Cytochrome p450 inhibitory properties of common efflux transporter inhibitors.

Drug transporter inhibitors are important tools to elucidate the contribution of transporters to drug disposition both in vitro and in vivo. These inhibitors are often unselective and affect several transporters as well as drug metabolizing enzymes, which can make experimental results difficult to interpret with confidence. We therefore tested 14 commonly used P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug-resistance associated protein (MRP) inhibitors as inhibitors of cytochrome P450 (P450) enzyme activities using recombinant enzymes. A subset of P-gp and/or CYP3A inhibitors were selected (cyclosporin A, elacridar, ketoconazole, quinidine, reserpine, and tacrolimus) for a comparison of P450 inhibition in human microsomes and hepatocytes. Most P-gp inhibitors showed CYP3A4 inhibition, with potencies often in a similar range as their P-gp inhibition, as well as less potent CYP2C19 inhibition. Other P450 enzymes were not strongly inhibited except a few cases of CYP2D6 inhibition. MRP and BCRP inhibitors showed limited P450 inhibition. Some inhibitors showed less P450 inhibition in human hepatocytes than human liver microsomes, for example, elacridar, probably due to differences in binding, permeability limitations, or active, P-gp mediated efflux of the inhibitor from the hepatocytes. Quinidine was a potent P450 inhibitor in hepatocytes but only showed weak inhibition in microsomes. Quinidine shows an extensive cellular uptake, which may potentiate intracellular P450 inhibition. Elacridar, described as a potent and selective P-gp inhibitor, displayed modest P450 inhibition in this study and is thus a useful model inhibitor to define the role of P-gp in drug disposition without interference with other processes.

Functional ATP-binding cassette drug efflux transporters in isolated human and rat hepatocytes significantly affect assessment of drug disposition.

Freshly isolated hepatocytes are considered the gold standard for in vitro studies of hepatic drug disposition. To ensure a reliable supply of cells, cryopreserved human hepatocytes are often used. ABC-superfamily drug efflux transporters are key elements in hepatic drug disposition. These transporters are often considered lost after isolation of hepatocytes. In the present study, the expression and activity of ABC transporters BCRP, BSEP, P-gp, MRP2, MRP3, and MRP4 in human and rat cryopreserved hepatocytes were investigated. In commercially available human cryopreserved hepatocytes, all drug efflux transporters except human BCRP (hBCRP) exhibited similar expression levels as in fresh liver biopsies. Expression levels of hBCRP were 60% lower in cryopreserved human hepatocytes than in liver tissue, which could lead to, at most, a 2.5-fold reduction in hBCRP-mediated efflux. Fresh rat hepatocytes showed significantly lower levels of rat BCRP compared with liver expression levels; expression levels of other ABC transporters were unchanged. ABC transporters in human cryopreserved cells were localized to the plasma membrane. Functional studies could demonstrate P-gp and BCRP activity in both human cryopreserved and fresh rat hepatocytes. Inhibiting P-gp-mediated efflux by elacridar in in vitro experiments significantly decreased fexofenadine efflux from hepatocytes, resulting in an increase in apparent fexofenadine uptake. The results from the present study clearly indicate that ABC transporter-mediated efflux in freshly isolated as well as cryopreserved rat and human hepatocytes should be taken into account in in vitro experiments used for modeling of drug metabolism and disposition.

Prediction of in vivo rat biliary drug clearance from an in vitro hepatocyte efflux model.

