Comparative kinetic analyses of interaction of inhibitors with Rauscher murine leukemia virus and human immunodeficiency virus reverse transcriptases.

The inhibitory effects of several nucleoside triphosphate analogs on Rauscher murine leukemia virus (RMuLV) and human immunodeficiency virus (HIV) type 1 reverse transcriptases (RTs) were studied. With RNA as the template, the apparent K(m) and apparent K(i) values of HIV RT toward its substrates and inhibitors are 12 to 500 times lower than the corresponding values for RMuLV RT. However, the k(i)/k(m) ratios (inhibition efficiencies) for HIV and RMuLV RTs'are similar for AZTTP (zidovudine triphosphate), d4TTP [3'-deoxythymidine-2'-ene-(3'-deoxy-2',3'-didehydrothymidine) triphosphate], PMEADP [9-(2-phosphonylmethoxyethyl)adenine diphosphate], FIAUTP [1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-iodouracil triphosphate], and HPMPCDP [(S)-1-(3-hydroxy-2-phosphylmethoxypropyl) cytosine diphosphate]. With DNA as the template, the K(m) values are similar for HIV and RMuLV RTs. However, the K(i)/K(m) values of HIV and RMuLV RTs are significantly different for ddCTP, ddATP, and 3TCTP (2',3'-dideoxy-3'-thiacytidine). The RTs of RMuLV and HIV are sufficiently different from one another that the kinetic inhibition constants for a particular antiviral compounds should be determined to indicate whether anti-RMuLV activity is likely to be predictive for the anti-HIV activity of the compound. This information, in conjunction with species-specific drug metabolism differences and tissue culture antiviral activity, is important in determining the suitability of a particular animal model.

Effects of SKF 108922, an HIV-1 protease inhibitor, on retrovirus replication in mice.

Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polypeptides in cultures of human T lymphocytes infected with human immunodeficiency virus type 1 (HIV-1) and therefore suppress the infectivity of HIV-1 in vitro. We have previously reported the antiviral activity in vitro of HIV-1 protease inhibitors against the C-type retrovirus Rauscher murine leukemia virus (RMuLV) and the lentivirus simian immunodeficiency virus (SIV). The same compounds which blocked the infectivity of HIV-1 also inhibited the infectivity of RMuLV and SIV in vitro. This report extends these findings by testing the antiviral activity of HIV-1 protease inhibitors in vivo in the RMuLV model. RMuLV-infected mice were treated twice a day (bid) with either an active (SKF 108922) or inactive (SKF 109273) compound for fourteen days by the intraperitoneal (i.p.) route. Compared with excipient control, SKF 108922, formulated with hydroxypropyl-beta-cyclodextrin (HPB), reduced virus-induced splenomegaly, viremia, and serum reverse transcriptase (RT) levels, while SKF 109273 was inactive. The HPB vehicle by itself enhanced replication of RMuLV. The effects of changing the formulation and the route of administration were examined. SKF 108922, formulated in HPB, had similar antiviral activity when administered by the i.p. or subcutaneous (SC) routes. However, SKF 108922 administered as a colloidal suspension in cholesterol sulfate (CS) had no detectable antiviral effect. Measurements of the circulating levels of the protease inhibitor in plasma explained this result. Plasma concentrations of SKF 108922 exceeded 1000 nM within 10 min after SC administration of the compound solubilized in HPB, but SKF 108922 was not detected in plasma after SC administration of the same dose formulated with CS. Information on optimal conditions for administering these agents should prove useful in guiding their clinical application Therefore, RMuLV should provide a good model for the preclinical evaluation and development of this class of agents for the treatment of HIV.

In vitro and in vivo enhancement of ddI activity against Rauscher murine leukemia virus by ribavirin.

