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The formulation developments and the in vivo assessment of Biopharmaceutical Classification System (BCS) class II drugs are challenging due to their low solubility and high permeability in the human gastrointestinal (GI) tract. Since the GI environment influences the drug dissolution of BCS class II drugs, the human GI characteristics should be incorporated into the in vitro dissolution system to predict bioperformance of BCS class II drugs. An absorptive compartment may be important in dissolution apparatus for BCS class II drugs, especially for bases (BCS IIb) because of high permeability, precipitation, and supersaturation. Thus, the in vitro dissolution system with an absorptive compartment may help predicting the in vivo phenomena of BCS class II drugs better than compendial dissolution apparatuses. In this study, an absorptive compartment (a biphasic device) was introduced to a gastrointestinal simulator. This addition was evaluated if this in vitro system could improve the prediction of in vivo dissolution for BCS class IIb drugs, ketoconazole and raloxifene, and subsequent absorption. The gastrointestinal simulator is a practical in vivo predictive tool and exhibited an improved in vivo prediction utilizing the biphasic format and thus a better tool for evaluating the bioperformance of BCS class IIb drugs than compendial apparatuses., (Copyright © 2018. Published by Elsevier Inc.)

Current interest in cocrystal development resides in the advantages that the cocrystal may have in solubility and dissolution compared with the parent drug. This work provides a mechanistic analysis and comparison of the dissolution behavior of carbamazepine (CBZ) and its 2 cocrystals, carbamazepine-saccharin (CBZ-SAC) and carbamazepine-salicylic acid (CBZ-SLC) under the influence of pH and micellar solubilization. A simple mathematical equation is derived based on the mass transport analyses to describe the dissolution advantage of cocrystals. The dissolution advantage is the ratio of the cocrystal flux to drug flux and is defined as the solubility advantage (cocrystal to drug solubility ratio) times the diffusivity advantage (cocrystal to drug diffusivity ratio). In this work, the effective diffusivity of CBZ in the presence of surfactant was determined to be different and less than those of the cocrystals. The higher effective diffusivity of drug from the dissolved cocrystals, the diffusivity advantage, can impart a dissolution advantage to cocrystals with lower solubility than the parent drug while still maintaining thermodynamic stability. Dissolution conditions where cocrystals can display both thermodynamic stability and a dissolution advantage can be obtained from the mass transport models, and this information is useful for both cocrystal selection and formulation development., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The purpose of this study was to assess the feasibility of a multicompartmental in vitro dissolution apparatus, gastrointestinal simulator (GIS), in assessing the drug dissolution of 2 commercially available oral dosage forms for itraconazole (ICZ). The GIS consists of 3 chambers, mimicking the upper gastrointestinal tract. In vitro dissolution of ICZ capsule or oral solution was evaluated in United States Pharmacopeia apparatus II and GIS. To investigate the suitability of fasted state simulated intestinal fluid (FaSSIF) to predict better in vivo, FaSSIF as well as phosphate buffer were used as dissolution media. Area under the dissolved drug amount-time curve (AUDC) was calculated for each dosage form in each apparatus, and the ratios of AUDCoral solution to AUDCcapsule were compared with human pharmacokinetic data. Based on this comparison, GIS with FaSSIF can adequately distinguish the pharmacokinetic profiles of 2 oral dosage forms for ICZ. Additionally, Caco-2 cell transepithelial transport study in combination with GIS revealed that improved drug dissolution by formulations resulted in enhanced permeation of ICZ through cell monolayer, suggesting the observed ICZ concentration in the GIS will directly reflect systemic exposure. These results indicate GIS would be a powerful tool to assess the formulations of ICZ as well as other Biopharmaceutics Classification System class II drug formulations., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The goal of this study was to improve the intestinal mucosal cell membrane permeability of the poorly absorbed guanidino analogue of a neuraminidase inhibitor, oseltamivir carboxylate (GOC) using a carrier-mediated strategy. Valyl amino acid prodrug of GOC with isopropyl-methylene-dioxy linker (GOC-ISP-Val) was evaluated as the potential substrate for intestinal oligopeptide transporter, hPEPT1 in Xenopus laevis oocytes heterologously expressing hPEPT1, and an intestinal mouse perfusion system. The diastereomers of GOC-ISP-Val were assessed for chemical and metabolic stability. Permeability of GOC-ISP-Val was determined in Caco-2 cells and mice. Diastereomer 2 was about 2 times more stable than diastereomer 1 in simulated intestinal fluid and rapidly hydrolyzed to the parent drug in cell homogenates. The prodrug had a 9 times-enhanced apparent permeability (P(app)) in Caco-2 cells compared with the parent drug. Both diastereomer exhibited high effective permeability (P(eff)) in mice, 6.32 ± 3.12 and 5.20 ± 2.81 × 10(-5) cm/s for diastereomer 1 and 2, respectively. GOC-ISP-Val was found to be a substrate of hPEPT1. Overall, this study indicates that the prodrug, GOC-ISP-Val, seems to be a promising