Intestinal Atp8b1 dysfunction causes hepatic choline deficiency and steatohepatitis.

Choline is an essential nutrient, and its deficiency causes steatohepatitis. Dietary phosphatidylcholine (PC) is digested into lysoPC (LPC), glycerophosphocholine, and choline in the intestinal lumen and is the primary source of systemic choline. However, the major PC metabolites absorbed in the intestinal tract remain unidentified. ATP8B1 is a P4-ATPase phospholipid flippase expressed in the apical membrane of the epithelium. Here, we use intestinal epithelial cell (IEC)-specific Atp8b1-knockout (Atp8b1IEC-KO) mice. These mice progress to steatohepatitis by 4 weeks. Metabolomic analysis and cell-based assays show that loss of Atp8b1 in IEC causes LPC malabsorption and thereby hepatic choline deficiency. Feeding choline-supplemented diets to lactating mice achieves complete recovery from steatohepatitis in Atp8b1IEC-KO mice. Analysis of samples from pediatric patients with ATP8B1 deficiency suggests its translational potential. This study indicates that Atp8b1 regulates hepatic choline levels through intestinal LPC absorption, encouraging the evaluation of choline supplementation therapy for steatohepatitis caused by ATP8B1 dysfunction.

A randomized trial to examine the impact of food on pharmacokinetics of 4-phenylbutyrate and change in amino acid availability after a single oral administration of sodium 4-phenylbutyrarte in healthy volunteers.

Urea cycle disorders (UCDs), inborn errors of hepatocyte metabolism, result in the systemic accumulation of ammonia to toxic levels. Sodium 4-phenylbutyrate (NaPB), a standard therapy for UCDs for over 20 years, generates an alternative pathway of nitrogen deposition through glutamine consumption. Administration during or immediately after a meal is the accepted use of NaPB. However, this regimen is not based on clinical evidence. Here, an open-label, single-dose, five-period crossover study was conducted in healthy adults to investigate the effect of food on the pharmacokinetics of NaPB and determine any subsequent change in amino acid availability. Twenty subjects were randomized to one of four treatment groups. Following an overnight fast, NaPB was administered orally at 4.3 g/m2 (high dose, HD) or 1.4 g/m2 (low dose, LD) either 30 min before or just after breakfast. At both doses, compared with post-breakfast administration, pre-breakfast administration significantly increased systemic exposure of PB and decreased plasma glutamine availability. Pre-breakfast LD administration attenuated plasma glutamine availability to the same extent as post-breakfast HD administration. Regardless of the regimen, plasma levels of branched-chain amino acids (BCAA) were decreased below baseline in a dose-dependent manner. In conclusion, preprandial oral administration of NaPB maximized systemic exposure of the drug and thereby its potency to consume plasma glutamine. This finding may improve poor medication compliance because of the issues with odor, taste, and pill burden of NaPB and reduce the risk of BCAA deficiency in NaPB therapy.

Cell-to-Medium Concentration Ratio Overshoot in the Uptake of Statins by Human Hepatocytes in Suspension, but Not in Monolayer: Kinetic Analysis Suggesting a Partial Loss of Functional OATP1Bs.

Suspended human hepatocytes (SHH) have long been used in assessing hepatic drug uptake, while plated human hepatocytes in short-term monolayer culture (PHH) have gained use in recent years. This study aimed to cross-evaluate SHH and PHH in measuring the hepatic uptake mediated by organic anion transporting polypeptide 1Bs (OATP1Bs). We compared the time courses of cell-to-medium (C/M) concentration ratios and initial uptake clearance values of the OATP1B substrates (pitavastatin, rosuvastatin, cerivastatin, pravastatin, dehydropravastatin, and SC-62807) between SHH and PHH. For all compounds except cerivastatin, the C/M ratios in SHH displayed an apparent overshoot (an initial increase followed by a decrease) during the 180-min uptake experiment, but not in PHH. Based on the literature evidence suggesting the possible internalization of OATP1Bs in primary hepatocytes, separate experiments measured the drug uptake after varying lengths of pre-incubation in the drug-free medium. The initial uptake clearances of pitavastatin and rosuvastatin declined in SHH beyond an apparent threshold time of 20-min drug-free pre-incubation, but not in PHH. Kinetic modeling quantitatively captured the decline in the active uptake clearance in SHH, and more than half of the active uptake clearances of pitavastatin and rosuvastatin were prone to loss during the 180-min uptake experiment. These results suggested a partial, time-delayed loss of the functional OATP1Bs in SHH upon prolonged incubation. Our results indicate that PHH is more appropriate for experiments where a prolonged incubation is required, such as estimation of unbound hepatocyte-to-medium concentration ratio (Kp,uu) at the steady-state.

