Kinetic and time-dependent features of sustained inhibitory effect of myricetin on folate transport by proton-coupled folate transporter.

Myricetin is a flavonoid that has recently been suggested to induce sustained inhibition of proton-coupled folate transporter (PCFT/SLC46A1), which operates for intestinal folate uptake. The present study was conducted to characterize the inhibitory effect in more detail, using human PCFT stably expressed in Madin-Darby canine kidney II cells, to gain information to cope with problems potentially arising from that. The kinetics of saturable folate transport was first assessed in the absence of myricetin in the cells pretreated with the flavonoid for 60 min. The pretreatment induced PCFT inhibition in a manner dependent on the concentration of myricetin, where the maximum transport rate was reduced by 35.5% and 83.1%, respectively, at its concentrations of 20 µM and 50 µM. The inhibitory effect was, however, less extensive at lower folate concentrations, because the Michaelis constant was also reduced similarly in a manner dependent on myricetin concentration. The inhibition was induced depending on the time of pretreatment and, after removal of myricetin (50 µM) upon the manifestation of an extensive inhibition at 60 min, reversed almost completely in 90 min. This rather short time required for recovery may suggest that the sustained inhibition of PCFT is of a reversible type.

Sustained inhibition of proton-coupled folate transporter by myricetin.

Myricetin is a flavonoid that has recently been suggested to interfere with the intestinal folate transport system. To examine that possibility, focusing on its sustained inhibitory effect on proton-coupled folate transporter (PCFT), the uptake of folate was examined in Caco-2 cells, in which PCFT is known to be in operation, in the absence of myricetin in the medium during uptake period after preincubation of the cells with the flavonoid (100 µM) for 1 h. This pretreatment induced an extensive and sustained reduction in the carrier-mediated component of folate uptake, which was attributable to a reduction in the maximum transport rate (Vmax). Although the affinity of the transporter for folate was increased at the same time as indicated by a reduction in the Michaelis constant (Km), the change in Km was overwhelmed in extent by that in Vmax. Consistent with the finding, folate transport by human PCFT stably expressed in Madin-Darby canine kidney II cells was reduced in a similar manner with simultaneous reductions in Vmax and Km by myricetin pretreatment. Attention may need to be given for a possibility that such a sustained inhibition of PCFT could potentially be a cause of the malabsorption of folate and also antifolate drugs.

Noncompetitive inhibition of proton-coupled folate transporter by myricetin.

Myricetin is a flavonoid that has recently been suggested to interfere with the intestinal folate transport system. The present study was conducted to examine that possibility, focusing on its inhibitory effect on proton-coupled folate transporter (PCFT) as the molecular entity of the transport system. The uptake transport of folate was first examined in the Caco-2 cell as an intestinal epithelial cell model, and its carrier-mediated component, of which the Michaelis constant (Km) was 0.407 µM, was found to be noncompetitively inhibited by myricetin with an inhibition constant (Ki) of 61 µM. Consistent with that, folate transport by human PCFT stably expressed in Madin-Darby canine kidney II (MDCKII) cells, of which the Km was 1.246 µM, was also noncompetitively inhibited by myricetin with a Ki of 130 µM. Thus, myricetin was suggested to inhibit intestinal folate transport by acting noncompetitively on PCFT, although the Km and Ki were similarly shifted to some extent to be smaller in Caco-2 cells. Finally, epigallocatechin-3-gallate was also suggested to act in a noncompetitive manner as an inhibitory flavonoid. Care may need to be taken, therefore, in the ingestion of myricetin and some flavonoids to maintain the absorption of folate and antifolate drugs.

Transcriptional regulation of PCFT by KLF4, HNF4α, CDX2 and C/EBPα: implication in its site-specific expression in the small intestine.

