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

2000 ◽  
Vol 44 (11) ◽  
pp. 3097-3100 ◽  
Author(s):  
Jose F. Rodriguez ◽  
Jorge L. Rodriguez ◽  
Jorge Santana ◽  
Hermes García ◽  
Osvaldo Rosario
Keyword(s):  
Human Immunodeficiency Virus ◽  
Highly Active Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Solid Phase ◽  
Parent Compound ◽  
Hiv Positive ◽  
Peripheral Blood Mononuclear ◽  
Limit Of Quantitation ◽  
Immunodeficiency Virus

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.

scholarly journals Quantitation of Intracellular Triphosphate of Emtricitabine in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Patients

1999 ◽  
Vol 43 (9) ◽  
pp. 2245-2250 ◽  
Author(s):  
Albert Darque ◽  
Gilles Valette ◽  
Frank Rousseau ◽  
Laurene H. Wang ◽  
Jean-Pierre Sommadossi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Anion Exchange ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Solid Phase ◽  
Peripheral Blood Mononuclear ◽  
Limit Of Quantitation ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells

ABSTRACT An analytical methodology combining solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) was developed to quantitate the intracellular active 5′-triphosphate (TP) of β-l-2′,3′-dideoxy-5-fluoro-3′-thiacytidine (emtricitabine) (FTC) in human peripheral blood mononuclear cells (PBMCs). The FTC nucleotides, including 5′-mono-, di-, and triphosphates, were successively resolved on an anion-exchange SPE cartridge by applying a gradient of potassium chloride. The FTC-TP was subsequently digested to release the parent nucleoside that was finally analyzed by HPLC with UV detection (HPLC-UV). Validation of the methodology was performed by using PBMCs from healthy donors exposed to an isotopic solution of [3H]FTC with known specific activity, leading to the formation of intracellular FTC-TP that was quantitated by an anion-exchange HPLC method with radioactive detection. These levels of FTC-TP served as reference values and were used to validate the data obtained by HPLC-UV. The assay had a limit of quantitation of 4.0 pmol of FTC-TP (amount on column from approximately 107 cells). Intra-assay precision (coefficient of variation percentage of repeated measurement) and accuracy (percentage deviation of the nominal reference value), estimated by using quality control samples at 16.2, 60.7, and 121.5 pmol, ranged from 1.3 to 3.3% and −1.0 to 4.8%, respectively. Interassay precision and accuracy varied from 3.0 to 10.2% and from 2.5 to 6.7%, respectively. This methodology was successfully applied to the determination of FTC-TP in PBMCs of patients infected with human immunodeficiency virus after oral administration of various dosing regimens of FTC monotherapy.

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

2004 ◽  
Vol 48 (2) ◽  
pp. 589-595 ◽  
Author(s):  
Cecile Le Saint ◽  
Raphael Terreux ◽  
Daniele Duval ◽  
Jacques Durant ◽  
Helene Ettesse ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Highly Active Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Exchange Chromatography ◽  
Therapeutic Monitoring ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus

ABSTRACT 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.

scholarly journals Intracellular Nucleotides of (−)-2′,3′-Deoxy-3′-Thiacytidine in Peripheral Blood Mononuclear Cells of a Patient Infected with Human Immunodeficiency Virus

1998 ◽  
Vol 42 (11) ◽  
pp. 2989-2995 ◽  
Author(s):  
Caroline Solas ◽  
Yu-Feng Li ◽  
Meng-Yu Xie ◽  
Jean-Pierre Sommadossi ◽  
Xiao-Jian Zhou
Keyword(s):  
Human Immunodeficiency Virus ◽  
Anion Exchange ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Solid Phase ◽  
Peripheral Blood Mononuclear ◽  
Limit Of Quantitation ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells

ABSTRACT An analytical methodology was developed to quantitate the intracellular nucleotides including mono-, di-, and triphosphates and the diphosphocholine derivative of (−)-2′,3′-deoxy-3′-thiacytidine (3TC) in human peripheral blood mononuclear cells (PBMCs). The procedure includes the resolution of 3TC nucleotides by solid-phase extraction (SPE) on an anion-exchange cartridge, with subsequent enzyme digestion of the resulting phosphates to the parent drug that is ultimately quantitated by high-performance liquid chromatography with UV detection (HPLC-UV). Validation was performed with PBMCs from healthy donors exposed to [3H]3TC, leading to the formation of intracellular nucleotides that were quantitated by anion-exchange HPLC with radioactive detection (HPLC-RA). These nucleotide levels served as reference values and were used for cross-validation with data obtained by HPLC-UV. An excellent correlation was established between the results obtained by HPLC-RA and those obtained by HPLC-UV, with a slope of the regression lines close to unity and intercepts near nullity as well as a correlation coefficient close to unity for all 3TC phosphates. The assay was characterized by a limit of quantitation below 1 ng (amount on column) with a precision (percentage of coefficient of variation of repeated measurement) ranging from 0.8 to 18.1% and an accuracy (deviation of the amount determined by HPLC-UV from the nominal reference value) varying from −14.8 to 19.4%. This methodology was successfully applied to determine the quantity of 3TC nucleotides in PBMCs of a patient infected with human immunodeficiency virus after oral administration of 3TC and stavudine.

