Effects of cytochrome P450 3A (CYP3A) and the drug transporters P-glycoprotein (MDR1/ABCB1) and MRP2 (ABCC2) on the pharmacokinetics of lopinavir.

BACKGROUND AND PURPOSE: Lopinavir is extensively metabolized by cytochrome P450 3A (CYP3A) and is considered to be a substrate for the drug transporters ABCB1 (P-glycoprotein) and ABCC2 (MRP2). Here, we have assessed the individual and combined effects of CYP3A, ABCB1 and ABCC2 on the pharmacokinetics of lopinavir and the relative importance of intestinal and hepatic metabolism. We also evaluated whether ritonavir increases lopinavir oral bioavailability by inhibition of CYP3A, ABCB1 and/or ABCC2. EXPERIMENTAL APPROACH: Lopinavir transport was measured in Madin-Darby canine kidney cells expressing ABCB1 or ABCC2. Oral lopinavir kinetics (+/- ritonavir) was studied in mice with genetic deletions of Cyp3a, Abcb1a/b and/or Abcc2, or in transgenic mice expressing human CYP3A4 exclusively in the liver and/or intestine. KEY RESULTS: Lopinavir was transported by ABCB1 but not by ABCC2 in vitro. Lopinavir area under the plasma concentration - time curve (AUC)(oral) was increased in Abcb1a/b(-/-) mice (approximately ninefold vs. wild-type) but not in Abcc2(-/-) mice. Increased lopinavir AUC(oral) (>2000-fold) was observed in cytochrome P450 3A knockout (Cyp3a(-/-)) mice compared with wild-type mice. No difference in AUC(oral) between Cyp3a(-/-) and Cyp3a/Abcb1a/b/Abcc2(-/-) mice was observed. CYP3A4 activity in intestine or liver, separately, reduced lopinavir AUC(oral) (>100-fold), compared with Cyp3a(-/-) mice. Ritonavir markedly increased lopinavir AUC(oral) in all CYP3A-containing mouse strains. CONCLUSIONS AND IMPLICATIONS: CYP3A was the major determinant of lopinavir pharmacokinetics, far more than Abcb1a/b. Both intestinal and hepatic CYP3A activity contributed importantly to low oral bioavailability of lopinavir. Ritonavir increased lopinavir bioavailability primarily by inhibiting CYP3A. Effects of Abcb1a/b were only detectable in the presence of CYP3A, suggesting saturation of Abcb1a/b in the absence of CYP3A activity.

Localization of activin subunits in early murine development determined by subunit-specific antibodies.

Activins are thought to be involved in early differentiation processes during amphibian and avian development. Little is known, however, about the role of activins in early developmental stages of higher vertebrates. In order to study activin protein expression in early murine development we have prepared polyclonal antibodies against synthetic peptides corresponding to C-terminal sequences of the murine beta A and beta B activin subunits. Both antibodies recognized specifically activin A and activin B in different immunological assays including ELISA and immunoblotting. Using these antibodies in immunofluorescence experiments a good correlation between mRNA and protein expression of the beta subunits was observed in different in vitro model systems for early murine development. In addition, beta A and beta B subunit protein localization appeared to be different in 3.5- and 4.5-day mouse blastocysts. The results presented here are consistent with a role for different activin forms in the regulation of distinct processes in early murine development.

Modulation of activin expression by type beta transforming growth factors.

Activins are potentially important regulators of early developmental processes in vertebrates. Although the different forms of activin appear to be differentially expressed during early amphibian, avian, and murine development, little is known about the factors that regulate their expression. In this study we report the qualitative effects of several growth and differentiation factors on the expression of inhibin subunits in three differentiated cell lines derived from P19 embryonal carcinoma cells. These cell lines include mesodermal (MES-1), neuroepithelial (EPI-7), and visceral endoderm-like (END-2) cell types, expressing both inhibin beta A and beta B subunit mRNAs. We have shown for the first time that this expression is modulated by transforming growth factor (TGF)beta 1 and TGF beta 2 but not significantly by other growth factors such as leukemia inhibitory factor or members of the fibroblast growth factor family (aFGF, bFGF, or kFGF). beta A mRNA expression is increased while beta B expression is simultaneously decreased by TGF beta. Furthermore, TGF beta increased the amount of bioactive activin secreted by MES-1 and END-2 cells. Inhibin alpha subunit mRNA expression is not affected by TGF beta. These results point to a possible role of type beta transforming growth factors as regulators of activin expression in embryonal cells.

Heat-induced unresponsiveness of heat shock gene expression is regulated at the transcriptional level.

The induction kinetics of the heat shock proteins hsp68, hsp70 and hsp84 were studied. Studies on hsp mRNA levels and protein synthetic rates, with or without the presence of actinomycin D, showed that regulation took place at the transcriptional level. Hsp mRNA induction was followed by a transient state of unresponsiveness. At the time point where the induced hsp mRNAs were decreasing again, hsp68, hsp70 and hsp84 mRNA could not be induced by a second, identical, heat shock. Hsp68 mRNA could be induced again 12-16 h after the first heat shock. Apparently, this state really seems to be a state of reduced sensitivity, since a higher heat dose could partially overcome this unresponsiveness.

Differentiation of aggregated murine P19 embryonal carcinoma cells is induced by a novel visceral endoderm-specific FGF-like factor and inhibited by activin A.

Aggregation of P19 embryonal carcinoma cells in the presence of a factor, secreted by the visceral endoderm-like cell line END-2, induces differentiation to cell types including visceral endoderm, mesoderm-derived muscle tissue and neurons. This factor is different from activin A, type beta transforming growth factors (TGF beta) and fibroblast growth factors (FGF) although its acid- and heat-lability and its stability in the presence of reducing agents resemble the properties of the FGFs. The END-2 factor is completely inhibited in its action by activin A. This inhibitory effect of activin A is not specific for the END-2 factor as retinoic acid (RA)-induced differentiation of aggregated P19 EC cells into neurons (10(-8) M RA) or mesoderm-derived muscle tissue (10(-9) M RA) is also completely inhibited by activin A. The results of this study suggest that the END-2 activity and activin A are intimately involved in the induction and regulation, respectively, of early differentiation processes in vertebrate embryogenesis.