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3"-Azido-3'-deoxythymidine (AZT) was the first approved drug for the treatment of the AIDS; however, despite its usefulness, AZT often produces side effects that require cessation of therapy. 2',3'-Dideoxycytidine (ddC) is a related anti-retroviral agent in advanced stages of clinical testing. A previous report demonstrated that AZT decreased the repair of UV-induced DNA strand breaks in mammalian cells after ultraviolet (UV) irradiation. We studied the effect of AZT and ddC on DNA repair from the hamster DHFR gene of the major UV-induced DNA lesion, cyclobutane pyrimidine dimers (CPDs). We conclude that neither AZT nor ddC inhibited DNA replication or the gene-specific repair of CPDs in the hamster DHFR gene after 8 or 24 hrs of repair incubation at concentrations of 25 microM and 10 microM, respectively.

3'-Amino-2',3'-dideoxycytidine (8) was directly synthesized from 2'-deoxycytidine. 2',3'-Dideoxy-3'-(N-acyl-L-phenylalanylamino)cytidines (acyl = butoxycarbonyl (9a), acetyl (9b), benzoyl (9c), and n-hexanoyl (9d)) were synthesized as chymotrypsin-activated prodrugs of 8. This N-protection was required for activation by chymotrypsin to 8. In vitro, compound 8 showed high cytotoxic activity against P388 cells, but the prodrugs 9a-d were ineffective. In vivo, however, these prodrugs showed much higher activity than 8 in mice bearing P388 cells.

MKC-442, a derivative of the non-nucleoside reverse transcriptase (RT) inhibitor 1-[(2-hydroxyethoxy)methyl)-6-(phenylthio)thymidine (HEPT), showed potent and selective inhibition of human immunodeficiency virus type 1 (HIV-1) replication in vitro, using a range of host-cell/virus systems including human peripheral blood mononuclear cells infected with primary clinical isolates. MKC-442 was evaluated in combination with the nucleoside analogues AZT, ddI and ddC, the non-nucleoside RT inhibitor nevirapine, the HIV-1 proteinase inhibitor Ro-31-8959, and the alpha-glucosidase 1 inhibitor, MDL-28,574, using a cell viability assay. Drug interactions were evaluated by the isobologram technique and by calculating combination indices. Notable synergistic inhibition of HIV-1 replication was observed when MKC-442 was combined with AZT and MDL-28,574 and moderate synergy with ddI. In combination with ddC, nevirapine or Ro-31-8959, only a slightly better than additive effect was observed. Impressive synergy was seen using the three-drug combinations of MKC-442, AZT and MDL-28,574 or MKC-442, AZT and Ro-31-8959. No additional cytotoxicity was observed as measured by [3H]thymidine incorporation by concanavalin A-stimulated peripheral blood mononuclear cells, when MKC-442 was combined with any of the above-mentioned compounds. The use of MKC-442 in a two- or three-drug combination regimen with other RT inhibitors, a proteinase inhibitor or an alpha-glucosidase 1 inhibitor should be considered for HIV-1-related chemotherapy.

Two dideoxynucleosides, 2',3'-dideoxy-beta-L-cytidine and 2',3'-dideoxy-beta-L-5-flurocytidine, containing unnatural L-configuration in their sugar moieties, were synthesized and assayed for antiviral activities. Both compounds were shown to possess potent anti-human immunodeficiency virus type 1 and antihepatitis B virus activities, while demonstrating no anti-herpes simplex viruses 1 and 2 activity. These two compounds exhibited in vitro cellular toxicities for several leukocytic cell lines and were shown to inhibit phytohemagglutinin-stimulated human peripheral blood mononuclear leukocyte proliferations. At inhibitory concentrations, both compounds caused accumulations of cells in the S phase. While demonstrating no obvious morphological toxicity in vivo in mice at concentrations of 75 and 150 mg/kg, 2',3'-dideoxy-beta-L-5-fluorocytidine- treated animals were shown to have considerable increases in CD4/CD8 double positive T lymphocyte population in their blood circulation.

2',3'-Dideoxycytidine (ddC) is a nucleoside analogue that inhibits HIV-1 replication in vitro and is currently used in AIDS therapy. This compound exerts a delayed cytotoxicity due to inhibition of mitochondrial DNA (mDNA) synthesis. We have found that long term exposure of U937 human monoblastoid cells to ddC allowed the selection of a drug-resistant cell line (U937-R) with 66% mDNA, normal ddC transport and altered deoxycytidine kinase kinetic properties. In this paper we show that U937-R cells contain an increased number of mitochondria per cell and a reduced copy number of mDNA/mitochondria. Furthermore, the intracellular concentrations of deoxycytidine 5'-triphosphate (dCTP) and 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) are also reduced although with a higher dCTP/ddCTP ratio in U937-R compared to the parental cells. This mechanism of drug resistance, with drug-resistance based on viral mutations, can provide an explanation for drug failure in antiviral therapy.

Ten different three-drug combinations have been analyzed for their ability to prevent HIV-induced cytopathic effects (CPEs) in a continuous human T-lymphoblastoid cell line. Agents acting at the same as well as at different sites in the HIV-1 replicative cycle were used. Each compound was analyzed at peak and trough plasma levels achieved in monotherapy and in the presence of HIV-1 strains 3B and MN at a viral inoculum varying from 1 x TCID50 (50% tissue culture inhibitory dose) to 1,000 x TCID50. Using a viral inoculum of 10 x TCID50 HIV-1 3B, it was determined that triple-drug combinations had greater antiviral activities than the corresponding double-drug combinations, which had greater antiviral activities than zidovudine (AZT) monotherapy. The most consistent triple-drug combination, demonstrating superior activity at all concentrations of virus, was AZT + dideoxyinosine + lamivudine which reduced the AZT IC95 (95% inhibitory concentration) by 208-, 57-, 133-, and 25-fold at of 1,000, 100, 10, and 1 x TCID50 HIV-1 3B, respectively, as compared with the IC95 for AZT monotherapy. For all antiviral regimens tested, higher viral inoculum resulted in less inhibition of viral replication and a higher IC95 for AZT. This observation argues for therapeutic intervention at an earlier stage in HIV infection, when viral burden is lower.

