The major goal of this proposal is to understand the metabolism of carbocylic 2'3' didehydro-2'3'-dideoxyguanosine (carbovir), alone and in combination with other anti-HIV agents, in various HIV- infected and uninfected human cell lines, and to study the effect of these drugs, alone and in combination, on nucleotide metabolism in these same cells. The anti-HIV agents that will be studied in combination with carbovir are 3'-azido-3'-deoxythymidine (AZT) and interferon (IFN) alpha. One primary objective of this proposal is to understand the mechanism responsible for the synergism seen between carbovir and AZT against HIV- replication. The following biochemical parameters will be determined in cells treated with carbovir alone and in combination with AZT or IFN alpha: 1) The metabolism of [3H]carbovir and [3H]AZT to the nucleoside mono, di, and triphosphate: 2) The metabolism of [3H]carbovir to other nucleoside analogs and their nucleotides; 3) The nucleoside kinase responsible for the metabolism of carbovir to nucleotides; 4) Natural ribo-and deoxyribo- nucleotide pools; 5) DNA, RNA, and protein synthesis; and 6) Interactions of carbovir triphosphate, AZT triphosphate, and IFN alpha at their potential targets isolated from human cells, or E. coli that express HIV reverse transcriptase. The potential targets that will be studied are HIV reverse transcriptase, DNA polymerase alpha, beta, and gamma, DNA primase, and ribonucleotide reductase. All biochemical effects will be correlated with antiviral activity and cytotoxicity of drug treatment. The knowledge of drug action on these biochemical parameters should aid in the understanding of the mechanism responsible for the synergism observed with carbovir plus AZT and AZT plus IFN alpha, and should also aid in the rational development of drug combinations for the treatment of AIDS. When carbovir has been thoroughly evaluated in these human cell lines, then its metabolism and it effect on endogenous nucleotide metabolism will be studied in mice treated with carbovir alone and in combination with AZT and/or IFN alpha. At various times after drug treatment the following tissues will be removed for study: blood; urine; bone marrow; peritoneal macrophage, intestinal tract, spleen, kidneys; and brain. In the blood and urine the amount of unchanged drug and any catabolities will be measured and their change over time will be followed. In the other tissues the presence of unchanged drug and any metabolites will be measured, and the effect of drug treatment on nucleotide pools and macromolecular synthesis will be determined. These studies should indicate the tissues in which the drug of any combination should be effective against HIV and should lead to an understanding of the mechanism of cytotoxicity. Such information should also lead to rational development of drug combinations to maximize anti-HIV activity and to minimize cytotoxicity.
