosphorylated thymidine (THY) but only slightly reduced that of phosphorylated AZT.
  • Figure 6 shows the inhibition of HIV-l in human M/M by AZT in combination with nitrobenzylthioinosine (NBTI). like
  • DPM an inhibitor of nucleoside transport.
  • the experiment was performed essentially as for Figure 1A except that the adherence-purified M/M cultures were prepared from freshly drawn donor blood, not cryopreserved cells. At the concentrations tested NBTI inhibited virus replication and potentiated the anti-HIV activity of AZT. Mean ⁇ S.E.M. of quadruplicate wells on day 11.
  • Figure 7 shows the inhibition of HIV-1 in human M/M by AZT in combination with papaverine.
  • Papaverine (PA) shares with DPM the ability to inhibit activity of cellular cyclic AMP phosphodiesterase. The experiment was performed as for Figure 6. At the concentrations tested papaverine inhibited virus replication and potentiated the anti-HIV activity of AZT. The pattern of effects was similar on days 13 and 21. Mean ⁇ S.E.M. of quadruplicate wells on day 11.
  • Figure 8 shows the inhibition of HIV-1 in human M/M by AZT in combination with mioflazine.
  • mioflazine is an inhibitor of nucleoside transport. The experiment was performed as was the experiment for Figure 6. At the concentrations tested, mioflazine Inhibited virus replication and potentiated the anti-HIV activity of AZT. MF: mioflazine. The pattern of effects was similar on days 13 and 21. Mean ⁇ S.E.M. of quadruplicate wells. DETAILED DESCRIPTION OF THE INVENTION
  • a pharmaceutical composition comprising an effective amount of an antiviral agent and an effective amount of a potentiating agent to inhibit viral replication in infected cells, wherein a combination of the antiviral and the potentiating agent produces greater viral inhibition than expected from the activity of each component alone, and pharmaceutically acceptable carrier.
  • Retroviruses or diseases caused thereby, against which the composition and the methods of the present invention can be used include HIV-1, HIV-2, HTLV-I. HTLV-II and the like.
  • the composition may include such additives as sterilants, adjuvants, non-toxic sterile buffers and the like, as are commonly used in such preparations and which are well known to one of ordinary skill in the art.
  • chain terminating drug refers to a compound or a precursor thereof, which, when inserted into an elongating nucleic acid chain, terminates the growth of the chain.
  • These drugs may act by additional mechanisms as well, for example, by competing for the enzymes active in phosphorylation of physiological nucleosides and nucleotides.
  • Base is selected from the group consisting of adenine, guanine, cytosine, thymine and analogues, derivatives or salts thereof which can be incorporated into growing DNA chains, which are metabolically processed to yield molecules that can be so incorporated or which work as functionally equivalent analogues of such molecules:
  • R is H, azido or another group which does not allow the 3'-attachment of the next nucleotide. thereby preventing chain elongation of retroviral DNA under the influence of reverse transcriptase; and X represents H, mono, di, tri or other phosphates and analogs thereof.
  • the ribose-based dideoxy-sugar is replaced by a different organic moiety in some of the functionally similar chain-terminating compounds, for example in adenylline, cytallene, and PMEA.
  • chain-terminator nucleosides are as follows: azidothymidine, 2', 3'-dideoxycytidine, 2', 3'-dideoxyadenosine, 2', 3'-dideoxythymidine, 2', 3'-dideoxyguanosine, 2', 3'-dideoxyinosine and the like.
  • the dideoxynucleotides include mono-, di- and triphosphatss of the dideoxynucleosides.
  • potentiating agent refers to dipyridamole (DPM) and to similar agents with overlapping mechanisms of action, including nitrobenzylthioinosine (NBTI); dilazep; lidoflazine; hexabendine; 7-bromo-1,5-dihydro-3, 6-dimethylimidazol-(2, 1, 6) quinazolin-2 (3H)-one, mioflazine and the like.