Impact of P-Glycoprotein-Mediated Active Efflux on Drug Distribution into Lumbar Cerebrospinal Fluid in Nonhuman Primates.

Estimation of unbound drug concentration in the brain (Cu,brain) is an essential part of central nervous system (CNS) drug development. As a surrogate for Cu,brain in humans and nonhuman primates, drug concentration in cerebrospinal fluid (CCSF) collected by lumbar puncture is often used; however, the predictability of Cu,brain by lumbar CCSF is unclear, particularly for substrates of the active efflux transporter P-glycoprotein (P-gp). Here, we measured lumbar CCSF in cynomolgus monkey after single intravenous administration of 10 test compounds with varying P-gp transport activities. The in vivo lumbar cerebrospinal fluid (CSF)-to-plasma unbound drug concentration ratios (Kp,uu,lumbar CSF) of nonsubstrates or weak substrates of P-gp were in the range 0.885-1.34, whereas those of good substrates of P-gp were in the range 0.195-0.458 and were strongly negatively correlated with in vitro P-gp transport activity. Moreover, concomitant treatment with a P-gp inhibitor, zosuquidar, increased the Kp,uu,lumbar CSF values of the good P-gp substrates, indicating that P-gp-mediated active efflux contributed to the low Kp,uu,lumbar CSF values of these compounds. Compared with the drug concentrations in the cisternal CSF and interstitial fluid (ISF) that we previously determined in cynomolgus monkeys, the lumbar CCSF were more than triple for two and all of the good P-gp substrates examined, respectively. Although lumbar CCSF may overestimate cisternal CSF and ISF concentrations of good P-gp substrates, lumbar CCSF allowed discrimination of good P-gp substrates from the weak and nonsubstrates and can be used to estimate the impact of P-gp-mediated active efflux on drug CNS penetration. SIGNIFICANCE STATEMENT: This is the first study to systematically evaluate the penetration of various P-glycoprotein (P-gp) substrates into lumbar cerebrospinal fluid (CSF) in nonhuman primates. Lumbar CSF may contain >3-fold higher concentrations of good P-gp substrates than interstitial fluid (ISF) and cisternal CSF but was able to discriminate the good substrates from the weak or nonsubstrates. Because lumbar CSF is more accessible than ISF and cisternal CSF in nonhuman primates, these findings will help increase our understanding of drug central nervous system penetration at the nonclinical stage.

Analysis of hiPSCs differentiation toward hepatocyte-like cells upon extended exposition to oncostatin.

The capability to produce and maintain functional human adult hepatocytes remains one of the major challenges for the use of in-vitro models toward liver cell therapy and industrial drug-screening applications. Among the suggested strategies to solve this issue, the use of human-induced pluripotent stem cells (hiPSCs), differentiated toward hepatocyte-like cells (HLCs) is promising. In this work, we propose a 31-day long protocol, that includes a final 14-day long phase of oncostatin treatment, as opposed to a 7-day treatment which led to the formation of a hepatic tissue functional for CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP3A4. The production of albumin, as well as bile acid metabolism and transport, were also detected. Transcriptome profile comparisons and liver transcription factors (TFs) motif dynamics revealed increased expression of typical hepatic markers such as HNF1A and of important metabolic markers like PPARA. The performed analysis has allowed for the extraction of potential targets and pathways which would allow enhanced hepatic maturation in-vitro. From this investigation, NRF1 and SP3 appeared as transcription factors of importance. Complex epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) patterns were also observed during the differentiation process. Moreover, whole transcriptome analysis highlighted a response typical of the one observed in liver regeneration and hepatocyte proliferation. While a complete maturation of hepatocytes was yet to be obtained, the results presented in this work provide new insights into the process of liver development and highlight potential targets aimed to improve in-vitro liver regeneration.

Modulation of blood-brain barrier function by a heteroduplex oligonucleotide in vivo.

The blood-brain barrier (BBB) is increasingly regarded as a dynamic interface that adapts to the needs of the brain, responds to physiological changes, and gets affected by and can even promote diseases. Modulation of BBB function at the molecular level in vivo is beneficial for a variety of basic and clinical studies. Here we show that our heteroduplex oligonucleotide (HDO), composed of an antisense oligonucleotide and its complementary RNA, conjugated to α-tocopherol as a delivery ligand, efficiently reduced the expression of organic anion transporter 3 (OAT3) gene in brain microvascular endothelial cells in mice. This proof-of-concept study demonstrates that intravenous administration of chemically synthesized HDO can remarkably silence OAT3 at the mRNA and protein levels. We also demonstrated modulation of the efflux transport function of OAT3 at the BBB in vivo. HDO will serve as a novel platform technology to advance the biology and pathophysiology of the BBB in vivo, and will also open a new therapeutic field of gene silencing at the BBB for the treatment of various intractable neurological disorders.