scholarly journals Response to Superantigen Stimulation in Peripheral Blood Mononuclear Cells from Children Perinatally Infected with Human Immunodeficiency Virus and Receiving Highly Active Antiretroviral Therapy

2004 ◽  
Vol 11 (5) ◽  
pp. 957-962 ◽  
Author(s):  
Thomas W. McCloskey ◽  
Viraga Haridas ◽  
Lucy Pontrelli ◽  
Savita Pahwa
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Antiretroviral Therapy ◽  
Highly Active Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Highly Active ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

ABSTRACT Our understanding of the pathogenesis of perinatal human immunodeficiency virus (HIV) infection is still evolving. We sought to characterize the response to the bacterial superantigen Staphylococcus enterotoxin B (SEB) of lymphocytes from HIV-infected children receiving treatment with highly active antiretroviral therapy (HAART). Using the flow cytometric methodology, we quantified apoptosis, proliferation, cytokine production, and activation antigen upregulation in CD4 and CD8 T lymphocytes following in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with SEB. The levels of proliferation, CD4 interleukin-2 (IL-2) production, CD8 gamma interferon (IFN-γ) production, and upregulation of CD69 expression by cells from HIV-infected children were indistinguishable from those by cells from controls. However, stimulation with SEB dramatically decreased the ratio of resting apoptotic cells to cycling apoptotic cells in the controls but not in the patients. In addition, unstimulated spontaneous apoptosis of CD4 T cells remained greater in the patients than in the controls. The percentages of IL-2-positive CD8 T cells and IFN-γ-positive CD4 T cells following SEB stimulation were significantly lower in the patients than in the controls. Our multiparameter approach was able to demonstrate differences in lymphocyte superantigen responsiveness in HIV-infected children receiving HAART in comparison to that in uninfected controls, notably, an apoptotic versus a proliferative response to stimulation.

scholarly journals Development of a New Cartridge Radioimmunoassay for Determination of Intracellular Levels of Lamivudine Triphosphate in the Peripheral Blood Mononuclear Cells of Human Immunodeficiency Virus-Infected Patients

1998 ◽  
Vol 42 (10) ◽  
pp. 2656-2660 ◽  
Author(s):  
Brian L. Robbins ◽  
Thu T. Tran ◽  
Frank H. Pinkerton ◽  
Fatima Akeb ◽  
Roger Guedj ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Nucleoside Analogs ◽  
Cross Reactivity ◽  
Active Metabolites ◽  
Rapid Separation ◽  
Peripheral Blood Mononuclear ◽  
Limit Of Quantitation ◽  
Immunodeficiency Virus

ABSTRACT A new sensitive method for the measurement of lamivudine triphosphate (3TC-TP), the active intracellular metabolite of lamivudine in human cells in vivo, has been established. The procedure involves rapid separation of 3TC-TP by using Sep-Pak cartridges, dephosphorylation to 3TC by using acid phosphatase, and measurement by radioimmunoassay using a newly developed anti-3TC serum. The radioimmunoassay had errors of less than 21% and a cross-reactivity of less than 0.016% with a wide variety of other nucleoside analogs. The limit of quantitation of the assay for intracellular 3TC-TP was 0.195 ng/ml (0.212 pmol/106 cells), and a cell sample of only 4 million cells was ample for the assay. This procedure, combined with our previously developed method for measuring zidovudine (ZDV) metabolite levels, proved capable of measuring 3TC-TP, ZDV monophosphate (ZDV-MP) and ZDV triphosphate (ZDV-TP) in human immunodeficiency virus (HIV)-infected subjects treated with combination 3TC and ZDV therapy. In seven subjects, intracellular 3TC-TP levels ranged from 2.21 to 7.29 pmol/106 cells, while intracellular ZDV-MP and ZDV-TP levels ranged from <0.01 to 1.76 and 0.01 to 0.07 pmol/106 cells, respectively. Concentrations of 3TC in plasma determined in these subjects ranged from 0.34 to 9.40 μM, which was about fivefold higher than ZDV levels in plasma of 0.04 to 1.4 μM. This is the first study to determine the intracellular levels of the active metabolites in HIV-infected subjects treated with this combination. These methods should prove very useful for in vivo pharmacodynamic studies of combination therapy.

