Hepatic Clearance Research Articles

Esterases Involved in the Rapid Bioconversion of Esmolol after Intravenous Injection in Humans.

Esmolol is indicated for the acute and temporary control of ventricular rate due to its rapid onset of action and elimination at a rate greater than cardiac output. This rapid elimination is achieved by the hydrolysis of esmolol to esmolol acid. It has previously been reported that esmolol is hydrolyzed in the cytosol of red blood cells (RBCs). In order to elucidate the metabolic tissues and enzymes involved in the rapid elimination of esmolol, a hydrolysis study was performed using different fractions of human blood and liver. Esmolol was slightly hydrolyzed by washed RBCs and plasma proteins while it was extensively hydrolyzed in plasma containing white blood cells and platelets. The negligible hydrolysis of esmolol in RBCs is supported by its poor hydrolysis by esterase D, the sole cytosolic esterase in RBCs. In human liver microsomes, esmolol was rapidly hydrolyzed according to Michaelis-Menten kinetics, and its hepatic clearance, calculated by the well-stirred model, was limited by hepatic blood flow. An inhibition study and a hydrolysis study using individual recombinant esterases showed that human carboxylesterase 1 isozyme (hCE1) is the main metabolic enzyme of esmolol in both white blood cells and human liver. These studies also showed that acyl protein thioesterase 1 (APT1) is involved in the cytosolic hydrolysis of esmolol in the liver. The hydrolysis of esmolol by hCE1 and APT1 also results in its pulmonary metabolism, which might be a reason for its high total clearance (170-285 mL/min/kg bodyweight), 3.5-fold greater than cardiac output (80.0 mL/min/kg bodyweight).

PULMONARY HYPERTENSION FOLLOWING TRANSJUGULAR INTRAHEPATIC PORTOSYSTEMIC SHUNT (TIPS) PLACEMENT DUE TO NEUROHORMONAL VASOACTIVE CAUSES

PULMONARY HYPERTENSION FOLLOWING TRANSJUGULAR INTRAHEPATIC PORTOSYSTEMIC SHUNT (TIPS) PLACEMENT DUE TO NEUROHORMONAL VASOACTIVE CAUSES

Modulation of vascular endothelial inflammatory response by proprotein convertase subtilisin-kexin type 9

Background and AimsEndotoxins carried within LDL are cleared from the circulation via hepatic LDL receptor (LDLR)-mediated endocytosis. Proprotein convertase subtilisin-kexin type 9 (PCSK9) reduces this clearance by down-regulating LDLR density on hepatocytes. In addition to hepatocytes, vascular endothelial cells also express receptor targets of PCSK9, including LDLR. Therefore, we hypothesized that PCSK9 may regulate vascular endothelial cell uptake of lipopolysaccharide (LPS) and alter the vascular endothelial cell inflammatory response. Methods and resultsWe found that LPS is internalized by human umbilical vein vascular endothelial cells (HUVECs) and LPS uptake dose-dependently increased with increasing LDL concentration. Intracellular LPS co-localized with LDL. PCSK9 and, separately, blocking antibodies against LDLR, dose-dependently decreased the vascular endothelial cell uptake of LPS and, furthermore, inhibition of endocytosis using Dynasore blocked LPS uptake. In contrast, blocking antibodies against TLR4 did not alter LPS uptake. PCSK9 decreased the LPS-induced proinflammatory response (IL-6 and IL-8 gene expression and protein secretion, and VCAM-1/ICAM-1 expression) in vascular endothelial cells. In addition, a decrease in PCSK9 and increase in LDLR, mediated by triciribine or siPCSK9, increased LPS uptake and the LPS-induced proinflammatory response. Similar results were also found in aortic vascular tissue from Pcsk9−/− mice after LPS injection. ConclusionsOur data suggest that, similar to PCSK9 treatment in hepatocytes, PCSK9 reduces vascular endothelial cell uptake of LPS via LDLR-mediated endocytosis. Consequently, PCSK9 decreases the LPS-induced proinflammatory response in vascular endothelial cells. These results raise the possibility that PCSK9 inhibition may have additional effects on vascular endothelial inflammation via this alternative pathway, beyond the known effects of PCSK9 inhibition on LDL lowering and hepatic endotoxin clearance.

