Analysis of the Endogenous Deoxynucleoside Triphosphate Pool in HIV-Positive and -Negative Individuals Receiving Tenofovir-Emtricitabine.

Tenofovir (TFV) disoproxil fumarate (TDF) and emtricitabine (FTC), two nucleos(t)ide analogs (NA), are coformulated as an anti-HIV combination tablet for treatment and preexposure prophylaxis (PrEP). TDF/FTC may have effects on the deoxynucleoside triphosphate (dNTP) pool due to their similar structures and similar metabolic pathways. We carried out a comprehensive clinical study to characterize the effects of TDF/FTC on the endogenous dNTP pool, from baseline to 30 days of TDF/FTC therapy, in both treatment-naive HIV-positive and HIV-negative individuals. dATP, dCTP, dGTP, and TTP were quantified in peripheral blood mononuclear cells (PBMC) with a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. Forty individuals (19 HIV-positive) were enrolled and underwent a baseline visit and then received TDF/FTC for at least 30 days. Longitudinal measurements were analyzed using mixed-model segmented linear regression analysis. The dNTPs were reduced by 14% to 37% relative to the baseline level within 3 days in both HIV-negative and HIV-positive individuals (P ≤ 0.003). These reductions persisted to various degrees at day 30. These findings indicate that dNTP pools are influenced by TDF/FTC therapy. This may alter cellular homeostasis and could increase the antiviral effect through a more favorable analog/dNTP ratio. Further work is needed to elucidate mechanisms, to evaluate the clinical significance of these findings, and to further probe differences between HIV-negative and HIV-positive individuals. (This study has been registered at ClinicalTrials.gov under identifier NCT01040091.).

High Plasma Concentrations of Zidovudine (AZT) Do Not Parallel Intracellular Concentrations of AZT-Triphosphates in Infants During Prevention of Mother-to-Child HIV-1 Transmission.

OBJECTIVES: Zidovudine (AZT) is mainly used to prevent mother-to-child HIV-1 transmission (PMTCT). Despite serious concerns on AZT-associated toxicity, there is little information on pharmacokinetics of intracellular AZT metabolites in infants. METHODS: We conducted a prospective study in 31 HIV-uninfected infants who received AZT for PMTCT. Blood samples were obtained from 14 infants on postdelivery days (PDD) 1, 7, 14, and 28 and from 17 infants at 0 and 4 hours after dosing on PDD-1. Plasma AZT concentrations (pAZT) and intracellular concentrations of AZT-monophosphate (icAZT-MP), diphosphate (icAZT-DP), and triphosphate (icAZT-TP) were determined. RESULTS: Plasma AZT and icAZT-MP concentrations were 2713 nmol/L and 79 fmol/10 cells in PDD-1, but decreased to 1437 nmol/L and 31 fmol/10 cells by PDD-28 (P = 0.02 and P = 0.07 for all PDDs, respectively), whereas those of icAZT-DP and icAZT-TP remained low throughout the sampling period (P = 0.29 and P = 0.61 for all PDDs, respectively) There were no differences in icAZT-TP between infants of the 2 mg/kg 4 times a day dose and 4 mg/kg twice daily dose (P = 0.25), whereas pAZT and icAZT-MP levels were higher in the latter (P < 0.01 and <0.01, respectively). The pAZT and icAZT-MP significantly increased from 0 to 4 hours after dosing (P < 0.001 and <0.001, respectively), whereas icAZT-DP, icAZT-TP levels were not changed (P = 0.41 and 0.33, respectively). CONCLUSIONS: The level of icAZT-TP did not change with age, time, or a single dose despite the wide range of pAZT concentration. A safer dosage needs to be determined because high pAZT levels do not parallel those of icAZT-TP.

Ultrasensitive method to quantify intracellular zidovudine mono-, di- and triphosphate concentrations in peripheral blood mononuclear cells by liquid chromatography-tandem mass spectrometry.

