Torreya grandis Kernel Oil Alleviates Loperamide-Induced Slow Transit Constipation via Up-Regulating the Colonic Expressions of Occludin/Claudin-1/ZO-1 and 5-HT3R/5-HT4R in BALB/c Mice.

SCOPE: Torreya grandis kernel has traditionally been used to remove intestinal parasites and increases intestinal motility. However, the effect of Torreya grandis kernel oil (TKO) on constipation has not yet been investigated. Therefore, mouse model is used to investigate the effect of TKO on slow transit constipation (STC) and its possible mechanism. METHODS AND RESULTS: The effects of TKO on intestinal motility of STC mice are evaluated by fecal weight, fecal water content, colon length, defecation test, and intestinal propulsion test. The mechanism of TKO alleviating STC is explored by detecting biochemical analysis, histological analysis, western blot, qRT-PCR, immunohistochemistry, and gut microbiota analysis. The results reveal that TKO effectively promotes defecation and intestinal motility, increases the level of endothelin-1, and restores the histopathological morphology of the colon under LOP pretreatment. The expression levels of occludin, claudin-1, and zonula occludens-1 (ZO-1) mRNA and protein are up-regulated in mice receiving TKO treatment. The colonic 5-hydroxytryptamine 3R/4R (5-HT3R/5-HT4R) expressions are also increased by TKO supplementation. Additionally, TKO rescues LOP-caused disorders of the gut microbiota. CONCLUSION: Consumption of TKO is beneficial to STC recovery, and it can alleviate LOP-induced STC by up-regulating the colonic expressions of Occludin/Claudin-1/ZO-1 and 5-HT3R/5-HT4R.

Hetong decoction relieves loperamide-induced constipation in rats by regulating expression of aquaporins.

OBJECTIVE: To investigate whether Hetong decoction (, HTT) alleviates constipation via regulating AQPs expression. METHODS: Constipation in rats was induced by loperamide, and rats were randomly assigned into model (saline), HHT-low (95 g/kg), HTT-medium (190 g/kg), HTT-high (380 g/kg) and positive control (mosapride) groups. Then the defecation function, the concentration of serum arginine vasopressin (AVP) and cyclic adenosine monophosphate (cAMP), and the expression of AQP3 and AQP8 in colon tissues were assessed. NCM460 colon cells with AQP3 and AQP8 knockdown or overexpression were exposed to serum from rats that received low or high dose of HTT, followed by detection of AQP3 and AQP8 expression. RESULTS: The model group showed lower fecal weight and water content, weaker intestinal transit, higher serum concentration of AVP and cAMP, increased proximal and distal AQP8 expression, increased proximal but decreased distal AQP3 expression. However, these trends were reversed in both the HTT group (low, medium and high dose) and the positive control group. In NCM460 cells, HTT dose-dependently stabilized AQP3 and AQP8 expression under AQP3/8 plasmid interference or overexpression. CONCLUSIONS: HTT relieves constipation in rats through regulating AQP3 and AQP8 expression.

Proteasome inhibition protects blood-brain barrier P-glycoprotein and lowers Aß brain levels in an Alzheimer's disease model.

BACKGROUND: Loss of P-glycoprotein (P-gp) at the blood-brain barrier contributes to amyloid-ß (Aß) brain accumulation in Alzheimer's disease (AD). Using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576), we previously showed that Aß triggers P-gp loss by activating the ubiquitin-proteasome pathway, which leads to P-gp degradation. Furthermore, we showed that inhibiting the ubiquitin-activating enzyme (E1) prevents P-gp loss and lowers Aß accumulation in the brain of hAPP mice. Based on these data, we hypothesized that repurposing the FDA-approved proteasome inhibitor, bortezomib (Velcade®; BTZ), protects blood-brain barrier P-gp from degradation in hAPP mice in vivo. METHODS: We treated hAPP mice with the proteasome inhibitor BTZ or a combination of BTZ with the P-gp inhibitor cyclosporin A (CSA) for 2 weeks. Vehicle-treated wild-type (WT) mice were used as a reference for normal P-gp protein expression and transport activity. In addition, we used the opioid receptor agonist loperamide as a P-gp substrate in tail flick assays to indirectly assess P-gp transport activity at the blood-brain barrier in vivo. We also determined P-gp protein expression by Western blotting, measured P-gp transport activity levels in isolated brain capillaries with live cell confocal imaging and assessed Aß plasma and brain levels with ELISA. RESULTS: We found that 2-week BTZ treatment of hAPP mice restored P-gp protein expression and transport activity in brain capillaries to levels found in WT mice. We also observed that hAPP mice displayed significant loperamide-induced central antinociception compared to WT mice indicating impaired P-gp transport activity at the blood-brain barrier of hAPP mice in vivo. Furthermore, BTZ treatment prevented loperamide-induced antinociception suggesting BTZ protected P-gp loss in hAPP mice. Further, BTZ-treated hAPP mice had lower Aß40 and Aß42 brain levels compared to vehicle-treated hAPP mice. CONCLUSIONS: Our data indicate that BTZ protects P-gp from proteasomal degradation in hAPP mice, which helps to reduce Aß brain levels. Our data suggest that the proteasome system could be exploited for a novel therapeutic strategy in AD, particularly since increasing Aß transport across the blood-brain barrier may prove an effective treatment for patients.