In vitro metabolism of the new antifungal dapaconazole using liver microsomes

Dapaconazole is a new antifungal imidazole that has been shown a high efficacy against several pathogenic fungi. This study aimed to investigate the interspecies variation in the in vitro metabolic profiles and in vivo hepatic clearance (CLH,in vivo) prediction of dapaconazole using liver microsomes from male Sprague Dawley rat, male Beagle dog and mixed gender human using a liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method. In addition, the produced metabolites were identified by ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS/MS). The microsomal protein concentration of 0.1 mg/mL and the incubation time of 10 min were employed for the kinetics determination, resulting in a sigmoidal kinetic profile for all species evaluated. The predicted CLH,in vivo was 6.5, 11.6 and 7.5 mL/min/kg for human, rat and dog, respectively. Furthermore, five metabolized products were identified. These findings provide preliminary information for understanding dapaconazole metabolism and the interspecies differences in catalytic behaviours, supporting the choice of a suitable laboratory animal for future pharmacokinetics and metabolism studies.

Sinusoidal Organic Anion-Transporting Polypeptide 1B1/1B3 and Bile Canalicular Multidrug Resistance-Associated Protein 2 Play an Essential Role in the Hepatobiliary Disposition of a Synthetic Cyclic Dinucleotide (STING Agonist)

The liver is central to the elimination of many drugs from the body involving multiple processes and understanding of these processes is important to quantitively assess hepatic clearance of drugs. The synthetic STING (STimulator of INterferon Genes protein) agonist is a new class of drugs currently being evaluated in clinical trials as a potential anticancer therapy. In this study, we used ML00960317 (synthetic STING agonist) to investigate the hepatobiliary disposition of this novel molecular entity. A bile-duct cannulated (BDC) rat study indicated that biliary excretion is the major route of elimination for ML00960317 (84% of parent dose in bile). The human biliary clearance using in vitro sandwich cultured human hepatocyte model predicted significant biliary excretion of ML00960317 (biliary excretion index (BEI) of 47%). Moreover, the transport studies using transporter expressing cell lines, hepatocytes, and membrane vesicles indicated that ML00960317 is a robust substrate of OATP1B1, OATP1B3, and MRP2. Using relative expression factor approach, the combined contribution of OATP1B1 (fraction transported (ft) = 0.62) and OATP1B3 (ft = 0.31) was found to be 93% of the active uptake clearance of ML00960317 into the liver. Furthermore, OATP1B1 and OATP1B3-mediated uptake of ML00960317 was inhibited by rifampicin with IC50 of 6.5 and 2.3 μM, respectively indicating an in vivo DDI risk (R value of 1.5 and 2.5 for OATP1B1 and OATP1B3, respectively). These results highlighted an important role of OATP1B1, OATP1B3, and MRP2 in the hepatobiliary disposition of ML00960317. These pathways may act as rate-determining steps in the hepatic clearance of ML00960317 thus presenting clinical DDI risk.Graphical

β-Cell Function, Hepatic Insulin Clearance, and Insulin Sensitivity in South Asian and Nordic Women After Gestational Diabetes Mellitus.

South Asian women have a higher risk of type 2 diabetes after gestational diabetes mellitus (GDM) than Nordic women; however, the mechanisms behind this difference remain unclear. We investigated insulin sensitivity, β-cell function, and hepatic insulin clearance in 179 South Asian and 108 Nordic women ∼17 months after GDM (mean age 35.3 years, BMI 29.1 kg/m2) by oral glucose tolerance test using deconvolution of C-peptide kinetics. Thirty-one percent of South Asian and 53% of Nordic participants were normoglycemic at the time of measurement. South Asian women had higher areas under the curve (AUCs) for glucose, prehepatic insulin, and peripheral insulin and lower insulin sensitivity, disposition index, and fasting hepatic insulin clearance than Nordic women. In the group with prediabetes or diabetes, South Asian women had similar AUCs for glucose and prehepatic insulin but a higher AUC for peripheral insulin, lower disposition index, and lower fasting hepatic insulin clearance than Nordic women. The waist-to-height ratio mediated ∼25-40% of the ethnic differences in insulin sensitivity in participants with normoglycemia. Overall, our novel data revealed that South Asian women with normoglycemia after GDM showed lower insulin secretion for a given insulin resistance and lower hepatic insulin clearance than Nordic women. South Asian women are at high risk of developing type 2 diabetes after GDM, and preventive efforts should be prioritized.

