A sensitive nonisotopic method for the determination of intracellular azidothymidine 5′-mono-, 5′-di-, and 5′-triphosphate

Toyoshima, Takuo; Kimura, Satoshi; Muramatsu, Shigeki; Takahagi, Hidekuni; Shimada, Kaoru

doi:10.1016/0003-2697(91)90470-e

Cited by 35 publications

(21 citation statements)

References 18 publications

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“…It is possible that these different levels are due to the methodology or to biological variability. As described in this work, we carefully addressed the questions of recovery and validation, which were clearly not addressed in previous work (9,16). The possibility of interindividual differences cannot be excluded, since Toyoshima et al (16) reported some variability between their two patients.…”

Section: Discussionmentioning

confidence: 99%

“…As described in this work, we carefully addressed the questions of recovery and validation, which were clearly not addressed in previous work (9,16). The possibility of interindividual differences cannot be excluded, since Toyoshima et al (16) reported some variability between their two patients. Although the two patients had AIDS and were asymptomatic, there is no plausible explanation for why the stages of their HIV infections would result in this difference.…”

Section: Discussionmentioning

confidence: 99%

“…ZDV anabolites were separated by using HPLC with a strong-anion-exchange (SAX) column (Patisil 10-SAX; Whatman) and a method similar to that described by Elion et al (4). The 16,000 x g for 10 min, and 100 ,ul of the supematant fluid was injected.…”

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confidence: 99%

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Intracellular zidovudine (ZDV) and ZDV phosphates as measured by a validated combined high-pressure liquid chromatography-radioimmunoassay procedure

Slusher

Kuwahara

Hamzeh

et al. 1992

Antimicrob Agents Chemother

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In vitro studies of zidovudine (ZDV) phosphorylation may not accurately reflect the in vivo dose-response relationship, which is crucial to determining the relationship between ZDV exposure, efficacy, and toxicity. However, measurement of ZDV phosphorylated anabolites in peripheral blood mononuclear cells (PBMCs) from ZDV-treated human immunodeficiency virus (HlV)-infected patients would be extremely useful in the more appropriate utilization of ZDV in the treatment of H1l infection. We developed a specific and sensitive combined high-pressure liquid chromatography (HPLC)-radioimmunoassay (RLA) procedure for the determination of ZDV, ZDV-monophosphate, ZDV-diphosphate, and ZDV-triphosphate in PBMCs taken from ZDV-treated HIV-infected patients. ZDV and its anabolites were extracted from washed, Ficoll-Paque-isolated PBMCs and then separated by HPLC using a strong anion-exchange column. 3'-Azido-3'-deoxythymidine (zidovudine) (ZDV) is a pyrimidine analog first synthesized in the mid 1960s (7) and shown to be active against human immunodeficiency virus (HIV) in 1985 (10). ZDV prolongs survival and reduces the rate of disease progression in HIV-infected patients (5). However, like several other anti-HIV nucleoside analogs, it is a prodrug and requires intracellular anabolic phosphorylation to be converted into its active form. ZDV enters cells by diffusion (8), after which it is first converted by a cellular thymidine kinase to ZDV-monophosphate (ZDV-MP). The ZDV-MP is then further phosphorylated by a cellular thymidylate kinase to ZDV-diphosphate (ZDV-DP). Although thymidylate kinase has a high affinity for ZDV-MP, it also has a low maximum rate of metabolism for its conversion to ZDV-DP, thus making conversion of ZDV-MP to ZDV-DP the rate-limiting reaction in the anabolic pathway (6). ZDV-DP is then further anabolized by other cellular nucleoside diphosphate kinases to ZDV-triphosphate (ZDV-TP). ZDV

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Intracellular zidovudine (ZDV) and ZDV phosphates as measured by a validated combined high-pressure liquid chromatography-radioimmunoassay procedure

Slusher

Kuwahara

Hamzeh

et al. 1992

Antimicrob Agents Chemother

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“…On the other hand, a recent study showed a linear relationship between ZDV-TP intracellular concentrations and an increase in the percent change in CD4 ϩ cells from baseline in HIV-infected adults (5). Furthermore, several stud- Several approaches have been reported for the individual determination of 13,[15][16][17][18]21,22,24). A recent approach was developed in which strong anionexchange-solid-phase extraction separated ZDV anabolites (ZDV-MP, ZDV-DP, and ZDV-TP), followed by enzyme digestion and quantification by radioimmunoassay (18).…”

Section: Highly Active Antiretroviral Therapy (Haart) Is the Standardmentioning

confidence: 99%

Simultaneous Quantitation of Intracellular Zidovudine and Lamivudine Triphosphates in Human Immunodeficiency Virus-Infected Individuals

