Intracellular Activation of 2',3'-Dideoxyinosine and Drug Interactions in Vitro

Kewn, Stephen; Hoggard, Patrick G.; Henry-Mowatt, Judith; Veal, Gareth J.; Sales, Sean D.; Barry, Michael; Back, David

doi:10.1089/088922299310692

Cited by 25 publications

(20 citation statements)

References 42 publications

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“…It is important to note that the ddATP content found in our PBMC model (picomole) is much higher than that reported in the literature (femtomole) (18,23,27). Such discrepancies are most likely attributable to the differences between the cellular models and the detection methods of ddATP contents (mainly HPLC/UV detection and radiolabeled experiments studies) used in these previous studies.…”

Section: Discussioncontrasting

confidence: 71%

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Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine

Saint

Terreux

Duval

et al. 2004

Antimicrob Agents Chemother

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Clinical failures of the highly active antiretroviral therapy could result from inefficient intracellular concentrations of antiviral drugs. The determination of drug contents in target cells of each patient would be useful in clinical investigations and trials. The purpose of this work was to quantify the intracellular concentration of ddATP, the active metabolite of dideoxyinosine (ddI), in peripheral blood mononuclear cells (PBMCs) of human immunodeficiency virus (HIV)-infected patients treated with ddI. We have raised antibodies against ddA-citrate, a stable isostere of ddATP selected on the basis of its structural and electronic analogies with ddATP. The anti-ddA-citrate antibodies recognized ddATP and ddA with nanomolar affinities and cross-reacted neither with any of the nucleotide reverse transcriptase inhibitors used in HIV therapy nor with their phosphorylated metabolites. The three phosphorylated metabolites of ddI (ddAMP, ddADP, and ddATP) were purified by anion exchange chromatography and the amount of each metabolite was determined by radioimmunoassay with or without prior phosphatase treatment. The intracellular levels of the three ddI metabolites were measured both in an in vitro model and in PBMCs of HIV-infected patients under ddI treatment. The possibility to measure intracellular levels of ddATP from small blood samples of HIV-infected patients treated with ddI could be exploited to develop individual therapeutic monitoring.Highly active antiretroviral therapy has been used successfully for treatment of human immunodeficiency virus (HIV) disease. The most common highly active antiretroviral therapy regimens consist of a combination of at least one protease inhibitor and two nucleoside reverse transcriptase inhibitors. Contrary to protease inhibitors, the expression of nucleoside reverse transcriptase inhibitor activity requires intracellular metabolism of the nucleoside precursor into its corresponding 5Ј-triphosphate nucleotide by the host cell kinases. The active metabolite (nucleoside reverse transcriptase inhibitor-triphosphate) competitively inhibits the HIV reverse transcriptase and acts as a chain terminator of the proviral DNA.The presence and activity of the intracellular kinases are highly dependent on the type and activation state of the target cell (37). Studies conducted in HIV-infected patients failed to establish a clear relationship between the plasma nucleoside reverse transcriptase inhibitor concentration and the antiviral efficiency of these drugs (3, 4, 18, 39). However, a clinical study showed a significant and linear relationship between the intracellular nucleoside reverse transcriptase inhibitor-triphosphate (zidovudine-triphosphate and lamivudine-triphosphate) concentrations, the percent change in CD4 ϩ cells and the rate of decline of HIV RNA in plasma (17). Thus, intracellular contents of active drugs in target cells seem to give a much better indication of therapeutic efficiency than plasma concentrations of drug precursors.The intracellular metabolism of ddI leads...

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Section: Discussioncontrasting

confidence: 71%

“…The intracellular ddATP levels were found to be the lowest among various ddNTPs (23,27). Thus, most of the technologies previously described were not sensitive enough to detect intracellular ddATP in HIV-infected patients.…”

Section: Discussionmentioning

confidence: 97%

Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine

Saint

Terreux

Duval

et al. 2004

Antimicrob Agents Chemother

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“…Approximately 3% of ddI intracellularly converts to ddATP, the active form of the drug (31,32). Therefore, to examine the toxicity of ddI, we examined the kinetics of incorporation of ddATP.…”

Section: Resultsmentioning

confidence: 99%

Toxicity of Antiviral Nucleoside Analogs and the Human Mitochondrial DNA Polymerase