Ribavirin has been reported to enhance the activity of ddI against HIV. We explored this enhancement of antiviral activity in Rauscher murine leukemia virus (RMuLV) models in vitro and in vivo. The significant finding in these studies was that combinations of the drugs exhibited virus titer reductions that were greater than would be expected if the drug interactions were simply additive. These effects were designated synergistic by the method of Prichard and Shipman (Prichard, M.N. and Shipman, C., Jr. (1990). A three-dimensional model to analyze drug-drug interaction, Antiviral Res. 14, 181-206). In addition to the antiviral synergy, we also observed some synergistic toxicity in the animal model.

Phosphatidylazidothymidine and phosphatidyl-ddC: assessment of uptake in mouse lymphoid tissues and antiviral activities in human immunodeficiency virus-infected cells and in Rauscher leukemia virus-infected mice.

During the early stages of human immunodeficiency virus (HIV) infection, although symptoms are absent and viral replication in peripheral blood mononuclear cells is low, substantial levels of HIV replication can be documented in lymphoid tissue [G. Pantaleo, C. Graziosi, J.F. Demarest, L. Butini, M. Montroni, C.H. Fox, J.M. Orenstein, D.P. Kotler, and A.S. Fauci, Nature (London) 362:355-358, 1993, and J. Embretsen, M. Zupancic, J.L. Ribas, A. Burke, P. Racz, K. Tenner-Tacz, and A.T. Haase, Nature (London) 362:359-362, 1993]. This observation suggests that earlier treatment of HIV infection may be indicated and that strategies for enhancing drug targeting to the lymphoid tissue reservoris of HIV infection may be beneficial. To address this issue, we synthesized dioleoylphosphatidyl-ddC (DOP-ddC) and dipalmitoylphosphatidyl-3'-azido-3'-deoxythymidine (DPP-AZT), phospholipid prodrugs which form lipid bilayers and which are readily incorporated into liposomes. The anti-HIV activity of DOP-ddC was similar to that of ddC in HIV type 1-infected HT4-6C cells, but DPP-AZT was considerably less active than AZT in HT4-6C cells. Liposomes containing DOP-[3H]ddC or DPP-[3H]AZT administered intraperitoneally to mice produced greater levels of total radioactivity over time in plasma, spleen, and lymphoid tissue relative to the results with [3H]ddC and [3H]AZT, respectively. DPP-AZT administered intraperitoneally in liposomes as a single daily dose to mice infected with Rauscher leukemia virus prevented increased spleen weight and reverse transcriptase levels in serum with a dose-response roughly comparable to that of AZT given continuously in the drinking water. DOP-ddC, DPP-AZT, and lipid conjugates of other antiretroviral nucleosides may provide higher levels of drug over time in plasma and in lymph nodes and spleen, important reservoirs of HIV infection, and may represent an interesting alternative approach to antiviral nucleoside treatment of AIDS.

Effects of tamoxifen, droloxifene and 17 beta-estradiol on Rauscher mouse leukemogenesis.

The effects of tamoxifen (TAM), its 3-hydroxy congener droloxifene (DROL) and 17 beta-estradiol were investigated on leukemogenesis induced in BALB/c mice by Rauscher murine leukemia virus (RLV). Multiple applications of each compound, in a dose-dependent manner, resulted in reduced virus titer in the serum, delayed onset of splenomegaly and significant prolongation of survival. Although 17 beta-estradiol proved most effective, prevention of disease was not achieved either by short- or long-term treatment with any of the drugs tested.

Differential antiviral activity of two TIBO derivatives against the human immunodeficiency and murine leukemia viruses alone and in combination with other anti-HIV agents.

