Bilateral diffuse pulmonary infiltration in a heart transplant recipient.

Pulmonary complications are not infrequent after heart transplantation. Kaposi sarcoma is a vascular tumor that can involve the skin as well as visceral organs. We describe a case of visceral and cutaneous Kaposi sarcoma that presented with diffuse bilateral pulmonary infiltration and breathlessness 6 month after heart transplantation. Following modulation of the immunosuppressive regimen and addition of chemotherapy, the patient had an excellent response and has had an uneventful 1-year follow-up.

Interplay between CYP3A-mediated metabolism and polarized efflux of terfenadine and its metabolites in intestinal epithelial Caco-2 (TC7) cell monolayers.

PURPOSE: To further characterize cytochrome P450 (CYP) and P-glycoprotein (Pgp) expression in monolayers of the Caco-2 cell clone TC7, a cell culture model of the human intestinal epithelium. To study the interplay between CYP3A and Pgp as barriers to intestinal drug absorption in TC7 cells using terfenadine and its metabolites as substrates. METHODS: mRNA expression of eight CYPs and Pgp was investigated in TC7 and parental Caco-2 (Caco-2p) cell monolayers using RT-PCR. The CYP3A kinetics was determined in microsomes from both cell lines. The transport, metabolism and efflux of terfenadine and its metabolites were investigated in TC7 monolayers. RESULTS: Both TC7 and Caco-2p cells expressed mRNA for Pgp and several important CYPs. However, mRNA for CYP3A4 was detectable anly from TC7 cells. The relative affinity of CYP3A for terfenadine metabolism in the two cell lines was comparable, but the maximum reaction rate in the TC7 cells was 8-fold higher. The rate of transport of terfenadine and its metabolites hydroxy-terfenadine (HO-T) and azacyclonol across TC7 monolayers was 7.1-, 3.5- and 2.1-fold higher, respectively, in the basolateral to apical direction than it was in the apical to basolateral (AP-BL) direction. Inhibition studies indicated that the efflux was mediated by Pgp. Ketoconazole increased the AP-BL transport terfenadine dramatically by inhibiting both terfenadine metabolism and Pgp efflux. CONCLUSIONS: Cell culture models such as TC7 provide qualitative information on drug interactions involving intestinal CYP3A and Pgp.

The role of an alpha-amino group on H+ -dependent transepithelial transport of cephalosporins in Caco-2 cells.

The role of an alpha-amino group on interaction with the intestinal and renal peptide carriers (PEPT 1 and PEPT 2, respectively) has been the subject of much investigation. Studies have differed in their conclusions about the role of an alpha-amino group on carrier-mediated absorption. Most studies have used brush-border membrane vesicles or perfused intestinal segments. These techniques enable the determination of membrane uptake and luminal disappearance, respectively, but not transepithelial transport. Transepithelial transport should be more predictive of absorption because it includes basolateral efflux, which could be the rate-limiting process in drug absorption. The objective of this study was to evaluate the influence of an alpha-amino group on PEPT 1-mediated transepithelial transport in Caco-2 cells. The apical-to-basolateral permeability coefficients of cephalosporins with or without a free alpha-amino group were determined in the presence and absence of a pH gradient. Permeability coefficients obtained under these conditions were used to calculate a permeability ratio (i.e. P(app) (pH 6.0)/P(app) (pH 7.4)), which should indicate whether PEPT 1 is involved in transport. For cephalosporins with an alpha-amino group (cephalexin, cefaclor, cefadroxil, cephradine, cephaloglycin) the permeability ratios ranged between 1.77 and 2.77. In contrast, the permeability ratios for cephalosporins without an alpha-amino group were 1 (approx.; range = 0.74-1.26). These data suggest that the presence of an alpha-amino group on cephalosporins increases their PEPT 1-mediated transepithelial transport in Caco-2 monolayers.

Comparison of CYP3A activities in a subclone of Caco-2 cells (TC7) and human intestine.

PURPOSE: To compare the activity of the CYP3A enzyme expressed by TC7, a cell culture model of the intestinal epithelial cell, to the activity of human intestinal CYP3A4, using terfenadine as a substrate. METHODS: The metabolism of terfenadine was investigated in intact cells and microsomal preparations from TC7, human intestine, and liver. The effect of two CYP3A inhibitors, ketoconazole and troleandomycin (TAO), on the metabolism of terfenadine was also examined. RESULTS: Only hydroxy-terfenadine was detected in TC7 microsomal incubations. In contrast, azacyclonol and hydroxy-terfenadine were detected in human intestinal and hepatic microsomal incubations. The Km values for hydroxy-terfenadine formation in TC7 cells, intestine and liver microsomes were 1.91, 2.5, and 1.8, microM respectively. The corresponding Vmax values were 2.11, 61.0, and 370 pmol/min/mg protein. Km values for azacyclonol in intestinal and hepatic samples were 1.44 and 0.82 microM and the corresponding Vmax values were 14 and 60 pmol/min/mg protein. The formation of hydroxy-terfenadine was inhibited by ketoconazole and TAO in human intestine and TC7 cell microsomes. The Km and Vmax values for terfenadine metabolism in intact TC7 cells were similar to those from TC7 cell microsomes. CONCLUSIONS: Our results indicate that TC7 cells are a potentially useful alternative model for studies of CYP3A mediated drug metabolism. The CYP3A expressed by TC7 cells is not CYP3A4, but probably CYP3A5, making this cell line suitable for studies of colonic drug transport and metabolism.

