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Based on recent genome-wide association studies, abacavir-induced hypersensitivity is highly associated with human leukocyte antigen (HLA)-B*57:01 allele. However, the underlying mechanism of this occurrence is unclear. To investigate the underlying mechanism, we developed HLA-B*57:01 transgenic mice and found that application of abacavir could cause CD8 T cell activation with elevation in PD1 expression; however, severe skin hypersensitivity was not observed. To eliminate the immunosuppressive effect of PD1, HLA-B*57:01 transgenic/PD1 knockout (01Tg/PD1) mice were generated by mating HLA-B*57:01 transgenic mice and PD1 knockout mice. Thereafter, 01Tg/PD1 mice were treated with abacavir. Similar to the above results, severe skin hypersensitivity was not observed. Therefore, we treated 01Tg/PD1 mice with an anti-CD4 antibody to deplete CD4 T cells, followed by abacavir topically and orally. Severe abacavir-induced skin hypersensitivity was observed in 01Tg/PD1 mice after depletion of CD4 T cells, in addition to significant CD8 T cell activation and dendritic cell maturation. Taken together, we succeeded in reproducing severe skin hypersensitivity in a mouse model. And we found that through the combined depletion of PD1 and CD4 T cells, CD8 T cells could be activated and could proceed to clonal proliferation, which is promoted by mature dendritic cells, thereby eventually inducing severe skin hypersensitivity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Antiretroviral therapy (ART) in pregnancy has dramatically reduced HIV vertical transmission rates. Consequently, there is a growing number of children that are HIV exposed uninfected (CHEUs). Studies suggest that CHEUs exposed in utero to ART may experience developmental delays compared to their peers. We investigated the effects of in utero ART exposure on perinatal neurodevelopment in mice, through assessment of developmental milestones. Developmental milestone tests (parallel to reflex testing in human infants) are reflective of brain maturity and useful in predicting later behavioral outcomes. We hypothesized that ART in pregnancy alters the in utero environment and thereby alters developmental milestone outcomes in pups. Throughout pregnancy, dams were treated with boosted-atazanavir combined with either abacavir/lamivudine (ATV/r/ABC/3TC), or tenofovir/emtricitabine (ATV/r/TDF/FTC), or water as control. Pups were assessed daily for general somatic growth and on a battery of tests for primitive reflexes including surface-righting, negative-geotaxis, cliff-aversion, rooting, ear-twitch, auditory-reflex, forelimb-grasp, air-righting, behaviors in the neonatal open field, and olfactory test. In utero exposure to either ART regimen delayed somatic growth in offspring and evoked significant delays in the development of negative geotaxis, cliff-aversion, and ear-twitch reflexes. Exposure to ATV/r/ABC/3TC was also associated with olfactory deficits in male and forelimb grasp deficits in female pups. To explore whether delays persisted into adulthood we assessed performance in the open field test. We observed no significant differences between treatment arm for males. In females, ATV/r/TDF/FTC exposure was associated with lower total distance travelled and less ambulatory time in the centre, while ATV/r/ABC/3TC exposure was associated with higher resting times compared to controls. In utero PI-based ART exposure delays the appearance of primitive reflexes that involve vestibular and sensory-motor pathways in a mouse model. Our findings suggest that ART could be disrupting the normal progress/maturation of the underlying neurocircuits and encourage further investigation for underlying mechanisms., Competing Interests: The authors have no conflicts of interest to disclose in relation to this work. LS reports personal fees from ViiV Healthcare for participation in a Women and Transgender Think Tank. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Various types of transgenic mice carrying either class I or II human leukocyte antigen (HLA) molecules are readily available, and reports describing their use in a variety of studies have been published for more than 30 years. Examples of their use include the discovery of HLA-specific antigens against viral infection as well as the reproduction of HLA-mediated autoimmune diseases for the development of therapeutic strategies. Recently, HLA transgenic mice have been used to reproduce HLA-mediated idiosyncratic drug toxicity (IDT), a rare and unpredictable adverse drug reaction that can result in death. For example, abacavir-induced IDT has successfully been reproduced in HLA-B*57:01 transgenic mice. Several reports using HLA transgenic mice for IDT have proven the utility of this concept for the evaluation of IDT using various HLA allele combinations and drugs. It has become apparent that such models may be a valuable tool to investigate the mechanisms underlying HLA-mediated IDT. This review summarizes the latest findings in the area of HLA transgenic mouse models and discusses the current challenges that must be overcome to maximize the potential of this unique animal model.

Highly active antiretroviral treatment has led to unprecedented efficacy and tolerability in people living with HIV. This effect was also observed in the central nervous system with the nowadays uncommon observation of dementias; yet in more recent works milder forms are still reported in 20-30% of optimally treated individuals. The idea of a subclinical neuronal toxicity induced by antiretrovirals has been proposed and was somehow supported by the late-emerging effects associated with efavirenz use. In this manuscript we are reviewing all the potential mechanisms by which antiretroviral drugs have been associated with in vitro, ex vivo, or in vivo toxicity to cells pertaining to the central nervous system (neurons, astrocytes, oligodendrocytes, and endothelial cells). These include direct or indirect effects and pathological pathways such as amyloid deposition, damage to small cerebral vessels, and impairment in neurotransmission. The aim of this review is therefore to provide a detailed description of the available literature in order to guide further clinical research for improving patients' neurocognition and quality of life.

Abacavir (ABC), zidovudine (AZT), and lamivudine (3TC) are nucleoside analog reverse transcriptase inhibitors (NRTIs) widely used as combination-based antiretroviral therapy against human immunodeficiency virus. Despite effective viral suppression using NRTI combinations, genotoxic potential of NRTIs can be increased when administered in combination. This study investigated the toxic and genotoxic potential of ABC when administered alone or in combination with AZT and/or 3TC using the somatic mutation and recombination test in Drosophila melanogaster. This test simultaneously evaluated two events related to carcinogenic potential: mutation and somatic recombination. The results indicated that ABC was responsible for toxicity when administered alone or in combination with AZT and/or 3TC. In addition, all treatment combinations increased frequencies of mutation and somatic recombination. The combination of AZT/3TC showed the lowest genotoxic activity compared to all combinations with ABC. Therefore, our results indicated that ABC was responsible for a significant portion of genotoxic activity of these combinations. Somatic recombination was the main genetic event observed, ranging from 83.7% to 97.7%.

Genome-wide association studies indicate that several idiosyncratic adverse drug reactions are highly associated with specific human leukocyte antigen (HLA) alleles. For instance, abacavir, a human immunodeficiency virus reverse transcriptase inhibitor, induces multiorgan toxicity exclusively in patients carrying the HLA-B*57:01 allele. However, the underlying mechanism is unclear due to a lack of appropriate animal models. Previously, we developed HLA-B*57:01 transgenic mice and found that topical application of abacavir to the ears induced proliferation of CD8+ lymphocytes in local lymph nodes. Here, we attempted to reproduce abacavir-induced liver injury in these mice. However, oral administration of abacavir alone to HLA-B*57:01 transgenic mice did not increase levels of the liver injury marker alanine aminotransferase. Considering the importance of innate immune activation in mouse liver, we treated mice with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist, plus abacavir. This resulted in a marked increase in alanine aminotransferase, pathological changes in liver, increased numbers of activated CD8+ T cells, and tissue infiltration by immune cells exclusively in HLA-B*57:01 transgenic mice. These results indicate that CpG oligodeoxynucleotide-induced inflammatory reactions and/or innate immune activation are necessary for abacavir-induced HLA-mediated liver injury characterized by infiltration of CD8+ T cells. Thus, we developed the first mouse model of HLA-mediated abacavir-induced idiosyncratic liver injury. Further investigation will show that the proposed HLA-mediated liver injury model can be applied to other combinations of drugs and HLA types, thereby improving drug development and contributing to the development of personalized medicine.