R82913 and R86183, two derivatives of tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO), were found to potently and selectively inhibit the replication and cell killing effects of a panel of biologically diverse laboratory and clinical strains of HIV-1. The two compounds exhibited significant activity in all human cell lines tested, as well as in fresh human peripheral blood lymphocytes and macrophages. One of these two compounds (R82913) was found to significantly inhibit the replication of a murine retrovirus (Rauscher murine leukemia virus) in both UV-XC plaque formation and virus yield reduction assays. R86183, despite differing from R82913 only in the positioning of a single chlorine molecule, was not active against the murine retrovirus but was 10-fold more potent in inhibiting HIV-1 replication. Combination antiviral assays with other reverse transcriptase inhibitors, including AZT, ddC, and carbovir, yielded synergistic anti-HIV activity with both TIBO derivatives. Additive to slightly synergistic results were obtained in combinations with ddI and phosphonoformic acid whereas additive to antagonistic activity was detected in combination with dextran sulfate.

Reduction of HIV-1 antigen production by phosphatidylcholine containing formulations via growth inhibition of HIV-1-infected cells.

Phosphatidylcholine (PC) and licensed formulations containing PC were tested for their influence on the proliferation and viability of cells permanently infected with HIV-1 (human immunodeficiency virus type 1). PC alone, as well as pharmaceutical formulations containing PC, selectively inhibited the growth of productively infected lymphoid cells. The strongest growth inhibition was observed with formulations containing PC, glycerol and triglyceride together. The growth inhibition was dose-dependent for HIV-1-infected cells. Additionally, PC-containing formulations dramatically reduced antigen production from peripheral blood mononuclear cells (PBMCs) infected in vitro with HIV-1. In vivo experiments with Rauscher-MuLV-infected mice showed that PC administered either intraperitoneally or orally was able to inhibit Rauscher-virus-induced splenomegaly. PC-containing formulations are currently used in man for supportive therapy at doses, which in vitro induced 50% growth inhibition of HIV-1-infected cells in vitro. Such doses have been used in man without side effects for many years. Thus, PC-containing formulations may be valuable for the treatment of HIV-1-infected individuals.

Antileukemic activity of Viva-Natural, a dietary seaweed extract, on Rauscher murine leukemia in comparison with anti-HIV agents, azidothymidine, dextran sulfate and pentosan polysulfate.

An antileukemic activity of partially purified polysaccharide of an edible seaweed. Viva-Natural, against Rauscher murine retrovirus-induced erythroleukemia has been demonstrated. This antileukemic effect is compared with standard anti-human immunodeficiency virus (HIV) agents, azidothymidine (AZT), dextran sulfate and pentosan polysulfate. Pretreatment with Viva-Natural, as an immunomodulator, on day 3 prior to the virus inoculation demonstrated definite prophylactic activity, while pretreatment with the other three anti-HIV agents showed no prophylactic activity. The replication of Rauscher virus in BALB/3T3 cell cultures accompanied by direct cytopathic effect (syncytia formation) was suppressed in the presence of Viva-Natural or the other anti-HIV agents in the culture medium. In spite of the antiviral potentials of the four agents in vitro, only Viva-Natural and AZT demonstrated therapeutic efficacy against Rauscher leukemia in mice.

Unsaturated and carbocyclic nucleoside analogues: synthesis, antitumor, and antiviral activity.