Duck hepatitis B virus (DHBV) DNA synthesis in congenitally infected ducks is inhibited by 2'-deoxycarbocyclic guanosine (2'-CDG). Three months of therapy reduces the number of infected hepatocytes at least 10-fold (W.S. Mason, J. Cullen, J. Saputelli, T.-T. Wu, C. Liu, W.T. London, E. Lustbader, P. Schaffer, A.P. O'Connell, I. Fourel, C.E. Aldrich, and A.R. Jilbert, Hepatology 19:393-411, 1994). The present study was performed to determine the kinetics of disappearance of infected hepatocytes and to evaluate the role of hepatocyte turnover in this process. Essentially all hepatocytes were infected before drug therapy. Oral treatment with 2'-CDG resulted in a prompt reduction in the number of infected hepatocytes. After 2 weeks, only 30 to 50% appeared to still be infected, and less than 10% were detectably infected after 5 weeks of therapy. To assess the possible role of hepatocyte turnover in these changes, 5-bromo-2'-deoxyuridine (BUdR) was administered 8 h before liver biopsy to label host DNA in hepatocytes passing through S phase, and stained nuclei were detected in tissue sections by using an antibody reactive to BUdR. The extent of nuclear labeling after 5 weeks was the same as that before therapy (ca. 1%). However, biopsies taken after 2 weeks of therapy showed a ca. 10-fold elevation in the number of nuclei labeled with BUdR. This result suggested that a rapid clearance of infected hepatocytes by 2'-CDG was caused not just by the inhibition of viral replication but also by an acceleration of the rate of hepatocyte turnover. To test this possibility further, antiviral therapy was carried out with another strong inhibitor of DHBV DNA synthesis, 5-fluoro-2',3'-dideoxy-3'-thiacytidine (524W), which did not accelerate hepatocyte turnover in ducks. 524W administration led to a strong inhibition of virus production but to a slower rate of decline in the number of infected hepatocytes, so that ca. 50% (and perhaps more) were still infected after 3 months of therapy. In addition, histopathologic evaluation of 2'-CDG-treated ducks revealed liver injury, especially at the start of therapy. No liver damage was observed during 524W therapy. These results imply that clearance of infected hepatocytes from the liver is correlated with hepatocyte turnover. Thus, in the absence of immune clearance or other sources for the accelerated elimination of infected hepatocytes, inhibitors of virus replication would have to be administered for a long period to substantially reduce the burden of infected hepatocytes in the liver.

With increasing awareness of the mitochondrial toxicity associated with certain 2',3'-dideoxynucleosides used in anti-human immunodeficiency virus therapy, procedures for quantitative analyses of drug effects on mitochondrial DNA (mtDNA) have assumed enhanced importance. For this reason we have developed a method to measure the copy numbers of mtDNA in cultured MOLT-4 cells. First a hybrid competitive DNA template was synthesized by conventional polymerase chain reaction (PCR), using two custom-synthesized 40-mer composite primers incorporating mitochondrial displacement loop sequences linked by a non-mitochondrial cDNA template (a 76-base pair sequence from the tat/rev region of human immunodeficiency virus cDNA). For the competitive assay, increasing known copy numbers of the hybrid competitive template were added as an internal control to samples containing total cellular DNA. With this approach, two competitive PCR products were generated, 1) a mitochondrial displacement loop-derived fragment (182 base pairs) and 2) a competitive DNA template-derived fragment (156 base pairs). Absolute quantitation was achieved by radiometric comparison of the relative amounts of the two products. To test the versatility of this method, varying amounts of competitive template (6.6 x 10(4) to 6.6 x 10(9) copies) were used with a fixed quantity of total cellular DNA taken from cells cultured for 9 days in the presence or absence of selected pyrimidine and purine dideoxynucleosides. The results showed that the copy number of cellular mtDNA is 823 +/- 71 copies/cell in MOLT-4 cells. Little selective depletion of mtDNA, compared with total cellular DNA, was seen with the purine dideoxynucleosides examined; however, when the cells were exposed to the pyrimidine dideoxynucleoside 2',3'-dideoxycytidine (50 nM) for 9 days, mtDNA content was specifically depleted, although total cellular DNA decreased by only 10%. Thus, in addition to the presently used methods of assessing mitochondrial impairment, i.e., Southern blot analysis and electron microscopy, the competitive PCR method provides a third and convenient assay, with particular applicability to determination of mtDNA in very small numbers of cells.

We attempted to detect drug-related HIV-1 pol gene mutations by selective polymerase chain reaction (PCR) using both proviral DNA and viral RNA isolated from patients (pts) with AIDS or ARC receiving antiretroviral therapy. Peripheral blood mononuclear cell (PBM)-associated proviral DNA and serum-derived viral RNA were obtained from eight patients before and after receiving an alternating regimen of AZT and ddC for 15-41 months or ddI monotherapy for 12-26 months. These specimens were examined for the presence of mutations at positions 70, 74, 215 and 219. We noted that selective PCR results can be ambiguous depending on the quantity of DNA template employed. We, therefore, used the minimal quantity of DNA templates that yielded evaluable PCR products in this study. For all the eight pairs of pre- and post-therapy proviral DNA samples, selective PCR results agreed with independently determined nucleotide sequences. Results of reverse transcription of serum-derived viral RNA followed by selective PCR differed in some cases from those using the proviral DNA. In particular, the use of serum viral RNA appeared to allow earlier detection of changes in drug-related mutations than the use of PBM-associated proviral DNA. We conclude that (i) selective PCR using the minimum and sufficient number of PBM-associated proviral DNA and serum viral RNA copies successfully detects the presence of known pol gene mutations; (ii) drug-related mutations may be distinguished earlier in virions in serum (or plasma) than in proviral DNA in PBM; and (iii) quantification of HIV-1 prior to selective PCR may be an important component in monitoring the therapy of HIV-1 infection.

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.

The inhibitory effects of the diphosphate of (S)-1-(3-hydroxy-2-phosphonylmethoxy-propyl)cytosine (HPMPCpp) toward human DNA polymerases beta and gamma were studied. The Ki values of HPMPCpp were compared with the Ki values of the triphosphates of 3'-azidothymidine (AZTTP), 2',3'-dideoxycytidine (ddCTP) and 5-iodo-2'-fluoroarabinosyluridine (FIAUTP). The Ki values toward DNA polymerase beta in increasing order were 1.32, 1.43, 140, and 520 microM for ddCTP, FIAUTP, AZTTP and HPMPCpp, respectively. The Ki values toward DNA polymerase gamma in increasing order were 0.034, 0.031, 18.3 and 299 microM for ddCTP, FIAUTP, AZTTP and HPMPCpp, respectively. Therefore, HPMPC would be expected to have less inhibitory effects on DNA repair (DNA polymerase beta) and mitochondrial DNA synthesis (DNA polymerase gamma) than ddC, FIAU or AZT.