Investigation of the impact of substrate selection on in vitro organic anion transporting polypeptide 1B1 inhibition profiles for the prediction of drug-drug interactions.

The risk assessment of organic anion transporting polypeptide (OATP) 1B1-mediated drug-drug interactions (DDIs) is an indispensable part of drug development. We previously reported that in vitro inhibitory potencies of several inhibitors on OATP1B1 depend on the substrates when prototypical substrates, estradiol-17ß-glucuronide (E2G), estrone-3-sulfate, and sulfobromophthalein were used as test substrates. The purpose of this study was to comprehensively investigate this substrate-dependent inhibition of OATP1B1 using clinically relevant OATP1B1 inhibitors and substrate drugs. Effects of cyclosporine A (CsA), rifampin, and gemfibrozil on OATP1B1-mediated uptake of 12 substrate drugs were examined in OATP1B1-expressing human embryonic kidney 293 cells. The Ki values (µM) for CsA varied from 0.0771 to 0.486 (6.3-fold), for rifampin from 0.358 to 1.23 (3.4-fold), and for gemfibrozil from 9.65 to 252 (26-fold). Except for the inhibition of torasemide uptake by CsA and that of nateglinide uptake by gemfibrozil, the Ki values were within 2.8-fold of those obtained using E2G as a substrate. Preincubation potentiated the inhibitory effect of CsA on OATP1B1 with similar magnitude regardless of the substrates. R values calculated based on a static model showed some variation depending on the Ki values determined with various substrates, and such variability could have an impact on the DDI predictions particularly for a weak-to-moderate inhibitor (gemfibrozil). OATP1B1 substrate drugs except for torasemide and nateglinide, or E2G as a surrogate, is recommended as an in vitro probe in the inhibition experiments, which will help mitigate the risk of false-negative DDI predictions potentially caused by substrate-dependent Ki variation.

Effects of one-time apple juice ingestion on the pharmacokinetics of fexofenadine enantiomers.

PURPOSE: We examined the effect of a single apple juice intake on the pharmacokinetics of fexofenadine enantiomers in healthy Japanese subjects. METHODS: In a randomized two phase, open-label crossover study, 14 subjects received 60 mg of racemic fexofenadine simultaneously with water or apple juice. For the uptake studies, oocytes expressing organic anion-transporting polypeptide 2B1 (OATP2B1) were incubated with 100 µM (R)- and (S)-fexofenadine in the presence or absence of 10 % apple juice. RESULTS: One-time ingestion of apple juice significantly decreased the area under the plasma concentration-time curve (AUC0-24) for (R)- and (S)-fexofenadine by 49 and 59 %, respectively, and prolonged the time to reach the maximum plasma concentration (t max) of both enantiomers (P < 0.001). Although apple juice greatly reduced the amount of (R)- and (S)-fexofenadine excretion into urine (Ae0-24) by 54 and 58 %, respectively, the renal clearances of both enantiomers were unchanged between the control and apple juice phases. For in vitro uptake studies, the uptake of both fexofenadine enantiomers into OATP2B1 complementary RNA (cRNA)-injected oocytes was significantly higher than that into water-injected oocytes, and this effect was greater for (R)-fexofenadine. In addition, apple juice significantly decreased the uptake of both enantiomers into OATP2B1 cRNA-injected oocytes. CONCLUSIONS: These results suggest that OATP2B1 plays an important role in the stereoselective pharmacokinetics of fexofenadine and that one-time apple juice ingestion probably inhibits intestinal OATP2B1-mediated transport of both enantiomers. In addition, this study demonstrates that the OATP2B1 inhibition effect does not require repeated ingestion or a large volume of apple juice.

Evaluation of Oatp and Mrp2 activities in hepatobiliary excretion using newly developed positron emission tomography tracer [11C]dehydropravastatin in rats.

