Inhibition of Organic Anion Transporting Polypeptide 1B1 and 1B3 by Betulinic Acid: Effects of Preincubation and Albumin in the Media

Betulinic acid (BA), a plant-derived pentacyclic triterpenoid, may interact with the members of the organic anion transporting polypeptide 1B subfamily. Here, we investigated the interactions of BA and its analogs with OATP1B1/3 and rat Oatp1b2 in vitro and in vivo. BA inhibited the activity of OATP1B1/3 and rat Oatp1b2 in vitro. Systemic exposure of atorvastatin was substantially altered with the intravenous co-administration of BA (20 mg/kg). Preincubation (incubation with inhibitors, followed by washout) with BA led to a sustained inhibition of OATP1B3, which recovered rapidly in the media containing 10% fetal bovine serum. The addition of albumin to the media decreased intracellular concentrations of BA and expedited the recovery of OATP1B3 activity following preincubation. For asunaprevir and cyclosporin A (previously known to inhibit OATP1B3 upon preincubation), the addition of albumin to the media shortened recovery time with asunaprevir, but not with cyclosporin A. Overall, our results showed that BA inhibits OATP1B transporters in vitro and may incur hepatic transporter-mediated drug interactions in vivo. Our results identify BA as another OATP1B3 inhibitor with preincubation effect and suggest that the preincubation effect and its duration is impacted by altered equilibrium of inhibitors between intracellular and extracellular space (e.g., albumin in the media).     

Contribution of equilibrative nucleoside transporters 1 and 2 to gemcitabine uptake in pancreatic cancer cells

Hepatic arterial infusion (HAI) chemotherapy is expected to be a more effective and safer method to treat the hepatic metastasis of pancreatic cancer than intravenous (iv) administration because of higher tumor exposure and lower systemic exposure. To clarify the uptake mechanism of nucleoside anticancer drugs, including gemcitabine (GEM), in pancreatic cancer, we investigated the uptakes of radiolabeled uridine (a general substrate of nucleoside transporters) and GEM in pancreatic cancer cell lines MIA‐PaCa2 and As‐PC1. Uridine uptake was inhibited by non‐labeled GEM and also by S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR; an inhibitor of equilibrative nucleoside transporters, ENTs) in a concentration‐dependent manner, suggesting that ENTs contribute to uridine uptake in pancreatic cancer cells. As for GEM, saturable uptake was mediated by high‐ and low‐affinity components with K_m values of micromolar and millimolar orders, respectively. Uptake was inhibited in a concentration‐dependent manner by NBMPR and was sodium ion‐independent. Moreover, the concentration dependence of uptake in the presence of 0.1 μM NBMPR showed a single low‐affinity site. These results indicated that the high‐ and low‐affinity sites correspond to hENT1 and hENT2, respectively. The results indicated that at clinically relevant hepatic concentrations of GEM in GEM‐HAI therapy, the metastatic tumor exposure of GEM is predominantly determined by hENT2 under unsaturated conditions, suggesting that hENT2 expression in metastatic tumor would be a candidate biomarker for indicating anticancer therapy with GEM‐HAI.     

Impact of individual behaviour change on the spread of emerging infectious diseases

Human behaviour plays an important role in the spread of emerging infectious diseases, and understanding the influence of behaviour changes on epidemics can be key to improving control efforts. However, how the dynamics of individual behaviour changes affects the development of emerging infectious disease is a key public health issue. To develop different formula for individual behaviour change and introduce how to embed it into a dynamic model of infectious diseases, we choose A/H1N1 and Ebola as typical examples, combined with the epidemic reported cases and media related news reports. Thus, the logistic model with the health belief model is used to determine behaviour decisions through the health belief model constructs. Furthermore, we propose 4 candidate infectious disease models without and with individual behaviour change and use approximate Bayesian computation based on sequential Monte Carlo method for model selection. The main results indicate that the classical compartment model without behaviour change and the model with average rate of behaviour change depicted by an exponential function could fit the observed data best. The results provide a new way on how to choose an infectious disease model to predict the disease prevalence trend or to evaluate the influence of intervention measures on disease control. However, sensitivity analyses indicate that the accumulated number of hospital notifications and deaths could be largely reduced as the rate of behaviour change increases. Therefore, in terms of mitigating emerging infectious diseases, both media publicity focused on how to guide people's behaviour change and positive responses of individuals are critical.     

Microbiota-derived uremic retention solutes: perpetrators of altered nonrenal drug clearance in kidney disease

Introduction: Scientific interest in the gut microbiota is increasing due to improved understanding of its implications in human health and disease. In patients with kidney disease, gut microbiota-derived uremic toxins directly contribute to altered nonrenal drug clearance. Microbial imbalances, known as dysbiosis, potentially increase formation of microbiota-derived toxins, and diminished renal clearance leads to toxin accumulation. High concentrations of microbiota-derived toxins such as indoxyl sulfate and p-cresol sulfate perpetrate interactions with drug metabolizing enzymes and transporters, which provides a mechanistic link between increases in drug-related adverse events and dysbiosis in kidney disease.

Areas covered: This review summarizes the effects of microbiota-derived uremic toxins on hepatic phase I and phase II drug metabolizing enzymes and drug transporters. Research articles that tested individual toxins were included. Therapeutic strategies to target microbial toxins are also discussed.

Expert commentary: Large interindividual variability in toxin concentrations may explain some differences in nonrenal clearance of medications. Advances in human microbiome research provide unique opportunities to systematically evaluate the impact of individual and combined microbial toxins on drug metabolism and transport, and to explore microbiota-derived uremic toxins as potential therapeutic targets.     