The K65R mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) encodes cross-resistance to 2',3'-dideoxycytidine (ddC), 2',3'-dideoxy-3'-thiacytidine (3TC), and 2',3'-dideoxyinosine (ddI). We characterized the in vitro sensitivities of recombinant wild type (wt) and K65R mutant RT to dideoxynucleoside triphosphate (ddNTP) inhibitors, using a variety of primer-templates. With poly(rA)-oligo(dT), the K65R mutant showed slight increases in Ki for ddTTP and 3'-azido, 3'-deoxythymidine triphosphate (AZTTP) compared to wt RT, but neither wt nor K65R RT was inhibited by ddCTP or ddATP. With poly(rI)-oligo(dC), the K65R mutant showed a 2-fold increase in Km for dCTP and a 20-fold increase in Ki for ddCTP compared to wt, whereas ddATP, ddTTP, and AZTTP failed to inhibit either enzyme. With a heteropolymeric primer-template, the K65R mutant showed 10-fold reduced sensitivities to ddCTP, 3TCTP, and ddATP, and 4-fold reduced sensitivity to AZTTP, compared to wt. In contrast, both enzymes were equally inhibited by ddTTP and ddGTP. HIV-1 cross-resistance to ddC/3TC/ddI resulting from the K65R mutation may therefore involve selective alterations in substrate/inhibitor recognition. Additionally, competitive inhibition by ddNTPs noncomplementary to the template base appears to be unimportant in the mechanism of inhibition of HIV-1 RT by dideoxynucleoside analogs.

When zidovudine and dideoxycytidine (ddC) were given on schedules of 1 week on drug and 1 week off, results differed substantially in effects on HIV (human immunodeficiency virus type 1)-induced immune activation. Zidovudine (200 mg every 4 h) caused marked lowering toward normal of serum neopterin and beta 2-microglobulin within 1 week. This effect was lost within 1 week off zidovudine. Intermittent ddC (0.03 mg/kg every 4 h) had a smaller 1-week effect but had a delayed cumulative suppressive effect on HIV-associated immune activation that was not seen with intermittent zidovudine therapy. Zidovudine and ddC given in alternating weeks had synergistic effects in the first 10 weeks (e.g., early and rapid reduction followed by cumulatively greater effects on immune cell activation). The identical sawtooth effect of intermittent zidovudine was also evident in serum HIV p24 antigen levels. This is consistent with the hypothesis that the increased serum levels of the immune activation markers seen in HIV infection reflect stimulatory effects of HIV viral components on immune system cells.

We have investigated the cellular accumulation, tissue distribution, and antihuman immunodeficiency virus activity of free dideoxycytidine (ddC) and liposomal ddC (L-ddC). We have found that L-ddC was more efficiently taken up than its free form by RAW 264.7 cells (a monocyte-macrophage cell line) (p < 0.01) while a comparable uptake was seen in U937 cells (a promonocytic cell line). In the rat, L-ddC accumulated preferentially in liver and spleen when injected intravenously (p < 0.01), and mostly in spleen when given intraperitoneally (p < 0.01). In contrast, free ddC was rapidly eliminated out of the body. Liposomal ddC showed a similar anti-HIV activity in comparison with free ddC in U937 cells. Given the fact that encapsulation of ddC in liposomes does not affect its anti-HIV activity but enhances its in vitro cellular accumulation and its in vivo distribution in reticuloendothelial system (RES) tissues, we conclude that ddC in liposomal formulation is a promising anti-HIV agent with a targeted action on the RES, which is considered a reservoir for dissemination of virus to other cells, tissues, and organs.

Reverse transcriptase-associated amino acid substitutions related to ddC, d4T, and nevirapine resistance have been found in isolates of human immunodeficiency virus type 1 (HIV-1) from patients treated with AZT only. Sequence analysis of 23 isolates documented the presence of 4 unexpected mutations at amino acid residues related to drug resistance. Two isolates contained an aspartic residue in codon 69 associated with ddC resistance, and another a change in codon 75 associated with resistance to d4T. The Y-to-C alteration in codon 181 associated with nevirapine resistance was observed in another isolate after serial passage in cell culture in the absence of drug. Changes in substitution patterns were also noted after serial passage of four AZT resistant isolates in cell culture without inhibitors. One of the strains showed changes in codons 67 and 70 to wild-type residues. Clonal analysis showed that this alteration occurred by the selection during cell culture passage of the wild-type genotype, which was present as a minority subpopulation in the initially resistant virus stock, rather than to genetic reversion. In summary, we present evidence documenting the presence of mutations associated with drug resistance in the absence of drug treatment and supporting the role played by gentic variability in the emergence of HIV-1 antiviral resistance.

The effects of hydroxyurea (HU), an inhibitor of ribonucleotide reductase, on the replication of human immunodeficiency virus type 1 (HIV-1) in activated peripheral blood mononuclear cells were studied. The inhibition of HIV-1 replication by HU alone was dose dependent, with a 90% inhibitory concentration of 0.4 mM, a plasma concentration tolerated by patients with oncological diseases. HU at lower concentrations (< 0.1 mM) was found to potentiate the antiviral activity of 2',3'-dideoxyinosine (ddl), 3'-azido-2',3'- dideoxythymidine, and 2',3'-dideoxycytidine against HIV-1, with the potentiation being ddl greater than 3'-azido-2',3'- dideoxythymidine = 2',3'-dideoxycytidine. In the presence of 0.1 mM HU, the 90% inhibitory concentration of ddl was reduced by 6-fold in activated peripheral blood mononuclear cells. The potentiating effect of HU on ddl action was time dependent, with the greatest inhibition of HIV-1 growth being seen when HU was present during and after virus adsorption, i.e., apparently coinciding with the time of proviral DNA synthesis. A brief incubation of activated cells with HU and ddl at low concentrations before virus exposure reduced p24 production by > 50%. Analyses using high performance liquid chromatography and enzymatic assays suggested that the greater degree of potentiation by HU of the action of ddl, compared with the other dideoxynucleosides, is due to the more effective inhibition by HU of dATP synthesis, compared with the synthesis of the other deoxynucleoside triphosphates (dGTP, dTTP, and dCTP). The present study suggests that, for appropriate agents, pharmacological reduction of deoxynucleoside triphosphate levels represents a potential therapeutic approach for inhibition of HIV-1 replication.

1. Zidovudine (3'-azido-2',3'-dideoxythymidine; AZT; ZDV) is a dideoxynucleoside analogue active against human immunodeficiency virus (HIV). We are currently investigating the intracellular metabolism of ZDV to its putative active triphosphate form (ZDV triphosphate) in peripheral blood mononuclear cells and a lymphoblastoid cell line (h1A2v2). 2. Optimal conditions for intracellular phosphate formation in peripheral blood mononuclear cells occurred following a 72 h preincubation with the mitogen phytohaemagglutinin at a concentration of 10 micrograms ml-1. ZDV was metabolized predominantly to the monophosphate with smaller amounts of the di- and triphosphate anabolites. There was considerable inter- and intraindividual variability in phosphate formation in peripheral blood mononuclear cells. A similar pattern of phosphorylation was seen with the h1A2v2 lymphoblastoid cell line with ZDV monophosphate being the major metabolite. 3. With increasing interest in combination nucleoside analogue therapy in HIV-positive patients it is important to know if an interaction occurs at the level of phosphorylation. Neither dideoxyinosine (ddI) or dideoxycytidine (ddC) significantly reduced the intracellular phosphorylation of ZDV in either peripheral blood mononuclear cells or h1A2v2 cells. In contrast thymidine always gave marked inhibition (e.g. at 2.0 microM, 89% inhibition of total phosphate formation in peripheral blood mononuclear cells and 79% in h1A2v2 cells). It is, therefore, unlikely that in vivo either ddI or ddC will perturb ZDV phosphorylation.