Loperamide induces excessive accumulation of bile acids in the liver of mice with different diets.

Loperamide is a non-prescription medicine normally used for the treatment of diarrhea. The abuse and misuse of loperamide have been demonstrated to have toxic effects on heart. It is still unclear whether the abuse of loperamide can cause hepatic toxicity. The C57BL/6 mice fed with high fat diet (HFD) or normal food diet (NFD) were administrated with loperamide (5 mg/kg/day) intragastrically once a day for two weeks, after that, the feces, blood, hepatic tissues and intestines were harvested for biochemical and histological detection, and the expression of genes related with lipid metabolism was further checked by qRT-PCR (quantitative real-time polymerase chain reaction) and Western blot. The administration of loperamide caused the constipation in mice fed with NFD or HFD. The content of bile acids was significantly reduced in the feces of mice treated with loperamide, but the content of bile acids was significantly increased in the liver of these mice. The results of H&E staining showed that loperamide administration caused the damage of hepatic tissues, especially for mice fed with HFD. The expression of genes related with the biosynthesis of cholesterol and bile acids, including Hmgcr, Lss, Sqle, Fdps, Idi1, Mvk, Cyp7a1 and Ch25h, was all upregulated in the liver of mice treated with loperamide. Conversely, the expression of Abcg5, Abcb11 and Abcc2, which encode genes for transporting cholesterols and bile acids from hepatocytes to bile respectively, was downregulated in the liver of mice treated with loperamide. At the same time, the expression of Fabp6 and Slc51a, which transport bile acids from intestinal lumen into the blood, was all upregulated in the ileum of mice treated with loperamide. The expression of SHP, which inhibits the transcription of Cyp7a1 in hepatocytes, was significantly downregulated in the hepatic tissues of mice treated with loperamide. These results demonstrated that administration of loperamide caused excessive accumulation of bile acids in the liver of mice via upregulating genes for biosynthesis of cholesterol and bile acid and downregulating genes for discharging cholesterol and bile acids in hepatocytes of mice, moreover, the downregulation of SHP in hepatic tissues might be one of the mechanisms of it, especially for mice fed with HFD.

DARK Classics in Chemical Neuroscience: Loperamide.

Loperamide, a popular and inexpensive over-the-counter antidiarrheal medicine, is a potent µ-opioid receptor agonist approved by the U.S. Food and Drug Administration (FDA). It has been on the market since 1976 and is relatively safe with no central nervous system-related side effects when used for a short period of time at the recommended therapeutic dose (2-8 mg/day). In recent years, loperamide has become notoriously known as the "poor man's methadone" for people with substance dependence due to the increase in loperamide overdoses from self-administered medication to treat opioid withdrawal symptoms. As a result, in 2018, the FDA decided to limit the available packaged dose of loperamide to stop prominent abuse. This review provides the synthesis and chemical properties of loperamide as well as the pharmacology and adverse effects of its use and the social effects of such abuse.

Investigation of the effects of axitinib on the pharmacokinetics of loperamide and its main metabolite N-demethylated loperamide in rats by UPLC-MS/MS.