Evaluation of a rapid, generic human gestational dose model

Chemical risk assessment considers potentially susceptible populations including pregnant women and developing fetuses. Humans encounter thousands of chemicals in their environments, few of which have been fully characterized. Toxicokinetic (TK) information is needed to relate chemical exposure to potentially bioactive tissue concentrations. Observational data describing human gestational exposures are unavailable for most chemicals, but physiologically based TK (PBTK) models estimate such exposures. Development of chemical-specific PBTK models requires considerable time and resources. As an alternative, generic PBTK approaches describe a standardized physiology and characterize chemicals with a set of standard physical and TK descriptors – primarily plasma protein binding and hepatic clearance. Here we report and evaluate a generic PBTK model of a human mother and developing fetus. We used a published set of formulas describing the major anatomical and physiological changes that occur during pregnancy to augment the High-Throughput Toxicokinetics (httk) software package. We simulated the ratio of concentrations in maternal and fetal plasma and compared to literature in vivo measurements. We evaluated the model with literature in vivo time-course measurements of maternal plasma concentrations in pregnant and non-pregnant women. Finally, we prioritized chemicals measured in maternal serum based on predicted fetal brain concentrations. This new model can be used for TK simulations of 859 chemicals with existing human-specific in vitro TK data as well as any new chemicals for which such data become available. This gestational model may allow for in vitro to in vivo extrapolation of point of departure doses relevant to reproductive and developmental toxicity.

Turmeric extract (Curcuma longa L.) regulates hepatic toxicity in a single ethanol binge rat model

Hepatic alcohol clearance is a key factor to overcome alcohol hangovers, and over the period, alcohol hangovers may lead to inflammation and oxidative stress. Natural food products with high antioxidant and anti-inflammatory effects might contribute to hepatic alcohol clearance, a hypothesis in this study. The present study aimed to evaluate the influence of turmeric (Curcuma longa L., Zingiberaceae) is an herbal product having antioxidant and anti-inflammatory activities, on alcohol metabolism using binge alcohol drinking rat model. In vivo investigations revealed that pretreatment with turmeric extract enhanced alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities upon binge ethanol (3 g/kg). Additionally, pretreatment with turmeric extract regulated CYP2E1 activity and levels of reactive oxygen species (ROS), Bax, Bcl-2, and inflammatory mediators like IL-1β, IL-6, and TNF-α. Moreover, turmeric extract upregulated superoxide dismutase, catalase, and glutathione peroxidase activities in liver tissues. Together, these observations shed light on the potential beneficial effects of turmeric extract against acute liver toxicity. The results offer an alternative natural functional food product, turmeric extract, to prevent the negative implications of binge drinking.

Population pharmacokinetics of unbound ceftriaxone in a critically ill population.

To develop a reliable 2-compartment population pharmacokinetic (PK) model for unbound ceftriaxone in a critically ill population and determine an optimal dosing regimen. This was a prospective, single-center, observational study of critically ill patients treated with ceftriaxone. Unbound serum ceftriaxone concentrations were measured using validated ultrafiltration and ultra-performance liquid chromatography-tandem mass spectrometry. PK analysis and dosing simulations were performed using an iterative 2-stage Bayesian fitting procedure and Monte Carlo simulations. The PK/pharmacodynamics (PD) target was attained when unbound serum ceftriaxone concentrations exceeded 4 times the minimum inhibitory concentration (MIC) for ≥60% of the dosing interval (ƒT>4xMIC ≥ 60%). 91 patients were enrolled, and 173 unbound ceftriaxone concentrations were acquired. The population PK parameter estimates were hepatic clearance 5.2±0.9L/h/1.85m2, the unbound renal clearance of ceftriaxone divided by the creatinine clearance 0.61±0.24, lean body mass corrected volume of distribution of the central compartment 0.82±0.21L/kg, and intercompartmental distribution rate constant from central to peripheral compartment 0.18±0.08h-1. Dosing simulations predicted ƒT>4 mg/L of 88% (95% CI: 69-100%) for 2,000 mg ceftriaxone once daily and ƒT>4 mg/L of 100% (95% CI: 100-100%) both for 1,000 mg twice daily and continuous infusion of 2,000 mg daily. We developed a reliable population PK model for unbound ceftriaxone in a critically ill population. Dosing simulations revealed ƒT>4 mg/L ≥60% for 1,000mg twice daily and 2,000 mg once daily or by continuous infusion.