  • NBTI nitrobenzylthioinosine
  • dilazep dilazep
  • lidoflazine hexabendine
  • 7-bromo-1,5-dihydro-3, 6-dimethylimidazol-(2, 1, 6) quinazolin-2 (3H)-one mioflazine and the like.
  • These agents are inhibitors of nucleoside and nucleobase transport.
  • Overlapping in effect on cyclic AMP phosphodiesterase are papaverine and mopidamol. Preferred among these compounds is DPM
  • the term "synergistic" or “potentiated” effect means that a combination of an antiviral agent and a potentiating agent produces greater antiviral effect than expected from the activity of each component alone.
  • Various cell types such as monocytes, T-lymphocytes. T-lyaphocyte tumors, macrophages, and the like could be used as the host cell for tests of viral infection or replication.
  • the viral activity, particularly of the HIV can be tested by p24 production. reverse transcriptase activity, trans-activator function, or other marker antigen production.
  • Use of a particular cell type or method of measuring viral activity is not the critical part of the invention and either in vitro or in vivo systems can be used for the testing of viral, activity.
  • the significant part of the invention is the discovery that a potentiated antiviral effect is produced by chain terminators and by the ⁇ -glucosida ⁇ e inhibitor castanospermine when administered with a nontoxic dosage of DPM or one of the other potentiating agents mentioned above. It is also significant that DPM does not potentiate the activity of AZT against human bone marrow cells in vitro. Bone marrow suppression is known to be the dose-limiting toxicity of AZT in humans. It may further be noted that the chain terminator and/or potentiating components can be administered in combination either as such or carried by liposomes or other delivery vehicles. Methods of preparing liposomes are well known to one of ordinary skill in the art and such techniques are not a critical part of the present invention. The potentiating agent can be administered as such or in a polymeric form.
  • Dipyridamole 2 ,6-bis-diethanolamino-4,8-di-piperidinopyrimido-(5,4d)-pyrimidine] was obtained from Sigma
  • AZT was prepared by Ash Stevens (Lot #HLR 0221) and obtained through the Developmental Therapeutics Branch. AIDS Program, NIAID.
  • ddC and ddCTP were obtained from Moravek Biochemicals (Brea, CA). a H-ddCTP was repurified by HPLC as described below.
  • Nitrobenzylthioinosine (NBTI; also abbreviated NBMPR) was obtained from Aldrich, Milwaukee. Wisconsin, and papaverine was obtained from Sigma. St. Louis, Missouri.
  • Adherence-Purified Monocyte/Hacro. hage ⁇ The cells were prepared and infected with HIV-l NIH/USA/1985/HTLV-III BaL , an HIV-1 isolate recovered from and propagated in M/M, as described by Gartner et al (1986, Science 233, 215-219).
  • peripheral blood mononuclear leukocytes were obtained from leukapheresed healthy, HIV-1 antibody-negative donors by fe Ficoll-Hypaque-separation. Purification by overnight adherence yielded M/M populations with >95% non-specific esterase-positive cells.
  • the purified cells were cryopreserved according to standard procedures in RPMI 1640 containing 20% heat-inactivated fetal calf serum (FCS) and 10% (final concentration) of DMSO. Prior to infection, the cells were thawed, washed to remove DMSO. and suspended in RPMI 1640 supplemented with 10% pooled human serum. 20% FCS and antibiotics. Infection was done by exposing 10 7 pelleted cells for 45-60 min at 37oC to 1 ml of virus inoculum containing 0.5-1.0 ⁇ 10 6 cpm RT activity.
  • FCS heat-inactivated fetal calf serum
  • D-MEM Dulbecco's modified Eagle's medium
  • ddCTP was incubated with uninfected, elutriated M/M (5 ⁇ 10 6 cells/well) under conditions mimicking those of the viral studies.