Proton-coupled folate transporter (PCFT), which is responsible for the intestinal uptake of folates and analogs, is expressed only in the proximal region in the small intestine. The present study was to examine its transcriptional regulation, which may be involved in such a unique expression profile and potentially in its alteration, using dual-luciferase reporter assays in human embryonic kidney (HEK) 293 cells. The luciferase activity derived from the reporter construct containing the 5'-flanking sequence of -1695/+96 of the human PCFT gene was enhanced most extensively by the introduction of Krüppel-like factor 4 (KLF4). The KLF4-induced luciferase activity was further enhanced by hepatocyte nuclear factor 4α (HNF4α) synergistically. To the contrary, caudal-type homeobox transcription factor 2 (CDX2) and CCAAT/enhancer-binding protein α (C/EBPα) extensively suppressed the luciferase activity induced by KLF4 alone and also that induced by KLF4 and HNF4α. Western blot analysis using the rat small intestine indicated uniform expression of KLF4 along the intestinal tract, proximal-oriented expression of HNF4α, distal-oriented expression of CDX2 and C/EBPα. These results suggest that the activity of PCFT promoter is basically induced by KLF4 and the gradiented expression profile of PCFT may be at least in part accounted for by those of HNF4α, CDX2 and C/EBPα.

Predictors of home death of home palliative cancer care patients: a cross-sectional nationwide survey.

OBJECTIVES: To identify factors influencing the place of death among home palliative cancer care patients, focusing on the role of nurses in terms of pre- and post-discharge from hospital to home care settings. DESIGN, SETTINGS AND PARTICIPANTS: A cross-sectional nationwide questionnaire survey was conducted at 1000 randomly selected homecare agencies in Japan. The questionnaires were completed by primary community nurses of home palliative patients just after their discharge. A total of 568 responses were analyzed (effective response rate, 69%). RESULTS: Multivariate logistic regression analysis revealed the following independent factors of place of death among those patients: desire for home death at referral by both patient and family caregiver; caregiver relationship to patient as daughter or daughter-in-law; totally bedridden functional status of patient; patient not suffering from depression and/or anxiety at referral; patients and caregivers duly informed about the dying process/death in detail, as well as instructed by community nurses about pain management and how to treat/prevent bedsores in home care settings. CONCLUSIONS: This study demonstrated the importance of both the hospital and community nurses' role in increasing the patients' chance of dying at home. Hospital nurses should support early transfer to home palliative care according to their assessment of the desire of patient/family caregiver for home death, the patients' clinical status, and caregivers' ability to provide patient care at home. Community nurses should inform patients/family caregiver in detail about the dying process/death just after discharge, relieve patient pain, treat/prevent bedsores, and instruct family caregivers on their symptom control.

Late referrals to home palliative care service affecting death at home in advanced cancer patients in Japan: a nationwide survey.

BACKGROUND: To identify factors influencing place of death among home palliative care patients with advanced cancer, focusing on the timing of referrals from hospital to home care settings. METHODS: A cross-sectional nationwide questionnaire survey was conducted on home palliative care patients at 1000 randomly selected home care agencies in Japan. A total of 568 responses were analyzed (effective response rate, 69%). RESULTS: Multivariate logistic regression analysis revealed that (i) predischarge health care supports in hospital (e.g. early referral 8 days or more before discharge; clear explanation by hospital staffs to patients and families regarding discharge to live and die at home) and (ii) postdischarge health care supports after transferring home care (e.g. signing a 24-h support insurance contract of network between primary physician and nurse as a home palliative care team; primary nurse consultation with primary physician >3 times during the first week after discharge) have an effect on place of death among home palliative care patients. CONCLUSION: An early and carefully coordinated referral support system for smooth discharge by hospital staffs as well as intensive and highly qualified support just after discharge by the home care team would help to increase the number of patients who could die at home.

Molecular and functional characteristics of proton-coupled folate transporter.

Proton-coupled folate transporter (PCFT) has recently been identified as the molecular entity of the carrier-mediated intestinal folate transport system. PCFT has been demonstrated to be most abundantly expressed in the upper small intestine, localizing at the brush border membrane of epithelial cells, transport folate and its analogs more efficiently at lower (acidic) pH by a H(+)-coupled cotransport mechanism, and have a high affinity for folate with a Michaelis constant (K(m)) of a few microM at pH 5.5 and somewhat lower affinities for reduced folates and methotrexate (MTX). A loss of PCFT function due to a homozygous mutation in its gene has been indicated to be responsible for hereditary folate malabsorption. Thus, PCFT has all the characteristics of the brush border H(+)-coupled cotransporter for folate and analogs, which has long been suggested to be present in the intestine. Furthermore, sulfasalazine was found to be a potent inhibitor of PCFT, suggesting that it is a risk factor that would cause malabsorption of folate and also MTX, when coadministered in the treatment of rheumatoid arthritis. Understanding the molecular and functional characteristics of PCFT should be important and helpful in exploring therapeutic strategies for folate malabsorption and in optimizing therapies using antifolate drugs.