Immune-mediated idiosyncratic drug toxicity (IDT) is a rare adverse drug reaction, potentially resulting in death. Although genome-wide association studies suggest that the occurrence of immune-mediated IDT is strongly associated with specific human leukocyte antigen (HLA) allotypes, these associations have not yet been prospectively demonstrated. In this study, we focused on HLA-B*57:01 and abacavir (ABC)-induced immune-mediated IDT, and constructed transgenic mice carrying chimeric HLA-B*57:01 (B*57:01-Tg) to determine if this in vivo model may be useful for evaluating immune-mediated IDT. Local lymph node assay (LLNA) results demonstrated that percentages of BrdU + , IL-2 + , and IFN-γ + in CD8 + T cells of ABC (50 mg/kg/day)-applied B*57:01-Tg mice were significantly higher than those in littermates (LMs), resulting in the infiltration of inflammatory cells into the ear. These immune responses were not observed in B*57:03-Tg mice (negative control). Furthermore, oral administration of 1% (v/v) ABC significantly increased the percentage of CD44 high CD62L low CD8 + memory T cells in lymph nodes and spleen derived from B*57:01-Tg mice, but not in those from B*57:03-Tg mice and LMs. These results suggest that B*57:01-Tg mice potentially enable the reproduction and evaluation of HLA-B*57:01 and ABC-induced immune-mediated IDT.

Antiretroviral (ARV) drug therapy, given during pregnancy for prevention of mother-to-child transmission of human immunodeficiency virus 1 (HIV-1), induces fetal mitochondrial dysfunction in some children. However, the persistence/reversibility of that dysfunction is unclear. Here we have followed Erythrocebus patas (patas) monkey offspring for up to 3 years of age (similar in development to a 15-year old human) after exposure of the dams to human-equivalent in utero ARV exposure protocols. Pregnant patas dams (3-5/exposure group) were given ARV drug combinations that included zidovudine (AZT)/lamivudine (3TC)/abacavir (ABC), or AZT/3TC/nevirapine (NVP), for the last 10 weeks (50%) of gestation. Infants kept for 1 and 3 years also received drug for the first 6 weeks of life. In offpsring at birth, 1 and 3 years of age mitochondrial morphology, examined by electron microscopy (EM), was compromised compared to the unexposed controls. Mitochondrial DNA (mtDNA), measured by hybrid capture chemiluminescence assay (HCCA) was depleted in hearts of patas exposed to AZT/3TC/NVP at all ages (P < 0.05), but not in those exposed to AZT/3TC/ABC at any age. Compared to unexposed controls, mitochondrial reserve capacity oxygen consumption rate (OCR by Seahorse) in cultured bone marrow mesenchymal fibroblasts from 3-year-old patas offspring was ∼50% reduced in AZT/3TC/ABC-exposed patas (P < 0.01), but not in AZT/3TC/NVP-exposed patas. Overall the data show that 3-year-old patas sustain persistent mitochondrial dysfunction as a result of perinatal ARV drug exposure. Environ. Mol. Mutagen. 57:526-534, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: NRTIs are essential components of HIV therapy with well-documented, long-term mitochondrial toxicity in hepatic cells, but whose acute effects on mitochondria are unclear. As acetaminophen-induced hepatotoxicity also involves mitochondrial interference, we hypothesized that it would be exacerbated in the context of ART., Methods: We evaluated the acute effects of clinically relevant concentrations of the most widely used NRTIs, alone or combined with acetaminophen, on mitochondrial function and cellular viability., Results: The purine analogues abacavir and didanosine produced an immediate and concentration-dependent inhibition of oxygen consumption and complex I and III activity. This inhibition was accompanied by an undermining of mitochondrial function, with increased production of reactive oxygen species and reduction of mitochondrial membrane potential and intracellular ATP levels. However, this interference did not compromise cell survival. Co-administration with concentrations of acetaminophen below those considered hepatotoxic exacerbated the deleterious effects of both compounds on mitochondrial function and compromised cellular viability, showing a clear correlation with diminished glutathione levels., Conclusions: The simultaneous presence of purine analogues and low concentrations of acetaminophen significantly potentiates mitochondrial dysfunction, increasing the risk of liver injury. This new mechanism is relevant given the liver's susceptibility to mitochondrial dysfunction-related toxicity and the tendency of the HIV infection to increase oxidative stress., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Today HIV-1 infection is recognized as a chronic disease with obligatory lifelong treatment to keep viral titers below detectable levels. The continuous intake of antiretroviral drugs however, leads to severe and even life-threatening side effects, supposedly by the deleterious impact of nucleoside-analogue type compounds on the functioning of the mitochondrial DNA polymerase. For detailed investigation of the yet partially understood underlying mechanisms, the availability of a versatile model system is crucial. We therefore set out to develop the use of Caenorhabditis elegans to study drug induced mitochondrial toxicity. Using a combination of molecular-biological and functional assays, combined with a quantitative analysis of mitochondrial network morphology, we conclude that anti-retroviral drugs with similar working mechanisms can be classified into distinct groups based on their effects on mitochondrial morphology and biochemistry. Additionally we show that mitochondrial toxicity of antiretroviral drugs cannot be exclusively attributed to interference with the mitochondrial DNA polymerase.

Aldehydes are highly reactive molecules, which can be generated during numerous physiological processes, including the biotransformation of drugs. Several non-P450 enzymes participate in their metabolism albeit alcohol dehydrogenase and aldehyde dehydrogenase are the ones most frequently involved in this process. Endogenous and exogenous aldehydes have been strongly implicated in multiple human pathologies. Their ability to react with biomacromolecules (e.g. proteins) yielding covalent adducts is suggested to be the common primary mechanism underlying the toxicity of these reactive species. Abacavir is one of the options for combined anti-HIV therapy. Although individual susceptibilities to adverse effects differ among patients, abacavir is associated with idiosyncratic hypersensitivity drug reactions and an increased risk of cardiac dysfunction. This review highlights the current knowledge on abacavir metabolism and discusses the potential role of bioactivation to an aldehyde metabolite, capable of forming protein adducts, in the onset of abacavir-induced toxic outcomes., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Abacavir is a nucleoside reverse transcriptase inhibitor marketed since 1999 for the treatment of infection with the human immunodeficiency virus type 1 (HIV). Despite its clinical efficacy, abacavir administration has been associated with serious and sometimes fatal toxic events. Abacavir has been reported to undergo bioactivation in vitro, yielding reactive species that bind covalently to human serum albumin, but the haptenation mechanism and its significance to the toxic events induced by this anti-HIV drug have yet to be elucidated. Abacavir is extensively metabolized in the liver, resulting in inactive glucuronide and carboxylate metabolites. The metabolism of abacavir to the carboxylate involves a two-step oxidation via an unconjugated aldehyde, which under dehydrogenase activity isomerizes to a conjugated aldehyde. Concurrently with metabolic oxidation, the two putative aldehyde metabolites may be trapped by nucleophilic side groups in proteins yielding covalent adducts, which can be at the onset of the toxic events associated with abacavir. To gain insight into the role of aldehyde metabolites in abacavir-induced toxicity and with the ultimate goal of preparing reliable and fully characterized prospective biomarkers of exposure to the drug, we synthesized the two putative abacavir aldehyde metabolites and investigated their reaction with the α-amino group of valine. The resulting adducts were subsequently stabilized by reduction with sodium cyanoborohydride and derivatized with phenyl isothiocyanate, leading in both instances to the formation of the same phenylthiohydantoin, which was fully characterized by NMR and MS. These results suggest that the unconjugated aldehyde, initially formed in vivo, rapidly isomerizes to the thermodynamically more stable conjugated aldehyde, which is the electrophilic intermediate mainly involved in reaction with bionucleophiles. Moreover, we demonstrated that the reaction of the conjugated aldehyde with nitrogen bionucleophiles occurs exclusively via Schiff base formation, whereas soft sulfur nucleophiles react by Michael-type 1,4-addition to the α,β-unsaturated system. The synthetic phenylthiohydantoin adduct was subsequently used as standard for LC-ESI-MS monitoring of N-terminal valine adduct formation, upon modification of human hemoglobin in vitro with the conjugated abacavir aldehyde, followed by reduction and Edman degradation. The same postmodification strategy was applied to investigate the products formed by incubation of abacavir with rat liver cytosol, followed by trapping with ethyl valinate. In both instances, the major adduct detected corresponded to the synthetic phenylthiohydantoin standard. These results suggest that abacavir metabolism to the carboxylate(s) via aldehyde intermediate(s) could be a factor in the toxic events elicited by abacavir administration. Furthermore, the availability of a reliable and fully characterized synthetic standard of the abacavir adduct with the N-terminal valine of hemoglobin and its easy detection in the model hemoglobin modifications support the usefulness of this adduct as a prospective biomarker of abacavir toxicity in humans., (© 2011 American Chemical Society)