N-(4-[18F]fluorobenzyl)cholylglycine, a potential tracer for positron emission tomography of enterohepatic circulation and drug-induced inhibition of ileal bile acid transport. A proof-of-concept PET/CT study in pigs

IntroductionEnterohepatic circulation (EHC) of conjugated bile acids is an important physiological process crucial for bile acids to function as detergents and signal carriers. Perturbation of the EHC by disease or drugs may lead to serious and life-threatening liver and gastrointestinal disorders. In this proof-of-concept study in pigs, we investigate the potential of N-(4-[18F]fluorobenzyl)cholylglycine ([18F]FBCGly) as tracer for quantitative positron emission tomography (PET) of the EHC of conjugated bile acids. MethodsThe biodistribution of [18F]FBCGly was investigated by PET/CT in domestic pigs following intravenous and intraileal administration of the tracer. Hepatic kinetics were estimated from PET and blood data using a 2-tissue compartmental model and dual-input of [18F]FBCGly. The ileal uptake of [18F]FBCGly was investigated with co-injection of nifedipine and endogenous cholyltaurine. Dosimetry was estimated from the PET data using the Olinda 2.0 software. Blood, bile and urine samples were analyzed for possible fluorine-18 labelled metabolites of [18F]FBCGly. Results[18F]FBCGly was rapidly taken up by the liver and excreted into bile, and underwent EHC without being metabolized. Both nifedipine and endogenous cholyltaurine inhibited the ileal uptake of [18F]FBCGly. The flow-dependent hepatic uptake clearance was estimated to median 1.2 mL blood/min/mL liver tissue. The mean residence time of [18F]FBCGly in hepatocytes was 4.0 ± 1.1 min. Critical organs for [18F]FBCGly were the gallbladder wall (0.94 mGy/MBq) and the small intestine (0.50 mGy/MBq). The effective dose for [18F]FBCGly was 36 μSv/MBq. ConclusionWe have shown that [18F]FBCGly undergoes EHC in pigs without being metabolized and that its ileal uptake is inhibited by nifedipine and endogenous bile acids. Combined with our previous findings in rats, we believe that [18F]FBCGly has potential as PET tracer for assessment of EHC of conjugated bile acids under physiological conditions as well as conditions with perturbed hepatic and ileal bile acid transport.

Unravelling the pharmacokinetics of aflatoxin B1: In vitro determination of Michaelis-Menten constants, intrinsic clearance and the metabolic contribution of CYP1A2 and CYP3A4 in pooled human liver microsomes.

Mycotoxins, fungal secondary metabolites, are ubiquitously present in food commodities. Acute exposure to high levels or chronic exposure to low levels has an impact on the human body. The phase I metabolism in the human liver, performed by cytochrome P450 (CYP450) enzymes, is accountable for more than 80% of the overall metabolism of exogenous and endogenous compounds. Mycotoxins are (partially) metabolized by CYP450 enzymes. In this study, in vitro research was performed on CYP450 probes and aflatoxin B1 (AFB1), a carcinogenic mycotoxin, to obtain pharmacokinetic data on AFB1, required for further experimental work. The CYP450 probes of choice were a CYP3A4 substrate, midazolam (MDZ) and a CYP1A2 substrate, phenacetin (PH) since these are the main metabolizing phase I enzymes of AFB1. Linearity experiments were performed on the three substrates indicating that linear conditions were achieved at a microsomal protein concentration and incubation time of 0.25 mg/ml and 5 min, 0.50 mg/ml and 20 min and 0.25 mg/ml and 5 min for MDZ, PH and AFB1, respectively. The Km was determined in human liver microsomes and was estimated at 2.15 μM for MDZ, 40.0 μM for PH and 40.9 μM for AFB1. The associated Vmax values were 956 pmol/(mg.min) (MDZ), 856 pmol/(mg.min) (PH) and 11,536 pmol/(mg.min) (AFB1). Recombinant CYP systems were used to determine CYP450-specific Michaelis–Menten values for AFB1, leading to a CYP3A4 Km of 49.6 μM and an intersystem extrapolation factor (ISEF) corrected Vmax of 43.6 pmol/min/pmol P450 and a CYP1A2 Km of 58.2 μM and an ISEF corrected Vmax of 283 pmol/min/pmol P450. An activity adjustment factor (AAF) was calculated to account for differences between microsome batches and was used as a correction factor in the determination of the human in vivo hepatic clearance for MDZ, PH and AFB1. The hepatic blood clearance corrected for the AAF CLH,B,MDZ,AAF, CLH,B,PH,AAF CLH,B,AFB1,AAF(CYP3A4) and CLH,B,AFB1,AAF(CYP1A2) were determined in HLM at 44.1 L/h, 21.7 L/h, 40.0 L/h and 38.5 L/h. Finally, inhibition assays in HLM showed that 45% of the AFB1 metabolism was performed by CYP3A4/3A5 enzymes and 49% by CYP1A2 enzymes.

