PubMed Health⌕ Search

Biomedical subjects

David J Back

Publications and source records attributed to David J Back.

At least 37 records · Page 2Linked to original sources
PubMed2003

Protein binding in antiretroviral therapies.

There is marked variability in the extent to which the three classes of antiretroviral (ARV) drugs bind to plasma proteins (<5 to >99%). Protease inhibitors (PIs), with the exception of indinavir, are more than 90% protein bound, mainly to alpha1-acid glycoprotein (AAG). Efavirenz, a nonnucleoside reverse transcriptase inhibitor (NNRTI), is more than 99% bound, mainly to albumin. Nucleoside reverse transcriptase inhibitors (NRTIs) are not highly protein bound. The pharmacological activity of ARV drugs is dependent on unbound drug entering cells that harbor the human immunodeficiency virus (HIV). There has been concern that changes in protein binding could impact on antiviral activity and management. However, for PIs and NNRTIs, and for many drugs given orally, altered plasma binding would not be expected to influence the average exposure to unbound (active) drug after chronic oral dosing. Nevertheless, there will be a change in the relationship between total and unbound concentrations that will be important if, as part of therapeutic drug monitoring, the total rather than the unbound drug is measured. Measuring drug concentrations that are needed to inhibit different HIV strains (wild type and drug resistant) in vitro could also cause confusion because most methods employ bovine serum in the assay medium, and unbound concentrations are not directly measured. Estimating unbound drug concentrations in human plasma and in incubation media can be highly method dependent and thus may affect the calculated IC50 (the concentration of drug that results in 50% inhibition of viral replication). Because inhibitory quotients (IQs = C(trough)/IC50) are becoming part of pharmacokinetic/pharmacodynamic (PK/PD) analyses of clinical trial data, the strengths and weaknesses of the methods used for the determination of unbound drug concentration in plasma and in vitro systems--ultracentrifugation, ultrafiltration, and equilibrium dialysis--need to be understood. Consensus on standard procedures must be reached. In June 2002, a panel of experts assembled by the Forum for Collaborative HIV Research met in Washington, DC, to review the basic principles of protein binding of ARV drugs, and to discuss the impact that changes in plasma protein binding may have on the PKs and activity of ARV drugs as well as on therapeutic drug monitoring. The purpose of the meeting was to discuss the following topics: (1) basic principles of protein binding and how changes in binding can impact on drug PKs and drug exposure in vivo, (2) variability in plasma protein binding among patients taking ARV drugs, (3) the impact of HIV infection and concomitant diseases on the extent of plasma protein binding, (4) the likelihood of clinically relevant drug interactions at the level of plasma protein binding, (5) the evidence that measuring unbound concentrations of ARV drugs in the plasma of patients gives more meaningful information than total drug concentration and, therefore, should be considered in routine therapeutic drug monitoring of ARV agents, (6) optimal method(s) for measuring the unbound concentration of drugs in vitro (for IC50 determination) and in vivo, and (7) future studies that need to be considered to fully understand the importance of plasma protein binding in therapeutic drug monitoring. This report summarizes the topics discussed at this meeting. It guides the reader through the discussions that allowed the panel to formulate a series of statements regarding the significance of plasma protein binding of ARV drugs when studied in vitro and in vivo. The roundtable participants also identified research priorities that are important for understanding the sources of inter- and intraindividual variability in protein binding in patients. These include obtaining data on unbound as well as on total concentrations in PK studies; looking at variants of AAG and whether they differ in binding affinity; and emphasizing the importance of developing a standard procedure for drug susceptibility assays used to determine IC50 values.

Animals↗
PubMed2003

Enzymatic assay for measurement of intracellular DXG triphosphate concentrations in peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected patients.