We developed a pravastatin derivative, sodium (3R,5R)-3,5-dihydroxy-7-((1S,2S,6S,8S)-6-hydroxy-2-methyl-8-((1-[(11)C]-(E)-2-methyl-but-2-enoyl)oxy)-1,2,6,7,8,8a-hexahydronaphthalen-1-yl)heptanoate ([(11)C]DPV), as a positron emission tomography (PET) probe for noninvasive measurement of hepatobiliary transport, and conducted pharmacokinetic analysis in rats as a feasibility study for future clinical study. Transport activities of DPV in freshly isolated rat hepatocytes and rodent multidrug resistance-associated protein 2 (rMrp2; human, MRP2)-expressing membrane vesicles were similar to those of pravastatin. Rifampicin diminished the uptake of DPV and pravastatin by the hepatocytes, with similar inhibition potency. [(11)C]DPV underwent biotransformation to produce at least two metabolites in rat, but metabolism of [(11)C]DPV occurred negligibly in human hepatocytes during a 90-minute incubation. After intravenous injection, [(11)C]DPV was mainly distributed to the liver and kidneys, where the tissue uptake clearances (CLuptake,liver and CLuptake,kidney) were blood-flow-limited (73.6 ± 4.8 and 24.6 ± 0.6 ml/min per kilogram, respectively). Systemic elimination of [(11)C]DPV was delayed in rifampicin-treated rat and an Mrp2-deficient mutant rat, Eisai hyperbilirubinemic mutant rat (EHBR). Rifampicin treatment decreased both CLuptake,liver and CLuptake,kidney of [(11)C]DPV by 30% (P < 0.05), whereas these parameters were unchanged in EHBR. Meanwhile, the canalicular efflux clearance (CLint,bile) of [(11)C]DPV, which was 12.2 ± 1.5 ml/min per kilogram in the control rat, decreased by 60% and 89% in rifampicin-treated rat and EHBR (P < 0.05), respectively. These results indicate that [(11)C]DPV is taken up into the liver by organic anion-transporting polypeptides (rodent, Oatps; human, OATP) and excreted into bile by Mrp2 in rat, and that rifampicin may inhibit Mrp2 as well as Oatps, and consequently increase systemic exposure of [(11)C]DPV. PET using [(11)C]DPV is feasible for studies prior to the future clinical investigation of OATP and MRP2 functionality, especially for personalized medicine.

Organic anion transporter 3 mediates the efflux transport of an amphipathic organic anion, dehydroepiandrosterone sulfate, across the blood-brain barrier in mice.

The present study investigated the efflux transport systems of organic anions across the blood-brain barrier (BBB) using dehydroepiandrosterone sulfate (DHEAS) as a probe. The elimination of DHEAS from the brain after microinjection into the cerebral cortex was characterized in wild-type mice and mice with deficiency of well characterized organic anion transporters, organic anion-transporting polypeptide 1a4 (Oatp1a4)/Slco1a4 and organic anion transporter 3 (Oat3)/Slc22a8, at the BBB. The saturable efflux of DHEAS from the brain was completely inhibited by probenecid, benzylpenicillin, and estrone-3-sulfate and moderately inhibited by taurocholate and p-aminohippurate (50-57%). Uptake of DHEAS and estrone-3-sulfate was greater in murine Oat3 cRNA-injected oocytes than that in water-injected oocytes. Efflux of these compounds from the brain was significantly delayed in Oat3(-/-) mice compared with that in wild-type mice, indicating that indeed Oat3 is functionally important in vivo. Furthermore, probenecid and taurocholate inhibited DHEAS efflux completely in Oat3(-/-) mice. Contrary to the past report in rats that suggested involvement of Oatp1a4, specific uptake of DHEAS and estrone-3-sulfate by murine Oatp1a4 was not detected in vitro, and efflux of both compounds from the brain was not altered in Oatp1a4(-/-) mice. There was no significant difference in the uptake of DHEAS by brain slices prepared from wild-type, Oatp1a4(-/-), and Oat3(-/-) mice. Taken together, these results suggest that Oat3 plays a significant role in the efflux of steroid conjugates across the BBB in mice and that the BBB also expresses other unknown organic anion transporters for the efflux of DHEAS. Transport mechanisms of organic anions at the BBB are far more diverse than they were assumed to be.

Effect of breast cancer resistance protein (Bcrp/Abcg2) on the disposition of phytoestrogens.

The effect of breast cancer resistance protein (Bcrp/Abcg2) on the disposition of the phytoestrogens daidzein, genistein, and coumestrol was investigated using Bcrp(-/-) mice. Expression of the genes for either mouse Bcrp or human BCRP in MDCK II cells induced apically directed transport of the three phytoestrogens, whereas their transcellular transport was identical in mock and LLC-PK1 cells expressing mouse Mdr1a. After oral administration, the plasma levels of daidzein and genistein were increased in Bcrp(-/-) mice, but only a minimal change was observed for coumestrol. At steady state, tissue-to-plasma concentration ratios of the three phytoestrogens in the brain and testis of wild-type mice were very small and similar to those of [(14)C]inulin, whereas those were significantly increased in the brain and testis of Bcrp(-/-) mice. The largest increases were observed with genistein (9.2- and 5.8-fold in the brain and testis, respectively). The distributions of genistein in the epididymis and fetus, but not the ovary, were also increased in Bcrp(-/-) mice. The Bcrp protein was localized in the luminal membrane of the endothelial cells in the testis and the body of the epididymis and in both the luminal and abluminal side of ducts in the head of the epididymis. These results suggest that Bcrp limits the oral availability and distribution into the brain and testis, epididymis, and fetus of phytoestrogens.

