Yayoi Hayashi

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 Km of 1.67 μM 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.

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.

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 NH4Cl 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 Km of 260 ± 10 and 3.01 ± 0.21 μM, 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.

Specific antiphospholipid antibodies as a predictive variable in patients with recurrent pregnancy loss

PROBLEM: Antiphospholipid antibodies (APLs) consist of very heterogenous autoantibodies. It has not been fully explored what kind of specificities are most relevant to recurrent pregnancy loss. Thus, we investigated the effects of specific APLs on recurrent aborters.

Human jejunal permeability of two polar drugs: cimetidine and ranitidine.

AbstractPurpose. To determine the human jejunal permeability of cimetidine and ranitidine using a regional jejunal perfusion approach, and to integrate such determinations with previous efforts to establish a baseline correlation between permeability and fraction dose absorbed in humans for soluble drugs. Methods. A sterile multi-channel perfusion tube, Loc-I-Gut®, was inserted orally and positioned in the proximal region of the jejunum. A solution containing cimetidine or ranitidine and phenylalanine, propranolol, PEG 400, and PEG 4000 was perfused through a 10 cm jejunal segment in 6 and 8 subjects, respectively. Results. The mean Peff (± se) of cimetidine and ranitidine averaged over both phases were 0.30 (0.045) and 0.27 (0.062) × 10−4cm/s, respectively, and the differences between the two were found to be statistically insignificant. The mean permeabilities for propranolol, phenylalanine, and PEG 400 averaged over both phases and studies were 3.88 (0.72), 3.36 (0.50), and 0.56 (0.08) × 10−4 cm/s, respectively. The differences in permeability for a given marker were not significant between phases or between the two studies. Conclusions. The 10-fold lower permeabilities found for cimetidine and ranitidine in this study, compared to propranolol and phenylalanine, appear to be consistent with their less than complete absorption in humans.

Human Intestinal Permeability of Piroxicam, Propranolol, Phenylalanine, and PEG 400 Determined by Jejunal Perfusion

AbstractPurpose. To determine the human jejunal permeabilities of compounds utilizing different transport mechanisms using a regional perfusion approach and to establish a standard procedure for determining drug permeability class to be used for the establishment of drug product bioequivalence standards. Methods. Six healthy male volunteers participated in this study. A multi-lumen perfusion tube was inserted orally and positioned in the proximal region of the jejunum. A solution containing piroxicam, phenylalanine, propranolol, PEG 400 and PEG 4000 was perfused through the intestinal segment at a rate of 3.0 ml/min. Perfusate samples were quantitatively collected every 10 minutes for two 100 minute periods with an intermediate wash out period to determine intra and intersubject variation. Results. The mean Peff (±SD) of piroxicam, phenylalanine, propranolol, and PEG 400 were 10.40 ± 5.93, 6.67 ± 3.42, 3.59 ± 1.60, 0.80 ± 0.46 × 10−4 cm/sec, respectively. The coefficient of variation for the intersubject variability, first and second perfusion periods were: piroxicam, 60.5% and 57.1%; phenylalanine, 52.8% and 57.8%: propranolol, 62.1 % and 44.6%; and PEG 400, 81.7% and 42.3%, indicating a slightly lower CV for the second perfusion period in the same subject. The intrasubject CVs between the two perfusion periods were: 19.4%, 21.3%, 23.6% and 41.0% respectively, indicating a smaller intraindividual variation for all compounds studied. Conclusions. Piroxicam, a nonpolar drug exhibited the highest permeability of the compounds studied. The intrasubject CV was lower than the intersubject CV, indicating consistent permeability estimation within subjects. The methodology is useful for permeability estimation regardless of absorption mechanism and can be used to establish a consistent data base of human permeabilities for estimation of human drug absorption and for establishing the biopharmaceutic permeability class of drugs.

Variability in cimetidine absorption and plasma double peaks following oral administration in the fasted state in humans: correlation with antral gastric motility

The role of gastrointestinal motility and pH in determining cimetidine bioavailability as well as double peaks in plasma profiles following oral administration, in the quiescent or active phase of antral motility, to humans in the fasted state was examined. Plasma cimetidine-time curves did not show the presence of double peaks in any subject following intravenous administration. The incidence of double peaks was 73% following oral administration and was independent of antral migrating motility complex phase. Further, it was found that oral administration of cimetidine in the quiescent phase resulted in significantly higher bioavailability and in other pharmacokinetic parameters compared to that obtained following administration in the active phase. Excellent linearity in plots of motility peaks vs. plasma peaks with slopes close to unity were evident for both quiescent (r(2)=0.93) and active phase (r(2)=0.97) administration. A total of 14 peaks out of 22 (10 subjects, 64%) and 20 out of 27 peaks (11 subjects, 74%), were accounted for in quiescent and active phase oral administration, respectively. The proximal occurrence of plasma peaks to antral motility peaks typical of phase III contractions strongly implies that motility patterns may be responsible for secondary maxima following oral cimetidine administration in the fasted state.

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.

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 AUC0–4h using them and the Bayesian method. The AUC0–4h 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 (C0), 1 h (C1), and 2 h (C2) after administration. Typical values for the absorption rate constant (ka), elimination rate constant (kel), 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 (R2 > 0.81), and accordingly, the predicted AUC0–4h values were in excellent agreement with those observed. The best predictability of AUC0–4h was found for the 3-point sampling strategy using C0, C1, and C2, closely followed by a 2-point sampling strategy using C1 and C2. The present findings suggest that a simplified strategy based on population pharmacokinetics can accurately predict AUC0–4h 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.

A prospective study on pregnancy risk of antiphospholipid antibodies in association with systemic lupus erythematosus

This prospective study is an attempt to address the issues of whether or not antiphospholipid antibodies (aPL) constitute a significant risk factor for pregnancy in individuals with systemic lupus erythematosus (SLE) and whether or not combination therapy of high-dose prednisolone (PSL) and low-dose acetylsalicylic acid (ASA) offers efficient control. Antibodies against six phospholipids were measured in sera of patients with stable SLE who had no severe complications before pregnancy, and were followed up during subsequent pregnancies. Four of 12 patients with SLE demonstrated aPL-positivity. Six of 8 patients without aPL had appropriate-for-date (AFD) live babies, the remaining two suffering intrauterine fetal death (IUFD) in the first trimester, one having a chromosome abnormality. Two aPL-positive patients treated only with 5-15 mg/day PSL during pregnancy ended in IUFD in the second trimester. In contrast, the other two patients treated with high-dose PSL and low-dose ASA each had AFD live babies at 38 weeks gestation. The results suggest that APL is a crucial risk factor in pregnancy with stable SLE. Combination therapy of high-dose PSL and low-dose ASA may enable aPL-positive patients with SLE to have AFD live babies.