Effect of glycerol-related compounds on carrier-mediated glycerol uptake in HCT-15 human colon cancer cell line.

The effect of several compounds, which are structurally analogous to glycerol, on carrier-mediated glycerol uptake was examined in HCT-15 cells to help clarifying the functional characteristics of the glycerol transport system. The carrier-mediated uptake of glycerol conformed to the Michaelis-Menten kinetics with a Michaelis constant of 21.1 microM and the tested compounds were all suggested to inhibit it competitively with the values of the inhibition constant (K(i)) in the increasing order as follows: monobutyrin (41.0 microM)< or =monoacetin (54.6 microM)

Functional characterization of PCFT/HCP1 as the molecular entity of the carrier-mediated intestinal folate transport system in the rat model.

Proton-coupled folate transporter/heme carrier protein 1 (PCFT/HCP1) has recently been identified as a transporter that mediates the translocation of folates across the cellular membrane by a proton-coupled mechanism and suggested to be the possible molecular entity of the carrier-mediated intestinal folate transport system. To further clarify its role in intestinal folate transport, we examined the functional characteristics of rat PCFT/HCP1 (rPCFT/HCP1) expressed in Xenopus laevis oocytes and compared with those of the carrier-mediated folate transport system in the rat small intestine evaluated by using the everted tissue sacs. rPCFT/HCP1 was demonstrated to transport folate and methotrexate more efficiently at lower acidic pH and, as evaluated at pH 5.5, with smaller Michaelis constant (K(m)) for the former (2.4 microM) than for the latter (5.7 microM), indicating its characteristic as a proton-coupled folate transporter that favors folate than methotrexate as substrate. rPCFT/HCP1-mediated folate transport was found to be inhibited by several but limited anionic compounds, such as sulfobromophthalein and sulfasalazine. All these characteristics of rPCFT/HCP1 were in agreement with those of carrier-mediated intestinal folate transport system, of which the K(m) values were 1.2 and 5.8 microM for folate and methotrexate, respectively, in the rat small intestine. Furthermore, the distribution profile of the folate transport system activity along the intestinal tract was in agreement with that of rPCFT/HCP1 mRNA. This study is the first to clone rPCFT/HCP1, and we successfully provided several lines of evidence that indicate its role as the molecular entity of the intestinal folate transport system.

Enhanced uptake of glycerol by butyrate treatment in HCT-15 human colon cancer cell line.

The HCT-15 human colon cancer cell line has a Na(+)-dependent carrier-mediated transport system for the uptake of glycerol. A similar transport system has been suggested to be present also in the small intestine and is of interest with regard to its role in the absorption of glycerol and possibly some structurally related compounds. To help clarifying functional characteristics of such glycerol transport systems, we examined the effect of butyrate, an agent known to facilitate the differentiation of cells, on glycerol uptake in HCT-15 cells. The uptake of glycerol (0.4 microM) was found to be about 5-fold greater in HCT-15 cells pretreated with butyrate (2 mM) for 24 h than in those untreated. The increase in the uptake by the butyrate treatment was due to an increase in the maximum transport rate. The effect of butyrate was almost completely suppressed when actinomycin D, an inhibitor of gene transcription, and cycloheximide, an inhibitor of protein synthesis, were added to the medium during the butyrate treatment. These results support the suggestion that a specific carrier protein is involved in glycerol uptake by HCT-15 cells and the carrier protein is one of those inducible by butyrate-induced cell differentiation.

Effect of Lactobacillus casei on the absorption of nifedipine from rat small intestine.

Lactobacillus casei Shirota strain (L. casei) has a modulating effect on the production of cytokines, which often play important roles in drug metabolism, in the inflamed intestinal mucosa. We evaluated the effect of L. casei administered orally in advance for 4 weeks on the absorption of nifedipine from the rat small intestine. The maximum concentration of nifedipine in plasma after administration into the intestinal loop (0.8 mg/kg) was significantly higher in L. casei-treated rats (3.26 microg/mL) than in those untreated rats (2.33 microg/mL) by 40%. Accordingly, the bioavailability of nifedipine was tended to be higher in the former, while the effect of L. casei on the disposition of intravenously administered nifedipine was negligible. We also found that the availability of nifedipine for the passage through the intestinal mucosa was significantly increased in L. casei-treated rats from the single-pass intestinal perfusion experiments. Therefore, it is likely that the exposure to nifedipine after its administration into rat intestine was increased by oral ingestion of L. casei due to an increase in absorption by increased intestinal availability (decreased metabolic extraction) during passage through the intestinal mucosa. This study has suggested that L. casei has some effect on the metabolic activity in the intestinal mucosa, though it seems to be only mild.

Functional characterization of human proton-coupled folate transporter/heme carrier protein 1 heterologously expressed in mammalian cells as a folate transporter.

The functional characteristics of human proton coupled folate transporter (hPCFT)/heme carrier protein (HCP) 1 were investigated. hPCFT/HCP1 expressed transiently in human embryonic kidney 293 cells mediated the transport of folate at an acidic extracellular pH of 5.5 in a manner independent of Na(+) and insensitive to membrane potential, but its transport activity was absent at near-neutral pH. Folate transport mediated by hPCFT/hHCP1 at pH 5.5 was saturable with a K(m) of 1.67 microM and extensively inhibited by reduced folates, such as folinate, 5-methyltetrahydrofolate, and methotrexate (MTX). Sulfobro-mophthalein and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid were also found to be potent inhibitors of hPCFT/hHCP1, but hemin was found to exhibit only minimal inhibitory effect. When expressed stably as a protein fused with green fluorescent protein (GFP-hPCFT/HCP1) in MDCKII cells, GFP-hPCFT/HCP1 was mainly localized at the apical membrane, and the cellular accumulation of MTX was higher from the apical side than from the basal side. These functional features of hPCFT/HCP1 are consistent with those of the well characterized carrier-mediated folate transport system in the small intestine, suggesting that hPCFT/HCP1 is responsible for the intestinal absorption of folate and also MTX. We also found that sulfasalazine is a potent inhibitor of hPCFT/HCP1, which would interfere with the intestinal absorption of MTX when coadministered in therapy for rheumatoid arthritis as well as folate.

Molecular identification and functional characterization of rat multidrug and toxin extrusion type transporter 1 as an organic cation/H+ antiporter in the kidney.

We have cloned and functionally characterized the rat ortholog of multidrug and toxin extrusion type transporter 1 (rMATE1). The mRNA of rMATE1 was strongly expressed in kidney and detectable in the various tissues such as brain, stomach, colon, lung, liver, spleen, skeletal muscle, and prostate. When stably expressed in HEK293 cells, rMATE1 could mediate the transport of tetraethylammonium (TEA) and cimetidine under the condition where the membrane potential was disrupted by a high concentration of potassium ion and intracellular pH was reduced by NH(4)Cl pretreatment. When extracellular pH was changed from 5.5 to 8.5, the transport of TEA by rMATE1 was greatest at pH 7.5. Kinetic analyses showed that the transports of TEA and cimetidine mediated by rMATE1 were both saturable with a K(m) of 260 +/- 10 and 3.01 +/- 0.21 muM, respectively. It was found that cimetidine is the most potent inhibitor of rMATE1, and many other organic cations, such as 1-methyl-4-phenylpyridinium, amiloride, imipramine, and quinidine, are also effective as inhibitors. Pretreatment of the cells expressing rMATE1 with p-chloromercuribenzene sulfonate significantly reduced TEA transport, but this effect was totally reversed by subsequent treatment with dithiothreitol. These results indicate that the functional nature of rMATE1 is consistent with that of the hypothetical organic cation/H(+) antiporter system in the brush-border membrane of the renal tubular epithelial cells. Accordingly, these results suggest that rMATE1 is an electroneutral and multispecific organic cation transporter energized by the trans-proton gradient, and plays a physiological role in renal secretion of organic cations, including clinically used cationic drugs.