oral anti-influenza agent that has sufficient stability at physiologically relevant pHs before absorption, significantly improved permeability via hPEPT1 and potentially rapid activation in the intestinal cells., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Bicarbonate is the main buffer in the small intestine and it is well known that buffer properties such as pKa can affect the dissolution rate of ionizable drugs. However, bicarbonate buffer is complicated to work with experimentally. Finding a suitable substitute for bicarbonate buffer may provide a way to perform more physiologically relevant dissolution tests. The dissolution of weak acid and weak base drugs was conducted in bicarbonate and phosphate buffer using rotating disk dissolution methodology. Experimental results were compared with the predicted results using the film model approach of (Mooney K, Mintun M, Himmelstein K, Stella V. 1981. J Pharm Sci 70(1):22-32) based on equilibrium assumptions as well as a model accounting for the slow hydration reaction, CO2 + H2 O → H2 CO3 . Assuming carbonic acid is irreversible in the dehydration direction: CO2 + H2 O ← H2 CO3 , the transport analysis can accurately predict rotating disk dissolution of weak acid and weak base drugs in bicarbonate buffer. The predictions show that matching the dissolution of weak acid and weak base drugs in phosphate and bicarbonate buffer is possible. The phosphate buffer concentration necessary to match physiologically relevant bicarbonate buffer [e.g., 10.5 mM (HCO3 (-) ), pH = 6.5] is typically in the range of 1-25 mM and is very dependent upon drug solubility and pKa ., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Development of an oral in vivo predictive dissolution medium for acid drugs with a pKa in the physiological range (e.g., Biopharmaceutics Classification System Class IIa) requires transport analysis of the complex in vivo CO2 /bicarbonate buffering system. In this report, we analyze this buffer system using hydrodynamically defined rotating disk dissolution. Transport analysis of drug flux was predicted using the film model approach of Mooney et al based on equilibrium assumptions as well as accounting for the slow hydration reaction, CO2 + H2 O → H2 CO3 . The accuracy of the models was compared with experimentally determined results using the rotating disk dissolution of ibuprofen, indomethacin, and ketoprofen. The equilibrium and slow hydration reaction rate models predict significantly different dissolution rates. The experimental results are more accurately predicted by accounting for the slow hydration reaction under a variety of pH and hydrodynamic conditions. Although the complex bicarbonate buffering system requires further consideration given its dynamic nature in vivo, a simplifying irreversible reaction (IRR) transport analysis accurately predicts in vitro rotating disk dissolution rates of several carboxylic acid drugs. This IRR transport model provides further insight into bicarbonate buffer and can be useful in developing more physiologically relevant buffer systems for dissolution testing., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

In vitro dissolution tests are performed for new formulations to evaluate in vivo performance, which is affected by the change of gastrointestinal (GI) physiology, in the GI tract. Thus, those environmental changes should be introduced to an in vitro dissolution test. Many studies have successfully shown the improvement of in vitro-in vivo correlations (IVIVC) by introducing those physiological changes into dissolution tests. The gastrointestinal simulator (GIS), a multicompartment in vitro dissolution apparatus, was developed to evaluate in vivo drug dissolution. A gastric-emptying rate along with transit rate are key factors to evaluate in vivo drug dissolution and, hence, drug absorption. Dissolution tests with the GIS were performed with Biopharmaceutical Classification System class I drugs at five different gastric-emptying rates in the fasted state. Computational models were used to determine in vivo gastric-emptying time for propranolol and metoprolol based on the GIS dissolution results. Those were compared with published clinical data to determine the gastric half-emptying time. In conclusion, the GIS is a practical tool to assess dissolution properties and can improve IVIVC., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The present monograph reviews data relevant to applying the biowaiver procedure for the approval of immediate-release multisource solid dosage forms containing codeine phosphate. Both biopharmaceutical and clinical data of codeine were assessed. Solubility studies revealed that codeine meets the "highly soluble" criteria according to World Health Organization (WHO), the European Medicines Agency (EMA), and the United States Food and Drug Administration (US FDA). Codeine's fraction of dose absorbed in humans was reported to be high (>90%) based on cumulative urinary excretion of drug and drug-related material following oral administration. The permeability of codeine was also assessed to be high in both Caco-2 monolayers and rat intestinal perfusion studies. The main risks associated with codeine, that is, toxicity (attributed to CYP2D6 polymorphism) and its abuse potential, are present irrespective of the dosage form, and do not need to be taken into account for bioequivalence (BE) considerations. Taken together, codeine is a class 1 drug with manageable risk and is a good candidate for waiver of in vivo BE studies., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