Synthesis,analytical characterization,and monoamine transporter activity of the new psychoactive substance 4‐methylphenmetrazine (4‐MPM), with differentiation from its ortho‐ and meta‐ positional isomers

The availability of new psychoactive substances (NPS) on the recreational drug market continues to create challenges for scientists in the forensic, clinical and toxicology fields. Phenmetrazine (3‐methyl‐2‐phenylmorpholine) and an array of its analogs form a class of psychostimulants that are well documented in the patent and scientific literature. The present study reports on two phenmetrazine analogs that have been encountered on the NPS market following the introduction of 3‐fluorophenmetrazine (3‐FPM), namely 4‐methylphenmetrazine (4‐MPM), and 3‐methylphenmetrazine (3‐MPM). This study describes the syntheses, analytical characterization, and pharmacological evaluation of the positional isomers of MPM. Analytical characterizations employed various chromatographic, spectroscopic, and mass spectrometric platforms. Pharmacological studies were conducted to assess whether MPM isomers might display stimulant‐like effects similar to the parent compound phenmetrazine. The isomers were tested for their ability to inhibit uptake or stimulate release of tritiated substrates at dopamine, norepinephrine and serotonin transporters using in vitro transporter assays in rat brain synaptosomes. The analytical characterization of three vendor samples revealed the presence of 4‐MPM in two of the samples and 3‐MPM in the third sample, which agreed with the product label. The pharmacological findings suggest that 2‐MPM and 3‐MPM will exhibit stimulant properties similar to the parent compound phenmetrazine, whereas 4‐MPM may display entactogen properties more similar to 3,4‐methylenedioxymethamphetamine (MDMA). The combination of test purchases, analytical characterization, targeted organic synthesis, and pharmacological evaluation of NPS and their isomers is an effective approach for the provision of data on these substances as they emerge in the marketplace.     

A tamoxifen derivative,N,N-diethyl-2-[4-(phenylmethyl) phenoxy] ethanamine,selectively targets P-glycoprotein-positive multidrug resistant Chinese hamster cells

DPPE, a tamoxifen derivative with antihistamine activity, was previously shown to potentiate the toxicity of chemotherapeutic drugs. Recently, a Phase III clinical study using doxorubicin with DPPE demonstrated significant increase in the overall survival of breast cancer patients. In this study we examined the effects of DPPE alone on the growth of drug sensitive and P-gp positive CHO cell line. Our results demonstrate DPPE is selectively toxic to P-gp positive cells and the sensitivity to DPPE alone correlated with the levels of P-gp expression. Moreover, in MDR cells, DPPE-induced apoptosis was significantly reduced with Bcl2 overexpression and in the presence of P-gp ATPase inhibitor, PSC833. Furthermore, knockdown of P-gp expression in MDR cells with P-gp-siRNA reversed DPPE sensitivity and increased their sensitivity to doxorubicin and taxol but not to cisplatin. The addition of DPPE to membrane fractions led to dose-dependent increase in P-gp ATPase that was inhibited with PSC833. Moreover, incubation of P-gp positive cells with DPPE led to a significant increase in superoxide levels and a drop in cellular ATP and GSH pools that were reversible with inhibitors of P-gp ATPase. The combined presence of DPPE and the mitochondria electron transport complex III inhibitor, antimycin A, synergized in their effects on the growth of MDR cells but had no effect on the growth of parental drug sensitive cells. Collectively, the results of this study provide a possible mechanism that may be relevant to the clinical results of DPPE in breast cancer trial and demonstrates DPPE as P-gp collateral sensitivity drug.     

Quantification of biliary excretion and sinusoidal excretion of 5(6)-carboxy-2′,7′-dichlorofluorescein (CDF) in cultured hepatocytes isolated from Sprague Dawley,Wistar and Mrp2-deficient Wistar (TR−) rats

Hepatic efflux of drug candidates is an important issue in pre-clinical drug development. Here we utilise a method which quantifies and distinguishes efflux of drugs at the canalicular and sinusoidal membranes in rat hepatocyte cultures. Bi-phasic kinetics of transport of 5(6)-carboxydichlorofluorescein (CDF) at the canalicular membrane was demonstrated in Sprague Dawley (SD) and Wistar (W) rat hepatocytes. The high affinity component (Km = 3.2 ± 0.8 μM (SD), 9.0 ± 3.1 μM (W)) was attributed to Mrp2-mediated transport, the low affinity component (Km = 192.1 ± 291.5 μM (SD), 69.2 ± 36.2 μM (W)) may be attributed to transport involving a separate Mrp2 binding site. Data from membranes (Hill coefficient (h) = 2.0 ± 0.5) and vesicles (h = 1.6 ± 0.2) expressing Mrp2 and from SD (h = 1.6 ± 0.4) and Wistar (h = 4.0 ± 0.6) hepatocytes suggests transport involves more than one binding site. In TR⁻ hepatocytes, CDF efflux was predominantly over the sinusoidal membrane (Km = 100.7 ± 36.0 μM), consistent with low abcc2 (Mrp2) expression and compensatory increase in abcc3 (Mrp3) expression. This report shows the potential of using this in vitro method to model changes in biliary excretion due to alterations in transporter expression.     

Digoxin net secretory transport in bronchial epithelial cell layers is not exclusively mediated by P-glycoprotein/MDR1

The impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe ³H-digoxin in air–liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin–Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. ³H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER = 11.4 or 3.0 were measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on ³H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in ³H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs.