Intracellular Accumulation of Human Immunodeficiency Virus Protease Inhibitors

Khoo, Saye; Hoggard, Patrick G.; Williams, Ian H.; Meaden, E. R.; Newton, Peggy; Wilkins, E.G.L.; Smith, Alan M.; Tjia, John; Lloyd, John William; Jones, Kevin F.; Beeching, Nicholas J.; Carey, Peter; Peters, Barry; Back, David

doi:10.1128/aac.46.10.3228-3235.2002

Cited by 69 publications

(79 citation statements)

References 20 publications

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“…Saquinavir, nelfinavir, and ritonavir all showed significant nonspecific binding, which was corrected for throughout this study to provide true kinetic parameters. Nelfinavir has previously been shown to exhibit a higher degree of intracellular accumulation compared with other PIs (Jones et al, 2001;Khoo et al, 2002). Significantly higher metabolic rates (corresponding to lower K m values) were observed when the kinetics of saquinavir, nelfinavir, and ritonavir in rat liver microsomes were compared with suspended rat hepatocytes, ranging from 6-fold lower for ritonavir and saquinavir to 30-fold lower for nelfinavir.…”

Section: Hepatocellular Uptake and Drug Metabolic Clearancementioning

confidence: 88%

Rate-Limiting Steps in Hepatic Drug Clearance: Comparison of Hepatocellular Uptake and Metabolism with Microsomal Metabolism of Saquinavir, Nelfinavir, and Ritonavir

Parker

Houston²

2008

Drug Metab Dispos

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ABSTRACT:The intrinsic metabolic clearance of saquinavir, nelfinavir, and ritonavir was determined over a range of concentrations (0.02-20 M) in both rat liver microsomes and fresh isolated rat hepatocytes in suspension. Clearance values were found to be concentration dependent for both systems, and at low concentrations, microsomal clearance was much greater (7-14-fold) than in hepatocytes. Kinetic parameters showed substantially lower microsomal K m values (5-42 nM) compared with suspended rat hepatocytes (34-270 nM) but similar scaled V max values (2-26 nmol/min/g liver). In the absence of metabolism (achieved by pretreating hepatocytes with a mechanism-based inhibitor of cytochrome P450), saquinavir, nelfinavir, and ritonavir were actively and rapidly taken up into hepatocytes (cell/medium concentration ratios of 306-3352), and intracellular unbound drug concentrations between 5-and 12-fold higher than extracellular unbound concentrations were achieved. Comparison of the rate of uptake into hepatocytes with the rate of metabolism in hepatocytes and microsomes indicates that the former is the rate-limiting step at low concentrations. The rate of metabolism saturates at lower concentrations (100-400-fold) than the rate of uptake; hence, at the high concentrations metabolic rate-limited clearance occurs. In conclusion, the clearance of saquinavir, nelfinavir, and ritonavir is extremely rapid, and it is proposed that in the case of hepatocytes and by inference in vivo, the rate of uptake limits the metabolic clearance of these three drugs.

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Section: Hepatocellular Uptake and Drug Metabolic Clearancementioning

confidence: 88%

Rate-Limiting Steps in Hepatic Drug Clearance: Comparison of Hepatocellular Uptake and Metabolism with Microsomal Metabolism of Saquinavir, Nelfinavir, and Ritonavir

Parker

Houston²

2008

Drug Metab Dispos

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“…2), with a loss of nearly 80% within 20 min. However as demonstrated previously for protease inhibitors (13), the use of ice-cold PBS and centrifugation at 4°C resulted in the retention of Ͼ80% of the intracellular drug at 60 min. Drug efflux from cells was therefore critically time and, more importantly, temperature dependent.…”

Section: Resultsmentioning

confidence: 99%

Sensitive Enzyme Immunoassay for Measuring Plasma and Intracellular Nevirapine Levels in Human Immunodeficiency Virus-Infected Patients

