Jose F. Rodriguez

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

ABSTRACT 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.

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.

Differential Extracellular and Intracellular Concentrations of Zidovudine and Lamivudine in Semen and Plasma of HIV-1–Infected Men

Objectives:To quantitate extracellular and intracellular zidovudine (ZDV) and lamivudine (3TC) concentrations in blood and semen of HIV-1-infected men. Design:Nonblind, single-center, open-label pharmacokinetic (PK) study in 14 subjects receiving ZDV plus 3TC. Methods:Paired blood and semen samples were obtained during 1 intensive visit and 3 single time point visits over 2 weeks. Extracellular ZDV and 3TC concentrations were measured in blood plasma (BP) and seminal plasma (SP), and intracellular ZDV and 3TC triphosphate (TP) concentrations were measured in isolated mononuclear cells using validated methods. HIV-1 RNA was measured in blood and semen. PK parameters were estimated using noncompartmental analysis. Results:Median (interquartile range [IQR]) SP/BP area under the time-concentration curve over the 12-hour dosing interval (AUC0-12h) ratios for ZDV and 3TC were 2.28 (1.48 to 2.97) and 6.67 (4.10 to 9.14), respectively, whereas individual SP/BP concentration ratios ranged from 1.9 to 91.4. Intracellular median (IQR) SP/BP AUC0-12h ratios for ZDV-TP and 3TC-TP were 0.36 (0.30 to 0.37) and 1.0 (0.62 to 1.30), respectively, whereas individual SP/BP concentration ratios ranged from 0.11 to 2.9. HIV-1 RNA was undetectable in both compartments. Conclusions:ZDV and 3TC SP exposures are 2- to 6-fold greater than BP exposures. Seminal ZDV-TP exposures are ∼40% of those found in peripheral blood mononuclear cells (PBMCs), whereas 3TC-TP exposures are similar to PBMC exposures. PK variability makes individual SP/BP ratios a suboptimal surrogate for genital tract exposure.

Intracellular Pharmacokinetics of Once versus Twice Daily Zidovudine and Lamivudine in Adolescents

ABSTRACT Zidovudine (ZDV) and lamivudine (3TC) metabolism to triphosphates (TP) is necessary for antiviral activity. The aims of this study were to compare ZDV-TP and 3TC-TP concentrations in adolescents receiving twice daily (BID) and once daily (QD) regimens and to determine the metabolite concentrations of ZDV and 3TC during chronic therapy on a QD regimen. Human immunodeficiency virus-infected patients (12 to 24 years) taking ZDV (600 mg/day) and 3TC (300 mg/day) as part of a highly active antiretroviral therapy regimen received QD and BID regimens of ZDV and 3TC for 7 to 14 days in a crossover design. Serial blood samples were obtained over 24 h on the QD regimen. Intracellular mono-, di-, and triphosphates for ZDV and 3TC were measured. The median ratio of BID/QD for ZDV-TP predose concentrations was 1.28 (95% confidence interval [CI] = 1.00 to 2.45) and for 3TC-TP was 1.12 (95% CI = 0.81 to 1.96). The typical population estimated half-lives (± the standard error of the mean) were 9.1 ± 0.859 h for ZDV-TP and 17.7 ± 2.8 h for 3TC-TP. Most patients had detectable levels of the TP of ZDV (24 of 27) and 3TC (24 of 25) 24 h after dosing, and half-lives on a QD regimen were similar to previously reported values when the drugs were given BID. Lower, but not significantly different, concentrations of ZDV-TP were demonstrated in the QD regimen compared to the BID regimen (P = 0.056). Although findings were similar between the BID and QD groups, the lower concentrations of ZDV and the number of patients below the level of detection after 24 h suggests that ZDV should continue to be administered BID.

HPLC–MS/MS method for the intracellular determination of ribavirin monophosphate and ribavirin triphosphate in CEMss cells

A sensitive and specific method using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination of ribavirin monophosphate (RBV-MP) and ribavirin triphosphate (RBV-TP) in cells has been developed and validated. In this method, ribavirin phosphorylated metabolites were extracted and separated by anion exchange solid phase extraction (SPE). The RBV-MP and RBV-TP fractions were dephosphorylated using acid phosphatase and further purified by phenyl boronate SPE prior to HPLC-MS/MS analysis. (13)C(5)-uridine was added as internal standard to obtain better accuracy and precision of the analysis. The MS/MS detector was optimized at multiple reaction monitoring (MRM) using positive electrospray ionization to detect 245-->113 and 250-->133 transitions for ribavirin and internal standard, respectively. The calibration curve was linear over a concentration range of 0.01-10 microg/mL with a limit of quantitation of 0.01 microg/mL. Mean inter-assay accuracy and precision for RBV-MP and RBV-TP quality control samples at 0.03, 0.3 and 8 microg/mL were 5% and 10%, respectively. This method was successfully used for the in vitro determination of RBV-MP and RBV-TP in CEM(ss) cells cultured with RBV.