Mitochondrial thymidine kinase 2 (TK2) is a key enzyme in the salvage of pyrimidine deoxynucleosides needed for mitochondrial DNA synthesis. TK2 phosphorylates thymidine (dThd), deoxycytidine (dCyd), and many other antiviral pyrimidine nucleoside analogs. Zidovudine (AZT) is the first nucleoside analog approved for anti-HIV therapy, and it is still used in combination with other drugs. One of the side effects of long-term treatment with nucleoside analogs is mitochondrial DNA depletion, which has been ascribed to competition by AZT for the endogenous dThd phosphorylation carried out by TK2. Here we studied the kinetics of AZT and 3'-fluorothymidine phosphorylation by recombinant human TK2 and the effects of these and other pyrimidine nucleoside analogs on the phosphorylation of dThd and dCyd. Thymidine analogs strongly inhibited dThd phosphorylation but not dCyd phosphorylation, which instead was stimulated ∼30%. We found that recombinant human TK2 contained the feedback inhibitor dTTP in a 1:1 molar ratio and that incubation with dThd and AZT could completely remove the enzyme-bound dTTP, but dCyd was less efficient in this regard. The release of feedback inhibitor by dThd and dThd analogs most likely accounts for the observed kinetics. Similar effects were also observed with native rat liver mitochondrial TK2, strongly indicating a physiologic role for this process, which most likely is an important factor in the mitochondrial toxicity observed with antiviral nucleoside analogs.

The most important factor limiting the success of an antiretroviral therapy regimes is toxicity. Toxicity can depend on a number of factors; some of these are intrinsic to the host and may not only affect the latter's outward appearance, but also determine the intensity these toxic effects may reach. The former is exemplified by idiosyncratic or hypersensitivity reactions, whereas the latter is usually appreciated in metabolic disturbances or fat redistribution syndromes. Some of the determinants of antiretroviral toxicity are genetic in origin and have been the subject of intense study in recent years. Some of these are linked to a single nucleotide polymorphism (SNP), whereas others depend on a complex interaction between multiple genes variations. One of these tests (HLA B*5701) is now being applied in clinical practice and widely used to prevent the risk of hypersensitivity reactions to abacavir. Many other genetic determinants of antiretroviral drug toxicity have been suggested as an explanation for nucleoside analogue toxicity; these include lactic acidosis, peripheral neuropathy and pancreatitis, and have also been suggested as a potential basis for the non-nucleoside toxicity derived from immunogenetic factors involved in nevirapine hypersensitivity to SNPs in efavirenz enzyme metabolism, amongst other things. Metabolic toxicity, mainly due to protease inhibitors (PIs) is far more complex and depends on the interaction of various genes. The same seems to be true for fat redistribution syndromes and atherosclerosis, although a clear picture of the genetic factors operating in these syndromes is yet to emerge. The ultimate goal of pharmacogenetics is to customize antiretroviral therapy by identifying the genes that can maximize efficacy whilst helping avoid known side effects of antiretroviral drugs.

Abacavir (ABC) is a guanosine nucleoside reverse transcriptase inhibitor (NRTI) with potent antiretroviral activity. Since NRTIs exhibit tissue-specific inhibition of mitochondrial DNA (mtDNA) synthesis, the ability of ABC to inhibit mtDNA synthesis in vivo was evaluated. Inbred wild-type (WT) and transgenic mice (TG) treated with ABC (3.125 mg/d p. o., 35 days) were used to define mitochondrial oxidative stress and cardiac function. Chosen TGs exhibited overexpression of HIV-1 viral proteins (NL4-3Deltagag/pol, non-replication competent), hemizygous depletion or overexpression of mitochondrial superoxide dismutase (SOD2(+/-) knock-out (KO) or MnSOD OX, respectively), overexpression of mitochondrially targeted catalase (MCAT), or double "knockout" deletion of aldehyde dehydrogenase activity (ALDH2 KO). Impact on mtDNA synthesis was assessed by comparing changes in mtDNA abundance between ABC-treated and vehicle-treated WTs and TGs. No changes in mtDNA abundance occurred from ABC treatment in any mice, suggesting no inhibition of mtDNA synthesis. Left ventricle (LV) mass and LV end-diastolic dimension (LVEDD) were defined echocardiographically and remained unchanged as well. These results indicate that treatment with ABC has no visible cardiotoxicity in these adult mice exposed for 5 weeks compared to findings with other antiretroviral NRTI studies and support some claims for its relative safety.

Based on the promising drug resistance profile and potent anti-HIV activity of beta-d-3'-azido-2',3'-dideoxyguanosine, a series of purine modified nucleosides were synthesized by a chemical transglycosylation reaction and evaluated for their antiviral activity, cytotoxicity, and intracellular metabolism. Among the synthesized compounds, several show potent and selective anti-HIV activity in primary lymphocytes., (Published by Elsevier Ltd.)

Positron emission tomography (PET) using F-18 fluoro-3'-deoxy-3-L-fluorothymidine (FLT) offers noninvasive assessment of cell proliferation in vivo. The most important application refers to the evaluation of tumour proliferative activity, representing a key feature of malignancy. Most data to date suggest that FLT is not a suitable biomarker for staging of cancers. This is because of the rather low fraction of tumour cells that undergo replication at a given time with subsequently relatively low tumour FLT uptake. In addition, generally, the high FLT uptake in liver and bone marrow limits the diagnostic use. We describe the current status on preclinical and clinical applications of FLT-PET including our own experience in brain tumours. The future of FLT-PET probably lies in the evaluation of tumour response to therapy and more importantly, in the prediction of early response in the course of treatment. The level of FLT accumulation in tumours depends on thymidine kinase 1 activity and on the therapy-induced activation of the salvage pathway and expression of nucleoside transporters. Therefore, cytostatic agents that cause arrest of the cell cycle in the S-phase may initially increase FLT uptake rather than reducing the tumour cell accumulation. In addition, agents that block the endogenous thymidine pathway may lead to overactivity of the salvage pathway and increase tumour FLT uptake. In contrast, many therapeutic agents inhibit both pathways and subsequently reduce tumour FLT uptake. Further studies comparing FLT with F-18 fluorodeoxyglucose-PET will be important to determine the complementary advantage of FLT-PET in early cancer therapy response assessment. Further research should be facilitated by simplified synthesis of FLT with improved yields and an increasing commercial availability.