A series of unsaturated analogues of nucleosides were prepared and their cytotoxic, antitumor, and antiviral activities were investigated. Alkylation of cytosine with (E)-1,4-dichloro-2-butene gave chloro derivative 2f, which was hydrolyzed to alcohol 2h. Cytosine, adenine, 2-amino-6-chloropurine, thymine, and (Z)-1,4-chloro-2-butene gave compounds 4c-f, which, after hydrolysis, afforded alcohols 4a, 4b, 4g, and 4h. Alkenes 4d and 4e were cyclized to heterocycles 12 and 13. Alkylation of 2,6-diaminopurine with 1,4-dichloro-2-butyne led to chloro derivative 6a, which was hydrolyzed to alcohol 6b. Allenic isomerization of 6b gave compound 5c. Chloro derivatives 2e-g, 4c-f, 5d, and 6c-e as well as pyrimidine oxacyclopentenes 9c and 9d are slow-acting inhibitors of murine leukemia L1210 of IC50 10-100 microM. The most active were analogues 4c, 4d, 4e, and 6e (IC50 10-20 microM). The corresponding hydroxy derivatives were less active of inactive. Inhibition of macromolecular synthesis with compounds 4c, 4d, 6e, 9c, and 9d follows the order: DNA greater than RNA greater than or equal to protein. Cytotoxic effects of 4c, 6e, and 9d are not reversed with any of the four basic ribonucleosides or 2'-deoxyribonucleosides. Inhibitory activity of cytosine derivative 9c is reversed with uridine and 2'-deoxyuridine but not with the corresponding cytosine nucleosides. Zone assays in several tumor cell lines show that active compounds are cytotoxic agents with little selectivity for tumor cells. Analogue 6c showed 16.7% ILS in leukemia P388/o implanted ip in mice at 510 and 1020 mg/kg, respectively. Cytallene (5b) and 6'beta-hydroxyaristeromycin (10) exhibited significant activity against Friend and Rauscher murine leukemia viruses. The rest of the hydroxy derivatives, with the exception of 4a, were moderately effective or inactive as antiviral agents. None of the chloro derivatives or oxacyclopentenes exhibited an antiviral effect at noncytotoxic concentrations. Z-Olefin 4b and 2-aminoadenallene (5c) are substrates for adenosine deaminase.

Castanospermine vs. its 6-O-butanoyl analog: a comparison of toxicity and antiviral activity in vitro and in vivo.

Inhibitors of glycoprotein processing, such as castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine), have been shown previously to inhibit human immunodeficiency virus type 1 (HIV-1) with acceptable toxicity in cultured human cells. In prior experiments, we have tested the toxicity and antiviral efficacy of castanospermine in mice infected with the Rauscher murine leukemia virus (RLV). When compared with 3'-azido-3'-deoxythymidine (AZT, zidovudine), castanospermine was less effective and more toxic. Since the 6-O-butanoyl analog of castanospermine was previously found to have a more favorable activity profile than the parent compound against HIV-1 in cultured cells, we compared the antiviral efficacy of both compounds in parallel in vitro and in vivo in the RLV system. Plaque formation in the XC assay was inhibited with a 50% inhibitory concentration (IC50) of 2.4 microM for the 6-O-butanoyl analog of castanospermine, as compared to 9 microM for castanospermine. For both compounds, concentrations resulting in significant cytotoxicity were about ten times higher. Both compounds significantly decreased HIV-1 env-induced syncytium formation in a novel in vitro assay. In RLV-exposed mice, the 6-O-butanoyl analog showed no advantage over the parent compound: both curves for toxicity as well as antiviral efficacy were super-imposable. We conclude that the 6-O-butanoyl analog of castanospermine as well as castanospermine itself are active antiviral agents in mice and that prolonged oral administration is tolerable. However, in comparison to AZT, their antiviral activity profiles are less favorable.

N-carboxymethylchitosan-N,O-sulfate as an anti-HIV-1 agent.

N-carboxymethylchitosan-N-O-sulfate (NCMCS), a sulfated polysaccharide derivative of chitin, inhibited the propagation of the human immunodeficiency virus type 1 (HIV-1) in human CD4+ cells and that of Rauscher murine leukemia virus (RLV) in murine fibroblasts. A dose-dependent inhibition of both viruses was observed without significant cytotoxicity. NCMCS blocked the binding of HIV-1 to human CD4+ target cells and competitively inhibited HIV-1 reverse transcriptase. Thus, NCMCS may prevent HIV-1 infection by inhibiting viral adsorption to the CD4 receptor and reverse transcription of the viral genome.

Different effect of benzylacyclouridine on the toxic and therapeutic effects of azidothymidine in mice.

