In vivo and in vitro models for determination of antiviral activity and resistance
Author: Ljungdahl Ståhle, Ewa
Date: 1997-12-12
Location: Föreläsningssalen vid Mikrobiologiskt och Tumörbiologiskt Center, Doktorsringen 13, Karolinska Institutet
Time: 9.00
Department: Inst för mikrobiologi, tumör- och cellbiologi / Dept of Microbiology, Tumor and Cell Biology
Abstract
The AIDS epidemic has been the driving force for the discovery of substances that inhibit the replication of human immunodeficiency virus (HIV). Much interest has earlier focused on a single gene product central to the life cycle of HIV and to all other retroviruses: the reverse transcriptase (RT), the enzyme responsible for synthesis of DNA from viral RNA. Analogues to the five naturally occurring nucleosides can inhibit the replication of retroviruses in cell culture to various degrees. Another enzyme critical for HIV replication is the viral protease. Inhibition of this enzyme leads to production of immature noninfectious viral progeny. The aim of this thesis was to develop and use methods required for the preclinical development and laboratory testing of antiviral drugs.
In order to monitor in vivo challenge models for retrovirus infection, we developed assays to identify viral antigens and immune responses to them. The antibody assay was based on a synthetic peptide, the sequence of which corresponds to a segment of the env protein of HIV-2 which cross reacts with SIV. The peptide was successfully used to detect IgM antibodies from days 13-16 after SIV infection and anti-SIV IgG antibody levels around days 16-20 which attained a plateau after two months.
In the development of new drugs it is imperative to use animal models for the study of therapies against AIDS and HIV infection. In this study monkeys were infected with 10-100 monkey infectious doses of SIV SM and treated for ten days with foscamet or one of four different nucleoside analogues. Foscamet, zidovudine, fluorothymidine, didanosine, and stavudine significantly delayed the appearance of SIV SM p24/26 antigen. Foscarnet also decreased IgM and delayed IgG antibodies. In our studies, infection was not prevented by any of the drugs investigated, but later studies of fluorothymidine have prevented infection when smaller infectious doses have been used.
A major clinical problem in the management of AIDS is the presence of neuropsychiatric symptoms. It is therefore valuable if a drug against HIV penetrates the blood-brain barrier. Microdialysis is a method of sampling extracellular fluid containing free(not protein-bound) drugs in the brain and other tissues. Using microdialysis, the concentration of the nucleoside analogues fluorothymidine and zidovudine in the brain were found to be approximately one-third those in the muscle and plasma in monkeys and one-fifth in rats. The in vivo unbound concentrations of both drugs in the brain, muscle and venous blood exceeded those reported to inhibit HIV replication in vitro. The method allows comparisons of the efficacy of antiviral drugs in penetrating the blood-brain barrier.
Herpesvirus infections are common among AIDS patients; more than half of them had evidence of disseminated cytomegalovirus infection at autopsy. A method for measurement of antiviral sensitivity of primary isolates of CMV was developed. The assay includes simultaneous virus titration and determination of the 50 % inhibitory concentration(IC5O). The absorbance obtained in the enzyme-linked immunosorbent assay correlated linearly to the number of plaques in a plaque reduction assay. The IC5O of antiviral compounds was strongly dependent on the virus dose, except for resistant isolates, where the IC50 did not change with the viral dose. Clinical isolates exhibited different IC50 values for ganciclovir in different cell types indicating that intracellular factors are important for drug metabolism.
The effect of drug combinations may also be primarily evaluated in vitro. Combinations of RT and protease inhibitors not only target two different proteins but also affect two different stages of the viral life cycle of HIV. Combinations of foscarnet and lamivudine, both inhibitors of RT, showed clear synergy and this effect was further pronounced using two more potent foscarnet analogues. A weak or no synergy was found when foscarnet was combined with the protease inhibitors indinavir, saquinavir or ritonavir.
In order to monitor in vivo challenge models for retrovirus infection, we developed assays to identify viral antigens and immune responses to them. The antibody assay was based on a synthetic peptide, the sequence of which corresponds to a segment of the env protein of HIV-2 which cross reacts with SIV. The peptide was successfully used to detect IgM antibodies from days 13-16 after SIV infection and anti-SIV IgG antibody levels around days 16-20 which attained a plateau after two months.
In the development of new drugs it is imperative to use animal models for the study of therapies against AIDS and HIV infection. In this study monkeys were infected with 10-100 monkey infectious doses of SIV SM and treated for ten days with foscamet or one of four different nucleoside analogues. Foscamet, zidovudine, fluorothymidine, didanosine, and stavudine significantly delayed the appearance of SIV SM p24/26 antigen. Foscarnet also decreased IgM and delayed IgG antibodies. In our studies, infection was not prevented by any of the drugs investigated, but later studies of fluorothymidine have prevented infection when smaller infectious doses have been used.
A major clinical problem in the management of AIDS is the presence of neuropsychiatric symptoms. It is therefore valuable if a drug against HIV penetrates the blood-brain barrier. Microdialysis is a method of sampling extracellular fluid containing free(not protein-bound) drugs in the brain and other tissues. Using microdialysis, the concentration of the nucleoside analogues fluorothymidine and zidovudine in the brain were found to be approximately one-third those in the muscle and plasma in monkeys and one-fifth in rats. The in vivo unbound concentrations of both drugs in the brain, muscle and venous blood exceeded those reported to inhibit HIV replication in vitro. The method allows comparisons of the efficacy of antiviral drugs in penetrating the blood-brain barrier.
Herpesvirus infections are common among AIDS patients; more than half of them had evidence of disseminated cytomegalovirus infection at autopsy. A method for measurement of antiviral sensitivity of primary isolates of CMV was developed. The assay includes simultaneous virus titration and determination of the 50 % inhibitory concentration(IC5O). The absorbance obtained in the enzyme-linked immunosorbent assay correlated linearly to the number of plaques in a plaque reduction assay. The IC5O of antiviral compounds was strongly dependent on the virus dose, except for resistant isolates, where the IC50 did not change with the viral dose. Clinical isolates exhibited different IC50 values for ganciclovir in different cell types indicating that intracellular factors are important for drug metabolism.
The effect of drug combinations may also be primarily evaluated in vitro. Combinations of RT and protease inhibitors not only target two different proteins but also affect two different stages of the viral life cycle of HIV. Combinations of foscarnet and lamivudine, both inhibitors of RT, showed clear synergy and this effect was further pronounced using two more potent foscarnet analogues. A weak or no synergy was found when foscarnet was combined with the protease inhibitors indinavir, saquinavir or ritonavir.
Issue date: 1997-11-21
Publication year: 1997
ISBN: 91-628-2716-2
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