The epidemic of loperamide abuse and misuse in the patients for the alternative to opioids has become an increasing worldwide concern and has led to considerations about the potential for drug-drug interactions between loperamide and other combined drugs, especially inhibitors of cytochrome P450 (CYP450) enzymes, such as axitinib. This study assessed the effects of axitinib on the metabolism of loperamide and its main metabolite N-demethylated loperamide in rats and in rat liver microsomes (RLM), human liver microsomes (HLM) and recombinant human CYP3A4*1. The concentrations of both compounds were determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The exposures (AUC(0-t), AUC(0-∞) and Cmax) of loperamide and N-demethylated loperamide showed a conspicuous increase when loperamide was co-administered with axitinib. The Tmax of loperamide increased while CLz/F decreased under the influence of axitinib. In vitro, axitinib inhibited loperamide metabolism with the IC50 of 18.34 µM for RLM, 1.705 µM for HLM and 1.604 µM for CYP3A4*1, and it was confirmed as a non-competitive inhibitor in all enzymes. Taken together, the results indicated that axitinib had an obvious inhibitory impact on loperamide metabolism both in vivo and in vitro. Thus, more attention should be paid to the concurrent use of loperamide and axitinib to reduce the risk of unexpected clinical outcomes.

Measuring ligand efficacy at the mu-opioid receptor using a conformational biosensor.

The intrinsic efficacy of orthosteric ligands acting at G-protein-coupled receptors (GPCRs) reflects their ability to stabilize active receptor states (R*) and is a major determinant of their physiological effects. Here, we present a direct way to quantify the efficacy of ligands by measuring the binding of a R*-specific biosensor to purified receptor employing interferometry. As an example, we use the mu-opioid receptor (µ-OR), a prototypic class A GPCR, and its active state sensor, nanobody-39 (Nb39). We demonstrate that ligands vary in their ability to recruit Nb39 to µ-OR and describe methadone, loperamide, and PZM21 as ligands that support unique R* conformation(s) of µ-OR. We further show that positive allosteric modulators of µ-OR promote formation of R* in addition to enhancing promotion by orthosteric agonists. Finally, we demonstrate that the technique can be utilized with heterotrimeric G protein. The method is cell-free, signal transduction-independent and is generally applicable to GPCRs.

Retina Compatible Interactions and Effective Modulation of Blood Ocular Barrier P-gp Activity by Third-Generation Inhibitors Improve the Ocular Penetration of Loperamide.

Effective drug delivery to the deeper ocular tissues remains an unresolved conundrum mainly due to the expression of multidrug resistance efflux proteins, besides tight junction proteins, in the blood ocular barriers (BOBs). Hence, the purpose of the current research was to investigate the ability of the third-generation efflux protein inhibitors, elacridar (EQ), and tariquidar (TQ), to diminish P-glycoprotein (P-gp) mediated efflux transport of loperamide (LOP), a P-gp substrate, across the BOB in Sprague Dawley rats. Initially, Western blot analysis confirmed the expression of P-gp in the iris-ciliary bodies and the retina choroid in the wild type rats. Next, the ocular distribution of LOP, in the presence and absence of EQ/TQ (at 2 doses), was evaluated. The significantly higher aqueous humor/plasma (DAH) and vitreous humor (VH)/plasma (DVH) distribution ratios of LOP in the rats pretreated with EQ or TQ demonstrated effective inhibition of P-gp activity in the BOB. Interestingly, the modulation of P-gp activity by EQ/TQ was more pronounced at the lower dose. The normal functioning and architecture of the retina, as indicated by electroretinography studies, confirmed the cytocompatibility of LOP and EQ/TQ interactions at the doses tested.

A Conformationally Gated Model of Methadone and Loperamide Transport by P-Glycoprotein.

P-glycoprotein (Pgp) is a multidrug resistance transporter that limits the penetration of a wide range of neurotherapeutics into the brain including opioids. The diphenylpropylamine opioids methadone and loperamide are structurally similar, but loperamide has about a 4-fold higher Pgp-mediated transport rate. In addition to these differences, they showed significant differences in their effects on Pgp-mediated adenosine triphosphate (ATP) hydrolysis. The activation of Pgp-mediated ATP hydrolysis by methadone was monophasic, whereas loperamide activation of ATP hydrolysis was biphasic implying methadone has a single binding site and loperamide has 2 binding sites on Pgp. Quenching of tryptophan fluorescence with these drugs and digoxin showed competition between the opioids and that loperamide does not compete for the digoxin-binding site. Acrylamide quenching of tryptophan fluorescence to probe Pgp conformational changes revealed that methadone- and loperamide-induced conformational changes were distinct. These results were used to develop a model for Pgp-mediated transport of methadone and loperamide where opioid binding and conformational changes are used to explain the differences in the opioid transport rates between methadone and loperamide.