scholarly journals Modulation of Human Immunodeficiency Virus (HIV)-Specific Immune Response by Using Efavirenz, Nelfinavir, and Stavudine in a Rescue Therapy Regimen for HIV-Infected, Drug-Experienced Patients

2002 ◽  
Vol 9 (5) ◽  
pp. 1114-1118 ◽  
Author(s):  
Daria Trabattoni ◽  
Sergio Lo Caputo ◽  
Mara Biasin ◽  
Elena Seminari ◽  
Massimo Di Pietro ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Highly Active Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Rescue Therapy ◽  
Interleukin 2 ◽  
Transcriptase Inhibitor ◽  
Peripheral Blood Mononuclear ◽  
Therapy Regimen ◽  
Immunodeficiency Virus ◽  
Ifn Γ

ABSTRACT Analysis of the virologic and immunomodulatory effects of an association of efavirenz (EFV), nelfinavir (NFV), and stavudine (d4T) was performed in 18 human immunodeficiency virus (HIV)-infected and highly active antiretroviral therapy (HAART)-experienced patients who failed multiple therapeutic protocols. Patients (<500 CD4+ cells/μl; >10,000 HIV copies/ml) were nonnucleoside reverse transcriptase inhibitor (NNRTI)-naive and were treated for 10 months with EFV (600 mg/day) in association with NFV (750 mg three times daily) and d4T (30 or 40 mg twice daily). Measurement of HIV peptide- and mitogen-stimulated production of interleukin-2 (IL-2), gamma interferon (IFN-γ), IL-4, and IL-10 as well as quantitation of mRNA for the same cytokines in unstimulated peripheral blood mononuclear cells were performed at baseline and 2 weeks (t1), 2 months (t2), and 10 months (t3) into therapy. The results showed that HIV-specific (but not mitogen-stimulated) IL-2 and IFN-γ production was augmented and IL-10 production was reduced in patients who received EFV, NFV, and d4T. Therapy was also associated with a reduction in HIV RNA in plasma and an increase in CD4+ cell count. These changes occurred in the first year of therapy (t2 and t3) and were confirmed by quantitation of cytokine-specific mRNA. Therapy with EFV, NFV, and d4T increases HIV-specific type 1 cytokine production as well as CD4 counts and reduces plasma viremia. This therapeutic regimen may be considered for use in cases of advanced HIV infection.

scholarly journals Effect of Shipment, Storage, Anticoagulant, and Cell Separation on Lymphocyte Proliferation Assays for Human Immunodeficiency Virus-Infected Patients

1998 ◽  
Vol 5 (6) ◽  
pp. 804-807 ◽  
Author(s):  
Adriana Weinberg ◽  
Rebecca A. Betensky ◽  
Li Zhang ◽  
Graham Ray
Keyword(s):  
Human Immunodeficiency Virus ◽  
Whole Blood ◽  
Highly Active Antiretroviral Therapy ◽  
Cell Separation ◽  
Lymphocyte Proliferation ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Varicella Zoster ◽  
Immunodeficiency Virus ◽  
Proliferation Assays

ABSTRACT Lymphocyte proliferation assays (LPA), which can provide important information regarding the immune reconstitution of human immunodeficiency virus (HIV)-infected patients on highly active antiretroviral therapy, frequently involve shipment of specimens to central laboratories. In this study, we examine the effect of stimulant, anticoagulant, cell separation, storage, and transportation on LPA results. LPA responses of whole blood and separated peripheral blood mononuclear cells (PBMC) to different stimulants (cytomegalovirus, varicella-zoster virus, candida and tetanus toxoid antigens, and phytohemagglutinin) were measured using fresh specimens shipped overnight and frozen specimens collected in heparin, acid citrate dextrose (ACD), and citrate cell preparation tubes (CPT) from 12 HIV-infected patients and uninfected controls. Odds ratios for positive LPA responses were significantly higher in separated PBMC than in whole blood from ACD- and heparin-anticoagulated samples obtained from HIV-infected patients and from ACD-anticoagulated samples from uninfected controls. On separated PBMC, positive responses were significantly more frequent in fresh samples compared with overnight transportation for all antigens and compared with cryopreservation for the candida and tetanus antigens. In addition, viral antigen LPA responses were better preserved in frozen PBMC compared with specimens shipped overnight. CPT tubes yielded significantly more positive LPA results for all antigens, irrespective of the HIV patient status compared with ACD, but only for the candida and tetanus antigens and only in HIV-negative controls compared with heparin. Although HIV-infected patients had a significantly lower number of positive antigen-driven LPA responses compared with uninfected controls, most of the specimen processing variables had similar effects on HIV-positive and -negative samples. We conclude that LPA should be performed on site, whenever feasible, by using separated PBMC from fresh blood samples collected in either heparin or ACD. However, if on-site testing is not available, optimal transportation conditions should be established for specific antigens.

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