Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity

Sandwich-cultured hepatocytes (SCH) are a powerful in vitro tool that can be utilized to study hepatobiliary drug transport, species differences in drug transport, transport protein regulation, drug-drug interactions, and hepatotoxicity. This review provides an up-to-date summary of the SCH model, including a brief history of, and introduction to, the use of SCH, as well as methodology to evaluate hepatobiliary drug disposition. A summary of the literature that has utilized this model to examine the interplay between drug metabolizing enzymes and transport proteins, drug-drug interactions at the transport level, and hepatotoxicity as a result of altered hepatic transport also is provided

An Experimental Approach To Evaluate the Impact of Impaired Transport Function on Hepatobiliary Drug Disposition Using Mrp2-Deficient TR – Rat Sandwich-Cultured Hepatocytes in Combination with Bcrp Knockdown

Breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) are members of the ATP binding cassette (ABC) transporter family located in the canalicular membrane of hepatocytes that mediate biliary excretion of many drugs and endogenous compounds. BCRP and MRP2 have overlapping substrate profiles. Predicting drug disposition in the setting of altered transport function has important clinical significance. This investigation was designed to establish an in vitro model system to evaluate the impact of impaired Mrp2 and Bcrp function on hepatobiliary drug disposition. To achieve Bcrp knockdown by RNA interference (RNAi), sandwich-cultured hepatocytes (SCH) from Mrp2-deficient (TR–) and wild-type (WT) rats were infected with adenoviral vectors to express shRNA targeting Bcrp (Ad-siBcrp) at multiplicity of infection (MOI) of 1–10. MOI of 5 was identified as optimal. At MOI of 5, viral infection as well as WT or TR– status was statistically significant predictors of the rosuvastatin (RSV) biliary excretion index (BEI), consistent with the known role of Bcrp and Mrp2 in the biliary excretion of RSV in vivo in rats. Relative to WT rat SCH, marginal mean BEI (%) of RSV in TR– rat SCH decreased by 28.6 (95% CI: 5.8–51.3). Ad-siBcrp decreased marginal mean BEI (%) of RSV by 13.3 (7.5–9.1) relative to SCH infected with adenoviral vectors expressing a nontargeting shRNA (Ad-siNT). The BEI of RSV was almost ablated in TR– rat SCH with Bcrp knockdown (5.9 ± 3.0%) compared to Ad-siNT-infected WT rat SCH (45.4 ± 6.6%). These results demonstrated the feasibility of Bcrp knockdown in TR– rat SCH as an in vitro system to assess the impact of impaired Bcrp and Mrp2 function. At MOI of 5, viral infection had minimal effects on RSV total accumulation, but significantly decreased marginal mean taurocholate total accumulation (pmol/mg of protein) and BEI (%) by 9.9 (7.0–12.8) and 7.5 (3.7–11.3), respectively, relative to noninfected SCH. These findings may be due to off-target effects on hepatic bile acid transporters, even though no changes in protein expression levels of the hepatic bile acid transporters were observed. This study established a strategy for optimization of the knockdown system, and demonstrated the potential use of RNAi in SCH as an in vitro tool to predict altered hepatobiliary drug disposition when canalicular transporters are impaired

Estimating aboveground carbon density and its uncertainty in Borneo's structurally complex tropical forests using airborne laser scanning

Borneo contains some of the world's most biodiverse and carbon-dense tropical forest, but this 750 000 km(2) island has lost 62% of its old-growth forests within the last 40 years. Efforts to protect and restore the remaining forests of Borneo hinge on recognizing the ecosystem services they provide, including their ability to store and sequester carbon. Airborne laser scanning (ALS) is a remote sensing technology that allows forest structural properties to be captured in great detail across vast geographic areas. In recent years ALS has been integrated into statewide assessments of forest carbon in Neotropical and African regions, but not yet in Asia. For this to happen new regional models need to be developed for estimating carbon stocks from ALS in tropical Asia, as the forests of this region are structurally and composition-ally distinct from those found elsewhere in the tropics. By combining ALS imagery with data from 173 permanent forest plots spanning the lowland rainforests of Sabah on the island of Borneo, we develop a simple yet general model for estimating forest carbon stocks using ALS-derived canopy height and canopy cover as input metrics. An advanced feature of this new model is the propagation of uncertainty in both ALS- and ground-based data, allowing uncertainty in hectare-scale estimates of carbon stocks to be quantified robustly. We show that the model effectively captures variation in aboveground carbon stocks across extreme disturbance gradients spanning tall dipterocarp forests and heavily logged regions and clearly outperforms existing ALS-based models calibrated for the tropics, as well as currently available satellite-derived products. Our model provides a simple, generalized and effective approach for mapping forest carbon stocks in Borneo and underpins ongoing efforts to safeguard and facilitate the restoration of its unique tropical forests.Peer reviewe

cGMP-Dependent Protein Kinase Type I Is Implicated in the Regulation of the Timing and Quality of Sleep and Wakefulness