P-glycoprotein Restricts Ocular Penetration of Loperamide across the Blood-Ocular Barriers: a Comparative Study in Mdr1a Knock-out and Wild Type Sprague Dawley Rats.

The current research was undertaken to determine the existence and magnitude of P-glycoprotein (P-gp) expression on the blood-ocular barriers by studying the ocular penetration of loperamide, a specific P-gp substrate, in P-gp (Mdr1a) knock-out (KO) and wild type (WT) Sprague Dawley rats. A clear, stable, sterile solution of loperamide (1 mg/mL), for intravenous administration, was formulated and evaluated. Ocular distribution was studied in P-gp KO and WT rats following intravenous administration of loperamide (at two doses). The drug levels in plasma, aqueous humor (AH), and vitreous humor (VH) samples were determined with the aid of UHPLC-Q-TOF-MS/MS, and the AH/plasma (D AH ) and VH/plasma (D VH ) distribution ratios were estimated. Electroretinography (ERG), ultrastructural analyses, and histology studies were carried out, in both KO and WT rats, to detect any drug-induced functional and/or structural alterations in the retina. Dose-related loperamide levels were observed in the plasma of both WT and KO rats. The loperamide concentrations in the AH and VH of KO rats were significantly higher compared to that observed in the WT rats, at the lower dose. However, a marked increase in the D AH and D VH was noted in the KO rats. ERG, ultrastructure, and histology studies did not indicate any drug-induced toxic effects in the retina under the test conditions. The results from these studies demonstrate that P-gp blocks the penetration of loperamide into the ocular tissues from the systemic circulation and that the effect is more pronounced at lower plasma loperamide concentrations.

Exploiting Bacterial Pathways for BBB Crossing with PLGA Nanoparticles Modified with a Mutated Form of Diphtheria Toxin (CRM197): In Vivo Experiments.

Drugs can be targeted to the brain using polymeric nanoparticles (NPs) engineered on their surface with ligands able to allow crossing of the blood-brain barrier (BBB). This article aims to investigate the BBB crossing efficiency of polymeric poly lactide-co-glycolide (PLGA) NPs modified with a mutated form of diphtheria toxin (CRM197) in comparison with the results previously obtained using PLGA NPs modified with a glycopeptide (g7-NPs). Different kinds of NPs, covalently coupled PLGA with different fluorescent probes (DY405, rhodamine-B base and DY675) and different ligands (g7 and CRM197) were tested in vivo to assess their behavior and trafficking. The results highlighted the possibility to distinguish the different kinds of simultaneously administered NPs and to emphasize that CRM-197 modified NPs and g7-NPs can cross the BBB at a similar extent. The analysis of BBB crossing and of the neuronal tropism of CRM197 modified NPs, along with their BBB crossing pathways were also developed. In vivo pharmacological studies performed on CRM197 engineered NPs, loaded with loperamide, underlined their ability as drug carriers to the CNS.

Factors Governing P-Glycoprotein-Mediated Drug-Drug Interactions at the Blood-Brain Barrier Measured with Positron Emission Tomography.