It has been reported that in vitro uridine (Urd) can reverse azidothymidine (AZT) cytotoxicity without decreasing anti-human immunodeficiency virus (HIV) activity. Our studies in mice have shown that daily oral doses of benzylacyclouridine (BAU), an inhibitor of Urd breakdown, also reduces AZT hematologic toxicity, presumably by elevating the plasma concentration of Urd. We now extend these murine studies and report the effect of various doses of exogenous Urd, various doses of BAU, or the combination of BAU and Urd, administered daily, on AZT-induced toxicity. In mice receiving concomitant AZT, daily doses of Urd of 1,000 to 2,000 mg/kg increase peripheral reticulocytes and slightly reduce AZT-induced hematologic toxicity. However, the range of effective doses is narrow, and higher doses of Urd (greater than 3,000 mg/kg/d) significantly enhance hematologic toxicity. At its most effective dose, (2,000 mg/kg/d), Urd produces 28% mortality. In contrast, BAU doses up to 300 mg/kg/d reduced AZT-related hematologic toxicity in a dose-dependent manner without mortality. Higher daily doses of BAU and the combination of BAU with low doses of Urd were not more effective. Studies conducted in mice infected with the Rauscher murine leukemia virus (RLV) indicate that BAU does not impair the antiretroviral effect of AZT when administered at doses that reduce AZT-induced anemia and leukopenia. These findings may be significant for the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex.

Differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular DNA and RNA polymerases.

Four flavonoids, 5,6,7-trihydroxyflavone (baicalein), 3,3',4',5,7-pentahydroxyflavone (quercetin), 3,3',4',5,6,7-hexahydroxyflavone (quercetagetin) and 3,3',4',5,5',7-hexahydroxyflavone (myricetin), were found to be potent inhibitors of reverse transcriptases from Rauscher murine leukemia virus (RLV) and human immunodeficiency virus (HIV). Under the reaction conditions employed, any one of these flavonoids almost completely inhibited the activity of RLV reverse transcriptase at a concentration of 1 microgram/ml. HIV reverse transcriptase was inhibited by 100%, 100%, 90% and 70% in the presence of 2 micrograms/ml quercetin, myricetin, quercetagetin and baicalein, respectively. The mode of inhibition of these flavonoids was competitive (RLV reverse transcriptase) or partially competitive (HIV reverse transcriptase) with respect to the template.primer complex, (rA)n.(dT), and noncompetitive with respect to the triphosphate substrate, dTTP. The Ki values for RLV reverse transcriptase were found to be 0.37 microM and 0.08 microM for baicalein and quercetin, respectively and those for HIV reverse transcriptase were 2.52 microM, 0.52 microM, 0.46 microM and 0.08 microM for baicalein, quercetin, quercetagetin and myricetin, respectively. Comparative studies with other flavonoids (hydroxyflavones, dihydroxyflavones and polyhydroxyflavones and flavanones) carried out to clarify the structure/activity relationships, revealed that the presence of both the unsaturated double bond between positions 2 and 3 of the flavonoid pyrone ring, and the three hydroxyl groups introduced on positions 5, 6 and 7, (i.e. baicalein) were a prerequisite for the inhibition of reverse transcriptase activity. Removal of the 6-hydroxyl group of baicalein required the introduction of three additional hydroxyl groups at positions 3, 3' and 4' (quercetin), to afford a compound still capable of inhibiting the reverse transcriptase activity. Quercetagetin which contains the structures of both baicalein and quercetin, and myricetin which has the structure of quercetin with an additional hydroxyl group on the 5' position also proved strong inhibitors of reverse transcriptase activity. The inhibition by baicalein of reverse transcriptase is highly specific, whereas quercetin and quercetagetin were also strong inhibitors of DNA polymerase beta and DNA polymerase I, respectively. Myricetin was also a potent inhibitor of both DNA polymerase alpha and DNA polymerase I.

Interferon-alpha and 3'-azido-3'-deoxythymidine are highly synergistic in mice and prevent viremia after acute retrovirus exposure.

