Modulation of P-glycoprotein function in human lymphocytes and Caco-2 cell monolayers by HIV-1 protease inhibitors.
OBJECTIVE
To determine the effect of the protease inhibitors ritonavir, nelfinavir and indinavir on the P-glycoprotein (P-gp)-mediated transport of saquinavir in Caco-2 cell monolayers. To study the modulation of P-gp function in human lymphocytes by saquinavir, ritonavir, nelfinavir and indinavir.


METHODS
We examined the effect of the protease inhibitors on P-gp function in human lymphocytes by using Rhodamine 123 (Rh 123; a fluorescent substrate of P-gp) by flow cytometry. Efflux of Rh 123 correlates with P-gp function and inhibition of P-gp results in dye retention. Verapamil, a P-gp modulator and inhibitor of active transport at 4 degrees C was used as a positive control. The transport of [14C]saquinavir (1 microM) across Caco-2 cell monolayers was investigated, alone and in the presence of verapamil and ketoconazole (500 microM) and the protease inhibitors at 100 microM. Caco-2 cells are an in vitro model of the intestinal epithelium that is widely used for the study of P-gp function. The transport of saquinavir was determined in both the apical to basolateral (AP-BL) and basolateral to apical (BL-AP) directions.


RESULTS
Saquinavir and ritonavir (10 microM) markedly inhibited Rh 123 efflux with an increase in fluorescence intensity similar to that obtained with verapamil. A small but statistically significant increase in fluorescence intensity was observed with nelfinavir; however indinavir did not modulate Rh 123 efflux. In Caco-2 cells the apparent permeability coefficient for BL-AP efflux of saquinavir exceeded that for AP-BL efflux by a factor of 26: this is indicative of an active efflux pump. Known P-gp modulators caused a decrease in BL-AP efflux and an increase in AP-BL transport. The protease inhibitors displayed some P-gp modulation with ritonavir having the most potent effect.


CONCLUSIONS
We have demonstrated that saquinavir is a substrate for P-gp and that ritonavir, nelfinavir and indinavir modulate P-gp function in both human lymphocytes and Caco-2 cells.
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Department of Pharmacology and Therapeutics, University of Liverpool, UK.
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D006801:Humans; D019469:Indinavir / Q000494:pharmacology; D008214:Lymphocytes / Q000378:metabolism*; D019888:Nelfinavir / Q000494:pharmacology; D020112:Rhodamine 123; D019438:Ritonavir / Q000494:pharmacology; D019258:Saquinavir / Q000493:pharmacokinetics*; D013696:Temperature; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D017320:HIV Protease Inhibitors; D020112:Rhodamine 123; D019469:Indinavir; D014700:Verapamil; D019888:Nelfinavir; D019258:Saquinavir; D019438:Ritonavir