The adenosine triphosphate-binding cassette transporter P-glycoprotein (ABCB1/Abcb1a) restricts at the blood-brain barrier (BBB) brain distribution of many drugs. ABCB1 may be involved in drug-drug interactions (DDIs) at the BBB, which may lead to changes in brain distribution and central nervous system side effects of drugs. Positron emission tomography (PET) with the ABCB1 substrates (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide and the ABCB1 inhibitor tariquidar has allowed direct comparison of ABCB1-mediated DDIs at the rodent and human BBB. In this work we evaluated different factors which could influence the magnitude of the interaction between tariquidar and (R)-[(11)C]verapamil or [(11)C]-N-desmethyl-loperamide at the BBB and thereby contribute to previously observed species differences between rodents and humans. We performed in vitro transport experiments with [(3)H]verapamil and [(3)H]-N-desmethyl-loperamide in ABCB1 and Abcb1a overexpressing cell lines. Moreover we conducted in vivo PET experiments and biodistribution studies with (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide in wild-type mice without and with tariquidar pretreatment and in homozygous Abcb1a/1b((-/-)) and heterozygous Abcb1a/1b((+/-)) mice. We found no differences for in vitro transport of [(3)H]verapamil and [(3)H]-N-desmethyl-loperamide by ABCB1 and Abcb1a and its inhibition by tariquidar. [(3)H]-N-Desmethyl-loperamide was transported with a 5 to 9 times higher transport ratio than [(3)H]verapamil in ABCB1- and Abcb1a-transfected cells. In vivo, brain radioactivity concentrations were lower for [(11)C]-N-desmethyl-loperamide than for (R)-[(11)C]verapamil. Both radiotracers showed tariquidar dose dependent increases in brain distribution with tariquidar half-maximum inhibitory concentrations (IC50) of 1052 nM (95% confidence interval CI: 930-1189) for (R)-[(11)C]verapamil and 1329 nM (95% CI: 980-1801) for [(11)C]-N-desmethyl-loperamide. In homozygous Abcb1a/1b((-/-)) mice brain radioactivity distribution was increased by 3.9- and 2.8-fold and in heterozygous Abcb1a/1b((+/-)) mice by 1.5- and 1.1-fold, for (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide, respectively, as compared with wild-type mice. For both radiotracers radiolabeled metabolites were detected in plasma and brain. When brain and plasma radioactivity concentrations were corrected for radiolabeled metabolites, brain distribution of (R)-[(11)C]verapamil and [(11)C]-N-desmethyl-loperamide was increased in tariquidar (15 mg/kg) treated animals by 14.1- and 18.3-fold, respectively, as compared with vehicle group. Isoflurane anesthesia altered [(11)C]-N-desmethyl-loperamide but not (R)-[(11)C]verapamil metabolism, and this had a direct effect on the magnitude of the increase in brain distribution following ABCB1 inhibition. Our data furthermore suggest that in the absence of ABCB1 function brain distribution of [(11)C]-N-desmethyl-loperamide but not (R)-[(11)C]verapamil may depend on cerebral blood flow. In conclusion, we have identified a number of important factors, i.e., substrate affinity to ABCB1, brain uptake of radiolabeled metabolites, anesthesia, and cerebral blood flow, which can directly influence the magnitude of ABCB1-mediated DDIs at the BBB and should therefore be taken into consideration when interpreting PET results.

Determination of loperamide in human plasma and saliva by liquid chromatography-tandem mass spectrometry.

A simple and sensitive liquid chromatography-tandem mass spectrometric method for quantification of loperamide in human plasma and saliva was developed and validated, and then successfully applied in pharmacokinetic clinical study to investigate and correlate bioavailability of Imodium(®) 2mg quartet tablet dose in both human plasma and saliva. Loperamide with labeled internal standard was extracted from its biological matrix by methanol as protein direct precipitant in single extraction step. Adequate chromatographic separation for analytes from plasma and saliva matrices was achieved using ACE C18 (50mm×2.1mm, 5µm) column, eluted by water/methanol/formic acid (30:70:0.1%, v/v), delivered isocratically at constant flow rate of 0.75ml/min. The method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to European guideline, and partial validation was applied on saliva, specificity, matrix effect, recovery, sensitivity, within and between day precision and accuracy. The calibration curve was linear through the range of 20-3000pg/ml in both plasma and saliva using a 50µl sample volume. The partial validation sections outcome in saliva was so close to those in plasma. The within- and between-day precisions were all below 8.7% for plasma and below 11.4% for saliva. Accuracies ranged from 94 to 105% for both matrices. In this study, 26 healthy volunteers participated in the clinical study, and 6 of gave their saliva samples in addition to plasma at the same time schedule. The pharmacokinetic parameters of Cmax, AUC0-t and AUC0-∞, Tmax and T1/2 in both plasma and saliva were calculated and correlated.

Gastrointestinal transfer: in vivo evaluation and implementation in in vitro and in silico predictive tools.