Recent studies show that patients presenting with cytochrome oxidase (COX) deficiency in infancy may have reduced mitochondrial DNA (mtDNA) in muscle. The human mitochondrial transcription factor A (h-mtTFA) may be an important regulator of both transcription and replication of mtDNA. h-mtTFA levels were investigated in cell lines which were either free of mtDNA (rho 0) or temporarily depleted by treatment with dideoxycytidine (ddC), and in tissue from three patients with mtDNA depletion and cytochrome oxidase deficiency. h-mtTFA was compared with other mitochondrial proteins such as pyruvate dehydrogenase and porin by Western blotting. The ratio of mtDNA and h-mtTFA mRNA to reference nuclear probes was measured by dual labelling of dot blots. The ratio of mtDNA to nuclear DNA in skeletal muscle was low in muscle in the three patients and in other tissues in one. h-mtTFA was low in cells depleted either permanently or transiently of mtDNA, and this reduction in h-mtTFA roughly paralleled mtDNA levels. Similarly, treatment of rho 0 cell lines with ddC induced a reduction in mtDNA as well as h-mtTFA protein. The relationship between h-mtTFA and mtDNA levels suggests that they may be causally linked. MtDNA depletion was accompanied by an increase in the level of h-mtTFA RNA in the cell lines but low levels in the patient. This suggests that either h-mtTFA regulates mtDNA levels, or that h-mtTFA expression may be regulated by a feedback mechanism initiated by MtDNA Depletion.

(-)-beta-L-2',3'-Dideoxycytidine (L-ddC) and (-)-beta-L-2',3'-dideoxy-5-fluorocytidine (L-FddC) have been reported to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in vitro. In the present study, the 5'-triphosphates of L-ddC (L-ddCTP) and L-FddC (L-FddCTP) were demonstrated to competitively inhibit HIV-1 reverse transcriptase (RT), with inhibition constants (KiS) of 2 and 1.6 microM, respectively, when a poly(rI).oligo(dC)10-15 template primer was used; in comparison Ki values for beta-D-2',3'-dideoxycytidine 5'-triphosphate (D-ddCTP) and beta-D-2',3'-dideoxy-5-fluorocytidine 5'-triphosphate (D-FddCTP) were 1.1 and 1.4 microM, respectively. Use of the mutant RT at position 184 (substitution of methionine to valine [M184V]), which is associated with resistance to beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), resulted in significant increases (50- to 60-fold) in Ki values for L-ddCTP and L-FddCTP, whereas the elevation in Ki values for D-ddCTP and D-FddCTP was moderate (2-fold). L-ddCTP and L-FddCTP did not inhibit human DNA polymerases alpha and beta up to 100 microM. In contrast, D-ddCTP and D-FddCTP inhibited human DNA polymerase beta, with Ki values of 0.5 and 2.5 microM, respectively. By using sequencing analysis, L-ddCTP and L-FddCTP exhibited DNA chain-terminating activities toward the parental HIV-1 RT, whereas they were not a substrate for the mutant M184V HIV-1 RT.L-ddC and L-FddC did not inhibit the mitochondrial DNA content of human cells up to a concentration of 10 microM, whereas D-ddC and D-FddC decreased the mitochondrial DNA content by 90% at concentrations of 1 and 10 microM, respectively. All of these results suggest that further development of L-ddC, and L-FddC in particular, is warranted as a possible anti-HIV candidate.

The synthesis of new potential PFA-BCH-189 conjugate analogues is described and their molecular structure clearly identified through NMR and mass spectra techniques. The anti-HIV-1 activity was determined according to the inhibition of syncytium formation in MT-4 cells, while the anti-HBV activity was determined in infected duck hepatocytes. Both antiviral activities of the PFA-BCH-189 conjugates were much lower than those of the parent BCH-189 (2',3'-dideoxy-3'-thiacytidine) (1). Whereas a prodrug effect, following cleavage and release of the free BCH-189 and PFA, cannot be ruled out, poor cellular permeation of the drug seems to be the most likely reason for the reduced activities against HIV and DHBV. The presence of the PFA moiety appears to be detrimental for both the anti-HIV and anti-DHBV activity of PFA-BCH-189 cases.

The effect of acivicin (ACV) on cell growth, cell cycle progression, and intracellular accumulation of 2',3'-dideoxycytidine (ddC) was examined. Treatment of H9 cells with ACV (1 microM) plus ddC (25 microM) for 24 hrs inhibited cell growth to 74 +/- 7%, arrested cells in the S phase, and increased the cellular ddC accumulation to 195-208% of control. The concentrations of ddCTP, the active form of ddC, was increased 3-4 fold in ACV treated cells.

We have shown that umbilical cord blood mononuclear cells (CBMC) are at least as sensitive as peripheral blood mononuclear cells (PBMC) for isolation of human immunodeficiency virus type 1 from the PBMC of infected individuals. Viral replication was more efficiently monitored by a p24 antigen capture assay than by a viral reverse transcriptase test, regardless of whether CBMC or PBMC were employed. We also found that CBMC and PBMC yielded similar results with regard to the susceptibility profiles of both wild-type and drug-resistant variants of human immunodeficiency virus type 1 for 3'-azido-3'-deoxythymidine, 2',3'-dideoxycytidine, and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine. Finally, viruses isolated on CBMC could be routinely grown on PBMC and vice versa.

The clinical significance of the reduced in vitro susceptibility of HIV to antiretroviral agents has been difficult to elucidate for nucleoside analogs such as zidovudine. However, the virological significance of resistance to nevirapine and other HIV-1-specific reverse transcriptase inhibitors has been established. With antiretroviral therapy, disease progression is not equivalent to drug failure, which is not equivalent to drug resistance. Clinical disease progression is only indirectly linked to HIV replication. Drug resistance is complex, and combining drugs does not appear to delay emergence of resistant strains of HIV although it may affect the specific amino acid substitutions. Drug resistance does appear to contribute to drug failure. The clinical trial ACTG 116B/117 found that the duration of prior zidovudine therapy was not related to the relative benefit of switching to didanosine. Preliminary results of analysis of resistant strains of HIV isolated from ACTG 116B/117 patients revealed that the relative hazard of progression was about threefold higher for patients with high-level resistance to zidovudine, syncytium-inducing biological phenotype, and an AIDS diagnosis at baseline. This study showed clearly that acquisition of an HIV strain with high-level resistance to zidovudine was a poor prognostic factor. Although nevirapine resistance emerges rapidly, preliminary data suggest that high dosages may be active against HIV even in the presence of resistant HIV strains. At the present time, viral resistance and biological phenotype are not useful in the management of individual patients.