5-Fluoro-2'-deoxyuridine (floxuridine, 5-FdUrd) and 5-fluorouracil (5-FU) are widely used for the treatment of colorectal cancers. The mechanisms of action of 5-FdUrd and 5-FU, as well as the biochemical pathway responsible for their metabolism, are well understood. Identification of every metabolite and achieving mass balance by conventional UV absorption-based HPLC analysis are not feasible because the metabolites beyond 5-FU in the 5-FdUrd metabolic pathway are undetectable by UV light. We therefore established a mass spectrometry method, designed for fast and convenient analysis, for simultaneously measuring 5-FdUrd, 5-FU, and their metabolites. Linearity, precision and accuracy were validated in the concentration ranges studied for each compound. Hydrolysis studies of 5-FdUrd and amino acid mono ester prodrugs of 5-FdUrd in Capan-2 cell homogenates were carried out and the achievement of mass balance was established with this method (recovery of 5'-O-l-leucyl-FdUrd was 96.6-108.2% and that of 5-FdUrd was 79.4-117.4%). This simple LC-MS method achieves reliable quantitation and mass balance of 5-FdUrd, 5-FU, and their metabolites and can be effectively utilized for further kinetic studies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A quasi-equilibrium mass transport analysis has been developed to quantitatively explain the solubility-permeability interplay that exists when using cyclodextrins as pharmaceutical solubilizers. The model considers the effects of cyclodextrins on the membrane permeability (P(m)) as well as the unstirred water layer (UWL) permeability (P(aq)), to predict the overall effective permeability (P(eff)) dependence on cyclodextrin concentration (C(CD)). The analysis reveals that: (1) UWL permeability markedly increases with increasing C(CD) since the effective UWL thickness quickly decreases with increasing C(CD); (2) membrane permeability decreases with increasing C(CD), as a result of the decrease in the free fraction of drug; and (3) since P(aq) increases and P(m) decreases with increasing C(CD), the UWL is effectively eliminated and the overall P(eff) tends toward membrane control, that is, P(eff) approximately P(m) above a critical C(CD). Application of this transport model enabled excellent quantitative prediction of progesterone P(eff) as a function of HP beta CD concentrations in PAMPA assay, Caco-2 transepithelial studies, and in situ rat jejunal-perfusion model. This work demonstrates that when using cyclodextrins as pharmaceutical solubilizers, a trade-off exists between solubility increase and permeability decrease that must not be overlooked; the transport model presented here can aid in striking the appropriate solubility-permeability balance in order to achieve optimal overall absorption., ((c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association)

Cidofovir (HPMPC) is a broad-spectrum antiviral agent, currently used to treat AIDS-related human cytomegalovirus retinitis. Cidofovir has recognized therapeutic potential for orthopox virus infections, although its use is hampered by its inherent low oral bioavailability. Val-Ser-cyclic HPMPC (Val-Ser-cHPMPC) is a promising peptide prodrug which has previously been shown by us to improve the permeability and bioavailability of the parent compound in rodent models (Eriksson et al., 2008. Molecular Pharmaceutics 5, 598-609). Puromycin-sensitive aminopeptidase was partially purified from Caco-2 cell homogenates and identified as a prodrug activating enzyme for Val-Ser-cHPMPC. The prodrug activation process initially involves an enzymatic step where the l-Valine residue is removed by puromycin-sensitive aminopeptidase, a step that is bestatin-sensitive. Subsequent chemical hydrolysis results in the generation of cHPMPC. A recombinant puromycin-sensitive aminopeptidase was generated and its substrate specificity investigated. The k(cat) for Val-pNA was significantly lower than that for Ala-pNA, suggesting that some amino acids are preferred over others. Furthermore, the three-fold higher k(cat) for Val-Ser-cHPMPC as compared to Val-pNA suggests that the leaving group may play an important role in determining hydrolytic activity. In addition to its ability to hydrolyze a variety of substrates, these observations strongly suggest that puromycin-sensitive aminopeptidase is an important enzyme for activating Val-Ser-cHPMPC in vivo. Taken together, our data suggest that puromycin-sensitive aminopeptidase makes an attractive target for future prodrug design.