Carrier-mediated transport of glycerol in the perfused rat small intestine.

Studies using the closed loop and everted sacs of the rat small intestine recently prompted us to suggest that carrier-mediated transport is involved in the intestinal absorption of glycerol. Although it could be mediated by a novel carrier system, little information is available. The aim of the present study was to kinetically characterize carrier-mediated glycerol transport in the perfused rat small intestine to help in identifying the carrier involved and to explore the possibility that the carrier might be used as a pathway for oral drug delivery and a target for drug development. In situ single-pass perfusion was conducted using a 10-cm midgut segment of the male Wistar rat, and the absorption of [3H]glycerol was evaluated by its disappearance from the intestinal lumen. The absorption of glycerol was saturable and significantly reduced by removing Na+ from the perfusion solution, suggesting the involvement of a Na+-dependent carrier-mediated transport system. The concentration-dependent absorption profile was successfully analyzed by assuming Michaelis-Menten type carrier-mediated transport and simultaneous passive (diffusive) transport. The maximum transport rate (J(max)) was 77.0 pmol/s/cm2 and the Michaelis constant (K(m)) was 1.04 mM, giving a J(max)/K(m) of 7.39 x 10(-5) cm/s. The membrane permeability coefficient for passive transport (P(m,d)) was 6.89 x 10(-5) cm/s, slightly smaller than J(max)/K(m). Therefore, it could be the major mechanism of intestinal glycerol absorption in the low concentration range where carrier-mediated transport conforms to linear kinetics represented by J(max)/K(m). Furthermore, carrier-mediated glycerol transport was found to be inhibited by glycerol 3-phosphate, monoacetin and diglycerol, indicating that the carrier system may be shared by these structural analogues. Thus, the present study has successfully demonstrated and characterized carrier-mediated glycerol transport in the perfused rat small intestine which is a physiologically relevant model.

Glycerol uptake in HCT-15 human colon cancer cell line by Na(+)-dependent carrier-mediated transport.

It has recently been suggested that an Na(+)-dependent carrier-mediated transport system is involved in intestinal glycerol absorption. Such a transport system is of general interest as a possible pathway of drug delivery and a target of drug development. However, the Na(+)-dependent mechanism of cellular glycerol uptake has not been fully clarified in the small intestine or in any other organ. The purpose of the present study was to examine glycerol uptake in the HCT-15 human colon cancer cell line, which was found to be able to perform Na(+)-dependent glycerol uptake, to determine the transport characteristics and help identify such glycerol transport systems. The uptake of glycerol in HCT-15 cells was highly saturable with a Michaelis constant of 15.0 microM and a maximum uptake rate of 11.9 pmol/min/mg protein, accompanied by minimal unsaturable transport; it was reduced markedly under Na(+)-free conditions, indicating Na+ requirement. Glycerol uptake was also reduced by 2,4-dinitrophenol, a metabolic inhibitor. These results suggest that a carrier-mediated glycerol transport system, which is Na(+)-dependent and secondarily active, is present in HCT-15 cells. The transport system could be specific for glycerol and some analogous compounds with hydroxyl groups, since glycerol uptake was inhibited by some alcohols and compounds related to glycerol, such as 1,2-propanediol and glycerol 3-phosphpate. However, it may represent a high affinity transport system, which is different from the one in the small intestine, because the Michaelis constant of 15.0 microM is about 50-fold lower than that observed in the rat small intestine. In conclusion, this is the first study to demonstrate an Na(+)-dependent carrier-mediated glycerol transport in an established cell line. This will help in identifying a group of Na(+)-dependent glycerol transport systems and elucidating their transport mechanisms, although the one found in HCT-15 cells in this study seems to be different from one previously found in the rat small intestine.