Well-established techniques are available to predict in vivo hepatic uptake and metabolism from in vitro data, but predictive models for biliary clearance remain elusive. Several studies have verified the expression and activity of ATP-binding cassette (ABC) efflux transporters central to biliary clearance in freshly isolated rat hepatocytes, raising the possibility of predicting biliary clearance from in vitro efflux measurements. In the present study, short-term plated rat hepatocytes were evaluated as a model to predict biliary clearance from in vitro efflux measurements before major changes in transporter expression known to take place in long-term hepatocyte cultures. The short-term cultures were carefully characterized for their uptake and metabolic properties using a set of model compounds. In vitro efflux was studied using digoxin, fexofenadine, napsagatran, and rosuvastatin, representing compounds with over 100-fold differences in efflux rates in vitro and 60-fold difference in measured in vivo biliary clearance. The predicted biliary clearances from short-term plated rat hepatocytes were within 2-fold of measured in vivo values. As in vitro efflux includes both basolateral and canalicular effluxes, pronounced basolateral efflux may introduce errors in predictions for some compounds. In addition, in vitro rat hepatocyte uptake rates corrected for simultaneous efflux predicted rat in vivo hepatic clearance of the biliary cleared compounds with less than 2-fold error. Short-term plated hepatocytes could thus be used to quantify hepatocyte uptake, metabolism, and efflux of compounds and considerably improve the prediction of hepatic clearance, especially for compounds with a large biliary clearance component.

The impact of solute carrier (SLC) drug uptake transporter loss in human and rat cryopreserved hepatocytes on clearance predictions.

Cryopreserved hepatocytes are often used as a convenient tool in studies of hepatic drug metabolism and disposition. In this study, the expression and activity of drug transporters in human and rat fresh and cryopreserved hepatocytes was investigated. In human cryopreserved hepatocytes, Western blot analysis indicated that protein expression of the drug uptake transporters [human Na(+)-taurocholate cotransporting polypeptide (NTCP), human organic anion transporting polypeptides (OATPs), human organic anion transporters, and human organic cation transporters (OCTs)] was considerably reduced compared with liver tissue. In rat cryopreserved cells, the same trend was observed but to a lesser extent. Several rat transporters were reduced as a result of both isolation and cryopreservation procedures. Immunofluorescence showed that a large portion of remaining human OATP1B1 and OATP1B3 transporters were internalized in human cryopreserved hepatocytes. Measuring uptake activity using known substrates of OATPs, OCTs, and NTCP showed decreased activity in cryopreserved as compared with fresh hepatocytes in both species. The reduced uptake in cryopreserved hepatocytes limited the in vitro metabolism of several AstraZeneca compounds. A retrospective analysis of clearance predictions of AstraZeneca compounds suggested systematic lower clearance predicted using metabolic stability data from human cryopreserved hepatocytes compared with human liver microsomes. This observation is consistent with a loss of drug uptake transporters in cryopreserved hepatocytes. In contrast, the predicted metabolic clearance from fresh rat hepatocytes was consistently higher than those predicted from liver microsomes, consistent with retention of uptake transporters. The uptake transporters, which are decreased in cryopreserved hepatocytes, may be rate-limiting for the metabolism of the compounds and thus be one explanation for underpredictions of in vivo metabolic clearance from cryopreserved hepatocytes.

Evaluation of Heterologous Effects of Travel Vaccines in Colorectal Cancer: A Database Study and a Cautionary Tale.

Background and Aims: Recently, cholera vaccine use was shown to be associated with a reduced risk of death in patients with colorectal cancer (CRC). However, evidence on heterologous effects of travel vaccines is limited. The aim of this study was to study heterologous effects of travel vaccines in patients with CRC. Methods: We performed a retrospective database study on a cohort of CRC patients in Sweden and their postdiagnostic use of travel medications between July 2005 and December 2017. We obtained data from national registries on number of CRC diagnosis, death from CRC or other causes, age at diagnosis, and postdiagnostic use of travel vaccines and malaria prophylaxis. The Cox regression model was used to calculate incidence rate and incidence rate ratios of CRC-related and all-cause mortality by postdiagnostic travel medication status. Results: Two hundred ninety-five patients exposed to travel vaccines and malaria prophylaxis and 73,466 patients not exposed to travel medications were identified. CRC-related mortality was lowered in the exposed patients compared to the unexposed patients, irrespective of the travel medications used. The incidence rate ratios for CRC-related mortality and overall mortality were comparable. Conclusion: We postulated that patients in better health were likely to travel more frequently than patients with poor health, leading to a healthy user bias. The results suggested the same, as similar reduced mortality risks were found for all the investigated travel medications, lowering the biological plausibility of truly protective effect from post-therapeutic use of any of the travel medication studied. We advocate the use of multiple negative exposure controls and to exercise caution while drawing conclusions from travel vaccine research.