Experiments were performed to investigate the impact of zidovudine (AZT), lamivudine (3TC), and abacavir (ABC) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK), using cell cloning assays for assessing the effects of individual drugs/drug combinations in (1) TK6 human lymphoblastoid cells exposed in vitro and (2) splenic lymphocytes from male CD-1 mice exposed transplacentally on days 12-18 of gestation. In TK6 cells, dose-related increases in HPRT and TK mutant frequencies were found following 3 days of exposure to AZT or 3TC alone (33, 100, or 300 microM), or to equimolar amounts of AZT-3TC. Compared with single drug exposures, AZT-3TC coexposures generally yielded enhanced elevations in HPRT and TK mutant frequencies. Mutagenicity experiments with ABC alone, or in combination with AZT-3TC, were complicated by the extreme cytotoxicity of ABC. Exposure of cells either to relatively high levels of AZT-3TC short-term (100 microM, 3 days), or to peak plasma-equivalent levels of AZT-3TC for an extended period (10 microM, 30 days), resulted in similar drug-induced mutagenic responses. Among sets of mice necropsied on days 13, 15, or 21 postpartum, Hprt mutant frequencies in T-cells were significantly elevated in the AZT-only (200 mg/kg bw/day) and AZT-3TC (200 mg AZT + 100 mg 3TC/kg bw/day) groups at 13 days of age. These results suggest that the mutagenicity by these nucleoside analogs is driven by cumulative dose, and raises the question of whether AZT-3TC has greater mutagenic effects than AZT alone in perinatally exposed children., ((c) 2006 Wiley-Liss, Inc.)

Background: Some nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) combinations cause additive or synergistic interactions in vitro and in vivo., Methods: We evaluated the mitochondrial toxicity of tenofovir (TFV), emtricitabine (FTC) and abacavir as carbovir (CBV) alone, with each other, and in combination with additional NRTIs. HepG2 human hepatoma cells were incubated with TFV, FTC, CBV, didanosine (ddl), stavudine (d4T), lamivudine (3TC) and zidovudine (AZT) at concentrations equivalent to 1 and 10x clinical steady-state peak plasma levels (C(max)). NRTIs were also used in double and triple combinations. Cell growth, lactate production, intracellular lipids, mtDNA and the mtDNA-encoded respiratory chain subunit II of cytochrome c oxidase (COXII) were monitored for 25 days., Results: TFV and 3TC had no or minimal toxicity. FTC moderately reduced hepatocyte proliferation independent of effects on mtDNA. ddl and d4T induced a time- and dose-dependent loss of mtDNA and COXII, decreased cell growth and increased levels of lactate and intracellular lipids. CBV and AZT strongly impaired hepatocyte proliferation and increased lactate and lipid production, but did not induce mtDNA depletion. The dual combination of TFV plus 3TC had only minimal toxicity; TFV plus FTC slightly reduced cell proliferation without affecting mitochondrial parameters. All other combinations exhibited more pronounced adverse effects on mitochondrial endpoints. Toxic effects on mitochondrial parameters were observed in all combinations with ddI, d4T, AZT or CBV. TFV and 3TC both attenuated ddI-related cytotoxicity, but worsened the effects of CBV and AZT., Conclusions: The data demonstrate unpredicted interactions between NRTIs with respect to toxicological endpoints and provide an argument against the liberal use of NRTI cocktails without first obtaining data from clinical trials.

A series of 5-(trifluoroethoxymethyl)-2',3'-dideoxyuridines and 5-[bis(trifluoroethoxy)-methyl]-2',3'-dideoxyuridines have been prepared and screened for antiviral activity. The conformations of these compounds are discussed on the bases of NOE studies and the MO calculations. Modelling and NOE studies suggest both syn- and anti conformations for these 5-(2,2,2-trifluoroethoxymethyl)- and 5-[bis(2,2,2-trifluoroethoxy)-methyl]- derivatives. The NOE parameters are also suggested to be more attributable to the nature of the fluorine atom than to structural or conformational changes. Compounds 17, 26 and 30 showed some activity in anti-HIV-1 and anti-HIV-2 assays, but the compounds were devoid of activity against HSV and human rhinovirus. The compounds tested exhibited low cytotoxicity and were inactive against a bank of cancer cells in vitro.

Enantiomers of 4'-aza-2',3'-dideoxynucleosides have been prepared by two different synthetic approaches, on the basis of 1,3-dipolar cycloaddition of a chiral nitrone. Cytotoxicity and apoptotic activity have been investigated. (5'S)-5-Fluoro-1-isoxazolidin-5-yl-1H-pyrimidine-2,4-dione [(-)-AdFU], while showing low level of cytotoxicity, is a good inductor of apoptosis on lymphoid and monocytoid cells, acting as a strong potentiator of Fas-induced cell death.

Medivir is developing alovudine, an anti-HIV compound for the potential treatment of drug-resistant HIV infection. A phase IIa trial involving patients with multiresistant HIV was initiated in October 2001 and by July 2002, the trial had been completed.

Abacavir (ZIAGEN) is a reverse transcriptase inhibitor marketed for the treatment of HIV-1 infection. A small percentage of patients experience a hypersensitivity reaction indicating immune system involvement and bioactivation. A major route of metabolism for abacavir is oxidation of a primary betagamma unsaturated alcohol to a carboxylic acid via an aldehyde intermediate. This process was shown to be mediated in vitro by human cytosol and NAD, and subsequently the alphaalpha and gamma2gamma2 human isoforms of alcohol dehydrogenase (ADH). The alphaalpha isoform effected two sequential oxidation steps to form the acid metabolite and two isomers, qualitatively reflective of in vitro cytosolic profiles. The gamma2gamma2 isozyme generated primarily an isomer of abacavir, which was minor in the alphaalpha profiles. The aldehyde intermediate could be trapped in incubations with both isozymes as an oxime derivative. These metabolites can be rationalized as arising via the aldehyde which undergoes isomerization and further oxidation by the alphaalpha enzyme or reduction by the gamma2gamma2 isozyme. Non-extractable abacavir protein residues were generated in cytosol, and with alphaalpha and gamma2gamma2 incubations in the presence of human serum albumin (HSA). Metabolism and residue formation were blocked by the ADH inhibitor 4-methyl pyrazole (4-MP). The residues generated by the alphaalpha and gamma2gamma2 incubations were analyzed by SDS-PAGE with immunochemical detection. The binding of rabbit anti-abacavir antibody to abacavir-HSA was shown to be dependent on metabolism (i.e. NAD-dependent and 4-MP sensitive). The mechanism of covalent binding remains to be established, but significantly less abacavir-protein residue was detected with an analog of abacavir in which the double bond was removed, suggestive of a double bond migration and 1,4 addition process.

Clinical studies with tenofovir disoproxil fumarate, an oral prodrug of the nucleotide analog tenofovir, recently approved for the treatment of HIV, have demonstrated antiviral activity and good tolerability in HIV-infected patients. In order to better understand the cytotoxicity profile of tenofovir relative to the other nucleoside reverse transcriptase inhibitors (NRTIs), the in vitro effects of these agents were evaluated in various human cell types. Tenofovir inhibited the proliferation of liver-derived HepG2 cells and normal skeletal muscle cells with CC(50) values of 398 and 870 microM, respectively. In comparison, ZDV, ddC, ddI, d4T, and abacavir all showed lower CC(50) values in these two cell types. Evaluation of hematopoietic toxicity revealed that tenofovir was less cytotoxic towards erythroid progenitor cells (CC(50)>200 microM) than ZDV, d4T, and ddC (CC(50)=0.06-5 microM). Despite some degree of donor-to-donor variability, the inhibitory activity of the tested NRTIs against myeloid cell lineage, in the order of decreasing severity, was consistently ddC>ZDV>d4T>tenofovir>3TC. Finally, tenofovir showed substantially weaker effects on proliferation and viability of renal proximal tubule epithelial cells than cidofovir, a related nucleotide analog with the potential to induce renal tubular dysfunction. In conclusion, tenofovir exhibited weak cytotoxic effects in all cell types tested with less in vitro cytotoxicity than the majority of NRTIs currently used for the treatment of HIV disease.