The di/tri-peptide transporter h-PEPT1 plays an important role in the oral absorption of di/tri-peptides and numerous drugs. Inflammatory conditions may influence intestinal xenobiotic transporter function; however, the effects of inflammation on h-PEPT1 have not been well described. This study was conducted to determine the effects of the inflammatory cytokine interferon-gamma (IFN-gamma) on h-PEPT1 mediated dipeptide absorption. Caco-2 monolayers were grown on permeable supports. The effective apical-to-basolateral permeability (P(eff)) of glycylsarcosine (Gly-Sar) was measured following incubation with IFN-gamma or control media. Additional experiments were conducted at 4 degrees C, and with escalating concentrations of Gly-Sar. h-PEPT1 expression was determined using semiquantitative RT-PCR. IFN-gamma 50 ng/ml increased Gly-Sar P(eff) 28.6% compared to controls (p=0.03). In experiments conducted at 4 degrees C, Gly-Sar P(eff) decreased 39.6% in IFN-gamma treated cells (p=0.003) and 28.4% in controls (p=0.006). In controls and IFN-gamma treated cells, concentration dependent transport was seen with escalating concentrations of Gly-Sar. Compared to controls, IFN-gamma 50 and 100 ng/ml increased h-PEPT1 mRNA expression by 14.2% and 11.5%, respectively (p=0.019). In summary, IFN-gamma increases h-PEPT1 expression and permeation of the dipeptide Gly-Sar in Caco-2 monolayers. These findings imply that intestinal absorption of peptides and peptidomimetic drugs may be increased in certain inflammatory conditions.

This work was to investigate the effects of particle size and paddle speed on the particle diffusional layer thickness h(app) in a USP dissolution apparatus II. After the determination of the powder dissolution rates of five size fractions of fenofibrate, including <20, 20-32, 32-45, 63-75, and 90-106 microm, the present work shows that the dependence of h(app) on particle size follows different functions in accordance with the paddle speed. At 50 rpm, the function of h(app) is best described by a linear plot of h{app} = 9.91sqrt d-23.31 (R(2) = 0.98) throughout the particle diameter, d, from 6.8 to 106 microm. In contrast, at 100 rpm a transitional particle radius, r, of 23.7 microm exists, under which linear relationship h(app) = 1.59r (R(2) = 0.98) occurs, but above which h(app) becomes a constant of 43.5 microm. Thus, h(app) changes not only with particle size, but also with the hydrodynamics under standard USP configurations, which has been overlooked in the past. Further, the effects of particle size and paddle speed on h(app) were combined using dimensionless analysis. Within certain fluid velocity/particle regime, linear correlation of h(app)/d with the square-root of Reynolds number (d\varpi/upsilon){1/2}, that is, h{app}/d = 1.5207 - 9.25 x 10{- 4} (d\varpi/n){1/2} (R(2) = 0.9875), was observed.