Nuclear Receptor Pathways Mediating the Development of Boar Taint.

The nuclear receptors PXR, CAR, and FXR are activated by various ligands and function as transcription factors to control the expression of genes that regulate the synthesis and metabolism of androstenone and skatole. These compounds are produced in entire male pigs and accumulate in the fat to cause the development of a meat quality issue known as boar taint. The extent of this accumulation is influenced by the synthesis and hepatic clearance of androstenone and skatole. For this reason, PXR, CAR, and FXR-mediated signaling pathways have garnered interest as potential targets for specialized treatments designed to reduce the development of boar taint. Recent research has also identified several metabolites produced by gut microbes that act as ligands for these nuclear receptors (e.g., tryptophan metabolites, short-chain fatty acids, bile acids); however, the connection between the gut microbiome and boar taint development is not clear. In this review, we describe the nuclear receptor signaling pathways that regulate the synthesis and metabolism of boar taint compounds and outline the genes involved. We also discuss several microbial-derived metabolites and dietary additives that are known or suspected nuclear receptor ligands and suggest how these compounds could be used to develop novel treatments for boar taint.

Validation of Methods for in Vitro-in Vivo Extrapolation Using Hepatic Clearance Measurements in Isolated Perfused Fish Livers.

In vitro biotransformation assays using hepatocytes or liver subcellular fractions, combined with in vitro-in vivo extrapolation (IVIVE) models, have been proposed as an alternative to live fish bioconcentration studies. The uncertainty associated with IVIVE approaches to date has been attributed to assay protocols, model assumptions, or variability of in vivo data. An isolated perfused trout liver model that measures hepatic clearance has been proposed for validating IVIVE predictions in the absence of other confounding factors. Here, we investigated the hepatic clearances of five chemicals (pyrene, phenanthrene, 4-n-nonlyphenol, deltamethrin, and methoxychlor) in this model and compared measured rates to values predicted from published in vitro intrinsic clearances for validation of IVIVE models. Additionally, we varied protein concentrations in perfusates to test binding assumptions of these models. We found that measured and predicted hepatic clearances were in very good agreement (root mean squared error 16.8 mL h-1 g-1) across three levels of protein binding and across a more diverse chemical space than previously studied within this system. Our results show that current IVIVE methods can reliably predict in vivo clearance rates and indicate that discrepancies from measured bioconcentration factors might be driven by other processes, such as extrahepatic biotransformation, etc., and help streamline optimization efforts to the processes that truly matter.

Improved Predictability of Hepatic Clearance with Optimal pH for Acyl-Glucuronidation in Liver Microsomes

The purpose of this study was to investigate the optimal pH for acyl-glucuronidation formation with carboxylic acid-containing compounds in human and rat liver microsomes to improve the predictability of their hepatic clearance. The optimal pH for acyl-glucuronidation of all 17 compounds was around pH 6.0 in human and rat liver microsomes. Correlation analysis was done with the predicted in vitro intrinsic clearance (CLint,in vitro) and in vivo intrinsic clearance (CLint,in vivo) calculated from available reported data of total clearance (CLtot) of 11 compounds in humans. For 8 of the 11 compounds, under the pH 6.0 condition, the CLint,in vitro were within 1/3 to 3-fold error of the observed CLint,in vivo whereas, the error was within 1/3 to 3-fold of the observed CLint,in vivo for only 3 of the 11 under the pH 7.4 condition. The intracellular pH in human and rat hepatocytes decreased in the presence of a carboxylic acid-containing compound. These findings suggest that acyl-glucuronidation in liver microsomes at pH 6.0 is closer to physiological conditions in the presence of carboxylic acid compounds, and thus, use of this pH condition is important for physiological interpretation and predictability of intrinsic clearance.