Glycerol absorption by Na+-dependent carrier-mediated transport in the closed loop of the rat small intestine.

The absorption of glycerol was examined using the closed loop of the rat small intestine in situ to clarify the transport mechanism. The absorption of glycerol, evaluated by its disappearance from the intestinal lumen, was saturable and reduced under the Na(+)-free conditions, suggesting the involvement of an Na(+)-dependent carrier-mediated transport system. Furthermore, glycerol absorption was selectively inhibited by several alcohols, among which 1,3-propanediol caused the greatest inhibition, and also by glycerol-3-phosphate and voglibose, which are alcohol-related compounds analogous to glycerol. Several other compounds that did not inhibit glycerol absorption included D-glucose and L-ascorbate, which are known to be transported by specific carriers. Therefore, the carriers for these two compounds do not seem to be involved in glycerol absorption. It is likely that the carrier-mediated transport system involved in glycerol absorption is specific to glycerol and, possibly, some analogous compounds with hydroxyl groups. Thus, the present study has provided in situ evidence for the presence of an Na(+)-dependent carrier-mediated transport system for glycerol in the rat small intestine. It would be interesting to examine the possibility that the carrier-mediated glycerol transport system could be involved in drug absorption and also that it could be used for oral drug delivery.

Functional characterization of the carrier-mediated transport system for glycerol in everted sacs of the rat small intestine.

The mechanism of intestinal glycerol transport was investigated by using the in vitro everted sac method involving the rat small intestine. The uptake of glycerol into everted sacs was saturable with a Michaelis constant (K(m)) of 0.77 mM and a maximum transport rate (J(max)) of 11.5 nmol/min/100 mg wet tissue weight (wtw), suggesting the involvement of carrier-mediated transport, and was accompanied by unsaturable transport (passive transport) with a membrane permeability clearance (CL(m,d)) of 4.9 microl/min/100 mg wtw. The carrier-mediated uptake of glycerol was inhibited by the removal of Na(+) and also by the addition of 2,4-dinitrophenol (DNP) and sodium azide (NaN(3)), which are metabolic inhibitors. These results suggest that the carrier-mediated glycerol transport is Na(+)-dependent and secondary active. Since glycerol uptake was also inhibited by p-chloromercuribenzene sulfonate (pCMBS), a thiol-modifying reagent, cysteine residues, which have a thiol group, seem to play an important role in the function of the carrier. We further found that glycerol uptake was selectively inhibited by glycerol-3-phosphate, chloramphenicol and voglibose, which are alcohol-related compounds analogous to glycerol. Several other compounds that did not inhibit glycerol uptake included D-glucose and 5-fluorouracil, which are known to be transported by specific carriers, and none of the selective inhibitors of glycerol uptake inhibited the uptake of D-glucose and 5-fluorouracil. Therefore, the carriers for these two compounds do not seem to be involved in glycerol uptake. It is likely that the carrier-mediated transport system involved in glycerol uptake is specific to glycerol and, possibly, some analogous compounds with hydroxyl groups. It would be interesting to examine the possibility that the carrier-mediated glycerol transport system might be involved in drug absorption and also that it might be used for oral drug delivery.

Dose adjustment strategy for oral microemulsion formulation of cyclosporine: population pharmacokinetics-based analysis in kidney transplant patients.

