Biological studies on inhibitors of human immunodeficiency virus

The toxicities induced by dideoxynucleoside analogs (ddNs, RT inhibitors) are due to the incorporation of ddNs triphosphates into the cellular or mitochondrial DNA of the host cells. Our hypothesis was that carboxyphosphonyl (CP) analogs of nucleosides should have a better toxicity profile because these 'pharmacophores' would not require "activation" by host enzymes for anti-HIV activity. The antiviral activity of CP analogs against HIV and AZT-resistant variants was determined by focal immunoassay (FIA), reverse transcriptase (RT) assay and cytopathic inhibition (MTT) assay. The antiviral activity of CP analogs is greatly influenced by the assay methodologies, virus stains and cell lines used. CP-deoxyuridine (CP-dUrd), CP-deoxyinosine (CP-dIno) and CP-deoxythymidine (CP-dThd) were selective inhibitors of HIV replication. The concentrations required to inhibit HIV-1 strain IIIB and AZT-resistant variants replication by 50% (ED50) was in the range of 0.15 μM to 0.55 μM by RT assay. These compounds were found to have moderate activity against HIV strains when tested using FIA and MTT assays. The antiviral activity of CP-ddNs was determined by different assay methods. CP analogs in general were less potent than ddNs. CP-AZT was found to have good antiviral activity. Foscarnet, AZT, ddC, ddI and d4T were used as drug controls. CP-dNs have low acute cytotoxicity. The cytotoxicity of CP-ddNs was 4 to 85 fold lower than the parent ddNs for different cell lines. The uptake and efflux of [6-3H]CP-dUrd was studied using CEM cells. The highest intracellular concentration of CP-dUrd attained was 1.8 pmole/106 cells after 72 hours of incubation with 50 μm of compound. The half-life of CP-dUrd in CEM cells was 25 hours. CP-dUrd was stable during the incubation period. No binding of CP-dUrd to plasma proteins occurred. The pharmacokinetic studies of [6-3]CP-dUrd (i.p. administration) in mice indicated that CP-dUrd was slowly absorbed, homogeneously distributed in body tissues and slowly excreted after extensive metabolism in the body. Acute and delayed cytotoxicities of active CP analogs were evaluated using CEM cells. CP-dUrd, CP-dIno and CP-AZT did not exhibit acute or delayed cytotoxicity. Control drug ddC exhibited acute and delayed cytotoxicity in CEM cells at 1 μM and 0.125 μM, respectively. The concentrations of AZT, CP-dUrd and CP-dIno required to inhibit 50% colony formation (IC50) of murine granulocyte-macrophage (CFU-GM) were 2.5 μM, 250 μM and 160 μM, respectively. CP-dUrd has low mammalian toxicity [LD50 (mouse) ≈ 1000 mg/kg, i. p.]