Azoulay

Nevers

Créminon

et al. 2004

Antimicrob Agents Chemother

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We have developed an enzyme immunoassay to measure nevirapine (NVP) in plasma and peripheral blood mononuclear cells. Anti-NVP polyclonal antibodies were raised in rabbits by using a synthetic NVP derivative coupled to keyhole limpet hemocyanin as the immunogen, and the enzyme tracer was prepared by chemically coupling the NVP derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates with a 100-pg ml ؊1 limit of detection and thus ϳ100 times more sensitive than previously published techniques. The plasma assay was performed directly without extraction (in this case, a 500-pg ml ؊1 limit of detection was observed) on a minimum of 30 l of plasma. This assay shows good precision and efficiency, since recovery from human plasma and cell extracts spiked with NVP ranged between 87 and 104%, with coefficients of variation of <10%. A pharmacokinetic analysis of plasma NVP was performed for seven patients infected with human immunodeficiency virus (HIV), and it gave results similar to published findings. Intracellular concentrations of NVP were measured in cultured human T-lymphoblastoid cells and peripheral blood mononuclear cells from HIV-infected patients. The results indicated a very low intracellular/extracellular concentration ratio (0.134), thus demonstrating the absence of intracellular drug accumulation. This is the first intracellular assay of a nonnucleoside reverse-transcriptase inhibitor, and this method could be useful in monitoring plasma and intracellular NVP levels in HIV-infected patients.Nevirapine (Viramune) (NVP) is a nonnucleoside reversetranscriptase inhibitor indicated for the treatment of human immunodeficiency virus (HIV) type 1 infection. It represents an attractive option for patients who prefer a protease-sparing regimen, because it can be taken twice daily (200 mg b.i.d.) and ingested without food restrictions. The drug binds to viral reverse transcriptase and blocks polymerase activity by disrupting the catalytic site (16). Consequently, nevirapine must enter cells to inhibit viral replication, and it is important to consider the intracellular drug concentration in peripheral blood mononuclear cells (PBMC) and other compartments, as the distribution of antiviral drugs from the plasma into cells and tissues is dependent on many complex factors, including affinities for cells versus plasma components or drug transporters (9, 19). As a consequence, the intracellular levels may be very different from those recorded in plasma. Knowledge of the intracellular distribution may help in understanding the mechanisms that are involved in the evolution of drug resistance and the development of sanctuary sites.Several high-performance liquid chromatographic (HPLC) assays combined with UV detection (6, 10, 22) or tandem mass spectrometry (14,24) for the quantitative determination of NVP in plasma have been described. However, these methods are characterized by a relatively high limit of quantification (10 ng ml Ϫ...

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“…Plasma was separated from 5 ml of whole blood (centrifugation, 700 ϫ g; 10 min), and peripheral blood mononuclear cells (PBMCs) were isolated from the remaining 20 ml by density gradient centrifugation (700 ϫ g, 25 min, 4°C) at each sampling time, as described previously (12,21,28). PBMCs were washed in cold phosphate-buffered saline and centrifuged (700 ϫ g, 6 min, 4°C).…”

Section: Methodsmentioning

confidence: 99%

“…HIV Ther., abstr. 3.14, 2001), and some have described intracellular SQV/r pharmacokinetics following twice-daily regimens (28,21) or a saquinavir soft-gel formulation (G. Peytavin, R. Landman, C. Lamotte, F. Mentre, J. Gerbe, E. Dohin, F. Boue, G. Spiridon, M. A. Valantin, C. Michelet, E. Bouvet, and P. Yeni, 2nd Int. Workshop Clin.…”

mentioning

confidence: 99%

Intracellular and Plasma Pharmacokinetics of Saquinavir-Ritonavir, Administered at 1,600/100 Milligrams Once Daily in Human Immunodeficiency Virus-Infected Patients

Ford

Boffito

Wildfire

et al. 2004

Antimicrob Agents Chemother

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Ritonavir-boosted saquinavir (SQV/r) is currently licensed as a twice-daily regimen. Reducing the pill burden with once-daily dosing may improve adherence. Intracellular concentrations of drugs must be related to the clinical efficacy of protease inhibitors. The aims of the study were to determine the cellular and plasma saquinavir and ritonavir concentrations, to determine the half-lives (t 1/2 s) of the drugs in each compartment, and to examine relationships between drug accumulation and lymphocyte subset P glycoprotein (P-gp) expression. Venous blood samples from 12 human immunodeficiency virus-infected patients receiving a hard-gel formulation of SQV/r (1,600/100 mg once daily) were collected at 2, 6, 12, and 24 h after dosing. Peripheral blood mononuclear cells were separated by density gradient centrifugation, and P-

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Intracellular Accumulation of Human Immunodeficiency Virus Protease Inhibitors

Cited by 69 publications

References 20 publications

Rate-Limiting Steps in Hepatic Drug Clearance: Comparison of Hepatocellular Uptake and Metabolism with Microsomal Metabolism of Saquinavir, Nelfinavir, and Ritonavir

Rate-Limiting Steps in Hepatic Drug Clearance: Comparison of Hepatocellular Uptake and Metabolism with Microsomal Metabolism of Saquinavir, Nelfinavir, and Ritonavir

Sensitive Enzyme Immunoassay for Measuring Plasma and Intracellular Nevirapine Levels in Human Immunodeficiency Virus-Infected Patients

Intracellular and Plasma Pharmacokinetics of Saquinavir-Ritonavir, Administered at 1,600/100 Milligrams Once Daily in Human Immunodeficiency Virus-Infected Patients

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