INTRODUCTION: The purpose of this study was to explore the transfer of drug solutions from stomach to small intestine and its impact on intraluminal drug concentrations in humans. The collected intraluminal data were used as reference to evaluate simulations of gastrointestinal transfer currently implemented in different in vitro and in silico absorption models. METHODS: Gastric and duodenal concentrations of the highly soluble and non-absorbable compound paromomycin were determined following oral administration to 5 healthy volunteers under the following conditions: fasted state, fed state and fed state in the presence of a transit-stimulating (domperidone) or transit-inhibiting (loperamide) agent. Based on the obtained intraluminal concentration-time profiles, gastrointestinal transfer (expressed as the half-life of gastric emptying) was analyzed using physiologically-based parameter estimation in Simcyp®. Subsequently, the observed transfer profiles were used to judge the implementation of gastrointestinal transfer in 2 in vitro simulation tools (the TNO Intestinal Model TIM-1 and a three-compartmental in vitro model) and the Simcyp® population-based PBPK modeling platform. RESULTS: The observed duodenal concentration-time profile of paromomycin under fasting conditions, with a high average Cmax obtained after 15 min, clearly indicated a fast transfer of drug solutions from stomach to duodenum (estimated gastric half-life between 4 and 13 min). The three-compartmental in vitro model adequately reflected the in vivo fasted state gastrointestinal transfer of paromomycin. For both TIM-1 and Simcyp®, modifications in gastric emptying and dilutions were required to improve the simulation of the transfer of drug solutions. As expected, transfer from stomach to duodenum was delayed in the fed state, resulting in lower duodenal paromomycin concentrations and an estimated gastric half-life between 21 and 40 min. Administration of domperidone or loperamide as transit-stimulating and transit-inhibiting agent, respectively, did not affect the fed state gastric half-life of emptying. CONCLUSION: For the first time, the impact of gastrointestinal transfer of solutions on intraluminal drug concentrations was directly assessed in humans. In vitro and in silico simulation tools have been validated and optimized using the in vivo data as reference.

Blood-brain barrier pharmacoproteomics-based reconstruction of the in vivo brain distribution of P-glycoprotein substrates in cynomolgus monkeys.

The aim of this study was to investigate whether in vivo drug distribution in brain in monkeys can be reconstructed by integrating four factors: protein expression levels of P-glycoprotein (P-gp)/multidrug resistance protein 1 at the blood-brain barrier (BBB), in vitro transport activity per P-gp molecule, and unbound drug fractions in plasma and brain. For five P-gp substrates (indinavir, quinidine, loperamide, paclitaxel, and verapamil) and one nonsubstrate (diazepam), in vitro P-gp transport activities were determined by measuring transcellular transport across monolayers of cynomolgus monkey P-gp-transfected LLC-PK1 and parental cells. In vivo P-gp functions at the BBB were reconstructed from in vitro P-gp transport activities and P-gp expression levels in transfected cells and cynomolgus brain microvessels. Brain-to-plasma concentration ratios (Kp,brain) were reconstructed by integrating the reconstructed in vivo P-gp functions with drug unbound fractions in plasma and brain. For all compounds, the reconstructed Kp,brain values were within a 3-fold range of observed values, as determined by constant intravenous infusion in adult cynomolgus monkeys. Among four factors, plasma unbound fraction was the most sensitive factor to species differences in Kp,brain between monkeys and mice. Unbound brain-to-plasma concentration ratios (Kp,uu,brain) were reconstructed as the reciprocal of the reconstructed in vivo P-gp functions, and the reconstructed Kp,uu,brain values were within a 3-fold range of in vivo values, which were estimated from observed Kp,brain and unbound fractions. This study experimentally demonstrates that brain distributions of P-gp substrates and nonsubstrate can be reconstructed on the basis of pharmacoproteomic concept in monkeys, which serve as a robust model of drug distribution in human brain.

Multilayer spheroids to quantify drug uptake and diffusion in 3D.

There is a need for new quantitative in vitro models of drug uptake and diffusion to help assess drug toxicity/efficacy as well as new more predictive models for drug discovery. We report a three-dimensional (3D) multilayer spheroid model and a new algorithm to quantitatively study uptake and inward diffusion of fluorescent calcein via gap junction intercellular communication (GJIC). When incubated with calcein-AM, a substrate of the efflux transporter P-glycoprotein (Pgp), spheroids from a variety of cell types accumulated calcein over time. Accumulation decreased in spheroids overexpressing Pgp (HEK-MDR) and was increased in the presence of Pgp inhibitors (verapamil, loperamide, cyclosporin A). Inward diffusion of calcein was negligible in spheroids that lacked GJIC (OVCAR-3, SK-OV-3) and was reduced in the presence of an inhibitor of GJIC (carbenoxolone). In addition to inhibiting Pgp, verapamil and loperamide, but not cyclosporin A, inhibited inward diffusion of calcein, suggesting that they also inhibit GJIC. The dose response curves of verapamil's inhibition of Pgp and GJIC were similar (IC50: 8 µM). The method is amenable to many different cell types and may serve as a quantitative 3D model that more accurately replicates in vivo barriers to drug uptake and diffusion.