The rationale for combining anti-HIV-1 agents is to provide more complete viral suppression, to limit the emergence of drug resistance during chronic viral replication, and to provide more effective antiretroviral treatment even when mixtures of drug-resistant and drug-sensitive strains are present. In vitro experiments reveal increased suppression with multiple-drug therapy, but viral breakthrough occurs after prolonged time in culture even during triple-drug therapy. Clinical results available to date indicate that drugs should be given simultaneously for optimal benefit. There appears to be a rationale for early initiation of combination therapy before the onset of increased viral burden and the emergence of syncytium-inducing viral variants. The results of ACTG protocol 155 revealed benefit of zidovudine and zalcitabine over monotherapy with either agent in patients with CD4+ cell counts > or = 150 cells/mm3. However, further clinical studies will be necessary before firm recommendations can be made about the indications for combination antiretroviral therapy in HIV-1-infected individuals at different stages of disease. Ultimately, we need better drugs, in combination, which significantly impact on HIV-1 burden to achieve more complete viral suppression and to reduce selection of drug-resistant viral variants.

The synthesis of potential "combined prodrugs" where phosphonoformic acid (PFA) or phosphonoacetic acid (PAA) was attached to the 5'-O or N4 position of 2',3'-dideoxy-3'-thiacytidine (BCH-189) is described. The anti-HIV-1 activity of 11 analogues which included carboxylic ester or phosphoric ester linkages of PFA or PAA to BCH-189 was determined in MT-4 cells. Of these compounds, the IC50 of analogues 3, 4, 6, and 7 ranged from 0.2 to 100 microM, while IC50 for BCH-189 in this system was 0.1 microM. In vitro hydrolysis of the various esters or amides in human plasma indicated that these agents were relatively stable in the presence of plasma esterases with t1/2 values of up to 120 min. Moreover, lipophilicity of these compounds (partition coefficient) was determined in order to establish correlation between lipophilicity and diffusion of BCH-189 analogues into the cells. The active compounds may exert their effects by extracellular or intracellular hydrolysis to the corresponding antiviral agent BCH-189, but intrinsic anti-HIV-1 activity of some of PAA and PFA adducts, themselves, may also be involved.

Although several studies have shown that 3'-azido-3'-deoxythymidine (AZT) is not toxic for CD4+ lymphocytes, its effect on CD8+ cells has never been studied in a systematic way. We purified CD8+ cells from the peripheral blood mononuclear cells of both human immunodeficiency virus (HIV)-seronegative and HIV-infected individuals by means of magnetic beads that had been coated with monoclonal antibodies. We report that AZT, but not two other nucleosides tested, inhibited the interleukin-2-dependent proliferation of CD8+ lymphocytes in a dose-dependent manner. No such effect was observed with regard to CD4(+)-enriched populations. The AZT-mediated antiproliferative effect did not appear to be related to either the CD4+ count or to prior treatment with this drug in the case of HIV-seropositive subjects.

We have selected a human immunodeficiency virus type 1 (HIV-1) mutant strain with a moderate (sevenfold) level of resistance to the nucleoside analog 2',3'-didehydro-2',3'-dideoxythymidine (D4T or stavudine). After serial passage of the HXB2 strain of HIV-1 in MT4 cells, a novel mutation involving two nucleotide substitutions in codon 75 of the viral reverse transcriptase, altering valine to threonine, was seen. When introduced into a wild-type HIV-1 background by site-directed mutagenesis, the T-75 mutation conferred cross-resistance to the dideoxynucleosides dideoxyinosine and dideoxycytosine as well as to 2',3'-didehydro-2',3'-dideoxycytosine.

The L enantiomer of 2',3'-dideoxycytidine (DDC) was recently shown to inhibit selectively human immunodeficiency virus type 1 (HIV-1) in vitro. In the current study, the potent anti-HIV activity of L-DDC was confirmed and extended to several HIV-1 and HIV-2 strains in various cell culture systems, including primary human lymphocytes and macrophages. Furthermore, its 5-fluoro congener, beta-L-2',3'-dideoxy-5-fluorocytidine (L-FDDC), was found to have more potent anti-HIV activity and a higher therapeutic index in acutely infected human peripheral blood mononuclear cells. These compounds had no marked activity against HIV-1 isolates resistant to the oxathiolane pyrimidine nucleosides (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC] and (-)-beta-L-2',3'-dideoxy-3'-thiacytidine, but 3'-azido-3'-deoxythymidine (AZT)-resistant viruses were susceptible to L-DDC and L-FDDC. Cytotoxicity studies with human myeloid progenitor cells indicated that L-DDC and L-FDDC had median inhibitory concentrations comparable to those of AZT, DDC, and FDDC, but L-DDC and L-FDDC were significantly less toxic than AZT, DDC, and FDDC when erythroid progenitor cells were used. L-FDDC had the highest selectivity indices (6,000 and 9,000 for erythroid and myeloid progenitor cells, respectively) of all the compounds evaluated. Further preclinical development of L-FDDC is warranted in order to determine its potential usefulness in the treatment of HIV infections.

Objective: To determine the in vitro susceptibility of primary clinical isolates and laboratory strains of HIV-1 to XM323., Methods: The AIDS Clinical Trials Group/US Department of Defense p24 antigen-based consensus assay was used to determine in vitro susceptibility of 57 primary clinical isolates and three laboratory strains of HIV-1 to XM323, zidovudine, zalcitabine (ddC), and didanosine (ddI)., Results: The concentrations of compound required to inhibit viral p24 antigen production by 50% [median inhibitory concentration (IC50)] for nucleosides were as follows: zidovudine, 0.001-->5 microM; ddC, < 0.01-0.23 microM; ddI, 0.2-->25 microM). Against both nucleoside susceptible and resistant isolates XM323 exhibited potent inhibition with IC50 values of < 0.02-0.27 microM and IC90 values of 0.03-1.17 microM., Conclusions: XM323 is a potent inhibitor of diverse clinical isolates of HIV-1 in vitro and represents a novel class of non-peptidyl inhibitors of HIV-1 protease.