We have studied gene expression profiles of intestinal transporters in model animals and humans. Total RNA was isolated from duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mice, rats, and humans were about 60% of 22,690 sequences, 40% of 8739, and 47% of 12,559, respectively. A total of 86 genes involving transporters expressed in mice, 50 genes in rats, and 61 genes in humans were detected. Mice exhibited abundant mRNA expressions for peptide transporter HPT1, amino acid transporters CSNU3, CT1 and ASC1, nucleoside transporter CNT2, organic cation transporter SFXN1, organic anion transporter NBC3, glucose transporter SGLT1, and fatty acid transporters FABP1 and FABP2. Rats showed high expression profiles of peptide transporter PEPT1, amino acid transporters CSNU1 and 4F2HC, nucleoside transporter CNT2, organic cation transporter OCT5, organic anion transporter SDCT1, glucose transporter GLUT2 and GLUT5, and folate carrier FOLT. In humans, the highly expressed genes were peptide transporter HPT1, amino acid transporters LAT3, 4F2HC and PROT, nucleoside transporter CNT2, organic cation transporter OCTN2, organic anion transporters NADC1, NBC1 and SBC2, glucose transporters SGLT1 and GLUT5, multidrug resistance-associated protein RHO12, fatty acid transporters FABP1 and FABP2, and phosphate carrier PHC. Overall these data reveal diverse transcriptomic profiles for intestinal transporters among these species. Therefore, this transcriptional data may lead to more effective use of the laboratory animals as a model for oral drug development.

DX-9065a, a newly synthesized anticoagulant that selectively inhibits factor Xa, is a zwitterion and has characteristics of high water solubility and low lipophilicity. We predicted the fraction absorbed (Fa) of DX-9065a to be approximately 15-35% in humans, based on the boundary layer theory using the intestinal perfusion method in rats. However, human oral bioavailability was 2-3% in clinical trials, and the result of actual human bioavailability was lower than that of the predicted Fa. Thus, in this report, the reason for low oral bioavailability of DX-9065a was examined by in vitro and in vivo experiments. The factors affecting oral bioavailability of DX-9065a were not the hepatic first-pass effect, degradation of the drug in intestinal fluid, nor the interaction of the drug with the intestinal mucin. Furthermore, no effect of P-gp efflux was observed. Oral absorption of the drug in rats with bile duct ligation was significantly higher than that in normal rats with bioavailability of 17 and 3%, respectively. It was confirmed that bile acids inhibited DX-9065a absorption because DX-9065a interacted with bile acids to form insoluble complexes. The results suggest that the complex formation of DX-9065a with bile acids in the intestinal tract is an important factor affecting absorption of DX-9065a.

Small intestine epithelial cell lines (TR-SIE), which are established from the small intestine of transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), were used to characterize the mRNA expression of small intestine transporters. TR-SIE cells had a polygonal morphology and expressed cytokeratin protein and villin mRNA. Although the large T-antigen was strongly expressed at 33 degrees C, this was reduced at 37 and 39 degrees C. Concomitantly, the cell growth was arrested at 37 and 39 degrees C compared with that at 33 degrees C, suggesting that TR-SIE cells are conditionally immortalized cell lines. RT-PCR analysis revealed that TR-SIE cells expressed ABCB1 (mdr1a and mdr1b), ABCB4 (mdr2), ABCC2 (mrp2), ABCC6 (mrp6), ABCG1, ABCG2 (bcrp/mxr), Slc21a7 (Oatp3), Slc15a1 (PepT1), and Slc16a1 (Mct1). Conditionally immortalized rat small intestine epithelial cell lines were established from tsA58 Tg rats and expressed the mRNA of intestinal transporters.

The objective of this study was to assess the contributions of surfactant-mediated solubility and micellar diffusivity on the ability of surfactant to enhance drug dissolution. The following model was derived to predict the degree to which surfactants enhance griseofulvin dissolution: phi = 1 + (fm/ff).((D(D-M)2/3)/(DD2/3)) where phi is the degree of surfactant-mediated dissolution enhancement, fm is the fraction of the drug in micelle, and ff is the fraction of free drug, and DD and D(D-M) are the diffusivities of free drug and drug-loaded micelles, respectively. The Wood apparatus was used to measure the dissolution of griseofluvin in the presence of the anionic surfactant sodium dodecyl sulfate (SDS), the cationic surfactant cetyl trimethyl ammonium bromide (CTAB), and the neutral surfactants Tween 80 and Cremophor EL. DD was estimated using the Levich equation. D(D-M) was measured using dynamic light scattering. Griseofulvin solubility was evaluated in SDS, CTAB, Tween 80, and Cremophor EL at the surfactant concentrations used in the dissolution studies. DD was 11.0 x 10(-6) cm2/s. D(D-M) was 1.29 x 10(-6) cm2/s, 0.956 x 10(-6) cm2/s, 0.569 x 10(-6) cm2/s, and 0.404 x 10(-6) cm2/s for griseofulvin-loaded micelles of SDS, CTAB, Tween 80, and Cremophor EL, respectively. At the highest surfactant concentrations studied, griseofulvin solubility increased 107-fold, 31-fold, fourfold, and threefold for SDS, CTAB, Tween 80, and Cremophor EL. Dissolution into SDS and CTAB were markedly enhanced, but only about one-third as much as solubility enhancement. Dissolution enhancement in the presence of SDS and CTAB were in excellent agreement with model predicted values, with prediction error less than 12%. The model predicted dissolution into Tween 80 and Cremophor EL to be minimally enhanced, as was observed, although the model underpredicted dissolution into these two neutral surfactants. The derived model predicted surfactant-mediated dissolution and reflects dissolution enhancement to be promoted by surfactant-enhanced solubility, but limited by the relatively slow diffusion of drug-loaded surfactant micelles., (Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:2064-2075, 2004)