Anti-HIV activity and cytotoxicity were tested for novel phosphonate derivatives of AZT, d4T and ddA. For d4T phosphonate derivatives the most active was 2',3'-Dideoxy-2',3'-didehydrothymidine 5'-isopropylphosphite and among the AZT phosphonate derivatives highest activity was shown by 2',3'-Dideoxy-3'-azidothymidine 5'-cyclohexylphosphite.

Perinatal treatment with 3'-azido-3'-deoxythymidine (AZT) has been found to reduce the rate of maternal-infant transmission of HIV; however, AZT is genotoxic in mammalian cells in vitro and induces tumors in the offspring of mice treated in utero. The purpose of the present study was to investigate the relationships between incorporation of AZT into DNA, and the frequency and spectrum of mutations at the HPRT locus of the human lymphoblastoid cell line, TK6, following in vitro exposures to AZT. Cells were cultured in medium containing 0 or 300 microM AZT for 1, 3, or 6 day(s) (n = 5/group). The effects of exposure duration on incorporation of AZT into DNA and HPRT mutant frequency were determined using an AZT radioimmunoassay and a cell cloning assay, respectively. AZT accumulated in DNA in a supralinear manner, approaching a plateau at 6 days of treatment (101.9 +/- 14.7 molecules AZT/10(6) nucleotides). After 3 days of AZT exposure, HPRT mutant frequency was significantly increased (1.8-fold, p = 0.016) compared to background (mutant frequency = 3.78 x 10(-6)). Multiplex PCR amplification of genomic DNA was used to determine the frequency of exon deletions in HPRT mutant clones from untreated cells versus AZT-treated cells. Molecular analyses of AZT-induced mutations revealed a significant difference in the frequency of total gene deletions (44/120 vs. 18/114 in controls, p = 0.004 by the Mann-Whitney U-statistic). In fact, the Chi-square test of homogeneity demonstrate that the differences between the control and AZT-treatment groups is attributed mainly to this increase in total gene deletion mutations (p = 0.00001). These data indicate that the primary mechanism of AZT mutagenicity in human TK6 cells is through the production of large deletions which occur as a result of AZT incorporation into DNA and subsequent chain termination. The data imply that perinatal chemoprophylaxis with AZT may put children of HIV-infected women at potential risk for genetic damage.

Purpose: The objective of this study was to evaluate a dual action prodrug concept wherein an unnatural myristic acid analogue is coupled via an ester moiety to the 5'-position of FLT or AZT. Subsequent intracellular cleavage of the prodrug ester would simultaneously release FLT or AZT that could inhibit reverse transcriptase (RT), and the myristic acid analogue that could inhibit myristoyl-CoA:protein N-myristoyltransferase (NMT)., Methods: Cytotoxicity (2.2.15 cell culture), and anti-hepatitis B activity of 5'-O-myristoyl analogue prodrug derivatives of FLT and AZT (2-8) were evaluated in vitro using human liver hepatitis B virus (HBV) producing 2.2.15 cell lines., Results: The 5'-O-(12-methoxydodecanoyl) ester derivatives of AZT (2, EC(50) = 2. 7 +/- 0.3 microM; CC(50)= 727 +/- 19 microM) and FLT (4, EC(50)= 2.8 +/- 0.3 microM; CC(50)= 186 +/- 20 microM) were the most effective anti-hepatitis B virus (anti-HBV) compounds of this series in a replication assay. In the series of 5'-O-myristic acid analogue ester prodrug derivatives of FLT, the relative anti-HBV potency order was MeO(CH(2))(11)CO(2)- > N(3)(CH(2))(11)CO(2)- and Br(CH(2))(11)CO(2)- > EtS(CH(2))(n)CO(2)- (n = 10 or 11) > Me(CH(2))(12)CO(2)- (myristoyl)., Conclusions: The in vitro data suggest that the 5'-O-myristoyl analogue prodrug concept offers a potential drug design approach to design dual acting antiviral agents, with superior pharmacokinetic, biodistribution, reduced cytotoxicity and/or increased efficacy. In this regard, the 5'-O-(12-methoxydodecanoyl) prodrug ester of 3'-thia-3'-deoxythymidine (3TC) may offer the greatest potential for the treatment of HBV infection.

Various 2,3-dideoxy-2,2-difluoro-L-glycero-pentofuranosyl nucleosides were synthesized via the key intermediate, 5-O-benzoyl-2,3-dideoxy-2,2-difluoro-L-glycero-pentofuranose (6). 2,3-O-Isopropylidene-L-glyceraldehyde was coupled with ethyl bromodifluoroacetate under Reformatsky conditions to obtain the diastereomeric mixture of ethyl (4S)-3-hydroxy-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro propionate (1). Treatment of compound 1 with carbon disulfide, sodium hydride and methyl iodide followed by reduction afforded ethyl (4S)-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-difluoro propionate (3). Compound 3 was treated with 5% HCl in ethanol, followed by refluxing in benzene under Dean-Stark conditions, to afford the lactone 4. The compound 4 was protected and reduced to afford the key intermediate 6. For the synthesis of pyrimidine derivatives 8-21, compound 6 was converted to the mesylate 7 and condensed with various silyl protected pyrimidine bases. The inosine and adenine derivatives 38-41 were obtained from compound 6 and 6-chloropurine using standard procedures. Compounds 22-35 and 38-41 were evaluated for their antiviral activity against HIV-1, HBV, HSV-1 and HSV-2, and for cellular toxicity. None of the synthesized compounds showed any significant activity or toxicity. Single-crystal X-ray structure of 1-(2,3-dideoxy-2,2-difluoro-beta-L-glycero-pentofuranosyl)-5-iodocytosin e (34) suggested a 2'-exo/3'-endo conformation for the carbohydrate moiety.

Carbovir (CBV) is a guanine nucleoside analog with potent in vitro anti-HIV activity. A prodrug of CBV is currently being evaluated in clinical trials as a potential agent for the treatment of AIDS. The ability of CBV to inhibit mitochondrial DNA synthesis in intact CEM cells was evaluated in the present study, because most of the currently available anti-HIV nucleoside analogs have significant toxicities that result from their inhibition of mitochondrial DNA synthesis. No delayed cytotoxicity was observed in CEM cells treated with 50 microM CBV for 4 weeks. In addition, CBV at concentrations as high as 1 mM did not cause a decline in mitochondrial DNA levels and only minimally increased the concentration of lactic acid in the medium. In contrast to these results with CBV, treatment of CEM cells with 0.5 microM 2',3'-dideoxycytidine resulted in delayed cytotoxicity, a decrease in mitochondrial DNA content and increases in lactic acid levels in the medium. These results indicated that treatment of CEM cells with CBV did not result in the inhibition of mitochondrial DNA synthesis and suggested that treatment of AIDS patients with CBV, or a prodrug of CBV, would not result in some of the toxicities seen with the other anti-HIV nucleoside analogs.

This in vivo study examines the ability of 5'-amino-5'-deoxythymidine (5'-AdThd) to modulate 5-iododeoxyuridine (IdUrd) cellular metabolism in two human colon cancer xenografts (HT 29 and HCT-116), two actively proliferating normal mouse tissues (bone marrow and intestine), and a quiescent normal mouse tissue (liver). 5'-AdThd is a thymidine analogue that at low concentrations (<30 micrometer) can increase thymidine kinase activity, which is the rate-limiting enzyme for activation of IdUrd. We reported recently that the in vitro incubation of HT 29 and HCT-116 cells in 5'-AdThd + IdUrd resulted in an enhancement of 5-iodo-2'-dUTP pools, IdUrd DNA incorporation, and subsequent radiosensitization compared with incubation with IdUrd alone (Clin. Cancer Res., 1: 407-416, 1995). These in vitro effects were more significant in the radioresistant cell line HT 29. Using a 6-day continuous infusion of IdUrd (50 or 100 mg/kg/day) and/or 5'-AdThd (200 mg/kg/day), no increase in systemic toxicity (percentage of body weight loss) was observed in athymic nude mice with 5'-AdThd alone or when combined with IdUrd. There was significant dose-dependent, systemic toxicity with IdUrd, which was reversible within 3 days of completing the lower-dose IdUrd infusion. However, a comparison of plasma levels during the 6-day continuous infusion of IdUrd +/- 5'-AdThd showed a significant interaction of IdUrd and 5'-AdThd, resulting in higher plasma levels by day 6 of both compounds and the principal metabolites, iodouracil and deoxyuridine, which is consistent with nonlinear saturating effects on dihydrouracil dehydrogenase. Coadministration of IdUrd and 5'-AdThd resulted in an increase in the percentage of IdUrd DNA incorporation in the two proliferating normal tissues, which was significant only with the lower IdUrd dose. No effect on IdUrd DNA incorporation was found in normal liver at either IdUrd dose +/- 5'-AdThd. Similar to our in vitro data, the continuous infusion of IdUrd and 5'-AdThd showed a significant effect by increasing the percentage of IdUrd DNA incorporation in HT-29 xenografts at both IdUrd doses, whereas coadministration of 5'-AdThd had no such effect in HCT-116 xenografts.