Travel vaccines are strongly associated to reduced mortality in prostate cancer patients - a real effect or residual confounding?

Repurposing of existing drugs and vaccines for diseases that they were not originally intended for is a promising research field. Recently there has been evidence that oral cholera vaccine might be used in the treatment of inflammatory disease and some common cancers. Specifically, Ji et al showed that the administration of cholera vaccine after a prostate cancer diagnosis reduced prostate cancer specific mortality rates by almost 50%. In a cohort of men from Stockholm, Sweden, with more detailed cancer data and a higher coverage of exposure to vaccine, we replicated these findings using a marginal structural Cox model. We showed that administration of cholera vaccine after prostate cancer diagnosis is associated with a significant reduction in mortality (HR 0.46, 95% CI 0.31-0.69, p-value 0.0001) even after adjusting for all known confounders. However, the same effect (or even stronger) could be seen for several other traveling vaccines and malaria prophylaxis. Therefore, we conclude that this effect is most likely due to a healthy traveler bias and is an example of residual confounding.

Dental hygiene education for nursing staff in a nursing home for older people.

AIM: This paper is a report of a study evaluating the effect of a repeated education programme for nursing staff in a home for older people. BACKGROUND: A strong relationship exists between oral infections and general health complications (especially aspiration pneumonia) among nursing home residents and hospitalized older people. Thus, nursing staff need to be educated in oral hygiene measures. METHODS: Forty-three nursing home resident older people (12 men, 31 women, age range 69-99 years) were included in a dental hygiene and gingivitis evaluation using gingival bleeding scores and modified plaque scores. Evaluation was conducted before and 3 weeks after a repeated dental hygiene education for nursing staff at a nursing home in Sweden in 2008. Dental hygiene education had been given 1.5 years previously. FINDINGS: Forty-one residents (12 men and 29 women) were available for evaluation after the repeated dental hygiene education (one died, one had had teeth extracted). There was a reduction in gingival bleeding scores (P < 0.001), and in plaque scores (P < 0.001). CONCLUSION: Repeated dental hygiene education improves the dental hygiene among nursing home resident older people. In order to succeed it may be necessary to address attitudes and perceptions towards oral care in such a dental hygiene education programme for nursing staff. Improved oral hygiene contributes to reducing the incidence of healthcare-associated pneumonia among nursing home resident older people, and thus to reduced healthcare costs.

Evaluation of dental hygiene education for nursing home staff.

AIM: This paper is a report of a study evaluating the long-term effects on the oral hygiene status of older nursing home residents one and a half years after dental hygiene education was given to the staff. BACKGROUND: A strong relationship exists between oral infections and general health complications (especially aspiration pneumonia) among nursing home residents and hospitalized older people. It is therefore important to educate nursing home staff in oral hygiene measures and to follow up the effects of the education over time. METHODS: Dental plaque measurements were conducted at a Swedish nursing home in 2006-2008. Forty-one residents (12 men, 31 women, aged 69-99 years) fulfilled the inclusion criteria and participated in a dental hygiene evaluation 1.5 years after dental hygiene education was given to the staff at the nursing home. Plaque index scores (year 2008) were compared to those soon after the education (year 2006). FINDINGS: After the dental hygiene education in 2006, 60 nursing home residents (14 men, 46 women) were available for plaque index measurements, whereas 41 residents (12 men, 29 women) were available 1.5 years later. The median plaque index scores were 17.0 (n = 60) in 2006, and 18.0 (n = 41) in 2008 (Mann-Whitney U-test, P > 0.05). CONCLUSION: Dental hygiene education for nursing home staff is important to maintain an adequate level of oral hygiene among older nursing home residents over time. Follow-up of dental hygiene education for nursing home staff is recommended to maintain a sufficient level of oral hygiene among the residents.

A survey of attitudes and perceptions toward oral hygiene among staff at a geriatric nursing home.

