The diverse secretory and absorptive functions of the intestinal epithelium are conducted by a mixed population of absorptive cells and mucus-producing goblet cells as the major cell types. In order to approach the main characteristics in an in vitro model, a coculture system of absorptive Caco-2 cells and mucus-secreting HT29-MTX cells was developed and the permeability of a range of different drugs was tested. Variable goblet cell frequency can be achieved, preserving a significant barrier to drug transport and maintaining the differentiated features of both cell types. Absorption rates for actively transported drugs are rather underestimated in the cell culture model when compared to in vivo data. However, a good correlation with fraction absorbed in humans was attained separating the range of passively transported drugs into two groups of well-absorbable compounds with Peff > or = 10 x 10(-6) cm/s and drugs that are absorbed 40-70% with Peff = 0.1-1 x 10(-5) cm/s. A permeability of Peff < 0.1 x 10(-5) cm/s is suggested for low absorbable drugs.

The FDA has published recommendations for sponsors who wish to request a waiver of in vivo bioavailability (BA) or bioequivalence (BE) studies for immediate release (IR) solid oral dosage forms based on the Biopharmaceutics Classification System (BCS). Biowaivers can be requested for IR formulations in which the active ingredient is shown to be a BCS class I drug: that is, a drug showing high permeability and high solubility over a pH range of 1-7.5. For permeability determinations, a variety of experimental methods can be used, such as the rat in situ single pass perfusion or Caco-2 cell culture models, once the suitability of the particular method is established. Following the recommended procedure for assessing the suitability of permeability determinations, we determined the permeability of 20 test drugs using the in situ single pass perfusion model in rats. The test compounds were coperfused through jejunal intestinal segments with an internal permeability reference standard (metoprolol) over a 90 min time period. Sample analysis was performed by HPLC, and the ratio of the effective permeability, Peff (cm/s), of test compound to that of metoprolol was determined. To address the question of test drug permeabilities that approach that of the internal standard, we propose that a statistical analysis such as the "0.8-1.25 rule" used for in vivo or in vitro bioequivalence studies provide guidance for permeability classification using the in situ single pass perfusion model. We developed a method using the 90% confidence interval of the permeability ratio of the test to internal reference standard in order to differentiate between high and low permeability compounds. This analysis allowed for the proper permeability classification of all of the test compounds and suggests a robust means for assessing drug permeability classification.

Gemcitabine, a clinically effective nucleoside anticancer agent, is a polar drug with low membrane permeability and is administered intravenously. Further, extensive degradation of gemcitabine by cytidine deaminase to an inactive metabolite in the liver affects its activity adversely. Thus, strategies that provide both enhanced transport and high metabolic bioevasion would potentially lead to oral alternatives that may be clinically useful. The objective of this study was to evaluate whether amino acid ester prodrugs of gemcitabine would (a) facilitate transport across intestinal membranes or across cells that express hPEPT1 and (b) provide resistance to deamination by cytidine deaminase. 3'-Monoester, 5'-monoester, and 3',5'-diester prodrugs of gemcitabine utilizing aliphatic (L-valine, D-valine, and L-isoleucine) and aromatic (L-phenylalanine and D-phenylalanine) amino acids as promoieties were synthesized and evaluated for their affinity and direct hPEPT1-mediated transport in HeLa/hPEPT1 cells. All prodrugs exhibited enhanced affinity (IC(50): 0.14-0.16 mM) for the transporter. However, only the 5'-L-valyl and 5'-L-isoleucyl monoester prodrugs exhibited (a) increased uptake (11.25- and 5.64-fold, respectively) in HeLa/hPEPT1 cells compared to HeLa cells and (b) chemical stability in buffers, that were comparable to valacyclovir, a commercially marketed oral amino acid ester prodrug. The widely disparate enzymatic bioconversion profiles of the 5'-L-valyl and 5'-L-isoleucyl prodrugs in Caco-2 cell homogenates along with their significant resistance to deamination by cytidine deaminase suggest that the disposition of gemcitabine following oral administration would be controlled by the rate of bioconversion following transport across the intestinal epithelial membrane. The combined results also suggest that it may be possible to modulate these characteristics by the choice of the amino acid promoiety.

Dipeptide monoester prodrugs of floxuridine were synthesized, and their chemical stability in buffers, resistance to glycosidic bond metabolism, affinity for PEPT1, enzymatic activation and permeability in cancer cells were determined and compared to those of mono amino acid monoester floxuridine prodrugs. Prodrugs containing glycyl moieties were the least stable in pH 7.4 buffer (t1/2 < 100 min). The activation of all floxuridine prodrugs was 2- to 30-fold faster in cell homogenates than their hydrolysis in buffer, suggesting enzymatic action. The enzymatic activation of dipeptide monoester prodrugs containing aromatic promoieties in cell homogenates was 5- to 20-fold slower than that of other dipeptide and most mono amino acid monoester prodrugs (t1/2 ∼ 40 to 100 min). All prodrugs exhibited enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase compared to parent floxuridine. In general, the 5′-O-dipeptide monoester floxuridine prodrugs exhibited higher affinity for PEPT1 than the corresponding 5′-O-mono amino acid ester prodrugs. The permeability of dipeptide monoester prodrugs across Caco-2 and Capan-2 monolayers was 2- to 4-fold higher than the corresponding mono amino acid ester prodrug. Cell proliferation assays in AsPC-1 and Capan-2 pancreatic ductal cell lines indicated that the dipeptide monoester prodrugs were equally as potent as mono amino acid prodrugs. The transport and enzymatic profiles of 5′-l-phenylalanyl-l-tyrosyl-floxuridine, 5′-l-phenylalanyl-l-glycyl-floxuridine, and 5′-l-isoleucyl-l-glycyl-floxuridine suggest their potential for increased oral uptake, delayed enzymatic bioconversion and enhanced resistance to metabolism to 5-fluorouracil, as well as enhanced uptake and cytotoxic activity in cancer cells, attributes that would facilitate prolonged systemic circulation for enhanced therapeutic action.