In the present study, a competitive ELISA technique was developed to specifically quantitate 3'-amino-3'-deoxythymidine (AMT), a toxic catabolite of 3'-azido-3'-deoxythymidine (AZT) detected in serum from AZT-treated patients. In order to eliminate cross-reacting AZT, serum sample was extracted with ethylacetate and then AMT was acetylated (Ac-AMT). A 5'-hemisuccinate-AMT-horseradish peroxidase conjugate was used as a tracer in the presence of anti-AMT rabbit antibodies which were raised against a 5' hemisuccinate-AMT-bovine serum albumin immunogen. Bound/free separation was achieved with an anti-rabbit IgG mouse monoclonal antibody insolubilized onto a microtiter plate. The limit of quantification of Ac-AMT was as low as 0.4 ng/ml in serum samples. This ELISA technique was applied for monitoring AMT plasma levels in patients receiving AZT therapy. The intra and inter-individual variations of the AZT/AMT plasma concentration ratios underlined the need for such a specific test in studying the formation of this toxic catabolite.

It would be desirable to develop antiviral agents that can be targeted to liver to enhance their antiviral effects and reduce nonhepatic toxicity. 2',3'-Dideoxyguanosine (ddG) has been found to be a potent and selective antihepatitis B agent both in vitro and in vivo. To evaluate ddG and its liver-targeted analog, we synthesized a series of phosphatidyl-ddGs and incubated them with 2.2.15 cells, which chronically produce hepatitis B virus. 1,2-Dipalmitoylphosphatidyl-dideoxyguanosine (DPP-ddG) inhibited the production of hepatitis B virus (HBV) DNA in the culture medium by 90% at 4.5 mumol/L versus 9.1 mumol/L for ddG, while the liposome vehicle itself had no effect. To compare the efficacy of free ddG with its lipid prodrug in vivo, we treated woodchucks that were experimentally infected with woodchuck hepatitis virus (WHV) for 4 weeks by intraperitoneal injection of 2.6 mumol/kg/d of free ddG or liposomes containing 2.6 mumol/kg/d of DPP-ddG. Liposomal DPP-ddG reduced serum WHV DNA by 23- to 46-fold at the end of the fourth week, while free ddG reduced serum WHV DNA by 2.2- to 10.4-fold. Treatment with small unilamellar liposomes containing DPP-ddG is substantially more effective than free ddG in reducing WHV-DNA levels in serum in WHV-infected woodchucks. The data suggest that the use of lipid prodrugs to target the liver may be useful in enhancing antiviral therapy of hepatitis.

beta-D-Uridine protected human granulocyte-macrophage lineage cells in both semi-solid (granulocyte-macrophage colony-forming units, CFU-GM) and liquid cultures against the toxic effects of 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-3'-deoxythymidine (FLT) and a combination of AZT and FLT, without impairment of the activities of these respective drugs against human immunodeficiency virus (HIV) replication. In addition, beta-D-uridine also protected human CFU-GM against toxicity of the in vivo AZT metabolite, 3'-amino-3'-deoxythymidine (AMT). Beta-L-uridine and alpha-D-uridine, two stereoisomers of the natural form, and the base uracil, were unable to protect cells against either AZT or FLT toxicity, whereas beta-D-uridine-5'-bis(SATE)phosphotriester, a prodrug of beta-D-uridine-5'-monophosphate, successfully protected cells against AZT toxic effects, suggesting that beta-D-uridine needs to be metabolized to its nucleotides to exert a pharmacological effect. These data suggest in addition that AZT, FLT and AMT share a common target site(s) of toxicity involved in myelosuppression.

Patients infected with the human immunodeficiency virus experienced severe hematopoietic toxicity after treatment with the deoxynucleoside analog 3'-fluorothymidine (FLT). Using several methods for the analysis of genome integrity, including histochemical staining of the 3' ends of DNA and both conventional and pulsed-field agarose gel electrophoresis, we demonstrated that FLT caused extensive DNA fragmentation in CEM cells that was not observed when these cells were treated with other, less toxic thymidine analogs. In addition, a distinctive pattern of small DNA fragments that is characteristic of cells in the process of programmed cell death was observed in the genomic DNA of CEM cells treated with FLT. We conclude that FLT induces DNA fragmentation and apoptosis in a human cell line of hematopoietic origin, and we offer this observation as a possible explanation for the severe toxicity of FLT observed in vivo.

Inhibition of in vitro colony formation of human hematopoietic progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid) by the antiviral nucleoside drugs alovudine, zalcitabine, zidovudine, ganciclovir, stavudine, didanosine, lamivudine, and acyclovir was measured. Significant correlations between in vitro 50% inhibitory concentrations and the daily human exposures (area under the concentration-time curve from 0 to 24 h; in micromolar.hour) of these chronically administered drugs in human immunodeficiency virus-positive patients that induced neutropenia or anemia were demonstrated by both linear regression and Spearman rank-order analyses. These quantitative correlations allow estimation of the exposure at which bone marrow toxicity may occur with candidate compounds.

As the incidence of human immunodeficiency virus (HIV) infection has increased in women over the past decade, the need for safe, effective therapy during pregnancy has increased concomitantly. Although dideoxynucleosides such as 3'-deoxy-3'-azidothymidine (AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxycytidine (ddC), and 2',3'-didehydro-3'-deoxythymidine have been approved for use in the general population, the administration, efficacy, and toxicity of these compounds during pregnancy and development are now being investigated. Initial human studies suggest that maternal use of AZT during pregnancy is well tolerated by both mother and child and provides a promising degree of protection from vertical HIV transmission to the infant. In vitro and animal models have greatly increased our understanding of the distribution and toxicity resulting from fetal dideoxynucleoside exposure. AZT, ddI, and ddC rapidly cross the placenta by simple diffusion but with different rates of transfer. In vivo data confirm the differential transfer of these compounds with AZT fetal exposure approximately twice that of ddI or ddC. Active phosphorylated metabolites have been detected in placental tissue after in vitro perfusion with AZT. The active triphosphate has not been detected in placental perfusion studies or in the fetal rhesus monkey 3 h after maternal exposure to ddI or ddC. Although in vitro and in vivo laboratory animal studies suggest the potential for toxicity with preimplantation exposure, the risk for teratogenic events after postimplantational exposure appears to be low at therapeutically effective concentrations of these dideoxynucleosides.