The aim of this survey was to test the impact of an oral hygiene educational model on attitudes and perceptions toward oral hygiene among nursing home staff members. A pilot questionnaire was distributed to the nursing staff before and after a course on oral hygiene at a geriatric nursing home in Stockholm in 2008. The nursing staff was of the opinion that they had sufficient time to carry out oral hygiene tasks but considered such tasks unpleasant, mainly because of unwillingness and resistance from the residents. These attitudes and perceptions among the nursing staff did not change significantly after oral hygiene education. Future oral hygiene educational models need to be developed with an aim to alter the perceptions and behavior of the nursing home staff.

Dental hygiene education for nursing staff.

The aim of this study was to describe a new dental hygiene education program for nursing staff and to report experiences from the program at a nursing home in Stockholm, Sweden (2006). This strategy comprises 3 steps. The first is individual instruction for nursing staff about oral care for patients and hands-on training in toothbrushing technique using an electric toothbrush. The second step was small discussion groups of 4 to 8 nursing staff, led by a dental hygienist and a psychologist. The third step was a theoretical lecture focusing on the associations among dental hygiene, oral health, and general health among the elderly. During the dental hygiene education program, a negative attitude toward oral care was noted among members of the nursing staff, although they did consider oral care important for their patients. Increased self-confidence of staff in providing oral care was noted after completing the dental hygiene education program. Nursing staff members stated that they had received more detailed knowledge about oral care during the program. This dental hygiene education program appears to result in increased knowledge and interest in oral hygiene tasks among the nursing staff and may lead to improved dental hygiene among nursing home residents.

Prediction and identification of drug interactions with the human ATP-binding cassette transporter multidrug-resistance associated protein 2 (MRP2; ABCC2).

The chemical space of registered oral drugs was explored for inhibitors of the human multidrug-resistance associated protein 2 (MRP2; ABCC2), using a data set of 191 structurally diverse drugs and drug-like compounds. The data set included a new reference set of 75 compounds, for studies of hepatic drug interactions with transport proteins, CYP enzymes, and compounds associated with liver toxicity. The inhibition of MRP2-mediated transport of estradiol-17beta-D-glucuronide was studied in inverted membrane vesicles from Sf9 cells overexpressing human MRP2. A total of 27 previously unknown MRP2 inhibitors were identified, and the results indicate an overlapping but narrower inhibitor space for MRP2 compared with the two other major ABC efflux transporters P-gp (ABCB1) and BCRP (ABCG2). In addition, 13 compounds were shown to stimulate the transport of estradiol-17beta-D-glucuronide. The experimental results were used to develop a computational model able to discriminate inhibitors from noninhibitors according to their molecular structure, resulting in a predictive power of 86% for the training set and 72% for the test set. The inhibitors were in general larger and more lipophilic and presented a higher aromaticity than the noninhibitors. The developed computational model is applicable in an early stage of the drug discovery process and is proposed as a tool for prediction of MRP2-mediated hepatic drug interactions and toxicity.

Substrate and method dependent inhibition of three ABC-transporters (MDR1, BCRP, and MRP2).

Drug transport and drug-drug interactions (DDI) with human ABC transporters are generally investigated in mammalian cell lines or inverted membrane vesicles from insect cells (Sf9) overexpressing the transporter of interest. In this study, we instead used membrane vesicles from human embryonic kidney cells (HEK293) overexpressing wild type MDR1/Pgp (ABCB1), BCRP (ABCG2), and MRP2 (ABCC2) with the aim to study the concentration dependent inhibition of shared and prototypic probe substrates. We first investigated 15 substrates and identified estrone-17-beta-glucorinide (E17G) as shared substrate. Nine specific and general inhibitors were then studied using E17G and prototypic probe substrates. The results were compared with those previously obtained in Sf9 vesicles and cell lines of canine (MDCKII) and human (Saos-2) origin. For the majority of inhibitors, Ki values differed <10-fold between E17G and probe substrates. Significant differences in Ki values were observed for about one third of the inhibitors. The transport inhibition potencies in HEK293 vesicles were in good agreement with those obtained in Sf9 vesicles. Large differences were found in the inhibition potencies observed in the vesicular systems compared to the cellular systems. Nevertheless, the rank order correlations between the different experimental systems were generally good. Our study provides further information on substrate dependent inhibition of ABC-transporters, and suggests that simple ranking of compounds can be used as a tier one approach to bridge results obtained in different experimental systems.