Rodrı́guez

Rodriguez²,

Santana

et al. 2000

Antimicrob Agents Chemother

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Highly active antiretroviral therapy (HAART) is the standard treatment for infection with human immunodeficiency virus (HIV).The most common HAART regimen consists of the combination of at least one protease inhibitor (PI) with two nucleoside reverse transcriptase inhibitors (NRTIs). Contrary to PIs, NRTIs require intracellular activation from the parent compound of their triphosphate moiety to suppress HIV replication. Simultaneous intracellular determination of two NRTI triphosphates is difficult to accomplish due to their relatively small concentrations in peripheral blood mononuclear cells (PBMCs), requiring large amounts of blood from HIV-positive patients. Recently, we described a method to determine intracellular zidovudine triphosphate (ZDV-TP) concentrations in HIV-infected patients by using solid-phase extraction and tandem mass spectrometry. The limit of quantitation (LOQ) for ZDV-TP was 0.10 pmol, and the method was successfully used for the determination of ZDV-TP in HIV-positive patients. In this study, we enhanced the aforementioned method by the simultaneous quantitation of ZDV-TP and lamivudine triphosphate (3TC-TP) in PBMCs from HIV-infected patients. The LOQ for 3TC-TP was 4.0 pmol, with an interassay coefficient of variation and an accuracy of 7 and 12%, respectively. This method was successfully applied to the simultaneous in vivo determination of the ZDV-TP and 3TC-TP pharmacokinetic profiles from HIV-infected patients receiving HAART.Highly active antiretroviral therapy (HAART) has been used successfully for treatment of human immunodeficiency virus (HIV) since the discovery of protease inhibitors (PIs) (3,4,20). HAART treatment includes a broad category of antiretroviral drug combinations with the goals of decreasing plasma HIV-1 RNA levels below the limit of detection, limiting disease progression, and delaying the appearance of resistant mutants (12). The most common HAART regimen consists of the combination of one PI with two nucleoside reverse transcriptase inhibitors (NRTIs). This triple drug combination has shown dramatic improvements in viral suppression over the combination of the two nucleosides zidovudine and lamivudine (ZDV and 3TC, respectively) (8-10).Contrary to PIs, NRTIs require intracellular activation from the parent compound of their triphosphate (TP) moiety to suppress HIV replication. ZDV and 3TC are not active against HIV; they need to be metabolized to 5Ј-ZDV-TP (ZDV-TP) and 5Ј-3TC-TP (3TC-TP) to act as competitive inhibitors of HIV reverse transcriptase or be incorporated into the viral genome (2,7,11,23). Studies conducted with HIV-infected populations have not established any relationship between ZDV or 3TC concentrations in plasma and the efficacy of these agents (19). On the other hand, a recent study showed a linear relationship between ZDV-TP intracellular concentrations and an increase in the percent change in CD4 ϩ cells from baseline in HIV-infected adults (5). Furthermore, several stud- Several approaches have been reported for the individual determinatio...

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“…It has been observed that the cellular amounts of zidovudine diphosphate (AZT-DP) and AZT-TP are significantly smaller than the corresponding AZT and AZT-MP levels (approximately 1:100) (Wattanagoon et al, 2000;Barry et al, 1996;Rodman et al, 1996;Aweeka et al, 2007;Toyoshima et al, 1991;Furman et al, 1986;Arnér et al, 1992;Flynn et al, 2007). The levels of di-and triphosphates in the rest of the body insignificantly influence the kinetics of AZT and AZT-MP (Chow et al, 1997).…”

Section: Detailed Physiologically Based Pharmacokinetic Model Of Zidomentioning

confidence: 99%

Pharmacokinetic–pharmacodynamic relationship of NRTIs and its connection to viral escape: An example based on zidovudine

Kleist¹,

Huisinga²

2009

European Journal of Pharmaceutical Sciences

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In HIV disease, the mechanisms of drug-resistance are only poorly understood. Incomplete suppression of HIV by antiretroviral agents is suspected to be a main reason. The objective of this in silico study is to elucidate the pharmacokinetic origins of incomplete viral suppression, exemplified for zidovudine (AZT) as a representative of the key class of nucleoside reverse transcriptase inhibitors (NRTIs). AZT, like other NRTIs, exerts its main action through its intracellular triphoshate (AZT-TP) by competition with natural thymidine triphosphate. We developed a physiologically based pharmacokinetic (PBPK) model describing the intracellular pharmacokinetics of AZT anabolites and subsequently established the pharmacokinetic-pharmacodynamic relationship. The PBPK model has been validated against clinical data of different dosing schemes. We reduced the PBPK model to derive a simple threecompartment model for AZT and AZT-TP that can readily be used in population analysis of clinical trials. A novel machanistic, and for NRTIs generic effect model has been developed that incorporates the primary effect of AZT-TP and potential secondary effect of zidovudine monophosphate. The proposed models were used to analyze the efficacy and potential toxicity of different dosing schemes for AZT. Based on the mechanism of action of NRTIs, we found that drug heterogeneities due to temporal fluctuations can create a major window of unsuppressed viral replication. For AZT, this window was most pronounced for a 600mg/once daily dosing scheme, in which insufficient viral suppression was observed for almost half the dosing period.

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A sensitive nonisotopic method for the determination of intracellular azidothymidine 5′-mono-, 5′-di-, and 5′-triphosphate

Cited by 35 publications

References 18 publications

Intracellular zidovudine (ZDV) and ZDV phosphates as measured by a validated combined high-pressure liquid chromatography-radioimmunoassay procedure

Intracellular zidovudine (ZDV) and ZDV phosphates as measured by a validated combined high-pressure liquid chromatography-radioimmunoassay procedure

Simultaneous Quantitation of Intracellular Zidovudine and Lamivudine Triphosphates in Human Immunodeficiency Virus-Infected Individuals

Pharmacokinetic–pharmacodynamic relationship of NRTIs and its connection to viral escape: An example based on zidovudine

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