Many dideoxynucleosides that are effective against human immunodeficiency virus (HIV) also are potent inhibitors of mitochondrial DNA (mtDNA) synthesis, and the resulting mtDNA decrease could be responsible for the delayed clinical toxicity sometimes observed with these drugs. The following compounds have been examined for their toxicity to human lymphoid CEM cells, and their ability to suppress mtDNA content: 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyinosine (ddI) and 2',3'-dideoxyguanosine (ddG); and their 2'-beta-fluoro analogs; beta-F-ddC, beta-F-ddA, beta-F-ddI and beta-F-ddG. Two other fluoro analogs, 5-F-ddC and 2'-beta,5-di-F-ddC were also examined. The ratio of C-IC50 (concentration that inhibited cell growth by 50%) to mt-IC50 (concentration that inhibited mtDNA synthesis by 50%) was determined for each compound. The rank-order of this ratio was ddC > 5-F-ddC >> ddA > ddI > ddG > beta-F-ddC > beta-F-ddA > beta-F-ddG with the highest ratios indicating the greatest potential for delayed toxicity. In comparison with ddC, beta-F-ddC and beta-F-ddA were 5,000 and 22,000 times less potent, respectively, in suppressing cellular mtDNA content, while their anti-HIV potencies were decreased only modestly relative to their unfluorinated parent compounds. beta-F-ddI and 2'-beta,5-di-F-ddC produced neither cellular toxicity nor mtDNA suppression at concentrations of 500 and 1000 microM, respectively. Lactic acid, the product of compensatory glycolysis that results from the inhibition of mitochondrial oxidative phosphorylation, was measured after cells were treated with these compounds. There appears to be a concentration-related correlation between the increase of lactic acid and the extent of mtDNA inhibition for the compounds examined.

5-Chloro-2',3'-dideoxy-3'-fluorouridine (935U83) is a selective anti-human immunodeficiency virus (HIV) agent. When tested in phytohemagglutinin-stimulated normal human peripheral blood lymphocytes against fresh clinical isolates of HIV type 1 (HIV-1) obtained from patients naive to AZT (3'-azido-3'-deoxythymidine [zidovudine]), 935U83 inhibited virus growth with an average 50% inhibitory concentration (IC50) of 1.8 microM; corresponding IC50s were 0.10 microM for FLT (3'-deoxy-3'-fluorothymidine) and 0.23, 0.49, and 0.03 microM for the approved agents AZT, ddI (2',3'-dideoxyinosine), and ddC (2',3'-dideoxycytosine), respectively. Importantly, 935U83 retained activity against HIV strains that were resistant to AZT, ddI, or ddC. Of additional interest, we were unable to generate virus which was resistant to 935U83 by passaging either HXB2 (AZT-sensitive) or RTMC (AZT-resistant) strains in the presence of high concentrations of 935U83. The anabolic profile of 935U83 was similar to that of AZT, and 935U83 triphosphate was a potent inhibitor of HIV-1 reverse transcriptase. Pharmacokinetic evaluation showed good oral bioavailability (86% in mice and 60% in monkeys) and less extensive metabolism to the glucuronide relative to AZT. 935U83 showed low toxicity. In an in vitro assay for toxicity to a human erythrocyte progenitor, erythroid burst-forming unit (BFU-E), the IC50 for 935U83 (> 400 microM) was more than 1,000-fold those of FLT (0.07 microM) and AZT (0.30 microM). Mild reversible reductions in erythrocytes and associated parameters were seen in mice dosed orally with 2,000 mg of 935U83 per kg per day for 1 and 6 months. In monkeys dosed orally with up to 700 mg/kg/day for 1 and 6 months, the only possible treatment-related finding was cataracts in 1 of 12 animals given the intermediate dose of 225 mg/kg/day. At the highest doses in mice and monkeys, maximal concentrations in plasma were more than 100-fold the anti-HIV IC50s against clinical isolates. This safety profile in animals compares very favorably with that of any of the anti-HIV drugs approved to date and has led us to begin evaluation of 935U83 in patients with HIV infection.

Six anti-AIDS drugs were assessed for in vivo genotoxicity and cytotoxicity at human clinical doses with the mouse bone marrow micronucleus assay. These included four dideoxynucleosides (azidothymidine, dideoxycytidine, dideoxyadenosine, and dideoxyinosine), an anthracycline antibiotic (doxorubicin), and a chelating agent (D-penicillamine). Cytological analysis of the mouse bone marrow cells revealed: (i) The dideoxynucleosides and D-penicillamine failed to induce significant number of micronuclei, and except for one of the five doses of dideoxyinosine, none of the dideoxynucleosides were cytotoxic at the doses tested. (ii) Doxorubicin induced micronuclei in a dose-dependent manner which was statistically significant at 4-times the clinical dose but was not cytotoxic at any of the doses tested.

Human immunodeficiency virus type 1 (HIV-1)-infected CEM cells were treated by the HIV-1-specific inhibitors bis-heteroarylpiperazine (BHAP), 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1 H)-on e (TIBO) R82913, nevirapine, and the N3-methylthymine derivative of [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro- 5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide) (TSAO-m3T), as single agents or in combination, at escalating concentrations. When used individually, the compounds led to the emergence of drug-resistant virus strains within two to five subcultivations. The resulting strains were designated HIV-1/BHAP, HIV-1/TIBO, HIV-1/Nev, and HIV-1/TSAO-m3T, respectively. The mutant viruses showed the following amino acid substitutions in their reverse transcriptase (RT): Leu-100-->Ile for HIV-1/BHAP; Lys-103-->Asn for HIV-1/TIBO; Val-106-->Ala for HIV-1/Nev; and Glu-138-->Lys for HIV-1/TSAO-m3T. Both the Tyr-181-->Cys and Val-106-->Ala mutations were found in another mutant emerging following treatment with nevirapine at escalating concentrations. The BHAP-resistant virus remained fully sensitive to the inhibitory effects of nevirapine and TSAO-m3T, whereas the TSAO-m3T-resistant virus remained fully sensitive to the inhibitory effects of nevirapine and BHAP. When different pairs of nonnucleoside RT inhibitors (i.e., BHAP plus TSAO-m3T, nevirapine plus TSAO-m3T, TIBO plus TSAO-m3T, nevirapine plus TIBO, and BHAP plus nevirapine) were used, resistant virus emerged as fast as with single-drug therapy. In all cases the Tyr-181-->Cys mutation appeared; the virus showed markedly reduced sensitivity to all HIV-1-specific inhibitors but retained sensitivity to 2',3'-dideoxynucleoside analogs such as zidovudine, ddC, and ddI. Our findings argue against simultaneous combination of two different nonnucleoside RT inhibitors that are unable to inhibit HIV-1 mutant strains containing the Tyr-181-->Cys mutation when administered as single drugs.

To evaluate analogues of the antiviral agent (R)-9-(3,4-dihydroxybutyl)guanine in which the side-chain C-3 hydroxyl oxygen is part of a five-membered ring, carbocyclic 3'-oxa-2',3'-dideoxyguanosine (4) and carbocyclic 3'-oxa-2',3'-dideoxy-7-deazaguanosine (5) have been synthesized in 17 and 14 steps, respectively, from 5-O-acetyl-1,2-O-isopropylidene-alpha-D-xylofuranose. Compounds 4 and 5 and their 6-chloro precursors were evaluated against a wide variety of DNA and RNA viruses. Only 4 showed any marginal activity and this was limited to HSV-1 and HSV-2. Even though 4 was less potent towards these latter two viruses than acyclovir, its mechanism and target of action is proposed to resemble that of acyclovir. The only toxicity observed for these compounds was observed in the cell growth assay with human embryonic lung cells.