  • Samples of the supernatants were centrifuged through Centrifree filters (Amicon Co., Danvers, MA) and analyzed for ddCTP metabolites by HPLC, using a VYDAC 303NT405 nucleotide column (Separations Group, Hesperia, CA). The column was eluted with a 0-50% linear gradient of 0.035 mM ammonium formate pH 4.65 and 0.5 M sodium phosphate pH 2.8 (formed over 10 min).
  • ddCTP Cellular uptake of ddCTP was assessed under similar conditions by incubating 1 ⁇ M a H-labeled ddCTP with uninfected, elutriated M/M cultures (5 ⁇ 10 6 cells/well), followed by thorough washing of the cells (4 times with ice-cold saline), detachment from the plates (with 0.5% Triton X-100), and measurement of cell-associated radioactivity.
  • the supernatants were removed and the wells quickly washed 3 times with iced DMEM containing 20 ⁇ M DPM (to block further transport). 0.9 ml of 0.5% Triton X-100 was then added to each well, and the plates were shaken for 30 minutes at room temperature. The contents were transferred to 1.5 ml polypropylene centrifuge tube (Eppendorf), and 0.1 ml of 50% trichloroacetic acid was added. The tubes were then spun in a Microfuge at maximum speed for 10 minutes. The supernatants were collected, neutralized with 1.2 ml 20% tri-n-octylamine (in trichlorotrifluoroethane), and vortex-mixed.
  • aqueous phase was transferred to another tube and lyophilized.
  • the sample was then redissolved in 120 ⁇ l of distilled water and vortex-mixed.
  • Samples (100 ⁇ l) were assayed by HPLC using the VYDAC column and elution scheme described above. Fifty fractions from each run were counted in scintillation vials. Generally, duplicate wells were harvested and processed separately for each time point.
  • Baseline 0 2 - production was assessed by adding superoxide dismutase (final concentration 600 ug/ml) to the wells to destroy any 0 2 - present; phorbol ester-stimulated production was assessed by adding phorbol myristate acetate (final concentration 10 ng/ml); spontaneous production was determined with no stimulus added.
  • Bone Marrow Cell Toxicity Standard procedures were used for the collection of bone marrow specimens from healthy volunteers and for the granulocyte/monocyte colony formation (CFU GM ) assay as described by Fine et al (1987, Clin. Oncol. 5. 489-495).
  • the cells were separated on Ficoll-Hypaque and suspended in McCoy's 5A medium supplemented with 20% heat-inactivated FCS. 2 mM glutamine. and 15% (v/v) HIV- negative colony stimulating factor (CSF) derived from supernatants of a human myelogenous leukemia line (P-38).
  • the cells were plated by the soft-agar method (Pike et al, 1970, J. Cell Phvsiol. 76, 77-84) at a final concentration of 0.3% agar with 20% FCS and 15% CSF. After 9-12 days of incubation, colonies of >40 cells were counted. Approximately 100 colonies formed from the 2 ⁇ 10 5 mononuclear cells plated.
  • the cells were stained by the method of Kubota et al (1980, Exp. Hematol. 8, 339-344) for morphological examination. For each donor the effects of AZT and DPM alone, as well as 2 to 10 different combinations with AZT/DPM ratios ranging from 0.16 to 20. were tested. Anti-HIV efficacy and cell cytotoxicitv in T-lymphobl astoid cell lines. Cells of the CD4+ CEM-SS and MT-2 T-lymphoblastoid lines were grown in RPMI-1640 containing 10% FCS.
  • the cells were then infected either with free HIV-l(III b ) stock or with HIV-1 (RF)-infected H9 cells and seeded in 96-well microtiter plates. After 7 days in culture, 50 ml of solution containing the tetrazolium salt "XTT" (1 mg/ml) and the electron acceptor phenazine methosulfate (0.01-0.02 mM) was added to each well. Uninfected cells or cells which are protected by drugs and have continued to proliferate produce a soluble orange formazan whose O.D. can be read at 450 nm.