The objective of this study is to evaluate the effect of acute renal or hepatic failure on the intestinal absorption of tacrolimus. Simultaneous perfusion study in rat small intestine revealed that the extent of absorption into blood vessels was decreased in the jejunum and the ileum of rat of acute renal failure due to the decrease in the uptake of tacrolimus into enterocytes. In contrast, there observed no significant changes in tacrolimus absorption in rat of acute hepatic failure. Since it has been reported that tacrolimus absorption is regulated mainly by Cytochrome P-450 (CYP) mediated metabolism in the jejunum, but by P-glycoprotein (P-gp) mediated efflux in the ileum, these factors might contribute to the changes in intestinal absorption of tacrolimus in rat of acute renal failure. Enzyme inhibitor, ketoconazole, was co-perfused with tacrolimus to specify the effect of CYP and P-gp. However, since ketoconazole failed to recover the permeability in the jejunum and ileum of rat of acute renal failure, it is considered that the changes in CYP or P-gp functions might not be involved in the decreased uptake of tacrolimus. This type of kinetic study in rats should be valuable to identify the precise mechanisms of drug absorption and the effects of various diseases on it, such as acute renal or hepatic failure.

We have cloned and functionally expressed a sodium-dependent human nucleoside transporter, hCNT2, from a CNS cancer cell line U251. Our cDNA clone of hCNT2 had the same predicted amino acid sequence as the previously cloned hCNT2 transporter. Of the several cell lines studied, the best hCNT2 transport function was obtained when transiently expressed in U251 cells. Na(+)-dependent uptake of [3H]inosine in U251 cells transiently expressing hCNT2 was 50-fold greater than that in non-transfected cells, and uptake in Na(+)-containing medium was approximately 30-fold higher than that at Na(+)-free condition. The hCNT2 displayed saturable uptake of [3H]inosine with K(m) of 12.8 microM and V(max) of 6.66 pmol/mg protein/5 min. Uptake of [3H]inosine was significantly inhibited by the purine nucleoside drugs dideoxyinosine and cladribine, but not by acyclic nucleosides including acyclovir, ganciclovir, and their prodrugs valacyclovir and valganciclovir. This indicates that the closed ribose ring is important for binding of nucleoside drugs to hCNT2. Among several pyrimidine nucleosides, hCNT2 favorably interacted with the uridine analogue floxuridine. Interestingly, we found that benzimidazole analogues, including maribavir, 5,6-dichloro-2-bromo-1-beta-D-ribofuranosylbenzimidazole (BDCRB), and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), were strong inhibitors of inosine transport, even though they have a significantly different heterocycle structure compared to a typical purine ring. As measured by GeneChip arrays, mRNA expression of hCNT2 in human duodenum was 15-fold greater than that of hCNT1 or hENT2. Further, the rCNT2 expression in rat duodenum was 20-fold higher than rCNT1, rENT1 or rENT2. This suggests that hCNT2 (and rCNT2) may have a significant role in uptake of nucleoside drugs from the intestine and is a potential transporter target for the development of nucleoside and nucleoside-mimetic drugs.