Optimizing DMPK Properties: Experiences from a Big Pharma DMPK Department.

BACKGROUND: The disposition of a drug is dependent on interactions between the body and the drug, its molecular properties and the physical and biological barriers presented in the body. In order for a drug to have a desired pharmacological effect it has to have the right properties to be able to reach the target site in sufficient concentration. This review details how drug metabolism and pharmacokinetics (DMPK) and physicochemical deliveries played an important role in data interpretation and compound optimization at AstraZeneca R&D in Södertälje, Sweden. METHODS: A selection of assays central in the evaluation of the DMPK properties of new chemical entities is presented, with guidance and consideration on assay outcome interpretation. Early in projects, solubility, LogD, permeability and metabolic stability were measured to support effective optimization of DMPK properties. Changes made to facilitate high throughput, efficient bioanalysis and the handling of large amounts of samples are described. Already early in drug discovery, we used an integrated approach for the prediction of the fate of drugs in human (early dose to man) based on data obtained from in vitro experiments. The early dose to man was refined with project progression, which triggered more intricate assays and experiments. At later stages, preclinical in vivo pharmacokinetic (PK) data was integrated with pharmacodynamics (PD) to allow predictions of required dose, dose intervals and exposure profile to achieve the desired effect in man. RESULTS AND CONCLUSIONS: A well-defined work flow of DMPK activities from early lead identification up to the selection of a candidate drug was developed. This resulted in a cost effective and efficient optimization of chemical series, and facilitated informed decision making throughout project progress.

Direct gas measurements indicate that the novel cyclooxygenase inhibitor AZD3582 is an effective nitric oxide donor in vivo.

1. AZD3582 [4-(nitrooxy)butyl-(2S)-2-(6-methoxy-2-naphthyl)propanoate] is a COX-inhibiting nitric oxide donor that inhibits COX-1 and COX-2. It is as effective as naproxen in models of pain and inflammation, but causes less gastroduodenal damage. Nitric oxide (NO) is generated from AZD3582 in vitro, and this study sought to show that the drug donates NO in vivo. 2. In anaesthetised male New Zealand white rabbits, the endogenous NO concentration in exhaled air was reduced by N(G)-nitro-L-arginine methyl ester (L-NAME) (30 mg kg(- 1) i.v.) from 33.5+/-1.0 ppb (mean+/-s.e.m.; n=6 per group) to 3.0+/-1.0 ppb, while increasing blood pressure and reducing heart rate. AZD3582 (0.2, 0.6, 2.0 or 6.0 micromol kg(- 1) min(- 1)) given 30 min after L-NAME increased the concentration of NO in exhaled air (P<0.05), decreased blood pressure and increased heart rate in a dose-dependent manner versus L-NAME control values. The peak mean NO concentration obtained was 44+/-8.0 ppb. 3. In in situ-perfused rabbit lungs, L-NAME (185 micromol l(- 1)) reduced the NO concentration in exhaled air from 106+/-13 to 4.0+/-0.4 ppb (n=5). Addition of AZD3582 (6 micromol min(- 1)) to the perfusate produced an initial rapid increase in the NO concentration in exhaled air, followed by a sustained, but lower plateau. Infusion of L-NAME increased, and AZD3582 decreased, pulmonary arterial pressure. 4. In both anaesthetised rabbits and in the perfused lungs, brief periods of hypoxia increased NO concentrations generated by AZD3582. 5. We conclude that, in rabbits, AZD3582 donates NO in vivo with characteristics similar to those reported for nitroglycerin and isosorbide nitrates

AZD3582 increases heme oxygenase-1 expression and antioxidant activity in vascular endothelial and gastric mucosal cells.