Johnson¹,

Ray²,

Hanes³

et al. 2001

Journal of Biological Chemistry

377

329

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To examine the role of the mitochondrial polymerase (Pol ␥) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol ␥ for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-␤-D-arabinofuranosyl)-5-iodouracil (FIAU), ␤-L-(؊)-2,3-dideoxy-3-thiacytidine (؊)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol ␥ holoenzyme in single turnover kinetic studies to measure K d (K m ) and k pol (k cat ) to estimate the specificity constant (k cat /K m ) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2,3-didehydro-2,3-dideoxythymidine (d4T) > > (؉)3TC > > (؊)3TC > PMPA > azidothymidine (AZT) > > Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s ؊1 for the series FIAU > (؉)3TC ϳ (؊)3TC > CBV > AZT > PMPA ϳ d4T > > ddA (ddI) > > ddC. Removal of ddC was too slow to measure (<0.00002 s ؊1 ). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (؊)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s ؊1 ) faster than it is removed (0.06 s ؊1 ) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.Current treatment of HIV 1 includes a mixture that generally consists of a combination of nucleoside and nonnucleoside analogs directed against HIV RT, plus an inhibitor of HIV protease. Treatment with this mixture allows patients to coexist with a low level of virus for years, but treatments are limited by the development of resistance of HIV to the drugs on the one hand and toxicity of nucleoside analogs on the other. Toxicity of nucleoside analogs is particularly troublesome for the long term management of the viral infection. Nucleoside analogs function as chain terminators to suppress viral replication by HIV-1 RT, and because HIV RT lacks a proofreading exonuclease, the specificity of nucleoside analogs toward HIV RT results from selective discrimination during incorporation and/or from removal by the proofreading exonuclease of the host DNA polymerase.Six nucleoside analogs have received Food and Drug Administration approval for treatment of HIV, and these analogs are illustrated with others...

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“…The ddNTPs are incorporated into the elongating DNA strands during synthesis of the DNA copy of the viral genome by RT, resulting in premature chain termination. The ddNTP to dNTP ratio critically determines drug activity and varies in different cells (Perno et al, 1994;Kewn, et al, 1999). Changes in the proportion of ddNTP to dNTP can affect the efficacy of the NRTIs, one reason for the different activities of a drug against the same virus in different cell types (Tsai et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

Differential Susceptibility of Retroviruses to Nucleoside Analogues

Rosenblum¹,

Patton²,

Grigg³

et al. 2001

Antivir Chem Chemother

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Retroviruses may cause diseases in their vertebrate hosts. They are distinguished by their common means of replication involving reverse transcription, a process inhibited by nucleoside reverse transcriptase inhibitors (NRTIs) and other compounds used in antiretroviral chemotherapy. Previous work on NRTIs has been limited to their effect on human immunodeficiency virus (HIV) (for review see Ho & Hitchcock, 1989; Weller, 1999) and little information exists regarding the efficacy and therapeutic potential of these drugs against other retroviruses. We have tested all six NRTIs licensed for HIV treatment [didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), zidovudine (AZT) and abacavir (ABC)] against seven retroviruses representative of the traditional subfamilies: Spumavirinae, Lentivirinae and the Oncovirinae. As expected, each drug showed a range of activities against the panel of retroviruses, some drugs inhibiting other viruses at concentrations well below those required for HIV. Overall, AZT was the most active inhibitor (IC50 range, 0.032–1.0 μM), being most active against the Spuma (foamy) viruses. Abacavir was inhibitory for HIV-1, MN strain (HIV-1 MN), amphotrophic murine leukemia virus (MLV-A) and simian foamy virus type 6 (SFV-6). The least effective inhibitor, 3TC (IC50 range, 0.32–>100 μM), was most potent against simian retrovirus types 1 and 2 (SRV-1, SRV-2) and HIV-1, but did not inhibit foamy viruses and MLV-A. Additionally, there were differences in the concentration of drug required to inhibit closely related viruses. Taken together, these data suggest that NRTIs have a wide spectrum of antiretroviral activity and the activity of compounds, even against closely related retroviruses, cannot be predicted.

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Intracellular Activation of 2',3'-Dideoxyinosine and Drug Interactions in Vitro

Cited by 25 publications

References 42 publications

Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine

Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine

Toxicity of Antiviral Nucleoside Analogs and the Human Mitochondrial DNA Polymerase

Differential Susceptibility of Retroviruses to Nucleoside Analogues

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