Four nucleoside analogues, 2',3'-dideoxyinosine (ddI), 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxycytosine (ddC) and 5-fluoro-2',3'-dideoxycytosine (5-F-ddC), were evaluated for their potential in vitro myelotoxic effects on normal murine hematopoietic progenitor cells. Myeloid granulocyte-macrophage colony-forming units (CFU-gm), erythroid burst-forming units (BFU-e) and colony-forming units (CFU-e) and megakaryocytic (CFU-meg) progenitors were exposed to the agents for 1 h prior to culture in Petri dish assays or continuously throughout the entire culture period. At 10 microM, both ddA and ddI were moderately toxic (2-36% colony inhibition) to murine CFU-gm, BFU-e, CFU-e and CFU-meg following either 1 h or continuous exposure. Colony inhibition for the progenitors ranged from 2-31% at 10 microM for 1 h ddC or 5-F-ddC exposure. Continuous exposure to ddC was highly myelotoxic to murine hematopoietic progenitors with 100 microM suppressing colony formation 82-89%. At the same concentration and exposure time, 5-F-ddC inhibited colony formation 56-67%. Our results demonstrate that 1 h and continuous exposures to ddA and ddI were similarly myelotoxic to murine hematopoietic cells regardless of exposure time. In contrast, continuous ddC or 5-F-ddC exposure was more toxic to murine progenitors than 1 h exposure to these agents.

2',3'-Didehydro-2',3'-dideoxythymidine (D4T) is a potent inhibitor of human immunodeficiency virus (HIV), with low hematological toxicity. In the present study, the cellular pharmacology of D4T was investigated in human bone marrow cells (BMC), in an attempt to understand the mechanism of the observed low bone marrow toxicity. After exposure of human BMC to 10 microM [3H]D4T for 24 hr, D4T-5'-triphosphate (D4T-TP) was the predominant metabolite, reaching a concentration of 0.3 pmol/10(6) cells. The D4T-5'-monophosphate levels were slightly lower, whereas the D4T-5'-diphosphate levels were about 6-fold lower than those of D4T-TP at 24 hr. Nucleic acids of human BMC exposed to 10 microM [3H]D4T for 24 hr were purified and analyzed by cesium sulfate density gradient centrifugation. No radioactivity was detected in the RNA region, whereas a limited amount was associated with the DNA region. The amount of label incorporated into DNA correlated with the extracellular D4T concentration and the length of incubation time. Enzymatic hydrolysis of radiolabeled DNA and subsequent analysis by high performance liquid chromatography demonstrated incorporation of both D4T and thymidine (dThd) into DNA. Degradation of D4T to thymine and subsequent formation of labeled dThd was also detected in human BMC. Pulse (24 hr)-chase (48 hr) experiments with 10 microM [3H]D4T demonstrated that the amount of radiolabel from D4T in DNA decreased over time during the chase. Under similar conditions, [3H]3'-azido-3'-deoxythymidine (AZT) incorporated into DNA of human BMC did not decrease during the chase. Although D4T-TP standard was demonstrated to be unstable at 37 degrees and neutral pH, D4T was much more stable in solution when incorporated into newly synthesized DNA isolated from human BMC, suggesting that enzymatic excision may be the mechanism for D4T removal from DNA. In summary, although higher concentrations of D4T-TP, compared with AZT-5'-triphosphate, are observed in human BMC, after exposure of cells to similar extracellular concentrations of parent drug, steady state levels of D4T incorporated into DNA are 10-50-fold lower, compared with AZT. Competition with dTTP formed by D4T metabolism and excision of D4T from DNA may be responsible, in part, for these effects. This study further demonstrates that incorporation of 2',3'-dideoxynucleosides into nuclear DNA of human BMC may be related to the ability of these anti-HIV agents to induce hematological side effects.

The anti-human immunodeficiency virus (-HIV) nucleoside analogs azidothymidine (AZT), dideoxycytidine (ddC), dideoxyinosine (ddl), dideoxydidehydrothymidine (D4T), and dideoxydidehydrocytidine (D4C) and the anticancer drug cytosine arabinoside (AraC) were compared for their effects on the mitochondrial DNA (mtDNA) content in a human lymphoblastoid cell line, CEM. The potency of these compounds in reducing mtDNA content was in the order of ddC greater than D4C greater than D4T greater than AZT greater than ddl. AraC did not have a significant effect on mtDNA content. All of the compounds tested, except AraC, stimulated lactic acid production at concentrations that inhibited mtDNA synthesis. The action of ddC and ddl occurred at concentrations that did not affect cell growth significantly in 4 days but retarded cell growth by day 6. D4T and D4C decreased mtDNA content by 50% at doses lower than those that inhibited cell growth by 50% in 4 days (ID50). However, AZT required a dose higher than the ID50 to exert similar effects on mtDNA content. The decrease of mtDNA content caused by ddC also occurred in nerve growth factor-treated PC12 cells, which differentiate to neuron-like cells upon treatment with nerve growth factor. The preferential inhibition of mtDNA, compared with cell growth, by some of these anti-HIV nucleoside analogs correlates well with their ability to cause drug-limiting delayed toxicity, such as peripheral neuropathy, in patients. These data suggest that the selective mitochondrial toxicity could be responsible for the delayed toxicity caused by these anti-HIV analogs.

Metabolic studies in humans have demonstrated that 3'-azido-3'-deoxythymidine (AZT) is primarily eliminated as its 5'-O-glucuronide (GAZT). However, no detailed cellular metabolic studies have been reported on the complete catabolic fate of AZT at the hepatic site. Because the liver is probably the major site of AZT catabolism, the metabolism and transmembrane distribution of AZT were evaluated in freshly isolated rat hepatocytes, a model for the study at the cellular level of biosynthetic, catabolic, and transport phenomena in the liver. Following exposure of cells to 10 microM [3H]AZT, the predominant intracellular catabolite was GAZT, which reached a concentration of approximately 22 microM by 60 min. Additionally, under nonreducing conditions substantial levels of two previously unidentified AZT catabolites that were formed at the hepatic site and were distinct from any known anabolites or catabolites were also detected. These catabolites were identified as 3'-amino-3'-deoxythymidine (AMT) by fast atom bombardment mass spectrometry and 3'-amino-3'-deoxythymidine glucuronide (GAMT) through specific enzymatic hydrolysis. However, AMT was not a substrate for uridine 5'-diphosphoglucuronyltransferase and GAMT was found to be a reductive product of GAZT. Studies using rat and human liver microsomes demonstrated that the rate of formation of AMT and GAMT increased in the presence of NADPH, suggesting the involvement of a NADPH-dependent enzyme system. Studies using human hematopoietic progenitor cells demonstrated that AMT was 5- to 7-fold more toxic to human colony-forming units granulocyte-macrophage and burst-forming units erythroid than was AZT. This study provides the first detailed catabolic profile of AZT at the hepatic site and emphasizes the critical role that the liver plays in drug clearance. Formation of AMT, a highly toxic catabolite of AZT, raises a question regarding the role of AMT in the cytotoxic effects of AZT observed in patients.

In this paper we describe the in vitro antiviral activity of the (-) enantiomer of carbocyclic 2',3'-deoxydidehydroguanosine, (-) carbovir, a nucleoside analogue that has selective and potent anti-HIV activity in a series of lymphocyte culture systems. The cellular cytotoxicity of this compound has also been evaluated in a number of systems and compared to the saturated dideoxynucleoside analogues AZT and ddC.

5'-Phosphites (5'-hydrogenphosphonates) of 3'-azido-2'-, 3'-dideoxynucleosides are shown to be effective inhibitors of the human immunodeficiency virus (HIV-1) in MT4 cell culture. 5'-Phosphite of 3'-azido-2', 3'-dideoxythymidine was the most active among these compounds and even a little more active as compared to the well-known anti-AIDS drug 3'-azido-2',3'-dideoxythymidine; at the same time 5'-phosphites of 3'-azido-2',3' -dideoxynucleosides with adenine, guanine and cytosine bases were more active than the corresponding nucleosides. The toxicity of all four phosphites was comparatively low and the equimolar mixture of all four phosphites was 2-3 fold less toxic than each of them separately. Data on the decreased toxicity of the phosphite mixture are explained from the viewpoint of a decreased pool disbalance of natural 2'-deoxynucleoside 5'-triphosphates in cells; a significant pool disbalance is developed in the case of 3'-azido-2',3'-dideoxythymidine action.