DXG ([2R-cis]-2-amino-1,9-dihydro-9-[2-[hydroxymethyl]-1,3-dioxolan-4-yl]-6H-purin-6-one) and its prodrug DAPD ([2R-cis]-4-[2,6-diamino-9H-purin-9-yl]-1,3-dioxolane-2-methanol; amdoxovir) are novel 2',3'-dideoxynucleosides (ddNs) displaying activity against human immunodeficiency virus type 1 (HIV-1). In this paper, we describe the development of an enzymatic assay for determining the intracellular active metabolite of DXG and DAPD, DXG triphosphate (DXGTP), in peripheral blood mononuclear cells (PBMCs) from HIV-infected patients. The assay involves inhibition of HIV reverse transcriptase (RT), which normally incorporates radiolabeled deoxynucleoside triphosphates (dNTPs) into a synthetic template primer. DXGTP (0.6 pmol) inhibited control product formation with or without a preincubation step. Inhibition was greatest when the template primer was most diluted. DAPDTP inhibited control product formation only at very high levels (50 pmol) and when a preincubation procedure was used. However, reduced template primer stability in assays using preincubation steps, coupled with potential interference by DAPDTP, led to the current assay method for DXGTP being performed without preincubation. Standard DXGTP inhibition curves were constructed. The presence of PBMC extracts or endogenous dGTP did not interfere with the DXGTP assay. Intracellular DXGTP and dGTP concentrations were determined in PBMCs from HIV-infected patients receiving oral DAPD (500 mg b.i.d.). Peak concentrations of DXGTP were obtained 8 h after dosing and were measurable through 48 h postdose. Levels of endogenous dGTP were also determined over 48 h. No direct relationship was observed between concentrations of DXGTP and dGTP. Quantification of DXGTP concentrations in PBMCs from patients receiving a clinically relevant dose of DAPD is possible with this enzymatic assay.

Dioxolanes↗
PubMed2003

Intracellular disposition and metabolic effects of zidovudine, stavudine and four protease inhibitors in cultured adipocytes.

OBJECTIVE: The pathogenesis of lipodystrophy caused by the HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs) is unclear. We have investigated the disposition of these drugs in adipocytes and the consequent effect on adipocyte metabolism and viability. DESIGN: Laboratory study utilizing two murine cell lines, 3T3-L1 and 3T3-F442A. METHODS: Intracellular NRTI phosphate and PI concentrations were determined by HPLC and HPLC-MS/MS, respectively. The cytotoxicity of the drugs was examined on the different adipogenic stages together with their effects on glucose uptake plus or minus insulin, and on glycerol and triglyceride levels. RESULTS: There was rapid intracellular accumulation and phosphorylation of [3H]-zidovudine and -stavudine to their phosphate metabolites in adipocytes. The NRTIs were not cytotoxic, did not affect preadipocyte protein synthesis and did not inhibit adipogenesis or induce lipolysis. PIs accumulated in adipocytes (nelfinavir>saquinavir>ritonavir>indinavir). All PIs, except indinavir, were cytotoxic and inhibited adipogenesis, increased lipolysis and impaired preadipocyte protein synthesis. PIs inhibited glucose uptake in the rank order: indinavir>saquinavir>ritonavir>nelfinavir. CONCLUSION: These data demonstrate that PIs may play a role in the insulin resistance observed in lipodystrophy by affecting glucose uptake, adipogenesis and lipolysis. NRTIs alone do not seem to have any effect on adipocyte metabolism despite undergoing phosphorylation to their triphosphorylated anabolites, although their effects in combination with PIs in perturbing adipocyte metabolism warrants further investigation.

Adipocytes↗
PubMed2002

Time-dependent changes in HIV nucleoside analogue phosphorylation and the effect of hydroxyurea.

BACKGROUND: Nucleoside analogues are activated to their triphosphates, which compete with endogenous deoxynucleoside triphosphate (dNTP) pools to inhibit HIV reverse transcriptase. Hydroxyurea has been administered with nucleoside analogues to modulate intracellular dNTP pools and thus the ratio of drug triphosphate:endogenous triphosphate. OBJECTIVES: To examine changes in drug activation over time and investigate the effects of hydroxyurea on intracellular phosphorylation of antiretroviral nucleoside analogues. PATIENTS: A total of 229 HIV-infected individuals receiving abacavir, lamivudine and zidovudine were randomly assigned to receive or not nevirapine and hydroxyurea. Twenty-four patients were recruited to an observational substudy measuring intracellular drug triphosphate and dNTP concentrations at 0, 2, 6, 12, 24 and 48 weeks. METHODS: Drugs were extracted from isolated peripheral blood mononuclear cells before analysis of endogenous dNTP and drug triphosphates by primer extension assays. RESULTS: Twenty-two out of 24 patients were followed to completion of the substudy. Hydroxyurea had no demonstrable effect on endogenous dNTP or drug triphosphate levels at any timepoint. However, the ratio of zidovudine triphosphate to endogenous deoxythymidine triphosphate was significantly increased with hydroxyurea. A significant decrease in lamivudine triphosphate (3TCTP) and the 3TCTP:endogenous deoxycytidine triphosphate ratio was seen over 48 weeks. In five patients who failed therapy in the first 24 weeks, significantly reduced 3TCTP was seen. CONCLUSION: Hydroxyurea does not affect measurable pools of endogenous nucleosides in vivo. Decreased lamivudine phosphorylation over time may provide a novel pharmacological explanation for the mechanism of resistance to this drug.