The present study aims to determine the population pharmacokinetic parameters of cyclosporine (CsA) after multiple oral administration of the microemulsion formulation, Neoral, in kidney transplant patients and to propose a limited sampling strategy to predict AUC(0-4h) using them and the Bayesian method. The AUC(0-4h) is a parameter that has recently been recommended as an index for the dose adjustment in therapeutic drug monitoring of CsA. Blood samples were obtained at the trough level and at hourly intervals up to 5 hours from 125 patients (78 male and 47 female) who were receiving Neoral twice daily, and whole-blood concentrations of CsA were measured. The population pharmacokinetic parameters were estimated using the NONMEM computer program and a linear two-compartment model with first-order absorption. The observed AUC0-4h and concentrations at different sampling times were compared with those computer-predicted by the Bayesian method, using the population pharmacokinetic parameters and 2 or 3 concentrations from those at 0 h (C(0)), 1 h (C(1)), and 2 h (C(2)) after administration. Typical values for the absorption rate constant (k(a)), elimination rate constant (k(el)), apparent volume of distribution for the central compartment (Vd/F), and oral clearance (CL/F) calculated by population pharmacokinetic analysis were 2.16 hours(-1), 0.547 hours(-1), 43.3 L, and 23.7 L/h, respectively. The CsA concentrations predicted using either the 2-point or 3-point sampling strategy exhibited an excellent correlation with the observed values (R(2) > 0.81), and accordingly, the predicted AUC(0-4h) values were in excellent agreement with those observed. The best predictability of AUC(0-4h) was found for the 3-point sampling strategy using C(0), C(1), and C(2), closely followed by a 2-point sampling strategy using C(1) and C(2). The present findings suggest that a simplified strategy based on population pharmacokinetics can accurately predict AUC(0-4h) from concentrations at 2 or 3 sampling time points, providing an excellent method for the daily dose adjustment of Neoral in routine clinical use for kidney transplant patients.

Absorption and metabolic extraction of diltiazem from the perfused rat small intestine.

The metabolic extraction of diltiazem was examined in conjunction with its absorption, using rat small intestine perfused in situ by the single-pass method, to clarify its intestinal metabolism. This is a topic of increasing interest which has not been fully clarified, particularly as far as the extent of metabolic extraction and the enzymes involved (cytochrome P450 (CYP) 3A and/or others) are concerned. The intestinal availability (Fi) of diltiazem was evaluated at steady-state by dividing the fraction absorbed into the mesenteric venous blood (Fa,b) by the fraction that disappeared from the intestinal lumen (Fa). The Fi of diltiazem (0.05 mM) was 0.126 and, hence, the extraction ratio (Ei=1-Fi) was 0.874, indicating that diltiazem undergoes extensive first-pass metabolism during its passage through the intestinal mucosa. The Ei was unchanged when the concentration was increased to 0.5 mM, suggesting that metabolism is linear over this concentration range. Thereafter, Ei decreased with concentration, demonstrating saturable metabolism, and reached an insignificant level at the highest concentrations of 30 and 50 mM. The decrease in Ei, or increase in Fi, was brought about by an increase in Fa,b (from about 0.02 to about 0.05) in the concentration range up to 10 mM and by a decrease in Fa (from about 0.15 to about 0.05) at concentrations higher than that. These results suggest that the extraction observed at the lower concentrations is almost solely attributable to metabolic extraction of a saturable nature. However, ketoconazole and cyclosporin A, which are specific CYP3A inhibitors, inhibited the metabolic extraction of diltiazem (0.05 mM) by only about 20% at the concentration (40 microM) at which they inhibited CYP3A almost completely, suggesting that the contribution of CYP3A to intestinal diltiazem metabolism is not marked. Thus, the present study demonstrates that diltiazem undergoes extensive first-pass metabolism in the rat small intestine, although the contribution of CYP3A seems to be relatively minor.

Saturable absorption of glycerol in the rat intestine.

The permeability of glycerol, a small hydrophilic solute, across the intestinal membrane would be low, if passive diffusion restricted to the paracellular route is the principal transport mechanism as generally assumed for this class of solutes. However, in the present study using a closed loop of rat small intestine in situ, we found that the absorption of glycerol was faster than that of urea, a probe solute widely assumed to permeate exclusively via the paracellular route. This finding is inconsistent with the paracellular permeation hypothesis, which predicts that the absorption of glycerol, which is larger than urea in terms of molecular size, could not be faster than that of urea. We also found that glycerol absorption was saturable. These findings suggest the involvement of carrier-mediated transport in intestinal glycerol absorption. Glycerol absorption in the colon was also saturable, suggesting the involvement of carrier-mediated transport, although it was much slower than that in the small intestine. Carrier-mediated glycerol transport might play an important role in absorbing glycerol liberated from dietary triglyceride. It would be interesting to further examine the possibility that a carrier-mediated glycerol transport system (or systems) might be involved in drug absorption and also that it might be utilized for oral drug delivery.