  • XTT tetrazolium salt
  • phenazine methosulfate 0.01-0.02 mM
  • PHA-stimulated human T-lymphocytes Mononuclear cells from a healthy volunteer were grown in the presence of phytohemagglutinin (PHA) (5 ⁇ g/ml) for 2 days and then stimulated with IL2 in RPMI 1640 with 15% FCS, 1% L-glutamine, and 0.1% gentamycin. After 3 days the cells were infected with various titers of HIV-1(III B ) for 90 minutes at
  • the 50% inhibitory doses (ID 50 ) for the tested drugs were calculated by plotting log[f a / ( l-f a ) ] versus log D (median-effect plot), where f a is the fraction affected (i.e., percent inhibition /100), and D is the drug dose [Chou et al, 1984, Adv. Enzyme Regul. 22, 27-55; Chou et al, 1987) in New Avenues in Developmental Cancer Chemotherapy, eds. Harrap, K. R. & Connors, T.A. (Bristol Myers Symposium Series, Academic Press, N.Y.), pp. 37-64].
  • Fig. 1A AZT effectively inhibited viral replication in cryopreserved. adherence-purified cells. Dipyridamole in the 0.08-10 ⁇ M range had little effect by itself but unexpectedly it greatly potentiated the antiviral efficacy of AZT. In the presence of 2 ⁇ M DPM, for example, the ID 50 and ID 95 levels of AZT (Table 1) were decreased by more than 5-fold and 10-fold, respectively (i.e.. to 18 and 8% of the values for AZT alone).
  • Fig. 1B shows that the potentiating influence of DPM, in relative terms, was even more pronounced in elutriated M/M cultures.
  • Table 2 shows the 0 2 - production by uninfected M/M treated with AZT and/or DPM. The principal significance of these data is that they showed no discernible
  • DPM has an intrinsic effect against HIV and that it significantly potentiates the antiviral action of AZT, ddC and ddCTP in human monocyte/macrophages.
  • DPM did not potentiate the marrow toxicity of AZT. and the in vitro therapeutic index was therefore unexpectedly increased.
  • DPM potentiated the anti-HIV activity of ddC, which enters cells primarily via DPM-sensitive nucleoside transport (Zimmerman et al. 1987, J. Blol. Chem. 262. 5748-5754).
  • ddC is more lipophilic than dC, the physiological nucleoside, because it has one less hydroxyl group. Therefore, a greater inhibitory effect of DPM on the transport of dC might explain the potentiation of ddC activity.
  • Results shown in Figure 4A indicate a beneficial effect of DPM on the AZT treatment of T-lymphoblastoid cell line (CEM- SS); DPM diminished the toxicity of AZT in these cells while increasing or not affecting its activity against HIV-l (HTLV- III RF ). Consequently, the in vitro therapeutic index increased in an unanticipated manner.
  • MT-2 cells another T- lymphoblastoid cell line, were more sensitive to toxic effects of both AZT and DPM.
  • Results presented in Figure 4B also show a surprising potentiation of castanospermine's anti-HIV activity in CEM-SS cells at high concentrations of DPM .
  • FIG. 5 shows results obtained in experiments for a H-thymidine and for a H-AZT.
  • DPM greatly decreased the appearance of phosphorylated thymidine species. whereas it had only a minor effect on the appearance of phosphorylated AZT.
  • Two independent experiments showed qualitatively similar inhibitory effects of DPM on the intracellular appearanpe of phosphorylated a H-thymidine.
  • DPM increased or did not change the amount of phosphorylated a H-AZT in the cells.
  • analogues of DPM were also evaluated for their intrinsic activity against HIV and for their ability to potentiate the activity of the chain-terminating antiviral agent AZT.
  • the nucleoside transport inhibitor NBTI had an intrinsic inhibitory effect on HIV-1 replication in human M/M and also potentiated the inhibitory effect of AZT.