Although zidovudine (AZT) is not the preferred antiretroviral drug for adult HIV-infected patients, it is still widely used in infants for both prevention of mother-to-infant HIV-1 transmission and treatment of HIV-infected children. However, it is difficult to measure intracellular concentrations of AZT metabolites in small blood samples due to their extremely low concentrations in peripheral blood mononuclear cells and interference by endogenous nucleotide triphosphates, residual plasma phosphates and electrolytes. We developed an ultrasensitive assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurement of intracellular concentrations of zidovudine (AZT)-monophosphate (AZT-MP), -diphosphate (AZT-DP) and -triphosphate (AZT-TP). The high sensitivity was due to the improvement of peripheral blood mononuclear cells extraction for complete removal of plasma and electrolytes, alkalization of LC buffer and use of alkaline-stable high performance liquid chromatography column and tetrabutylammonium hydroxide as the ion pair. Using this method, the lower limits of quantification of AZT, AZT-MP, -DP and -TP were 6, 6, 10 and 10 fmol per sample, respectively. Accuracy ranged 89-115% and precision was lower than 15% in the quantification range of 6-6000 fmol/sample for plasma AZT and intracellular AZT-MP and 10-10 000 fmol/sample for AZT-DP and -TP. The validation parameters met the international requirements. Among nine AZT-treated HIV-infected adult patients, five had low AZT-TP levels (<10 fmol/10(6) cells). Our assay has high sensitivity and is advantageous for evaluation of AZT phosphates in children and infants based on minimum blood sampling requirement.

Biphasic elimination of tenofovir diphosphate and nonlinear pharmacokinetics of zidovudine triphosphate in a microdosing study.

OBJECTIVE: Phase 0 studies can provide initial pharmacokinetics (PKs) data in humans and help to facilitate early drug development, but their predictive value for standard dosing is controversial. To evaluate the prediction of microdosing for active intracellular drug metabolites, we compared the PK profile of 2 antiretroviral drugs, zidovudine (ZDV) and tenofovir (TFV), in microdose and standard dosing regimens. STUDY DESIGN: We administered a microdose (100 µg) of C-labeled drug (ZDV or tenofovir disoproxil fumarate) with or without a standard unlabelled dose (300 mg) to healthy volunteers. Both the parent drug in plasma and the active metabolite, ZDV-triphosphate (ZDV-TP) or TFV-diphosphate (TFV-DP) in peripheral blood mononuclear cells (PBMCs) and CD4 cells were measured by accelerator mass spectrometry. RESULTS: The intracellular ZDV-TP concentration increased less than proportionally over the dose range studied (100 µg-300 mg), whereas the intracellular TFV-DP PKs were linear over the same dose range. ZDV-TP concentrations were lower in CD4 cells versus total PBMCs, whereas TFV-DP concentrations were not different in CD4 cells and PBMCs. CONCLUSIONS: Our data were consistent with a rate-limiting step in the intracellular phosphorylation of ZDV but not TFV. Accelerator mass spectrometry shows promise for predicting the PK of active intracellular metabolites of nucleosides, but nonlinearity of PK may be seen with some drugs.

Design, synthesis and studies of triphosphate analogues for the production of anti AZT-TP antibodies.

Triphosphates anabolites are the active chemical species of nucleosidic reverse transcriptase inhibitors in HIV-therapy. Herein, we describe (i) the design of stable triphosphate analogues of AZT using molecular modelling, (ii) their synthesis and (iii) their use for producing anti AZT-TP antibodies in the aim of developing an immunoassay for therapeutic drug monitoring.

High levels of zidovudine (AZT) and its intracellular phosphate metabolites in AZT- and AZT-lamivudine-treated newborns of human immunodeficiency virus-infected mothers.

Newborns from human immunodeficiency virus-infected mothers are given antiretroviral prophylaxis against mother-to-child transmission, including predominantly nucleoside reverse transcriptase inhibitors. Pharmacological monitoring of these drugs in newborns has so far been limited to plasma and cord blood. In this study, samples from newborns (up to 45 days old) treated with zidovudine (AZT) alone (n = 29) or in combination with lamivudine (3TC) (n = 20) were analyzed for both intracellular concentrations of phosphate metabolites in peripheral blood mononuclear cells and levels of parent drugs in plasma. Plasma AZT and intracellular AZT-monophosphate and AZT-triphosphate (TP) concentrations were significantly higher during the first 15 days of life (199 versus 52.7 ng/ml [P < 0.0001], 732 versus 282 fmol/10(6) cells [P < 0.0001], and 170 versus 65.1 fmol/10(6) cells [P < 0.0001], respectively) and then became comparable to those of adults. No difference in intracellular AZT metabolite concentrations was found when AZT- and AZT-3TC-treated groups were compared. Plasma 3TC levels (lower limit of quantification [LLOQ], 1,157 ng/ml; median, 412.5 ng/ml) were not associated with the newborn's age, gender, or weight. Intracellular 3TC-TP concentrations (LLOQ, 40.4 pmol/10(6) cells; median, 18.9 pmol/10(6) cells) determined for newborns receiving the AZT-3TC combination were associated with neither the age nor weight of the newborns. Concentrations in females were significantly higher (1.8-fold [P = 0.0415]) than those in males. Unexpectedly, newborns on AZT monotherapy whose mothers' treatment included 3TC displayed residual plasma 3TC and intracellular 3TC-TP levels up to 1 week after birth.