This study was undertaken to calculate the in vivo drug interactions between recombinant human interferon-alpha A/D (rHuIFN-alpha A/D) and 3'-azido-3'-deoxythymidine (AZT) in a quantitative model for retroviral viremia. When given as single agents, both AZT and rHuIFN-alpha A/D suppressed virus-induced splenomegaly in a dose-dependent fashion in mice inoculated with Rauscher murine leukemia virus (RLV). However, suppressive doses of single-agent AZT caused anemia after 20 days of therapy. Combining rHuIFN-alpha A/D with AZT allowed drastic dose reductions for each agent while maintaining greater than or equal to 93% inhibition of splenomegaly. No clinically significant toxicity was seen. Computer analysis with the isobologram technique and combination index method showed that these combination regimens were highly synergistic. A 20-day course of AZT + rHuIFN-alpha A/D started 4 h after virus exposure was protective against RLV viremia and disease. After cessation of therapy, the animals were resistant to rechallenge with fully infectious RLV. We conclude that prompt initiation of effective combination therapy after retroviral exposure prevented viremia and disease and led to protective immunity.

Synthesis and antiviral activity of several 2,5'-anhydro analogues of 3'-azido-3'-deoxythymidine, 3'-azido-2',3'-dideoxyuridine, 3'-azido-2',3'-dideoxy-5-halouridines, and 3'-deoxythymidine against human immunodeficiency virus and Rauscher-murine leukemia virus.

Several 2,5'-anhydro analogues of 3'-azido-3'-deoxythymidine (AZT), 3'-azido-2'3'-dideoxyuridine (AZU), 3'-azido-2'3'-dideoxy-5-bromouridine, 3'-azido-2',3'-dideoxy-5-iodouridine, and 3'-deoxythymidine and the 3'-azido derivative of 5-methyl-2'-deoxyisocytidine have been synthesized for evaluation as potential anti-HIV (human immunodeficiency virus) agents. These 2,5'-anhydro derivatives, compounds 13-17, demonstrated significant anti-HIV-1 activity with IC50 values of 0.56, 4.95, 26.5, 27.1, and 48 microM, respectively. Compared to that of the parent compounds AZT and AZU, the respective 2,5'-anhydro analogues, compounds 13 and 14, were somewhat less active. Whereas AZT was cytotoxic with a TCID50 of 29 microM, the toxicity of the 2,5'-anhydro derivative of AZT, compound 13, was reduced considerably to a TCID50 value of greater than 100 microM. The 2,5'-anhydro analogue of 5-methyl-2'-deoxyisocytidine also demonstrated anti-HIV-1 activity with an IC50 value of 12 microM. These compounds were also evaluated against Rauscher-Murine leukemia virus (R-MuLV) in cell culture. Among them, AZT, 3'-azido-2',3'-dideoxy-5-iodouridine, 3'-azido-2',3'-dideoxy-5-bromouridine, and 2,5'-anhydro-3'-azido-3'-deoxythymidine (13) were found to be most active, with IC50 values of 0.023, 0.21, 0.23, and 0.27 microM, respectively.

Significant differences in therapeutic responses to a human interferon-alpha B/D hybrid in Rauscher or Friend murine leukemia virus infections.

Treatment of mice infected with Rauscher (RMLV) or Friend (FMLV) murine leukemia viruses at an early stage of disease (beginning at day 0 and continuing every other day for 21 days) with 5 x 10(7) units/kg body weight of a cross-species-active recombinant human interferon-alpha B/D hybrid (rHuIFN-alpha B/D) was more effective in FMLV than in RMLV infections. In contrast, treatment with 5 x 10(7) units/kg body weight of IFN beginning as late as 15 days postinfection and continuing every other day for 21 days was more effective in RMLV than in FMLV infections. These differences were consistent with observed changes in circulating white blood cells, spleen weight, and reverse transcriptase levels. Additionally, biweekly long-term administration (beginning at day 0) of 5 x 10(6) units/kg of rHuIFN-alpha B/D (an ineffective treatment in short-term therapy) significantly prolonged the mean survival time of RMLV-infected mice, but only weakly prolonged that of FMLV-infected mice.