Transport inhibition of digoxin using several common P-gp expressing cell lines is not necessarily reporting only on inhibitor binding to P-gp.

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.

Advances in PET imaging of P-glycoprotein function at the blood-brain barrier.

Efflux transporter P-glycoprotein (P-gp) at the blood-brain barrier (BBB) restricts substrate compounds from entering the brain and may thus contribute to pharmacoresistance observed in patient groups with refractory epilepsy and HIV. Altered P-gp function has also been implicated in neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Positron emission tomography (PET), a molecular imaging modality, has become a promising method to study the role of P-gp at the BBB. The first PET study of P-gp function was conducted in 1998, and during the past 15 years two main categories of P-gp PET tracers have been investigated: tracers that are substrates of P-gp efflux and tracers that are inhibitors of P-gp function. PET, as a noninvasive imaging technique, allows translational research. Examples of this are preclinical investigations of P-gp function before and after administering P-gp modulating drugs, investigations in various animal and disease models, and clinical investigations regarding disease and aging. The objective of the present review is to give an overview of available PET radiotracers for studies of P-gp and to discuss how such studies can be designed. Further, the review summarizes results from PET studies of P-gp function in different central nervous system disorders.

Liquid chromatography-tandem mass spectrometry for analysis of intestinal permeability of loperamide in physiological buffer.

Analysis of in vitro samples with high salt concentrations represents a major challenge for fast and specific quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To investigate the intestinal permeability of opioids in vitro employing the Ussing chamber technique, we developed and validated a fast, sensitive and selective method based on LC-MS/MS for the determination of loperamide in HEPES-buffered Ringer's solution. Chromatographic separation was achieved with an Atlantis dC18 column, 2.1 mm×20 mm, 3 µm particle size and a gradient consisting of methanol/0.1% formic acid and ammonium acetate. The flow rate was 0.7 ml/min, and the total run time was 3 min. For quantification, two mass transitions for loperamide and a deuterated internal standard (methadone-d(3)) were used. The lower limit of loperamide quantification was 0.2 ng/ml. This new LC-MS/MS method can be used for the detection of loperamide in any experimental setup using HEPES-buffered Ringer's solution as a matrix compound.

[Evaluation of P-glycoprotein mediated in vitro loperamide biliary excretion with sandwich-cultured rat hepatocytes model].

An in vitro P-glycoprotein mediated drug biliary excretion model (B-Clear model) was developed and validated using sandwich-cultured rat hepatocytes (SCRH) and a model substrate rhodamine 123 (Rh123). SCRH formed functional bile canalicular networks after 5 days of culture. Rh123 (10 micromol x L(-1)) was then incubated with the SCRH in standard Ca+ Hanks buffer or Ca(2+)-free buffer. The cumulative cell uptake and canalicular efflux of Rh123 under Ca2+ and Ca(2+)-free conditions were measured with a LC-MS/MS method. The biliary excretion index (BEI) and instinct biliary clearance (CL(bile, int)) were calculated. To assess the effect of known P-gp inhibitors on the efflux of Rh123, cyclosporine A (CyA), tariquidar (TQD) or quinidine (QND) (10, 50 and 100 micromol x L(-1)) was pre-incubated separately with SCRH for 30 min, then co-incubated with Rh123. The BEI and CL(bile, int) of Rh123 obtained from the SCRH model were (17.8 +/- 1.3) % and (10.7 +/- 0.9) mL x min(-1) x kg(-1), respectively. All the three P-gp inhibitors showed a dose-dependent inhibition on the bile clearance of Rh123, indicating that the B-Clear model with SCRH was functional properly. The biliary excretion of loperamide (LPAD) and the role of P-gp were further investigated with this validated model. The BEI and CL(bile, int) for LPAD (20 micromol x L(-1)) were obtained after it was incubated with SCRH for 30 min, and found to be (12.9 +/- 1.2)% and (6.1 +/- 0.3) mL x min(-1) x kg(-1) respectively. The dose-dependent inhibition on LPAD biliary excretion by CyA, TQD or QND confirmed the major role of P-gp in LPAD canalicular efflux. The results suggested that the B-Clear model with SCRH would be a useful tool for evaluation of P-gp mediated efflux and drug-drug interaction.