Rapid elimination of cefaclor from the cerebrospinal fluid is mediated by a benzylpenicillin-sensitive mechanism distinct from organic anion transporter 3.

The purpose of this study was to investigate the carrier-mediated elimination of cephalosporins from the cerebrospinal fluid (CSF) via the choroid plexus. Cefaclor and cefalexin are structural analogs with similar lipophilicity, differing by only one functional group (cefaclor, -Cl; cephalexin, -CH(3)), and they are substrates of rat peptide transporter PEPT2 with similar transport activities. However, cefaclor was cleared from the CSF more rapidly than cefalexin after intracerebroventricular administration (the elimination rate constants were 0.11 and 0.050 min(-1), respectively). The elimination of cefaclor from the CSF was inhibited by benzylpenicillin, but not by glycylsarcosine (GlySar), whereas GlySar, but not benzylpenicillin, had an inhibitory effect on the elimination of cefalexin from the CSF. The uptake of cefaclor by the freshly isolated rat choroid plexus was saturable, with a K(m) value of 250 muM, and the uptake clearance corresponding to saturable components accounts for the major part of the in vivo clearance from the CSF (17 versus 26 mul/min, respectively). The uptake of cefaclor by the choroid plexus was inhibited by benzylpenicillin, but not by GlySar. However, the inhibitory effect of benzylpenicillin was weaker than expected from its own K(m) value, and furthermore, organic anion transporter (Oat)3 substrates (cimetidine or p-aminohippurate) had no effect. These results suggest that cefaclor and cefalexin are eliminated from the CSF by different transporters, and rapid elimination of cefaclor from the CSF is accounted for by a benzylpenicillin-sensitive mechanism distinct from Oat3. A slight modification of a single chemical group of cephalosporins can greatly affect the contribution of the transporters involved, and their duration in the CSF.

Characterization of uremic toxin transport by organic anion transporters in the kidney.

BACKGROUND: Harmful uremic toxins, such as indoxyl sulfate (IS), 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), indoleacetate (IA), and hippurate (HA), accumulate to a high degree in uremic plasma. IS has been shown to be a substrate of rat organic anion transporter 1 (rOat1) and rOat3. However, the contribution of rOat1 and rOat3 to the renal uptake transport process of IS and other uremic toxins in the kidney remains unknown. METHODS: The cellular uptake of uremic toxins was determined using stable transfectants of rOat1/hOAT1 and rOat3/hOAT3 cells. Also, the uptake of uremic toxins by rat kidney slices was characterized to evaluate the contribution of rOat1 and rOat3 to the total uptake by kidney slices using inhibitors of rOat1 (p-aminohippurate) and rOat3 (pravastatin and benzylpenicillin). RESULTS: Saturable uptake of IS, CMPF, IA, and HA by rOat1 was observed with Km values of 18, 154, 47, and 28 micromol/L, respectively, whereas significant uptake of IS and CMPF, but not of IA or HA, was observed in rOat3-expressing cells with Km values of 174 and 11 micromol/L, respectively. Similar parameters were obtained for human OAT1 and OAT3. Kinetic analysis of the IS uptake by kidney slices revealed involvement of two saturable components with Km1 (24 micromol/L) and Km2 (196 micromol/L) values that were comparable with those of rOat1 and rOat3. The Km value of CMPF uptake by kidney slices (22 micromol/L) was comparable with that of rOat3, while the corresponding values of IA and HA (42 and 33 micromol/L, respectively) were similar to those of rOat1. PAH preferentially inhibited the uptake of IA and HA by kidney slices, while pravastatin and benzylpenicillin preferentially inhibited the uptake of CMPF. The effect of these inhibitors on the uptake of IS by kidney slices was partial. CONCLUSIONS: rOat1/hOAT1 and rOat3/hOAT3 play major roles in the renal uptake of uremic toxins on the basolateral membrane of the proximal tubules. Both OAT1 and OAT3 contribute almost equally to the renal uptake of IS. OAT3 mainly accounts for CMPF uptake by the kidney, while OAT1 mainly accounts for IA and HA uptake.