Antiviral activity of a novel recombinant human interferon-alpha B/D hybrid.

The antiviral potential of a novel cross-species active, recombinant human interferon-alpha B/D hybrid (rHuIFN-alpha B/D), was evaluated for its efficiacy in cultured human monocytes and in several murine models of viral disease. When examined in 14-day-old human monocyte cultures, rHuIFN-alpha B/D was highly effective in preventing viral replication and cell destruction caused by herpes simplex virus type 1 (HSV-1/VR3). The effect observed with 100 units of this hybrid IFN was as good or higher than that observed with equivalent amounts of rHuIFN-alpha A or IFN-gamma. In addition, a single dose (5 X 10(7) U/kg) of rHuIFN-alpha B/D administered several hours after intranasal infection with HSV-1/VR3 suppressed pulmonary virus replication and prevented death due to interstitial pneumonia. Similarly, mice infected with a more aggressive strain of HSV-1 (McIntyre) were protected when this IFN preparation was administered at the time of virus infection and 1 day later. The anti-retroviral activity of rHuIFN-alpha B/D was examined in two murine leukemia retroviral models, Rauscher (RMLV) and Friend (FMLV), and a murine model of acquired immunodeficiency (LP-BM5). Treatment of RMLV or FMLV infected mice significantly prolonged mean survival times and the number of long-term FMLV survivors. These therapeutic effects were demonstrated when IFN was administered on the day of virus infection or as late as 3 days following infection. Transient reversal of the immunosuppressive effects induced by LP-BM5 infection was observed when rHuIFN-alpha B/D treatment was initiated at the time of virus infection. Moreover, when rHuIFN-alpha B/D was used together with azidothymidine (AZT), the effect of the combination was better than either drug alone.

Murine models for antiretroviral therapy.

Rauscher murine leukemia virus (RLV) infection of adult mice results in splenomegaly that is proportional to the virus titer. We have used this model to study the therapy of chronic viremia as well as chemoprophylaxis after viral exposure, both with single agents and with combination therapy. We showed that prompt initiation of effective antiviral therapy after viral exposure can prevent de novo infection. We have also developed a murine neurotropic retrovirus model which allows analysis of candidate antiviral agents for activity across the blood-brain barrier. This model can also be used to study therapeutic approaches to retroviral infections acquired at midgestation or during the neonatal period.

In vivo analysis of castanospermine, a candidate antiretroviral agent.

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine), an inhibitor of glycoprotein processing, has been shown to inhibit the human immunodeficiency virus type 1 (HIV-1) with acceptable toxicity in cultured cells. In contrast to reverse transcriptase inhibitors, castanospermine targets host enzymes. We have analyzed castanospermine in murine systems, using cultured cells as well as live animals. Plaque formation by Rauscher murine leukemia virus (RLV) was inhibited with a median inhibitory concentration (IC50) of 2 micrograms/ml. RLV-exposed BALB/c mice treated with a 20 day course of castanospermine starting 4 h postinoculation showed a dose-dependent inhibition of splenomegaly. Oral castanospermine therapy given to chronically RLV-infected mice prolonged median survival from 36 to 94 days when compared to untreated controls (p = 0.007). Castanospermine was better tolerated orally than intraperitoneally at the same dose. Toxic effects included weight loss, lethargy, and dose-dependent thrombocytopenia. At the highest intraperitoneal dose, lymphoid depletion occurred in thymus, spleen, and lymph nodes. We conclude that castanospermine is an active antiviral agent in animals and that prolonged oral administration is tolerable; however, when compared to 3'-azido-3'-deoxythymidine in the same murine system, castanospermine was less active and more toxic.