Plasma glutathione concentrations in children infected with human immunodeficiency virus

BACKGROUND Glutathione (GSH) is the principal intracellular defense against oxidants, and HIV-infected individuals tend to have subnormal concentrations in plasma. This GSH deficiency may contribute to the pathogenesis of disease progression. In the pediatric population correlations between GSH concentrations with clinical, immunologic and virologic disease profiles are scarce. OBJECTIVES The main objectives of this study were (1) to compare plasma GSH concentrations of HIV-infected children and healthy controls and (2) to correlate GSH values with clinical, immunologic and virologic disease indices. METHODS Twenty-four HIV-infected and 24 healthy control children entered the study. Plasma concentrations of total glutathione and related thiols were determined. RESULTS The difference in mean plasma GSH concentrations between HIV-infected (2.96 +/- 0.31 microM) and control (6.62 +/- 0.58 microM) groups was highly significant (P < 0.0001). Linear regression analyses in HIV-infected patients revealed significant correlations between GSH and both absolute CD4+ cell counts (r = 0.56, P = 0.004) and viral load measured as log HIV-RNA PCR (r = -0.49, P = 0.018). GSH concentrations did not significantly correlate with CDC clinical stage but were lower in HIV-infected patients with growth failure (1.60 +/- 0.54 microM) vs. non-growth failure (3.23 +/- 0.33 microM); P = 0.05. CONCLUSIONS This study confirmed that HIV-infected children are deficient in plasma GSH concentrations compared with healthy controls. We documented that low GSH concentrations in HIV-infected children are directly correlated with CD4+ cell counts and inversely correlated with viral loads. These findings support a possible role of GSH in the pathogenesis of HIV disease progression.

Concentraciones totales de homocisteína plasmática en pacientes puertorriqueños con cardiopatía isquémica

Introduccion y objetivos En Puerto Rico se ha comprobado que, a pesar de que la enfermedad coronaria es la principal causa de muerte, la poblacion tiene una incidencia menor de estas enfermedades que en los EE.UU. y posee una incidencia menor de taquicardia ventricular y muerte subita. Un factor que puede contribuir a la menor incidencia de enfermedades coronarias en Puerto Rico es que las concentraciones totales de homocisteina plasmatica (tHcys) en nuestra poblacion sean menores que en la poblacion de los EE.UU. Nuestro objetivo era medir tHcys en la poblacion puertorriquena con cardiopatia isquemica. Metodos Se midio aleatoriamente la tHcys en 70 pacientes puertorriquenos hospitalizados en el Centro Cardiovascular de Puerto Rico y el Caribe (Division UPR). Resultados La concentracion promedio de tHcys en estos pacientes resulto similar a la comunicada por el estudio de Framingham cuando es ajustada por edad (11,2 frente a 11,8 mmol/l). En la poblacion puertorriquena, los varones tenian una concentracion mayor de tHcys que las mujeres (11,7 frente a 9,5 mmol/l; p = 0,07). Ademas, no observamos un aumento en la concentracion de tHcys en pacientes diabeticos cuando se compararon con los pacientes no diabeticos (10,1 frente a 11,2 mmol/l; p = 0,74). Tampoco observamos una correlacion directa entre la concentracion de tHcys y las condiciones cardiacas medidas por angiografia coronaria (normal = 11,1 mmol/l; leve = 10,5 mmol/l; moderado = 10,9 mmol/l; severo = 10,5 mmol/l; Kruskal-Wallis = 0,45). Conclusion Estos resultados sugieren que la concentracion de tHcys no predice fiablemente la gravedad de las lesiones de cardiopatia isquemica en la poblacion puertorriquena.

Incidence and impact of undisclosed cocaine use in emergency department chest pain and trauma patients.

BackgroundOne of the highest rates of illicit cocaine consumption in Europe is in Spain. Our objective was to study the incidence and impact of undisclosed cocaine consumption in patients attending the emergency department (ED) for trauma or chest pain.MethodsWe analysed urine samples from consecutive patients attending the ED for trauma or chest pain to determine the presence of cocaine, cannabis, amphetamine/metaamphetamine and opioids by semiquantative tests with fluorescence polarization immunoassay (FPIA).ResultsThirty percent of eligible patients participated. Of 75 cases, 61.3% had trauma and 38.7% chest pain; 25% presented a positive test for drugs. Cocaine was present in 13.3% and cannabis in the same proportion. No differences were found regarding positive cocaine test and chief complaint, ED or hospital stay, or additional tests. Cocaine-positive patients were significantly younger.

Lack of Evidence for In Vivo Transformation of Zidovudine Triphosphate to Stavudine Triphosphate in Human Immunodeficiency Virus-Infected Patients

ABSTRACT The in vivo and in vitro determination of significant intracellular stavudine (d4T) triphosphate (d4TTP) concentrations in human immunodeficiency virus (HIV)-infected subjects and NS-1 cells treated with zidovudine (ZDV) has recently been reported. This study was conducted to corroborate these findings with in vivo samples from HIV-infected subjects taking ZDV and in vitro CEMSS cells incubated with different ZDV concentrations. Previously, we have reported on our validated high-performance liquid chromatography coupled with tandem mass spectrometry methodology for the simultaneous determination of d4TTP, lamivudine triphosphate, and ZDV triphosphate (ZDVTP) concentrations. Using this methodology, we monitored the d4TTP concentration in more than 100 samples from HIV-infected subjects treated with d4T. In addition, we simultaneously monitored the concentrations of d4TTP and ZDVTP in more than 500 samples from HIV-infected individuals who were taking ZDV. Finally, we performed in vitro studies by incubating CEMSS cells with 10 μM, 50 μM, and 100 μM ZDV and monitored the formation of d4TTP at 24 and 48 h. We could measure d4TTP concentrations from HIV-infected individuals with a limit of quantitation (LOQ) of 2.7 fmol/106 cells (total injection, 54 fmol). In the in vivo studies, we measured the d4TTP concentrations among patients receiving d4T treatment, but the samples from patients taking ZDV did not provide d4TTP concentrations above the LOQ. Furthermore, in vitro samples did not produce any signal for d4TTP, despite the detection of substantial ZDVTP concentrations in CEMSS cells. Thus, contrary to the previous report, we found no evidence for the in vivo or in vitro transformation of ZDVTP to d4TTP in HIV-infected subjects or CEMSS cells.