Current methodologies used for evaluation of intestinal permeability and absorption.

This review article will focus on the various techniques that are currently employed by drug discovery scientists in evaluating permeability/absorption of drug candidates during the drug candidate selection process. Various preclinical methodologies are available; each having advantages and disadvantages, but it is the judicious use of these techniques that can help identify drug candidates that will be well absorbed in humans. It is well recognized that the human intestinal permeability cannot be accurately predicted based on a single methodology (in vitro: tissue/cell culture, in situ, or in vivo).

International Transporter Consortium commentary on clinically important transporter polymorphisms.

This Commentary focuses on genetic polymorphisms in membrane transporters. We present two polymorphisms for which there is a compelling body of literature supporting their clinical relevance: OATP1B1 (c.521T>C, p.V174A, rs4149056) and BCRP (c.421C>A, p.Q141K, rs2231142). The clinical evidence demonstrating their role in variation in pharmacokinetics and pharmacodynamics is described along with their allele frequencies in ethnic populations. Recommendations for incorporating studies of transporter polymorphisms in drug development are provided, along with the regulatory implications.

Carrier-mediated intestinal absorption of valacyclovir, the L-valyl ester prodrug of acyclovir: 1. Interactions with peptides, organic anions and organic cations in rats.

The mechanism of intestinal transport of valacyclovir (VACV), the L-valyl ester prodrug of acyclovir, was investigated in rats using an in situ intestinal perfusion technique. VACV demonstrates an oral bioavailability that is three to five time greater than acyclovir, concentration dependent, and saturable in humans. Homogenate and perfused buffer stability results demonstrated that VACV was increasingly unstable with increasing pH. VACV was converted to ACV in a concentration dependent manner during a single pass through the intestinal segment. Perfusions were performed at 37 degrees C, pH 6.5, and under iso-osmotic conditions (290 +/- 10 mOsm L-1). Intestinal outlet concentrations were corrected for VACV that was converted to ACV during the perfusion. The effective dimensionless intestinal permeability (P*e) of VACV was concentration dependent, saturable (intrinsic Km = 1.2 +/- 0.7 mM), and significantly reduced (p < 0.05) in the presence of peptide analogues (amoxicillin, ampicillin, cefadroxil, and cephradine), by the organic anion, p-amino hippuric acid and by the organic cation quinine. VACV transport was not inhibited by classical nucleoside competitive substrates or inhibitors or by valine. These results suggest that H(+)-oligopeptide, H(+)-organic cation, and organic anion transporters are involved in the small intestinal uptake of VACV. The permeability of VACV in the colon was very low, indicating that VACV is predominantly absorbed from the small intestine. VACV P*e was not altered in the presence of glucose-induced convective fluid flow, suggesting that carrier-mediated, transcellular uptake is the predominant absorption pathway of VACV in rat small intestine. Based on these results, the oral bioavailability of VACV appears to be significantly influenced by the preabsorptive conversion of VACV to the poorly absorbed ACV, by the involvement of multiple transporters in VACV small-intestinal uptake, and by the low permeability of VACV in the colon.

Interactions of a nonpeptidic drug, valacyclovir, with the human intestinal peptide transporter (hPEPT1) expressed in a mammalian cell line.

The results of previous work performed in our laboratory using an in situ perfusion technique in rats and rabbit apical brush border membrane vesicles have suggested that the intestinal uptake of valacyclovir (VACV) appears to be mediated by multiple membrane transporters. Using these techniques, it is difficult to characterize the transport kinetics of VACV with each individual transporter in the presence of multiple known or unknown transporters. The purpose of this study was to characterize the interaction of VACV and the human intestinal peptide transporter using Chinese hamster ovary (CHO) cells that overexpress the human intestinal peptide transporter (hPEPT1) gene. VACV uptake was significantly greater in CHO cells transfected with hPEPT1 than in cells transfected with only the vector, pcDNA3. The optimum pH for VACV uptake was determined to occur at pH 7.5. Proton cotransport was not observed in hPEPT1/CHO cells, consistent with previously observed results in tissues and Caco-2 cells. VACV uptake was concentration dependent and saturable with a Michaelis-Menten constant and maximum velocity of 1.64 +/- 0.06 mM and 23.34 +/- 0.36 nmol/mg protein/5 min, respectively. A very similar Km value was obtained in hPEPT1/CHO cells and in rat and rabbit tissues and Caco-2 cells, suggesting that hPEPT1 dominates the intestinal transport properties of VACV in vitro. VACV uptake was markedly inhibited by various dipeptides and beta-lactam antibiotics, and Ki values of 12.8 +/- 2.7 and 9.1 +/- 1.2 mM were obtained for Gly-Sar and cefadroxil at pH 7.5, respectively. The present results demonstrate that VACV is a substrate for the human intestinal peptide transporter in hPEPT1/CHO cells and that although transport is pH dependent, proton cotransport is not apparent. Also, the results demonstrate that the hPEPT1/CHO cell system has use in investigating the transport kinetics of drugs with the human intestinal peptide transporter hPEPT1; however, the extrapolation of these transport properties to the in vivo situation requires further investigation.

Direct evidence for peptide transporter (PepT1)-mediated uptake of a nonpeptide prodrug, valacyclovir.

Xenopus laevis oocytes were used as a gene expression system to characterize the carrier-mediated transport of valacyclovir (vacv), the L-valine ester prodrug of the acyclic nucleoside acyclovir (acv). A significant increase in the uptake of [3H]vacv by Xenopus laevis oocytes injected with human intestinal peptide transporter (hPepT1) cRNA compared to the uptake by water injected oocytes indicated that vacv was translocated by hPepT1. Vacv uptake was found to be concentration dependent, saturable (K(m) = 5.94 +/- 1.91 mM and Jmax = 1.68 +/- 0.25 nmoles/hr/oocyte), pH dependent, and inhibited by various known substrates of hPepT1 but not by acv, valine or pentaglycine. Vacv also inhibited the uptake of 14C-glycylsarcosine, a known substrate of hPepT1, in a concentration-dependent manner (Ki = 4.08 +/- 1.02 mM). These results demonstrate that human intestinal peptide transporter hPepT1 has broad specificity since it recognizes vacv as a substrate even though it lacks a typical peptide bond.