Many effects of nitric oxide (NO) are mediated by the activation of guanylyl cyclases and subsequent production of the second messenger cyclic guanosine-3′,5′-monophosphate (cGMP). cGMP activates cGMP-dependent protein kinases (PRKGs), which can therefore be considered downstream effectors of NO signaling. Since NO is thought to be involved in the regulation of both sleep and circadian rhythms, we analyzed these two processes in mice deficient for cGMP-dependent protein kinase type I (PRKG1) in the brain. Prkg1 mutant mice showed a strikingly altered distribution of sleep and wakefulness over the 24 hours of a day as well as reductions in rapid-eye-movement sleep (REMS) duration and in non-REM sleep (NREMS) consolidation, and their ability to sustain waking episodes was compromised. Furthermore, they displayed a drastic decrease in electroencephalogram (EEG) power in the delta frequency range (1–4 Hz) under baseline conditions, which could be normalized after sleep deprivation. In line with the re-distribution of sleep and wakefulness, the analysis of wheel-running and drinking activity revealed more rest bouts during the activity phase and a higher percentage of daytime activity in mutant animals. No changes were observed in internal period length and phase-shifting properties of the circadian clock while chi-squared periodogram amplitude was significantly reduced, hinting at a less robust oscillator. These results indicate that PRKG1 might be involved in the stabilization and output strength of the circadian oscillator in mice. Moreover, PRKG1 deficiency results in an aberrant pattern, and consequently a reduced quality, of sleep and wakefulness, possibly due to a decreased wake-promoting output of the circadian system impinging upon sleep

Hepatic Basolateral Efflux Contributes Significantly to Rosuvastatin Disposition I: Characterization of Basolateral Versus Biliary Clearance Using a Novel Protocol in Sandwich-Cultured Hepatocytes

Transporters responsible for hepatic uptake and biliary clearance (CL(Bile)) of rosuvastatin (RSV) have been well characterized. However, the contribution of basolateral efflux clearance (CL(BL)) to hepatic and systemic exposure of RSV is unknown. Additionally, the appropriate design of in vitro hepatocyte efflux experiments to estimate CL(Bile) versus CL(BL) remains to be established. A novel uptake and efflux protocol was developed in sandwich-cultured hepatocytes (SCH) to achieve desired tight junction modulation while maintaining cell viability. Subsequently, studies were conducted to determine the role of CL(BL) in the hepatic disposition of RSV using SCH from wild-type (WT) and multidrug resistance-associated protein 2 (Mrp2)-deficient (TR(−)) rats in the absence and presence of the P-glycoprotein and breast cancer resistance protein (Bcrp) inhibitor elacridar (GF120918). RSV CL(Bile) was nearly ablated by GF120918 in TR(−) SCH, confirming that Mrp2 and Bcrp are responsible for the majority of RSV CL(Bile). Pharmacokinetic modeling revealed that CL(BL) and CL(Bile) represent alternative elimination routes with quantitatively similar contributions to the overall hepatocellular excretion of RSV in rat SCH under baseline conditions (WT SCH in the absence of GF120918) and also in human SCH. Membrane vesicle experiments revealed that RSV is a substrate of MRP4 (K(m) = 21 ± 7 µM, V(max) = 1140 ± 210 pmol/min per milligram of protein). Alterations in MRP4-mediated RSV CL(BL) due to drug-drug interactions, genetic polymorphisms, or disease states may lead to changes in hepatic and systemic exposure of RSV, with implications for the safety and efficacy of this commonly used medication

Adalimumab long-term safety: infections, vaccination response and pregnancy outcomes in patients with rheumatoid arthritis

Adalimumab has been used in patients with moderately to severely active rheumatoid arthritis (RA) for over 10 years and has a well-established safety profile across multiple indications. To update adverse events (AEs) of special interest from global adalimumab clinical trials in patients with RA. This analysis includes 15 132 patients exposed to adalimumab in global RA clinical trials. AEs of interest included overall infections, laboratory abnormalities and AEs associated with influenza vaccination. Pregnancy outcome data were collected from the Adalimumab Pregnancy Registry. Serious infections and tuberculosis occurred at a rate of 4.7 and 0.3 events/100 patient-years, respectively. Two patients experienced hepatitis B reactivation. No significant laboratory abnormalities were reported with adalimumab-plus-methotrexate compared with placebo-plus-methotrexate. Influenza-related AEs occurred in 5% of vaccinated patients compared with 14% of patients not vaccinated during the study. Relative risk of major birth defects and spontaneous abortions in adalimumab-exposed women were similar between that of unexposed women with RA and healthy women. This analysis confirms and expands the known safety profile of adalimumab and reports no additional safety risk of laboratory abnormalities, hepatitis B reactivation and pregnancy outcomes, including spontaneous abortions and birth defects. The benefits of influenza vaccination are reinforced. NCT00195663, NCT00195702, NCT00448383, NCT00049751, NCT00234845, NCT00650390, NCT00235859, NCT00647920, NCT00649545, NCT00647491, NCT00649922, NCT00538902, NCT00420927, NCT00870467, NCT00650156, NCT00647270, NCT01185288, NCT0118530