Adult↗
PubMed2002

Clinical use of lopinavir/ritonavir in a salvage therapy setting: pharmacokinetics and pharmacodynamics.

Lopinavir/ritonavir was administered to 35 HIV-infected patients after therapeutic failure with other protease inhibitors. The pharmacokinetics (trough concentrations) and baseline viral genotype were determined, together with the immunovirological outcome. The 22 responders had significantly higher mean lopinavir concentrations and lower baseline numbers of mutations. On multivariate analysis, a lopinavir concentration of 5.7 microg/ml or greater was an independent predictor of viral suppression over a 9-month follow-up period.

CD4 Lymphocyte Count↗
PubMed2002

Absence of opioid withdrawal symptoms in patients receiving methadone and the protease inhibitor lopinavir-ritonavir.

A study was designed to determine the interactions, both clinical and pharmacokinetic, between methadone and lopinavir-ritonavir. Results demonstrated a 36% reduction in the methadone area under the plasma concentration-time curve after the introduction of lopinavir-ritonavir, with no coincident symptoms of opioid withdrawal and no requirement for methadone dose adjustment.

Adult↗
PubMed2002

Response to antiretroviral treatment in HIV-1-infected individuals with allelic variants of the multidrug resistance transporter 1: a pharmacogenetics study.

BACKGROUND: HIV-1-infected patients vary considerably by their response to antiretroviral treatment, drug concentrations in plasma, toxic events, and rate of immune recovery. This variability could have a genetic basis. We did a pharmacogenetics study to analyse the association between response to antiretroviral treatment and allelic variants of several genes. METHODS: In 123 patients, we did PCR analyses of the gene for the multidrug-resistance transporter (MDR1), which codes for P-glycoprotein, of genes coding for isoenzymes of cytochrome P450, CYP3A4, CYP3A5, CYP2D6, and CYP2C19, and of the gene for the chemokine receptor CCR5. We measured concentrations in plasma of the antiretroviral agents efavirenz and nelfinavir by high-performance liquid-chromatography, and measured levels of P-glycoprotein expression, CD4-cell count, and HIV-1 viraemia. FINDINGS: Median drug concentrations in patients with the MDR1 3435 TT, CT, and CC genotypes were at the 30th, 50th, and 75th percentiles, respectively (p=0.0001). In patients with CYP2D6 extensive-metaboliser or poor-metaboliser alleles, median drug concentrations were at percentiles 45 and 62.5, respectively (p=0.04). Patients with the MDR1 TT genotype 6 months after starting treatment had a greater rise in CD4-cell count (257 cells/microL) than patients with the CT (165 cells/microL) and CC (121 cells/microL) genotype (p=0.0048), and the best recovery of naïve CD4-cells. INTERPRETATION: The polymorphism MDR1 3435 C/T predicts immune recovery after initiation of antiretroviral treatment. This finding suggests that P-glycoprotein has an important role in admittance of antiretroviral drugs to restricted compartments in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

The unbound percentage of saquinavir and indinavir remains constant throughout the dosing interval in HIV positive subjects.

AIMS: To measure the unbound plasma concentrations of saquinavir (SQV) and indinavir (IDV) and to relate them to the total plasma concentrations in order to establish the unbound percentage of protease inhibitors in vivo during a full dosage interval profile. METHODS: HIV-infected subjects (n = 35; median CD4 cell count = 340 x 10(6) cells l-1, range: 120-825; viral load < 50 copies ml-1 in 22/35) treated with SQV or IDV containing regimens were studied. Plasma drug samples were collected at 0, 2, 4, 8 and 12 h postdose for the twice daily regimens and 0, 1, 2, 4 and 8 h for the three times daily regimens. Ultra-filtration was used to separate unbound IDV and SQV in plasma and their respective concentrations were measured by a fully validated method using high performance liquid chromatography-mass spectometry (h.p.l.c.-MS/MS). RESULTS: Based on the ratio AUCunbound/AUCtotal, the median unbound percentage (95% CI for differences) of SQV and IDV from all the samples studied was 1.19% (0.99, 1.58%) and 36.3% (35.1, 44.2%), respectively. No significant difference was seen in the percentage binding of SQV between patients receiving SQV alone (median = 1.49%) or with ritonavir (median = 1.09%; P = 0.141; 95% CI for difference between medians = -0.145, 0.937) over the pharmacokinetic profile. Similarly, no significant difference was seen in the percentage binding of IDV in patients receiving IDV alone (median 35.2%) or with ritonavir (median = 41.3%; P = 0.069; 95% CI for difference between medians = -0.09, 15.4). The unbound concentrations of SQV (P < 0.0001; 95% CI for r(2) = 0.634, 0.815) and IDV (P < 0.0001; 95% CI for r(2) = 0.830, 0.925) remained constant as a proportion of total concentration over the full dosing profile. CONCLUSIONS: These in vivo data confirm previously published in vitro measurements of SQV and IDV protein binding. The unbound percentage of both protease inhibitors remained constant over the dosing interval.