Strain differences in mice highlight the role of DNA damage in neoplasia induced by low dietary folate.

In earlier work, we showed that low dietary folate induced intestinal tumors in BALB/c mice. In this study, our goal was to examine the effect of the same diets on a strain that is more resistant to tumorigenesis (C57Bl/6). We also questioned whether supplementation of the folate-deficient diet (FD) with betaine, an alternate methyl donor, would influence tumor formation. C57Bl/6 mice were fed the same diets [control diet (CD) with 2 mg folate/kg diet and FD with 0.3 mg folate/kg diet] as those in our previous study for 1 y, but they did not develop tumors. We also fed BALB/c mice the FD or FD supplemented with betaine for 1 y, but there was no change in tumor incidence. To determine the relative contributions of DNA damage and altered methylation patterns, we measured intestinal dUTP:dTTP ratios, phosphorylated histone H2AX (p-H2AX) staining, and global DNA methylation in both strains. Only BALB/c mice showed changes due to diet in dUTP:dTTP (from 2.19 +/- 0.20 in CD to 2.77 +/- 0.18 in FD; P = 0.05) and in p-H2AX staining (from 14.10 +/- 3.59% in CD to 22.40 +/- 2.65% in FD; P = 0.054). In BALB/c mice only, FD tended to have less (P = 0.06) global DNA methylation than CD. Although the FD increased plasma homocysteine and the betaine-supplemented FD lowered plasma homocysteine, the latter diet did not reduce tumor incidence. We conclude that plasma homocysteine is not likely to be associated with tumorigenesis in our model. However, DNA damage plays a critical role in initiating tumorigenesis when dietary folate is low and methylation changes may also be contributory.

Intracellular nucleoside triphosphate concentrations in HIV-infected patients on dual nucleoside reverse transcriptase inhibitor therapy.

BACKGROUND: Intracellular nucleoside reverse transcriptase inhibitor triphosphate (NRTI-TP) concentrations are crucial in suppressing HIV replication. Little is known about how commonly used dual-NRTI regimens affect the intracellular levels of NRTI-TPs, the active form of these drugs. This study investigates the effect of dual-NRTI therapy in intracellular NRTI-TP levels. METHODS: NRTI and NRTI-TP concentrations were evaluated in HIV-infected patients receiving either lamivudine (3TC) and stavudine (d4T) or lamivudine with zidovudine (ZDV); NRTI and NRTI-TP concentrations were determined using a validated HPLC/MS/MS method. Plasma HIV-1 RNA levels were determined at baseline and monthly to examine the relationship between NRTI-TP concentrations and plasma HIV-1 RNA. RESULTS: Forty-one subjects completed the study. 3TC-TP significantly increased between day 1 and week 28 from 1.48 to 5.00 pmol/10(6) peripheral blood mononuclear cells (PBMC; P < 0.0001). NRTI-TP concentrations for d4T and ZDV did not significantly increase, with values at week 28 of 0.011 and 0.02 pmol/10(6) PBMC, respectively. Mean NRTI-TP/plasma ratios were 3%, 0.007% and 0.05% for 3TC, d4T and ZDV, respectively. Linear relationships were observed between ZDV- and 3TC-TP and changes in plasma HIV-1 RNA. CONCLUSION: Of the three drugs studied, only 3TC-TP levels increased significantly between day 1 and week 28. ZDV-TP and 3TC-TP levels were unaffected by dual-NRTI therapy relative to monotherapy, regardless of the combination (3TC-ZDV or 3TC-d4T). Intracellular levels of d4T-TP were similar to previous reports for dual-NRTI therapy; however, in the case of d4T, these values appear lower than those achieved with d4T monotherapy.