AZD3582 [4-(nitrooxy)-butyl-(2S)-2-(6-methoxy-2-naphthyl)-propanoate] is a COX-inhibiting nitric oxide donator (CINOD). Incubation of human endothelial cells (derived from umbilical cord) with AZD3582 (10-100muM) led to increased expression of heme oxygenase (HO)-1 mRNA and protein. Heme oxygenase-1 (HO-1) is a crucial mediator of antioxidant and tissue-protective actions. In contrast, naproxen (a non-selective NSAID) and rofecoxib (a selective inhibitor of COX-2), did not affect HO-1 expression. Pre-treating endothelial cells with AZD3582 at concentrations that were effective at inducing HO-1 also reduced NADPH-dependent production of oxygen radicals. Antioxidant activity in the endothelial cells persisted after AZD3582 had been washed out from the incubation medium. When added exogenously to the cells at low micromolar concentrations, the HO-1 metabolite, bilirubin, virtually abolished NADPH-dependent oxidative stress. AZD3582-induced blockade of free-radical formation was reversed in the presence of the HO-1 inhibitor, tin protoporphyrin-IX (SnPP). Similar results were obtained in human gastric mucosal cells (KATO-III). Our results demonstrate that HO-1 is a novel target of AZD3582.

Oral gingival delivery systems from chitosan blends with hydrophilic polymers.

Chitosan blends with hydrophilic polymers including polyvinylalcohol (PVA), polyethyleneoxide (PEO) and polyvinylpyrrolidone (PVP), were investigated as candidates for oral gingival delivery systems. The bioavailabilty conferred by the chitosan blend delivery systems, as concluded from dog studies, was shown to be comparable to that based on chitosan alone, especially for those blends involving high molecular weight hydrophilic polymers. Results from differential scanning calorimetry and dynamic mechanical thermal analysis, Fourier transform infrared spectroscopy and tensile testing, indicated that the chitosan/PEO and chitosan/PVP blends showed evidence of miscibility in all blend ratios studied, while the chitosan/PVA blend only showed evidence of interaction for the (50:50) and (80:20) blends, but not for the (20:80) blend. However, even a phase separated system may show interesting and exploitable properties, as evidenced by the tensile testing data for the high molecular weight PVA blend (20:80). The study also indicated that chitosan blends were superior in other properties compared to chitosan alone. These included improved comfort and reduced irritation, ease of processing, improved film quality, improved flexibility, and enhanced dissolution. Blends of chitosan with different hydrophilic polymers could thus be promising candidates for formulation in oral mucosal delivery systems.

A common pathway of nitric oxide release from AZD3582 and glyceryl trinitrate.

4-(Nitrooxy)-butyl-(S)-2-(6-methoxy-2-naphthyl)-propanoate (AZD3582) is a cyclooxygenase (COX)-inhibiting nitric oxide donator (CINOD). It donates nitric oxide (NO) in biological systems through as yet unidentified mechanisms. cGMP, a marker of intracellularly generated NO, was increased up to 27-fold over basal levels by AZD3582 (1-30microM) in LLC-PK1 kidney epithelial cells. A 5h pretreatment with glyceryl tinitrate (GTN, 0.1-1microM) attenuated the cGMP response to a subsequent challenge with AZD3582 or GTN. Similarly, AZD3582 (10-30microM) pretreatment reduced the increase in cGMP on subsequent incubation with AZD3582 or GTN. In contrast, cGMP stimulation by SIN-1, which releases NO independently of enzymatic catalysis, remained unimpaired in cells pretreated with GTN or AZD3582. Our results demonstrate that AZD3582 decreases the sensitivity of the guanylyl cyclase/cGMP system to GTN and vice versa. This suggests that bioactivation pathways for organic nitrates, which involve enzymatic catalysis, may be responsible for NO donation from AZD3582.

Characterization of jejunal absorption and apical efflux of ropivacaine, lidocaine and bupivacaine in the rat using in situ and in vitro absorption models.