Adult↗
PubMed2002

Pharmacokinetics of saquinavir co-administered with cimetidine.

The present study evaluated the effect of cimetidine, a histamine H(2) receptor antagonist able to inhibit cytochrome P450 metabolism, on the steady-state pharmacokinetics of saquinavir soft gel. Twelve healthy volunteers (eight males and four females) participated in an open-label, double-phase pharmacokinetic study. Volunteers took saquinavir soft gel 1200 mg three times a day for 13 days and then saquinavir soft gel 1200 mg twice a day with cimetidine 400 mg twice a day from day 14 to 26. The pharmacokinetics of saquinavir on days 13 and 26 were compared. All 12 volunteers completed the study. The association of cimetidine with saquinavir soft gel 1200 mg twice a day resulted in a significant increase in saquinavir AUC(0-24) (120%; P = 0.023) and C(max) (179%; P = 0.019), whereas C(trough) did not differ significantly (32% increase; P = 0.272). Increased exposure to saquinavir was observed in healthy volunteers after co-administration with cimetidine. The most significant increase involved C(max). Further pharmacokinetic studies in HIV-infected subjects are warranted to confirm the boosting effect of cimetidine and to investigate any impact that the increase in saquinavir C(max) may have on intracellular accumulation of the drug.

Adult↗
PubMed2002

Intracellular pharmacology of nucleoside analogues and protease inhibitors: role of transporter molecules.

Antiretroviral agents target HIV replication within infected cells. It is therefore important to focus on the pharmacology of these drugs at their site of action rather than just in plasma. Activation of nucleoside analogues to a triphosphate is essential for antiretroviral activity. Following activation, by intracellular kinases, drug triphosphates compete with endogenous triphosphates for HIV reverse transcriptase. Methodologies to measure triphosphates in peripheral blood mononuclear cells from HIV patients have been described. This has allowed investigation of once-daily dosing regimens, drug interactions, modulation of intracellular activation and the bypassing of initial phosphorylation steps. Drug accumulation within a cell is a balance between influx and efflux. There is a growing body of evidence indicating that transport proteins are vitally important in regulating intracellular concentrations of antiretroviral drugs. Allelic variants, inhibition (or induction) are all potentially critical determinants of active drug present in the cell. It is hoped that understanding the intracellular pharmacology will improve long-term therapy and reduce the likelihood of cellular resistance in therapeutic failure.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Simultaneous determination of rifampicin and efavirenz in plasma.

Complex drug interactions involving antiretroviral agents and drugs for the management of opportunistic infections demand the monitoring of plasma drug concentrations to prevent treatment failure. The high occurrence of tuberculosis in HIV-infected subjects makes the management of HIV treatment complex. Rifampicin, a potent inducer of the cytochrome P 450 metabolic pathway, is a very active antituberculosis drug that accelerates the metabolism of protease inhibitors. Regimens containing efavirenz, a non-nucleoside reverse transcription inhibitor, could be an alternative, but efavirenz plasma concentrations may be altered after the coadministration of rifampicin. Efavirenz is also a cytochrome P 450 inducer and may alter rifampicin plasma levels. Due to the increasing need to monitor plasma concentrations in HIV patients with tuberculosis, a high-performance liquid chromatographic (HPLC) method has been developed to measure rifampicin and efavirenz at the same time in a small amount of sample. This HPLC method is highly sensitive and precise, suitable for pharmacokinetic studies or routine clinical monitoring of rifampicin and efavirenz simultaneously in HIV patients with tuberculosis.

Adult↗