Improved method to quantify intracellular zidovudine mono- and triphosphate in peripheral blood mononuclear cells by liquid chromatography-tandem mass spectrometry.

The determination of intracellular triphosphate metabolites of nucleoside analogs used in anti-HIV therapy is very challenging. Despite the well-known sensitivity and selectivity of LC-MS/MS, the measurement of the triphosphate metabolite of zidovudine (AZT-TP) remains difficult because of the interferences induced by endogenous nucleotides triphosphates. We describe a new approach that allows improved determination of AZT-TP simultaneously with AZT-monophosphate (MP). This was obtained, first, by monitoring a transition from the molecular ion of AZT-TP to a minor but very specific product ion. Then, the spiking of samples with a constant amount of AZT-TP allowed the signal to emerge from background, leading to increased sensitivity. Finally, the analytical run time was reduced to less than 10 min. The low limits of quantification were at 150 and 300 fmol per sample for AZT-TP and AZT-MP, respectively. Recoveries were higher than 85%. Inaccuracy and precision were lower than 10% and 15% (17% at the limit of quantification), respectively. The new method offers the possibility of determining simultaneously other nucleotide phosphates, as shown here for d4T-TP (the triphosphate metabolite of another nucleoside analog, stavudine or d4T) and 2'-deoxythymidine-5'-triphosphate or dTTP (the corresponding natural nucleotide triphosphate).

Quantitation of zidovudine triphosphate concentrations from human peripheral blood mononuclear cells by anion exchange solid phase extraction and liquid chromatography-tandem mass spectroscopy; an indirect quantitation methodology.

To facilitate the assessment of drug safety and determination of phamacokinetics, an anion exchange isolation of zidovudine triphosphate (ZDV-TP) from human peripheral blood mononuclear cells (hPBMC), coupled with dephosphorylation, desaltation, and detection by liquid chromatography-tandem mass spectroscopy (LC-MS-MS) was validated. hPBMCs were harvested from whole blood, lysed, and a suspension of intracellular ZDV-TP was produced. ZDV-TP was isolated from ZDV, ZDV-monophosphate (ZDV-MP), and ZDV-diphosphate (ZDV-DP), which were all present in the cell lysate, by performing a salt gradient anion exchange SPE. Isolated ZDV-TP was dephosphorylated with acid phosphatase to its parent drug form, ZDV. ZDV was then desalted and concentrated for tandem mass spectral detection. An LC-MS-MS methodology was developed and validated for the determination of molar ZDV directly corresponding to the intra-hPBMC molar ZDV-TP concentration. ZDV-TP concentrations were determined in femtomoles per million hPBMCs (fmol/10(6)cells). The assay was able to determine ZDV-TP concentrations accurately and precisely within the range of 5-640 fmol/10(6)cells with 10 million cells per sample analyzed. Inter- and intra-day accuracy and precision data for back calculated standards and quality controls fell within 15% of nominal. The assay correlated well with a previous ELISA method developed and validated in our laboratory, and has been successfully used to quantitate ZDV-TP concentrations in patients being routinely monitored and treated with ZDV.

Simultaneous quantitation of zidovudine and zidovudine monophosphate from plasma, amniotic fluid and tissues by micellar capillary electrophoresis.

Zidovudine (AZT) therapy given during pregnancy has been shown to reduce the vertical transmission of the human immunodeficiency virus (HIV) from mother to fetus. In order to investigate the efficacy of AZT, it is important to know the concentration of its active phosphorylated metabolites. We have developed the first CE method for the simultaneous quantitation of AZT and zidovudine monophosphate (AZT-MP) from rat plasma, amniotic fluid and fetal tissues. Sample extractions were performed by protein precipitation using acetonitrile for the plasma and amniotic fluids, while in fetal tissues solid phase extraction using Waters Oasis HLB extraction cartridges was used. Recoveries ranged from 78 to 92% for AZT, AZT-MP and 3'-azidouridine (internal standard, AZDU), in the three matrices. The optimum separation conditions were achieved using a 40 mm sodium dodecylsulfate (SDS) in 50 mm phosphate buffer (pH 7) with a run voltage of 15 kV. The CE system consists of a 75 microm i.d., 50 cm effective length uncoated fused silica capillary. The method was validated over the range 0.5-100 microg/ml ( micro g/g for tissues). Intra-day precision (RSD) and accuracy (%error) for AZT ranged from 0.13 to 11 and 0.68 to 11.1%, respectively, while for AZT-MP it ranged from 2.05 to 11.1 and 4.22 to 11.7%. Inter-day precision and accuracy for AZT ranged from 3.82 to 11.2 and 3.14 to 9.01%, while for AZT-MP it ranged from 3.9 to 9.32 and 3.44 to 9.37%, respectively. We also report the enzymatic dephosphorylation of AZT-MP in the placental tissue of rats. This new enzymatic pathway provides increased understanding of the mechanism of anti-viral transport in the rat during pregnancy.