Mitochondria play a prominent role in shaping intracellular calcium concentration ([Ca2+]i) transients in dorsal root ganglion neurons. Mitochondrial DNA polymerase is inhibited by antiviral compounds such as 2',3'-dideoxycytidine (ddC). Here, we test the hypothesis that ddC can alter mitochondrially mediated Ca2+ buffering in neurons. Chronic treatment of dorsal root ganglion cultures with ddC (1 microM) lowered mitochondrial DNA levels and decreased the mitochondrially mediated component of depolarization-induced [Ca2+]i transients. The inhibition increased in a time-dependent manner, reaching a maximum at 6 days. ddC did not affect small, action potential-evoked, [Ca2+]i transients that are predominantly buffered by Ca(2+)-ATPases, suggesting that ATP levels were not depleted. The drug did not inhibit whole-cell Ca2+ currents, indicating that the Ca2+ load was not affected. Thus, ddC produces a graded, time-dependent inhibition of mitochondrial function that is reflected, in part, by a decrease in the direct buffering of Ca2+ by mitochondria. This effect may contribute to the peripheral neuropathy that results from ddC treatment. Furthermore, ddC promises to be a useful tool to study the role of mitochondria in [Ca2+]i homeostasis and neurodegenerative processes.

Blood-derived macrophages were acutely infected with HIV and treated with a combination of leucocyte interferon (IFN) and five anti-HIV drugs. HIV growth was assayed by quantitation of p24 antigen in the supernatant and in some experiments by determination of reverse transcriptase activity. Both IFN and all drugs, with the exception of fusidic acid, inhibited HIV growth in a dose-dependent manner. IFN in combination with zidovudine, dideoxycytidine or fusidic acid exerted a synergistic effect on HIV titers, while IFN combined with dideoxyinosine or foscarnet had an additive effect.

Dideoxycytidine (ddC) inhibits the replication of hepatitis B virus (HBV) but its clinical use is limited by peripheral neuropathy. We synthesized dioleoylphosphatidyl-ddC (DOP-ddC), a phospholipid prodrug of ddC which forms lipid bilayers and is readily incorporated into liposomes. The 90% effective dose (ED90) of DOP-ddC was 18 microM vs. 7 microM for ddC. However, in HBV-infected human hepatoma cells (2.2.15 cells), DOP-ddC was less toxic in vitro. When liposomal DOP-[5,6-3H]ddC was administered intraperitoneally to mice, drug levels in liver were 40 times greater than [5,6-3H]ddC when expressed as area under curve. Liposomal DOP-ddC also provided higher levels of drug in lymph nodes and spleen, important accessory sites of HBV replication. Plasma levels of drug remained above the ED90 six times longer with DOP-ddC than with ddC. DOP-ddC levels in sciatic nerve, the major site of toxicity, were not significantly different from levels observed with free ddC. The phospholipid prodrug approach is a general one which may readily be applied to other antiviral nucleosides for HBV.

Combinations of ddC with either the ribonucleotide reductase inhibitor hydroxyurea (HU) or with the natural nucleoside thymidine have been investigated on the cycle of a defective (psi neo) Moloney Leukemia Virus (MoMLV) using 3T3 fibroblasts as host cells. In this experimental model, ddC displayed very poor antiviral action which was obvious given an IC50 value close to 100 microM, i.e. an efficiency about thirty thousand fold lower than that of AZT. Both HU and thymidine alone resulted in significant inhibition of MoMLV replication with IC50 values of 40 microM and 100 microM respectively. The combination of ddC with 50 microM HU lowered the IC50 of ddC by a factor of 10. A similar but more pronounced effect was obtained by combining ddC and 100 microM thymidine, which decreases the IC50 value of ddC by a factor of 50. Combining 40 microM ddC and 100 microM thymidine resulted in the quite complete inhibition of viral replication. These results show that in cell types with strongly restricted ddC action, combination treatment with compounds known to ultimately decrease dCTP biosynthesis leads to the restoration of efficient antiviral activity.

3'-Amino-2',3'-dideoxycytidine (3'-NH2-ddCyd) is a 3'-modified deoxycytidine analog that specifically inhibits DNA synthesis. Inhibition of chain elongation at the replication fork was examined utilizing a batch hydroxylapatite chromatography method. Exponentially growing cells were exposed to 3'-NH2-ddCyd and the diterpene aphidicolin for 9.5 hr at concentrations that inhibited DNA synthesis by approximately 60 and 90%, as determined by precursor uptake. Both agents demonstrated a concentration-dependent inhibition of pulse labeling of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) generated by a limited alkaline lysis procedure. Upon removal of drug, the rate of elongation of pulse-labeled DNA was similar to that of untreated cells at both concentrations of aphidicolin and at the low concentration of the amino analog. Under these conditions, no reduction in cell survival was observed using the clonogenic assay technique. However, at the high concentration of 3'-NH2-ddCyd, the rate of elongation following drug removal was one-third that of untreated cultures, and a 50% loss in cell viability was observed. Furthermore, upon incubation of purified dsDNA with the Klenow fragment of Escherichia coli DNA polymerase I or purified ssDNA with calf thymus terminal deoxynucleotidyl transferase, only DNA from cells treated with the high concentration of 3'-NH2-ddCyd served as a poor template for further synthesis. The results indicate that 3'-NH2-ddCyd, in a concentration-dependent manner, inhibits DNA synthesis by reducing the rate of chain elongation at the replication fork, which subsequently leads to a functional blocking of 3'-ends in DNA. The data suggest that there may be a relationship between loss of cell viability and reduction in the number of 3'-ends available for DNA replication.

Unlike herpes viruses, human immunodeficiency virus and other retroviruses do not encode specific enzymes required for the metabolism of the purine or pyrimidine nucleotides to their corresponding 5'-triphosphates. Therefore, 2',3'-dideoxynucleosides and acyclic nucleoside phosphonates must be phosphorylated and metabolized by host cell kinases and other enzymes of purine and/or pyrimidine metabolism. Different animal species (or even different cell types within one animal species) may differ in the efficiency of conversion of these drugs to their antivirally active metabolite(s). Three 2',3'-dideoxynucleosides are officially licensed for clinical use [i.e., zidovudine (3'-azido-2',3'-dideoxythymidine, AZT), didanosine (2',3'-dideoxyinosine, DDI) and zalcitabine (2',3'-dideoxycytidine, DDC)]. A number of other 2',3'-dideoxynucleoside analogues [among them stavudine (2',3'-didehydro-2',3'-dideoxythymidine, D4T), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC) and the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA)] are currently under clinical investigation and are candidate compounds for eventual licensing as anti-AIDS drugs. The metabolic pathways, antimetabolic effects and mechanism of antiviral action of these nucleoside analogues will be discussed.

