Glucose homeostasis depends critically on insulin that is secreted by pancreatic β-cells. Serum glucose, which is directly sensed by β-cells, stimulates depolarization- and Ca2+-dependent exocytosis of insulin granules. Here we show that pancreatic islets prominently express LRRC8A and LRRC8D, subunits of volume-regulated VRAC anion channels. Hypotonicity- or glucose-induced β-cell swelling elicits canonical LRRC8A-dependent VRAC currents that depolarize β-cells to an extent that causes electrical excitation. Glucose-induced excitation and Ca2+ responses are delayed in onset, but not abolished, in β-cells lacking the essential VRAC subunit LRRC8A. Whereas Lrrc8a disruption does not affect tolbutamide- or high-K+-induced insulin secretion from pancreatic islets, it reduces first-phase glucose-induced insulin secretion. Mice lacking VRAC in β-cells have normal resting serum glucose levels but impaired glucose tolerance. We propose that opening of LRRC8/VRAC channels increases glucose sensitivity and insulin secretion of β-cells synergistically with KATP closure. Neurotransmitter-permeable LRRC8D-containing VRACs might have additional roles in autocrine/paracrine signaling within islets.

BFE1224, prodrug of ravuconazole, is a novel, once-daily, oral, triazole antifungal drug, and currently in development for the treatment of onychomycosis. The clinical drug-drug interaction (DDI) potential of BFE1224 with cytochrome P450 (CYP) and transporter was assessed by using two types of cocktails in healthy subjects in separate clinical studies. The CYP and transporter cocktails consisted of caffeine/tolbutamide/omeprazole/dextromethorphan/midazolam used in study 1 and digoxin/rosuvastatin used in study 2. In addition, repaglinide was separately administered to the same subjects in study 2. There were no major effects on the pharmacokinetics of CYP and transporter substrates, except for an approximate threefold increase in midazolam exposure after oral administration of BFE1224. The clinical DDIs of BFE1224 were mild for CYP3A and minor for other major CYPs (CYP1A2/2C8/2C9/2C19/2D6) as well as those of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1, and OATP1B3.

Single-walled carbon nanotubes (SWCNTs) are made from a rolled single sheet of graphene with a diameter in the nanometer range. SWCNTs are potential carriers for drug delivery systems because antibodies or drugs can be loaded on their surface; however, their effect on the activities of cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the effect of two kinds of SWCNTs with different lengths (FH-P- and SO-SWCNTs) on human CYP activity. In addition, other nano-sized carbon materials, such as carbon black, fullerene-C60 , and fullerene-C70 were also evaluated to compare their effects on CYP activities. Ten CYP substrates (phenacetin, coumarin, bupropion, paclitaxel, tolbutamide, S-mephenytoin, dextromethorphan, chlorzoxazone, midazolam, and testosterone) were used. Testosterone 6β-hydroxylation and midazolam 1'-hydroxylation, which are catalyzed by both CYP3A4 and CYP3A5 in liver microsomes, were decreased by 25% and 45%, respectively, in the presence of 0.1 mg/mL SO-SWCNT. Dextromethorphan O-demethylation, which is catalyzed mainly by CYP2D6, was decreased by 40% in the presence of SO-SWCNT. Other CYP activities, however, were not attenuated by SO-SWCNT. FH-P-SWCNT, carbon black, fullerene-C60 , and fullerene-C70 at 0.1 mg/mL had no effect on CYP activities. The Ki values for testosterone 6β-hydroxylation, midazolam 1'-hydroxylation, and dextromethorphan O-demethylation in liver microsomes were 136, 34, and 56 μg/mL, respectively. SO-SWCNT was determined to be a competitive inhibitor of CYP3A4, CYP3A5, and CYP2D6. These results suggest that the effect of SO-SWCNT differs among CYP isoforms, and that the inhibition potency depends on the physicochemical properties of the nanocarbons.

Liver metabolism is commonly considered the major determinant in drug discovery and development. Many in vitro drug metabolic studies have been developed and applied to understand biotransformation. However, these methods have disadvantages, resulting in inconsistencies between in vivo and in vitro experiments. A major factor is that they are static systems that do not consider the transport process in the liver. Here we developed an in vitro dynamic metabolic system (Bio-PK metabolic system) to mimic the human pharmacokinetics of tolbutamide. Human liver microsomes (HLMs) encapsulated in a F127'-Acr-Bis hydrogel (FAB hydrogel) were placed in the incubation system. A microdialysis sampling technique was used to monitor the metabolic behavior of tolbutamide in hydrogels. The measured results in the system were used to fit the in vitro intrinsic clearance of tolbutamide with a mathematical model. Then, a PBPK model that integrated the corresponding in vitro intrinsic clearance was developed to verify the system. Compared to the traditional incubation method, reasonable PK profiles and the in vivo clearance of tolbutamide could be predicted by integrating the intrinsic clearance of tolbutamide obtained from the Bio-PK metabolic system into the PBPK model. The predicted maximum concentration (Cmax), area under the concentration-time curve (AUC), time to reach the maximum plasma concentration (Tmax) and in vivo clearance were consistent with the clinically observed data. This novel in vitro dynamic metabolic system can compensate for some limitations of traditional incubation methods; it may provide a new method for screening compounds and predicting pharmacokinetics in the early stages, supporting the development of compounds.