Development of a pharmacodynamic model for HIV treatment with nucleoside reverse transcriptase and protease inhibitors.

There is a need for models useful for predicting the efficacy of agents developed for treating human immunodeficiency virus (HIV) based on information obtained during the drug development process. A pharmacodynamic model that superimposes the pharmacokinetics of anti-HIV nucleoside reverse transcription (RT) and protease inhibitors over a previously published predator-prey model of HIV and CD4 dynamics was developed to address this need. This model was applied to in vitro measurements and patient-derived pharmacokinetics of the unbound antiviral drugs to simulate HIV-1 and CD4 counts versus time and dose. The primary mechanism for nucleoside RT inhibitors was assumed to be competitive inhibition of HIV-1-RT by the active nucleoside triphosphates (NTP). Cellular accumulation and breakdown rates of the NTP were estimated from previous in vivo pharmacokinetic studies. Median inhibition concentrations for the HIV-1 RT enzyme were estimated from previously published cell-free binding studies. The concentration of active protease inhibitor available for binding with HIV-1 protease was assumed equal to the unbound fraction in the plasma. The resulting simulations for mono- and dual nucleoside therapy with zidovudine and lamivudine single dose regimen with the protease inhibitor indinavir, produced similar HIV and CD4 response profiles to those reported in large Phase II and III clinical trials. Based on these findings this pharmacodynamic model can be applied to predict starting doses for a new agent based on simulated biological responses as a function of time for dosage regimens comprising one or two agents. However, the model overestimated the efficacy of highly effective drug combinations where all three agents are combined as in highly active anti-retroviral therapy.

Development of a direct assay for measuring intracellular AZT triphosphate in humans peripheral blood mononuclear cells.

Direct LC/MS/MS methods have recently been developed for measuring triphosphate anabolites of several nucleosidic reverse transcriptase inhibitor (NRTI) in peripheral blood mononuclear cells (PBMCs) from HIV-positive patients. Whereas AZT is one of the most-used NRTIs, no such method has been developed for AZT-TP, its active anabolite, mainly because of the presence of endogenous nucleotides that interfere with such an assay. In this paper, we first describe the development of two enzyme immunoassays (EIA) of AZT-TP in PBMCs: one directly measuring AZT-TP content; the other, measuring the nucleoside AZT after selective extraction of AZT-TP and dephosphorylation. The precision of these two assays was too low to achieve precise determination of AZT-TP in PBMC samples. Direct LC/MS/MS is not specific enough for AZT-TP, since at least two interfering endogenous nucleotides (same m/z ratio and fragment as well as retention time close to that of AZT-TP) are found in the intracellular medium of PBMCs. The off-line combination of immunoaffinity extraction (IAE) and LC/MS/MS proved to be a successful strategy allowing without dephosphorylation appropriate specificity and sensitivity (limit of quantification established as 9.3 fmol/10(6) cells) to determine AZT-TP in PBMCs from 7 mL of blood of HIV-infected patients. Validation of this IAE-LC/MS/MS method demonstrated CV percent for repeatability and intermediate precision lower than 15%. More than 150 samples/week can be analyzed by one analyst, making this method suitable for routine analysis during clinical studies.

Simultaneous quantitation of the 5'-triphosphate metabolites of zidovudine, lamivudine, and stavudine in peripheral mononuclear blood cells of HIV infected patients by high-performance liquid chromatography tandem mass spectrometry.