Mutants of feline immunodeficiency virus (FIV) were selected in cell culture in the continuous presence of 10 microM (each) 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI). These mutants (AIR-1 and AIR-3) displayed a 13-fold resistance to AZT but had less than a 2-fold decrease in susceptibility to ddI. Interestingly, the AIR mutants were cross-resistant to phosphonoformate (PFA) and were hypersensitive to 2',3'-dideoxycytidine (ddC). Mutants of FIV were also selected in the presence of 10 microM ddI alone (DIS-1, DIS-2c), and these displayed a two- to fourfold decrease in susceptibility to ddI. Like the mutants selected with the combination of AZT plus ddI, DIS-1 and DIS-2c were cross-resistant to PFA and were hypersensitive to ddC. However, they remained as susceptible as wild-type FIV to AZT. Thus, the mutants selected with the combination of AZT plus ddI have phenotypes which reflect those obtained by selection with these drugs individually.

beta-L-2',3'-Dideoxycytidine (beta-L-ddC) and beta-L-5-fluoro-2',3'-dideoxycytidine (5-F-beta-L-ddC) were prepared and shown to have potent activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). These compounds were compared with beta-D-2',3'-dideoxycytidine (beta-D-ddC) and two beta-L-oxathiolane nucleosides (beta-L-3'-thio-2',3'-dideoxycytidine and beta-L-5-fluoro-3'-thio-2',3'-dideoxycytidine) in terms of anti-HIV and anti-HBV activity, cytotoxicity, and development of HIV-1 resistance. Compared with beta-D-ddC, the beta-L-dideoxycytidine nucleosides had similar anti-HIV-1 activities, significantly greater anti-HBV activities, and decreased toxicities to a B-cell line, T-cell lines, and human bone marrow progenitor cells. HIV-1 strains resistant to beta-D-ddC were susceptible to the beta-L-ddC analogs. Compared with the beta-L-oxathiolane nucleosides, beta-L-ddC and 5-F-beta-L-ddC had similar anti-HIV-1 activities, decreased anti-HBV activities, and greater toxicities to B- and T-cell lines and bone marrow progenitor cells. There were similarities between the beta-L-ddC and beta-L-oxathiolane nucleosides in the rate of development and pattern of resistant HIV-1 selection. While the in vitro activity and cytotoxicity profiles of the beta-L-ddC nucleosides differed from those of the beta-D-ddC and beta-L-oxathiolane nucleosides, the data presented herein suggest that the sugar configuration of a dideoxynucleoside analog may play a major role in the rate of development and the pattern of HIV-1 resistance.

Various 2',3'-dideoxy-L-cytidine,2',3'-dideoxy-L-uridine, and 3'-deoxy-L-thymidine analogues have been synthesized and evaluated in vitro as potential anti-HIV and anti-HBV agents. Coupling of 1-O-acetyl-5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-L-ribofuranose (1) with silylated derivatives of 5-fluorocytosine, cytosine, 5-fluorouracil, uracil, and thymine in the presence of ethylaluminum dichloride gave the corresponding nucleosides 2, 3, 4, 5, 10, 11, 12, 16, 17, and 18 as a mixture of alpha- and beta-anomers, which were then deblocked to yield the corresponding 2',3'-dideoxy-L-5-fluorocytidine derivatives, 6 and 7, 2',3'-dideoxy-L-cytidine derivatives, 8 and 9, 2',3'-dideoxy-beta-L-fluorouridine (13), 2',3'-dideoxy-beta-L-uridine (14), and 3'-deoxy-L-thymidine derivatives, 15 and 19. Among these 2',3'-dideoxy-L-nucleoside analogues, 2',3'-dideoxy-beta-L-5-fluorocytidine (6, beta-L-FddC) was found to be the most active against HIV-1, which is approximately 3 and 4 times more active against HIV-1 in vitro than 2',3'-dideoxy-beta-D-cytidine (ddC) and 2',3'-dideoxy-beta-D-5-fluorocytidine (beta-D-FddC) with ED50 values of 0.5, 1.5, and 2 microM, respectively. The dose-limiting toxicity of ddC is severe neuropathy which may be caused by the inhibition of the synthesis of mitochondrial DNA. ddC has an IC50 value of 0.022 microM against host mitochondrial DNA synthesis. Conversely, the IC50 values for beta-L-FddC and beta-L-ddC are > 100 microM; therefore, neuropathy may not present itself to be a problem with beta-L-FddC and beta-L-ddC as chemotherapeutic agents. In addition, beta-L-FddC and 2',3'-dideoxy-beta-L-cytidine (8, beta-L-ddC) demonstrated equally potent activity against HBV in vitro by having the same ED50 value of 0.01 microM. Both beta-L-FddC and beta-L-ddC, which have an "unnatural" L-configuration in the sugar moiety, are approximately 1000 and 280 times more potent, respectively, against HBV than the D-configuration beta-D-FddC and ddC which have an ED50 values of 10 and 2.8 microM. In view of the potent antiviral activity of beta-L-FddC against both HIV-1 and HBV and potent antiviral activity of beta-L-ddC against HBV in vitro, their low cytotoxicity, and especially the negligible inhibitory effect on host mitochondrial DNA synthesis, beta-L-FddC and beta-L-ddC merit further development as potential anti-HIV and anti-HBV agents.

Human immunodeficiency virus type 1 (HIV-1) isolates resistant to zidovudine (ZDV) have previously been demonstrated to exhibit in vitro cross-resistance to other similar dideoxynucleoside agents which contain a 3'-azido group. However, cross-resistance to didanosine (ddI) or dideoxycytidine (ddC) has been less well documented. ZDV, ddI, and ddC susceptibility data have been collected from clinical HIV-1 isolates obtained by five clinical centers and their respective retrovirology laboratories. All subjects were treated only with ZDV. Clinical HIV-1 isolates were isolated, amplified, and assayed for drug susceptibility in standardized cultures of phytohemagglutinin-stimulated donor peripheral blood mononuclear cells obtained from healthy seronegative donors. All five cohorts showed a correlation between decreased in vitro susceptibility to ZDV and decreased susceptibility to ddI and ddC. For each 10-fold decrease in ZDV susceptibility, an average corresponding decrease of 2.2-fold in ddI susceptibility was observed (129 isolates studied; P < 0.001, Fisher's test of combined significance). Similarly, susceptibility to ddC decreased 2.0-fold for each 10-fold decrease in ZDV susceptibility (82 isolates studied; P < 0.001, Fisher's test of combined significance). These data indicate that a correlation exists between HIV-1 susceptibilities to ZDV and ddI or ddC for clinical HIV-1 isolates.

Combinations of the human immunodeficiency virus (HIV) Tat protein antagonist Ro 24-7429 with either the HIV protease inhibitor Ro 31-8959 or the HIV reverse transcriptase inhibitors AZT (3'-azido-3'-deoxythymidine), ddC (2',3'-dideoxycytidine), ddI (2',3'-dideoxyinosine), and nevirapine were synergistic or additive in reducing HIV type 1 p24 antigen production in CEM cells or inhibiting HIV type 1-induced syncytium formation in HT4-6C cells.