We quantified the effect of acute ethanol exposure (initial blood concentrations 0.7 g/L) on major drug metabolizing enzymes and p-glycoprotein. Sixteen healthy Caucasians participated in a randomized crossover study with repeated administration of either vodka or water. Enzyme/transporter activity was assessed by a cocktail of probe substrates, including caffeine (CYP1A2/NAT2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and digoxin (P-glycoprotein). The ratio of AUC0-t of dextromethorphan for ethanol/water coadministration was 1.95 (90% confidence interval (CI) 1.48-2.58). The effect was strongest in individuals with a CYP2D6 genotype predicting high activity (n = 7, ratio 2.66, 90% CI 1.65-4.27). Ethanol increased caffeine AUC0-t 1.38-fold (90% CI 1.25-1.52) and reduced intestinal midazolam extraction 0.77-fold (90% CI 0.69-0.86). The other probe drugs were not affected by ethanol. The results suggest that acute ethanol intake typically has no clinically important effect on the enzymes/transporters tested.

Corylin, an phenolic compound from Psoralea corylifolia, has been reported with various pharmacological properties but has poor bioavailability due to massive metabolism. In this study, twelve metabolites of corylin mainly involving in oxidation, hydration, glucuronidation and sulfation were detected in mice. Furthermore, the oxidation and hydration of corylin (M4) in human liver microsomes (HLM) and human intestine microsomes (HIM) were both efficient with high CLint (intrinsic clearance) values of 24.29 and 42.85 μL/min/mg, respectively. CYP1A1, 1B1 and 2C19 contributed most for M4 with the CLint values of 26.63, 33.09 and 132.41 μL/min/mg, respectively. Besides, M4 was strongly correlated with phenacetin-N-deacetylation (r = 0.885, p = 0.0001) and tolbutamide-4-oxidation (r = 0.727, p = 0.001) in twelve individual HLMs, respectively. In addition, corylin was efficiently glucuronidated (M7) in HLM (125.33 μL/min/mg) and in HIM (108.74 μL/min/mg). UGT1A1 contributed the most for M7 with the CLint value of 122.32 μL/min/mg. Meanwhile, M7 was significantly correlated with β-estradiol-3-O-glucuronidation (r = 0.742, p = 0.006) in twelve individual HLMs. Moreover, the metabolism of corylin showed marked species differences. Taken together, corylin was subjected to massive first-pass metabolism in liver and intestine, while CYP1A1, 1B1, 2C19 and UGT1A1 were the main contributors. Finally, the proposed metabolic pathway of corylin involed CYP and UGT isoforms were summarized, which could help to understand the metabolic fate of corylin in vivo.

Drug transporter and cytochrome P450 expression is regulated by shared nuclear receptors and, hence, an inducer should induce both, although the magnitude may differ. The objective of this study was to establish relative induction relationships between CYP3A and drug transporters (P-glycoprotein (P-gp), organic anion transporting polypeptide (OATP), and breast cancer resistance protein (BCRP)) or other P450s (CYP2C9 and CYP1A2) using ascending doses of the prototypical pregnane xenobiotic receptor (PXR) agonist, rifampin, to elicit weak, moderate, and strong PXR agonism. Healthy subjects received dabigatran etexilate, pravastatin, rosuvastatin, and a midazolam/tolbutamide/caffeine cocktail before and after rifampin 2, 10, 75, or 600 mg q.d. Unlike CYP3A, only moderate induction of P-gp, OATP, and CYP2C9 was observed and dose-dependent induction of P-gp, OATP, and CYP2C9 was always one drug-drug interaction category lower than observed for CYP3A, even when correcting for probe drug sensitivity. Data from this study establish proof-of-concept that P450 induction data can be leveraged to inform on the effect on transporters.

In vitro studies of human pancreatic islets are critical for understanding normal insulin secretion and its perturbations in diabetic β-cells, but the influence of islet preparation characteristics and organ donor attributes in such experiments is poorly documented. Preparations from normal donors were tested with a standardized protocol evaluating dynamic insulin secretion induced by glucose, tolbutamide, and cAMP (forskolin). Secretion rates, normalized to insulin content (fractional insulin secretion), were analyzed as a function of preparation and donor characteristics. Low purity (25-45%) of the preparation (n = 8) blunted the first phase of insulin secretion induced by glucose or tolbutamide and increased basal secretion, resulting in threefold lower stimulation index than in more pure (55-95%) preparations (n = 43). In these more pure preparations, cold ischemia time (1-13 h) before pancreas digestion did not impact insulin secretion. Islet size (estimated by the islet size index) did not influence the dynamics of secretion, but fractional insulin secretion rates were greater in large than small islets, and positively correlated with islet size. Age of the donors (20-68 years) had no influence on islet size and insulin content or on dynamics and amplitude of insulin secretion, which were also similar in islets from male and female donors. In contrast, islet size and islet insulin content (normalized for size), and basal or stimulated insulin secretion positively correlated with Body-Mass Index (19-33). These results contradict previous reports on the impact of donor age and islet size and point to possible confounding effects of donor BMI in insulin secretion studies with isolated human islets.

1. A novel method utilizing an internal standard in hepatocytes incubations has been developed and demonstrated to decrease the variability in the determination of intrinsic clearance (CLint) in this system. The reduced variability was shown to allow differentiation of lower elimination rate constants from noise. 2. The suggested method was able to compensate for a small but systematic error (0.5 µL/min/106 cells) caused by an evaporation of approximately 15% of the volume during the incubation time. 3. The approach was validated using six commercial drugs (ketoprofen, tolbutamide, phenacetin, etodolac and quinidine) which were metabolized by different pathways. 4. The suggested internal standard, MSC1815677, was extensively characterized and the acquired data suggest that it fulfills the requirements of an internal standard present during the incubation. The proposed internal standard was stable during the incubation and showed a low potential to inhibit drug metabolizing enzymes and transporters. With MSC1815677 we propose a novel simple, robust and cost-effective method to address the challenges in the estimation of low clearance in hepatocyte incubations.