A high-performance liquid chromatography (HPLC) method utilizing triple quadrupole mass spectrometry (MS) detection was developed and validated for the simultaneous measurement of the intracellular nucleoside 5'-triphosphate anabolites of zidovudine (ZDV-TP), lamivudine (3TC-TP), and stavudine (d4T-TP). These compounds were extracted from patient peripheral blood mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. Ion-exchange solid phase extraction (SPE) followed by enzymatic digestion with alkaline phosphatase was utilized to yield the measurable nucleoside forms of the nucleotides. Reversed phase C-18 SPE with addition of a nucleoside internal standard, 3'-azido-2',3'-dideoxyuridine (AzdU) allowed for the indirect measurement of the original 5'-triphosphate concentration by HPLC/MS/MS. Quantitation was performed from calibration curves generated from authentic 5'-triphosphate standards spiked in PBMCs from healthy volunteers. Analytical range for the three 5'-triphosphates was equivalent to 50-45,000 pg. Mean interassay accuracies for 3TC-TP, d4T-TP, and ZDV-TP (n > 90) were 99.4%, 100.1%, and 108.0%, respectively. Mean interassay precisions (%C.V.) for 3TC-TP, d4T-TP, and ZDV-TP (n > 90) were 8.8%, 10.4%, and 8.2%, respectively. Recovery of the extraction method was 79.2%, 83.1%, and 98.3% for 3TC-TP, d4T-TP, and ZDV-TP, respectively. This method can be utilized to measure the intracellular 5'-triphosphate levels in HIV infected patients receiving antiretroviral therapy containing the nucleoside reverse transcriptase inhibitors 3TC, d4T, or ZDV.

Simultaneous quantitation of intracellular zidovudine and lamivudine triphosphates in human immunodeficiency virus-infected individuals.

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.

Pharmacokinetics of zidovudine phosphorylation in human immunodeficiency virus-positive thai patients and healthy volunteers.

We assessed the pharmacokinetics of zidovudine (ZDV) in plasma and intracellular ZDV phosphate anabolites in peripheral blood mononuclear cells in Thai human immunodeficiency virus (HIV) type 1-infected patients and healthy volunteers. The plasma ZDV area under the concentration-time curve from 0 to 6 h (AUC(0-6)) was similar in patients and healthy volunteers (32.77 and 22.77 micromol/liter. h, respectively; confidence interval, -3.37 to 19. 92). Although the concentration of ZDV triphosphate (ZDVTP) was similar in the two groups, the ZDV monophosphate (ZDVMP) AUC(0-6) was significantly greater in HIV patients (1.12 pmol/10(6) cells) than in healthy volunteers (0.15 pmol/10(6) cells). In agreement with previously published data obtained with Caucasians, the significant difference in intracellular phosphorylation in Thai volunteers and HIV patients is primarily due to ZDVMP. Comparing the data from this study with the data obtained with Caucasians suggests no marked ethnic differences in ZDV phosphorylation.

Determination of zidovudine triphosphate intracellular concentrations in peripheral blood mononuclear cells from human immunodeficiency virus-infected individuals by tandem mass spectrometry.

Nucleoside reverse transcriptase inhibitors (NRTIs) used against the human immunodeficiency virus (HIV) need to be activated intracellularly to their triphosphate moiety to inhibit HIV replication. Intracellular concentrations of these NRTI triphosphates, especially zidovudine triphosphate (ZDV-TP), are relatively low (low numbers of femtomoles per 10(6) cells) in HIV-infected patient peripheral blood mononuclear cells. Recently, several methods have used either high-performance liquid chromatography (HPLC) or solid-phase extraction (SPE) coupled with radioimmunoassay to obtain in vivo measurements of ZDV-TP. The limit of detection (LOD) by these methods ranged from 20 to 200 fmol/10(6) cells. In this report, we describe the development of a method to determine intracellular ZDV-TP concentrations in HIV-infected patients using SPE and HPLC with tandem mass spectrometry for analysis. The LOD by this method is 4.0 fmol/10(6) cells with a linear concentration range of at least 4 orders of magnitude from 4. 0 to 10,000 fmol/10(6) cells. In hispanic HIV-infected patients, ZDV-TP was detectable even when the sampling time after drug administration was 15 h. Intracellular ZDV-TP concentrations in these patients ranged from 41 to 193 fmol/10(6) cells. The low LOD obtained with this method will provide the opportunity for further in vivo pharmacokinetic studies of intracellular ZDV-TP in different HIV-infected populations. Furthermore, this methodology could be used to perform simultaneous detection of two or more NRTIs, such as ZDV-TP and lamivudine triphosphate.

Systemic pharmacokinetics and cellular pharmacology of zidovudine in human immunodeficiency virus type 1-infected women and newborn infants.