HIF1 inhibition of the biflavonoids against pancreas cancer: drug-likeness, bioavailability, ADMET, PASS, molecular docking, molecular dynamics, and MM/GBSA calculations

Pancreatic cancer is an aggressive disease with a high death rate and is difficult to treat. This disease, in the most cases, is asymptomatic until it progresses to an advanced stage. Therefore, the search for bioactive molecules is urgent to combat pancreatic cancer. Then, this work analyzed the interaction potential of agathisflavone and amentoflavone molecules against the HIF1 target using the ADMET, molecular docking, and molecular dynamics simulations. More recent drug-likeness filters that combine physicochemical and physiological parameters have shown that high polar surface area (TPSA > 75 Å2) drives biflavonoids out of the toxic drug space of Pfizer dataset. Regarding the pharmacokinetic descriptors, it was possible to notice that Amentoflavone showed a better order of passive cell permeability (Papp = 8 × 10−6 cm/s) and better metabolic stability, biotransformed by aromatic hydroxylation reactions by the CYP3A4 isoenzyme on the human liver, that favor its hepatic clearance. The molecular docking and molecular dynamics simulations indicated the high interaction potential and stability between the ligands analyzed (highlighted the amentoflavone molecule), respectively. The MM/GBSA calculations showed that the amentoflavone ligand registered the highest ΔG binding value of −32.6957 kcal/mol with the HIF1 target. Then, this molecule may be used as a potential inhibitor of pancreatic cancer. In this perspective, the present work represents an initial step in the virtual bioprospecting a pharmacological tool for treating of pancreatic cancer. Communicated by Ramaswamy H. Sarma

Cluster Gauss-Newton method analyses of PBPK model parameter combinations of coproporphyrin-I based on OATP1B-mediated rifampicin interaction studies.

Coproporphyrin I (CP‐I) is an endogenous biomarker supporting the prediction of drug–drug interactions (DDIs) involving hepatic organic anion transporting polypeptide 1B (OATP1B). We previously constructed a physiologically‐based pharmacokinetic (PBPK) model for CP‐I using clinical DDI data with an OATP1B inhibitor, rifampicin (RIF). In this study, PBPK model parameters for CP‐I were estimated using the cluster Gauss–Newton method (CGNM), an algorithm used to find multiple approximate solutions for nonlinear least‐squares problems. Eight unknown parameters including the hepatic overall intrinsic clearance (CLint,all), the rate of biosynthesis (v syn), and the OATP1B inhibition constant of RIF(K i,u,OATP) were estimated by fitting to the observed CP‐I blood concentrations in two different clinical studies involving changing the RIF dose. Multiple parameter combinations were obtained by CGNM that could well capture the clinical data. Among those, CLint,all, K i,u,OATP, and v syn were sensitive parameters. The obtained K i,u,OATP for CP‐I was 5.0‐ and 2.8‐fold lower than that obtained for statins, confirming our previous findings describing substrate‐dependent K i,u,OATP values. In conclusion, CGNM analyses of PBPK model parameter combinations enables estimation of the three essential parameters for CP‐I to capture the DDI profiles, even if the other parameters remain unidentified. The CGNM also clarified the importance of appropriate combinations of other unidentified parameters to enable capture of the CP‐I concentration time course under the influence of RIF. The described CGNM approach may also support the construction of robust PBPK models for additional transporter biomarkers beyond CP‐I.

Regulation of Insulin Clearance by Non-Esterified Fatty Acids

Insulin stores lipid in adipocytes and prevents lipolysis and the release of non-esterified fatty acids (NEFA). Excessive release of NEFA during sustained energy supply and increase in abdominal adiposity trigger systemic insulin resistance, including in the liver, a major site of insulin clearance. This causes a reduction in insulin clearance as a compensatory mechanism to insulin resistance in obesity. On the other hand, reduced insulin clearance in the liver can cause chronic hyperinsulinemia, followed by downregulation of insulin receptor and insulin resistance. Delineating the cause–effect relationship between reduced insulin clearance and insulin resistance has been complicated by the fact that insulin action and clearance are mechanistically linked to insulin binding to its receptors. This review discusses how NEFA mobilization contributes to the reciprocal relationship between insulin resistance and reduced hepatic insulin clearance, and how this may be implicated in the pathogenesis of non-alcoholic fatty liver disease.