A human immunodeficiency virus type 1 variant resistant to zalcitabine (2',3'-dideoxycytidine [ddC]) was selected by sequential passage in the presence of increasing concentrations of ddC in peripheral blood mononuclear cell cultures. A mutation causing a lysine-to-arginine substitution was noted in reverse transcriptase (RT) codon 65 of this ddC-selected virus. A cloned mutant virus with this codon 65 mutation was constructed by using a novel PCR approach for site-directed mutagenesis. Characterization of this virus confirmed that the RT Lys-65-->Arg substitution was necessary and sufficient for a fourfold increase in the ddC 50% inhibitory concentration, as well as for resistance to didanosine (2',3'-dideoxyinosine [ddI]). Lys-65-->Arg and virus resistance to ddC and ddI also developed during therapy in isolates from one ddC-treated patient and two ddI-treated patients. Recombinant-expressed codon 65 mutant RT enzyme was resistant to ddCTP and ddATP in cell-free polymerase assays. Results of mutant enzyme studies are consistent with Lys-65-->Arg leading to changes in binding of the triphosphate forms of these nucleoside analogs to the RT. These data have implications for future studies of ddC resistance, particularly those aimed at defining its clinical relevance.

The technique of in vitro selection was used to generate variants of the human immunodeficiency virus type 1 that are resistant to 2',3'-dideoxycytidine (ddC). Most of the pol regions of such viruses, including the complete reverse transcriptase open reading frame and portions of flanking protease and integrase genes, were cloned and sequenced, using PCR-based procedures. Mutations were variously detected at amino acid site 65 (Lys-->Arg; AAA-->AGA) and at a previously reported codon, site 184 (Met-->Val; ATG-->GTG). We introduced the site 65 mutation into the pol gene of infectious, cloned HxB2-D DNA by site-directed mutagenesis in order to confirm by viral replication assay the importance of this site in conferring resistance to ddC. The recombinant virus possessed greater than 10-fold resistance against this compound in comparison with parental HxB2-D. Cross-resistance of approximately 20- and 3-fold, respectively, was detectable against the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine and 2',3'-dideoxyinosine but not against 3'-azido-3'-deoxythymidine. Combinations of the site 65 and 184 mutations did not yield levels of resistance higher than those attained with the site 65 mutation alone. The presence of the site 65 mutation was confirmed by PCR analysis of peripheral blood mononuclear cells from patients on long-term ddC therapy in 4 of 11 cases tested. Viruses that possessed a ddC resistance phenotype were isolated from subjects whose viruses contained the site 65 mutation in each of four instances. Four of these clinical samples were also demonstrated to possess the Met-184-->Val mutation, and one of them possessed both the Lys-65-->Arg and Met-184-->Val substitutions. Direct cloning and sequencing revealed the site 65 mutation in viruses isolated from these individuals.

Bisheteroarylpiperazine compounds are nonnucleoside reverse transcriptase inhibitors of human immunodeficiency virus type 1 (HIV-1). To provide a rationale for combination therapy with a second-generation bisheteroarylpiperazine, we investigated the effect of U-90152 in combination with 3'-azido-3'-deoxythymidine (AZT) or 2',3'-dideoxycytidine (ddC). HIV-1-infected cells were cultured in the presence of test compounds, and drug effects on p24 core antigen production were measured by an enzyme-linked immunosorbent assay. In a CD4+ T-cell line (H9) infected with HIV-1IIIB, the 50% effective concentrations for U-90152, AZT, and ddC were 6.0, 80.4, and 31.8 nM, respectively. In human peripheral blood mononuclear cells infected with the molecularly cloned clinical isolate HIV-1JRCSF, the 50% effective concentrations for U-90152, AZT, and ddC were 5.3, 5.9, and 25.0 nM, respectively. Over a range of drug concentrations (U-90152 and AZT at 0.3, 1, 3, 10, and 30 nM; ddC at 3, 10, 30, and 100 nM), U-90152 in combination with AZT or ddC synergistically inhibited the replication of a laboratory-adapted strain and a clinical isolate of HIV-1.

To ascertain whether combination therapy with zidovudine (AZT) and zalcitabine (ddC) delayed the emergence of AZT resistance, isolates of human immunodeficiency virus (HIV) were evaluated from 15 previously untreated patients with advanced HIV disease who received combination therapy. Eighteen sequential viral isolates were available from 15 patients who received > or = 6 months of combination therapy. Isolates from eight (73%) of 11 patients obtained between 24 and 48 weeks of therapy were AZT resistant [50% inhibitory concentration (IC50) > or = 0.45 microM; range, 0.45-2.0 microM]. Four of these eight patients yielded virus isolates that were highly AZT resistant (IC50 > 1.0 microM). No changes in ddC susceptibility were discerned. The median IC50 for ddC was 0.2 microM and ranged from 0.07 to 0.5 microM. The CD4 cell counts of patients with AZT-sensitive virus tended to have higher median areas under the curve (AUC) and greater increases compared with patients who had AZT-resistant virus. They also had higher means and ranges of the average CD4 cell counts at week 36 (p = 0.01) and week 48 (p = 0.04). These data would indicate that the previously described more sustained CD4 cell responses conferred by the addition of ddC to AZT therapy cannot be ascribed to delayed emergence of AZT resistance with combination therapy.

2',3'-Dideoxy-beta-L-5-fluorocytidine (beta-L-FddC) and 2',3'-dideoxy-beta-L-cytidine (beta-L-ddC), two nucleosides with "unnatural L-configuration," have been synthesized and found to have potent antiviral activity against hepatitis B virus (HBV) and human immunodeficiency virus type 1 (HIV-1) in vitro with very little toxicity. At 1 microM, both beta-L-ddC and beta-L-FddC inhibited the growth of HBV by more than 90%, while at the same concentration the D-configuration counterparts, 2',3'-dideoxy-beta-D-cytidine (ddC) and 2',3'-dideoxy-beta-D-5-fluorocytidine (beta-D-FddC), did not show antiviral activity against HBV. The order of anti-HIV-1 activity was beta-L-FddC > ddC; beta-D-FddC > beta-L-ddC. The dose-limiting toxicity of ddC is neuropathy which is believed to be caused by the inhibition of the synthesis of mitochondrial DNA. ddC severely inhibited the mitochondrial DNA synthesis of CEM cells yielding an IC50 value of 0.022 microM. Conversely, both beta-L-FddC and beta-L-ddC did not demonstrate any inhibition against mitochondrial DNA synthesis up to 100 microM concentration.