Orphenadrine and methimazole inhibit multiple cytochrome P450 enzymes in human liver microsomes.

The specificities of orphenadrine and methimazole on eight human liver P450 enzyme activities were evaluated by studying the extent of inhibition at different concentrations in two protocols: competitive inhibition and preincubation. In the competitive inhibition protocol, orphenadrine decreased CYP2B6 marker activity up to 45-57% in human liver microsomes and up to 80-97% in cell microsomes containing cDNA-expressed CYP2B6. Orphenadrine strongly decreased CYP2D6 marker activity by 80-90%. Orphenadrine also partially decreased the CYP1A2, CYP2A6, CYP3A4, and CYP2C19 marker activities. In the preincubation protocol, orphenadrine decreased the CYP2B6 activity in cDNA-expressed cell microsomes to completion. In human liver microsomes, orphenadrine strongly decreased the marker activities of CYP2B6, CYP2D6, as well as CYP2C9; and partially decreased the marker activities of CYP1A2, CYP2A6, CYP3A4, and CYP2C19. In the competitive inhibition protocol, methimazole had no effect on the marker activities of CYP2E1 and CYP2A6; slightly decreased CYP2D6 marker activity; partially decreased the marker activities of CYP2C19, CYP2C9, and CYP2B6; and dramatically decreased CYP3A4 marker activity. Methimazole decreased CYP1A2 marker activity at lower concentrations, but not at the highest concentration studied (1 mM). In the preincubation protocol, methimazole was shown to be a potent and nonspecific inhibitor of all the enzyme activities. Marker activities of CYP2C9, CYP2C19, and CYP3A4 were completely inhibited at relatively low concentrations. This study indicates orphenadrine cannot be used as a selective inhibitor of CYP2B6 in human liver microsomes and that methimazole is not a selective inhibitor of the flavin-containing monooxygenase in human liver microsomes.

Polarized transport of docetaxel and vinblastine mediated by P-glycoprotein in human intestinal epithelial cell monolayers.

The expression of the multidrug transporter P-glycoprotein has been studied in two human intestinal epithelial cell lines. No functional expression of P-glycoprotein was found in the differentiated HT29-18-C1 cell line. The expression of P-glycoprotein in the Caco-2 cell line was very high, as judged by immunoblotting and by active efflux of vinblastine. The polarized transport of vinblastine in the basolateral to apical direction was temperature and energy dependent, and was reduced by P-glycoprotein inhibitors such as verapamil, chlorpromazine and reserpine. This adds further evidence that the polarized transport of vinblastine across Caco-2 monolayers is mediated by P-glycoprotein. The anticancer drug docetaxel (Taxotere) was transported in a polarized manner: basolateral to apical permeability was 20-fold higher than in the reverse direction. This polarized transport was inhibited by verapamil, chlorpromazine and reserpine, thus demonstrating that docetaxel is a substrate of P-glycoprotein. The implications of these results for the pharmacokinetics and toxicity of taxoids are discussed.

In-vitro characterization of blood-brain barrier permeability to delta sleep-inducing peptide.

The diffusion of delta sleep-inducing peptide (DSIP) across the blood-brain barrier (BBB) has been investigated with an in-vitro model comprised of primary cultures of brain microvessel endothelial cell (BMEC) monolayers. The BMEC monolayers were mounted in a side-by-side diffusion apparatus and the transendothelial flux of DSIP analysed by HPLC with UV detection at 280 nm. The transendothelial flux of the peptide was linear with time and increasing concentrations of DSIP (non-saturable), but was not altered by reduced temperature. The apparent permeability coefficient for DSIP penetration of BMEC monolayers was in a range similar to water-soluble substances (e.g. fluorescein, fluorescein isothiocyanate dextrans) that penetrate the blood-brain barrier to a limited degree based on molecular weight. DSIP flux across the BMEC monolayers was also found to be bidirectional, insensitive to metabolic inhibitors, and not altered by high concentrations of tryptophan. Little degradation (apparent t1/2 about 10 h) of DSIP to major metabolites, tryptophan (trp) and des-trp DSIP, occurred over the time of the diffusion experiments. The results of these studies support and confirm observations in-vivo indicating that intact DSIP crosses the BBB by simple transmembrane diffusion.