Use of selective substrates and inhibitors to rapidly characterise batches of cryopreserved primary human hepatocytes for assessment of active uptake liability in drug discovery and development

The use of hepatocytes to predict human hepatic metabolic clearance is the gold standard approach. However whilst enzymes are well characterised, knowledge gaps remain for transporters. Furthermore, methods to study specific transporter involvement are often complicated by overlapping substrate specificity. Selective substrates and inhibitors would aid investigations into clinically relevant pharmacokinetic effects. However, to date no consensus has been reached. This work defines selective hepatic uptake transporter substrates and inhibitors for the six main human hepatocyte transporters (OATP1B1, OATP1B3, OATP2B1, NTCP, OAT2 & OCT1), and demonstrates their use to rapidly characterise batches of human hepatocytes for uptake transporter activity. Hepatic uptake was determined across a range of substrate concentrations, allowing the definition of kinetic parameters and hence active and passive components. Systematic investigations identified a specific substrate and inhibitor for each transporter, with no overlap between the specificity of substrate and inhibitor for any given transporter. Early characterisation of compound interactions with uptake transporters will aid in early risk assessment and chemistry design. Hence, this work further highlights the feasibility of a refined methodology for rapid compound characterisation for the application of static and dynamic models, for early clinical risk assessment and guidance for the clinical development plan.

New Insights into the Role of Insulin and Hypothalamic-Pituitary-Adrenal (HPA) Axis in the Metabolic Syndrome.

Recent data suggests that (pre)diabetes onset is preceded by a period of hyperinsulinemia. Consumption of the “modern” Western diet, over-nutrition, genetic background, decreased hepatic insulin clearance, and fetal/metabolic programming may increase insulin secretion, thereby causing chronic hyperinsulinemia. Hyperinsulinemia is an important etiological factor in the development of metabolic syndrome, type 2 diabetes, cardiovascular disease, polycystic ovarian syndrome, and Alzheimer’s disease. Recent data suggests that the onset of prediabetes and diabetes are preceded by a variable period of hyperinsulinemia. Emerging data suggest that chromic hyperinsulinemia is also a driving force for increased activation of the hypothalamic-adrenal-pituitary (HPA) axis in subjects with the metabolic syndrome, leading to a state of “functional hypercortisolism”. This “functional hypercortisolism” by antagonizing insulin actions may prevent hypoglycemia. It also disturbs energy balance by shifting energy fluxes away from muscles toward abdominal fat stores. Synergistic effects of hyperinsulinemia and “functional hypercortisolism” promote abdominal visceral obesity and insulin resistance which are core pathophysiological components of the metabolic syndrome. It is hypothesized that hyperinsulinemia-induced increased activation of the HPA axis plays an important etiological role in the development of the metabolic syndrome and its consequences. Numerous studies have demonstrated reversibility of hyperinsulinemia with lifestyle, surgical, and pharmaceutical-based therapies. Longitudinal studies should be performed to investigate whether strategies that reduce hyperinsulinemia at an early stage are successfully in preventing increased activation of the HPA axis and the metabolic syndrome.

Discovery of Novel Dual Adenosine A2A and A1 Receptor Antagonists with 1H-Pyrazolo[3,4-d]pyrimidin-6-amine Core Scaffold as Anti-Parkinson's Disease Agents.

New compounds with 1H-pyrazolo [3,4-d]pyrimidin-6-amine core scaffolds were synthesized and characterized in vitro to determine their affinity for human A2A and A1 receptors. Among the tested compounds, a few compounds displayed nanomolar binding affinities for both receptors. One particular compound, 11o, showed high binding activities (hA2A Ki = 13.3 nM; hA1 Ki = 55 nM) and full antagonism (hA2A IC50 = 136 nM; hA1 IC50 = 98.8 nM) toward both receptors. Further tests showed that 11o has low hepatic clearance and good pharmacokinetic properties in mice, along with high bioavailability and a high brain plasma ratio. In addition, 11o was associated with very low cardiovascular risk and mutagenic potential, and was well-tolerated in rats and dogs. When tested in an MPTP-induced mouse model of Parkinson’s disease, 11o tended to improve behavior. Moreover, 11o dose-dependently reversed haloperidol-induced catalepsy in female rats, with graded ED50 of between 3 and 10 mg/kg. Taken together, these results suggest that this potent dual A2A/A1 receptor antagonist, 11o, is a good candidate for the treatment of Parkinson’s disease with an excellent metabolic and safety profile.