Systemic and intracellular pharmacokinetics of zidovudine were determined for 28 human immunodeficiency virus type 1-infected pregnant women and their newborn infants. Plasma zidovudine and intracellular zidovudine monophosphate and triphosphate concentrations were determined in serial maternal samples and cord blood at delivery. Higher levels of cord blood zidovudine were associated with lower maternal zidovudine clearance and longer infusion times. Median levels of zidovudine monophosphate and triphosphate in maternal (1556 and 67 fmol/106 cells) and cord (1464 and 70 fmol/106 cells) blood were similar but highly variable. Intersubject pharmacokinetic variability for zidovudine is substantial, but intravenous therapy provides plasma concentrations and intracellular zidovudine triphosphate levels consistent with high antiviral activity. The substantial amount of intracellular zidovudine triphosphate in cord blood provides an explanation for the clinical success of zidovudine in reducing vertical transmission. Studies of simpler oral regimens of zidovudine can now be evaluated regarding the ability to achieve these pharmacologic end points associated with highly effective parenteral therapy.

Palindromic oligonucleotide-directed enzymatic determination of 2'-deoxythymidine 5'-triphosphate and 2'-deoxycytidine 5'-triphosphate in human cells.

A new method is presented for the determination of 2'-deoxythymidine 5'-triphosphate and 2'-deoxycytidine 5'-triphosphate concentrations within human cells based on a DNA polymerase reaction directed by a palindromic oligonucleotide precursor. Two 19-mer oligonucleotide precursors are employed that contain a common 8-mer palindromic sequence followed by a sequence-specific insertion site and a 5'-oligodeoxythymidylate tail. To conduct a measurement, two molecules of the 19-mer oligonucleotide precursor are first annealed to form a pair of symmetrical template-primer addition sites at their 3'-termini that are coded for the analyte of interest, present in limiting amounts. The Klenow fragment of Escherichia coli DNA polymerase I then elongates the template-primer by the addition of two molecules of the complementary deoxyribonucleotide analyte. Following the addition of the analyte molecules, the template-primer is extended with a 10-mer oligo(dA) tail in the presence of excess dATP and the Klenow fragment. The result is a 30-mer palindromic oligonucleotide that can be separated from any remaining 19-mer precursor and quantified by paired-ion HPLC using UV detection. Since the molar extinction coefficient of the 30-mer palindromic oligonucleotide is much larger than that of the nucleotide analyte alone, the UV signal is markedly enhanced, thereby increasing sensitivity. Details describing this method and the application of it to measure these analytes in as few as 2.5 x 10(6) human cells are presented.

Gamma-phosphate-substituted 2'-deoxynucleoside 5'-triphosphates as substrates for DNA polymerases.

Several 2'-deoxythymidine 5'-triphosphate and 3'-azido-2', 3'-dideoxythymidine 5'-triphosphate analogs containing a hydrophobic phosphonate group instead of the gamma-phosphate were synthesized and evaluated as substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus reverse transcriptases, human placental DNA polymerases alpha and beta, and calf thymus terminal deoxynucleotidyl transferase. They were efficiently incorporated into the DNA chain by the retroviral enzymes but were not utilized by the mammalian ones. Also, some gamma-ester and gamma-amide derivatives of dTTP and 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP) were synthesized and studied. They proved to be substrates for both the retroviral and mammalian enzymes under study. The Km values for incorporation of the dTTP derivatives into the DNA chain were close to those for dTTP and AZTTP. The Km for the AZTTP derivatives were one order of magnitude greater than those for dTTP and AZTTP. The results obtained indicate that HIV and avian myeloblastosis virus reverse transcriptases have no sterical obstacles for binding the triphosphate fragment bearing a bulky substituent at the gamma-position. Modification of the gamma-phosphate in AZTTP increased the selectivity of HIV reverse transcriptase inhibition versus DNA polymerase alpha. gamma-Methylphosphonate and gamma-phenylphosphonate were dephosphorylated in human serum much less rapidly than AZTTP. Besides, they were shown to be markedly more hydrophobic than AZTTP. Thus, replacement of the gamma-phosphate in AZTTP with gamma-phosphonate markedly alters its substrate properties toward some cellular DNA polymerases and blood dephosphorylating enzymes but does not change its substrate activity with respect to HIV reverse transcriptase.