  • papaverine gave similar results.
  • mioflazine also had an intrinsic inhibitory effect and potentiated the inhibitory effect of AZT.
  • Mioflazine like DPM, inhibits nucleoside transport, but it has the possible advantage of crossing the blood-brain barrier (Deckert et al, 1988. Life Sciences 42.
  • DPM or its analogues potentiate or act synergistically is most likely due to the well-established activities of DPM in blocking nucleoside transport and/or inhibiting cyclic AMP phosphodiesterase activity.
  • Plasma concentrations of DPM in excess of 10 ⁇ M can be sustained in humans. It is important to note, therefore, that the studies described in Example 1 demonstrated potentiation of dideoxynucleoside-mediated HIV inhibition at DPM concentrations of 2 ⁇ M and in some experiments much less (in 10%-20% fetal calf serum). DPM is largely protein-bound in blood. Hence, the free drug level at a given overall concentration is expected to be higher in tissue culture experiments than in vivo. The other potentiating agents are employed similarly to DPM.
  • the present invention now provides a chemotherapeutic method of treating AIDS or other viral diseases.
  • the method comprises administering to a "host afflicted with viral infection, including AIDS-virus infection. an effective amount of the drug-combination of the present invention to inhibit replication of the disease-causing virus.
  • the drug-combination can be administered by any suitable route such as oral, parenteral. intrathecal or the like and administered as often per day as tolerated. Uninfected individuals or those exposed to the virus can be treated in like manner to prevent infection.
  • Antiviral agents such as AZT, ddC and the like, can be administered in combination or in alternating schedule along with DPM and/or its analogues.
  • Table 1 Levels of AZT eliciting 50% and 95% inhibition of HiV-1 p24 antigen expression in adherence-purifiedhuman M/M cultures, in the presence andabsence of DPM. ID 50 and ID 95 values were calculated from the equations of the medianeffect plots (see Methods for data analysis), r, linear regression coefficients for the plots. The percentages in brackets relate the ID 50 and ID 95 values to those of AZT alone.

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Abstract

La présente invention décrit une combinaison d'un médicament synergétique en tant qu'inhibiteur effectif d'une infection virale, en particulier une infection rétrovirale. La combinaison de médicaments peut être un médicament antiviral tel que l'azidothymidine, 21, 31-didesoxycytidine ou son triphosphate, ainsi qu'un agent de potentialisation tel que le dipyridamole. Le médicament antiviral, l'agent de potentialisation ou une combinaison de ceux-ci peuvent être encapsulés dans des liposomes. Le médicament combiné est utile pour le traitement ou la prévention du SIDA et d'autres syndromes en relation avec le virus d'immunodéficience humaine.
EP19890906537 1988-05-16 1989-05-12 Chemotherapeutic composition for aids Ceased EP0416011A4 (en)

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AU654152B2 (en) * 1990-09-06 1994-10-27 Australian National University, The Immunosuppressant composition
US5567689A (en) * 1993-08-13 1996-10-22 The Uab Research Foundation Methods for increasing uridine levels with L-nucleosides
EP1352910A1 (fr) * 2002-04-10 2003-10-15 Grünenthal GmbH Nouveaux derives d'analogues de nitrobenzylthioinosine
EP2248523A1 (fr) * 2009-05-06 2010-11-10 Universität zu Köln Composés à utiliser dans le traitement de conditions cliniques résultant d'un déficit de cellules souches endothéliales

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US4235871A (en) * 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4515736A (en) * 1983-05-12 1985-05-07 The Regents Of The University Of California Method for encapsulating materials into liposomes
US4751224A (en) * 1983-07-21 1988-06-14 Brown University Research Foundation Treatment of metastasis
US4792558A (en) * 1987-05-29 1988-12-20 Merrell Dow Pharmaceuticals Inc. Castanospermine for inhibiting tumor metastasis
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