The objective of this research was to determine the characteristics of intestinal transport of PD 0158473 using in vitro Caco-2 cells as well as in situ single-pass rat intestinal perfusion models. Because apical (AP)-to-basolateral (BL) transport was greater (1.8- fold) than BL-to-AP transport in the Caco-2 cell model, identification of carrier system(s) involved was further investigated. Cellular uptake of PD 0158473 was concentration and/or pH dependent and significantly inhibited by substrates of dipeptide and monocarboxylic acid transporters. Although this compound is an analog of L-Phe and previous studies demonstrated a high affinity of this compound to large neutral amino acid transporter, the involvement of amino acid carriers did not appear to be significant in the Caco-2 cell model. Subsequently, in situ single-pass intestinal perfusion studies in rats demonstrated that intestinal absorption of PD 0158473 was not concentration dependent at a concentration range tested but significantly inhibited by various dipeptides as well as substrates of dipeptide transporters. The difference in the concentration-dependent transport of PD 0158473 between Caco-2 cells and the rat perfusion model could be explained by the difference in the affinity (apparent K(m)) of PD 0158473 between Caco-2 cells (107 microM) and rat tissue (>1 mM). The present study suggested that multiple transporters are involved in the transcellular transport of this amino-acid analogue compound, of which peptide transporters could play a major role., (Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2579-2587, 2002)

Our recent work on the intestinal metabolism and absorption of cholecystokinin analogs, sulfated C-terminal octapeptide (CCK8; Asp-Tyr(SO(3)H)-Met-Gly-Trp-Met-Asp-Phe(NH(2)) = DY(SO(3)H)MGWMDF(NH(2))) and tetrapeptide (CCK4; Trp-Met-Asp-Phe(NH(2)) = WMDF(NH(2))), was extended to investigate the degradative process of these analogs using rabbit jejunum brush-border membrane vesicles and to find a better enzyme-inhibitor system for intestinal absorption of peptide drugs. Various enzyme inhibitors and a lower pH buffer were applied to discover the major enzyme(s) involved in each process. Metabolic pathways showing degradative processes were proposed for both analogs. The major cleavage site occurs at the W(1)-M(2) for CCK4. At least three metabolic pathways occur independently for CCK8 and appear at peptides bonds between G(4)-W(5), M(6)-D(7), and D(7)-F(NH(2))(8). Many different enzymes of aminopeptidase, endopeptidase, angiotensin-converting enzyme, metalloenzyme, and others were involved in each process. Identification of more specific yet safe enzyme inhibitors and co-administration of various these inhibitors may lead to further enhancement in intestinal peptide absorption when administered orally.

Intestinal metabolism and poor permeability were known to be major barriers for oral absorption of large peptide drugs. Dimensionless wall permeability values of C-terminal octa- and tetra-peptides cholecystokinin analogs (CCK8 and CCK4) were estimated and found out to be greater than 1, suggesting no permeability-limited absorption for CCK analogs. Thus, a strategy employing enzyme inhibitors and a specific delivery site to improve the absorption was developed and tested with CCK8, followed by identification of metabolites of the analogs and their participating enzymes in rabbit brush-border membrane vesicles. Thiorphan and amastatin, a specific enzyme inhibitor for enkephalinase and aminopeptidase, respectively, in pH 4 buffer solution were coadministered with CCK8 to the ileum in fistulated rats. The absolute bioavailability (F) of CCK8 was 5.4% and increased to 19% in the presence of the enzyme inhibitors, while the F values following oral administration were close to zero. These results indicate that peptide oral delivery is possible.

The objective of this study is to investigate the role of P-glycoprotein (P-gp), a membrane efflux pump associated with multidrug resistance (MDR) and a known substrate for tacrolimus, in determining the regional intestinal permeability of tacrolimus in rats. Thus, isolated segments of rat jejunum, ileum, or colon were perfused with tacrolimus solutions containing polyethoxylated hydrogenated castor oil 60 surfactant, and with or without verapamil, a P-gp substrate used to reverse the MDR phenotype. The results indicated that the intrinsic permeability of tacrolimus in the jejunum, calculated on the basis of the concentration of non-micellized free tacrolimus, was quite high ( approximately 1.4 x 10(-4) cm/s). The apparent permeability (P(app)) in the jejunum was unaffected by the presence of verapamil; however, the P(app) in the ileum and the colon increased significantly in the presence of verapamil and were similar to the values observed in the jejunum. The results suggest that systemic absorption of tacrolimus from the gastrointestinal tract could be significantly affected by P-gp efflux mechanisms. It is also possible that differences in P-gp function at various intestinal sites in a subject or at a given intestinal site in various subjects could lead to large intra- and interindividual variability in bioavailability of tacrolimus following oral administration., (Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association .)