The purpose of this study was to characterize the rat jejunal passive transport and the possible active efflux of three local anaesthetics, ropivacaine, lidocaine and bupivacaine using two different absorption models, the in situ single-pass intestinal perfusion and the in vitro Ussing chamber model, as well as P-glycoprotein (Pgp)-mediated calcein transport inhibition in Caco-2 cells. Concentration and pH dependence, efflux inhibition by verapamil and digoxin and bi-directional permeability studies were performed to investigate the potential involvement of efflux carriers in the intestinal absorption of the local anaesthetics. In the jejunal perfusion the permeability of these agents appeared to be high, predicting complete intestinal absorption (>90%). There was no effect of the Pgp inhibitors on net absorption for any of the local anaesthetics in the two absorption models. However, in the Ussing chamber at an equal pH of 7.4 at mucosal and serosal sides, the observed jejunal permeability ratios (S-M)/(M-S), of 2.3, 1.8 and 3.0 for ropivacaine, lidocaine and bupivacaine, respectively, indicated at least some involvement of carrier-mediated intestinal secretion. This idea was supported in the calcein AM Pgp transport assay in which two of the tested local anaesthetic agents affected cellular calcein retention. As anticipated for these agents, the mucosal pH conditions were shown to largely affect the gut permeability. The jejunal permeabilities of the local anaesthetics as measured in the two absorption models fitted well in a model comparison that incorporated the permeabilities of six other structurally unrelated drugs. In conclusion, the jejunal permeability of ropivacaine, lidocaine and bupivacaine was high and although evidence was obtained for carrier-mediated intestinal efflux this process appeared not to have a significant influence on the rate and extent of in vivo intestinal absorption. Rather, passive diffusion of these agents seems to be the major mechanism for the intestinal absorption.

Regional transport and metabolism of ropivacaine and its CYP3A4 metabolite PPX in human intestine.

The major aim of this study was to investigate the CYP3A4 metabolism and polarized transport of ropivacaine and its metabolite 2',6'-pipecoloxylidide (PPX) in tissue specimens from the human small and large intestine. Ropivacaine has been shown to be effective in the treatment of ulcerative colitis in human colon. This study was conducted using a modified Ussing-chamber technique with specimens from jejunum, ileum and colon collected from 11 patients. The local kinetics of ropivacaine and PPX were assessed from their concentration-time profiles in mucosal and serosal compartments. The permeability (P(app)) in the absorptive direction for both ropivacaine and PPX increased regionally in the order jejunum < ileum < colon. Ropivacaine was not found to be subjected to any carrier-mediated intestinal efflux. However, the CYP3A4 metabolite left the human enterocyte in a polarized manner and both the extent of CYP3A4 metabolism of ropivacaine and the extrusion of its metabolite to the mucosal chamber were more efficient in jejunum than in ileum. P-glycoprotein was probably not involved in the metabolite extrusion. No other metabolite than PPX was found. This in-vitro study with human intestinal tissues provides new mechanistic insights into regional transport and metabolism of drugs.

Early identification of clinically relevant drug interactions with the human bile salt export pump (BSEP/ABCB11).

A comprehensive analysis was performed to investigate how inhibition of the human bile salt export pump (BSEP/ABCB11) relates to clinically observed drug-induced liver injury (DILI). Inhibition of taurocholate (TA) transport was investigated in BSEP membrane vesicles for a data set of 250 compounds, and 86 BSEP inhibitors were identified. Structure-activity modeling identified BSEP inhibition to correlate strongly with compound lipophilicity, whereas positive molecular charge was associated with a lack of inhibition. All approved drugs in the data set (n = 182) were categorized according to DILI warnings in drug labels issued by the Food and Drug Administration, and a strong correlation between BSEP inhibition and DILI was identified. As many as 38 of the 61 identified BSEP inhibitors were associated with severe DILI, including 9 drugs not previously linked to BSEP inhibition. Further, among the tested compounds, every second drug associated with severe DILI was a BSEP inhibitor. Finally, sandwich-cultured human hepatocytes (SCHH) were used to investigate the relationship between BSEP inhibition, TA transport, and clinically observed DILI in detail. BSEP inhibitors associated with severe DILI greatly reduced the TA canalicular efflux, whereas BSEP inhibitors with less severe or no DILI resulted in weak or no reduction of TA efflux in SCHH. This distinction illustrates the usefulness of SCHH in refined analysis of BSEP inhibition. In conclusion, BSEP inhibition in membrane vesicles was found to correlate to DILI severity, and altered disposition of TA in SCHH was shown to separate BSEP inhibitors associated with severe DILI from those with no or mild DILI.