In Vitro ADME and Preclinical Pharmacokinetics of Ulotaront, a TAAR1/5-HT1A Receptor Agonist for the Treatment of Schizophrenia.

PURPOSE: Ulotaront (SEP-363856) is a TAAR1 agonist with 5-HT1A agonist activity currently in clinical development for the treatment of schizophrenia. The objectives of the current study were to characterize the in vitro ADME properties, preclinical PK, and to evaluate the DDI potential of ulotaront and its major metabolite SEP-383103. METHODS: Solubility, permeability, plasma protein binding, CYP inhibition and induction, transporter inhibition and uptake studies were conducted in vitro. Phenotyping studies were conducted using recombinant human CYPs and FMOs, human liver microsomes and human liver homogenates. Preclinical plasma and brain pharmacokinetics were determined after a single intraperitoneal, intravenous, and oral administration of ulotaront. RESULTS: Ulotaront is a compound of high solubility, high permeability, and low binding to plasma proteins. Ulotaront metabolism is mediated via both NADPH-dependent and NADPH-independent pathways, with CYP2D6 as the major metabolizing enzyme. Ulotaront is an inducer of CYP2B6, and an inhibitor of CYP2D6, OCT1 and OCT2, while SEP-383103 is neither a CYP inducer nor a potent inhibitor of CYPs and human transporters. Ulotaront exhibits rapid absorption, greater than 70% bioavailability, approximately 3.5 L/kg volume of distribution, 1.5-4 h half-life, 12-43 ml/min/kg clearance, and good penetration across the blood-brain barrier in preclinical species. CONCLUSIONS: Ulotaront has been designated as a BCS1 compound by US FDA. The ability of ulotaront to penetrate the blood-brain barrier for CNS targeting has been demonstrated in mice and rats. The potential for ulotaront and SEP-383103 to act as perpetrators of CYP and transporter-mediated DDIs is predicted to be remote.

Prediction of Transporter-Mediated Rosuvastatin Hepatic Uptake Clearance and Drug Interaction in Humans Using Proteomics-Informed REF Approach.

Suspended, plated, or sandwich-cultured human hepatocytes are routinely used for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated hepatic clearance (CL) of drugs. However, these hepatocyte models have been reported to underpredict transporter-mediated in vivo hepatic uptake CL (CL uptake,in vivo ) of some drugs. Therefore, we determined whether transporter-expressing cells (TECs) can accurately predict the CL uptake,in vivo of drugs. To do so, we determined the uptake CL (CL int,uptake,cells ) of rosuvastatin (RSV) by TECs (organic anion transporting polypeptides/Na+-taurocholate cotransporting polypeptide) and then scaled it to that in vivo by relative expression factor (REF) (the ratio of transporter abundance in human livers and TEC) determined by liquid chromatography tandem mass spectrometry-based quantitative proteomics. Both the TEC and hepatocyte models did not meet our predefined success criteria of predicting within 2-fold the RSV CL uptake,in vivo value obtained from our positron emission tomography (PET) imaging. However, the TEC performed better than the hepatocyte models. Interestingly, using REF, TECs successfully predicted RSV CL int,uptake,hep obtained by the hepatocyte models, suggesting that the underprediction of RSV CL uptake,in vivo by TECs and hepatocytes is due to endogenous factor(s) not present in these in vitro models. Therefore, we determined whether inclusion of plasma (or albumin) in TEC uptake studies improved IVIVE of RSV CL uptake,in vivo It did, and our predictions were close to or just fell above our lower 2-fold acceptance boundary. Despite this success, additional studies are needed to improve transporter-mediated IVIVE of hepatic uptake CL of drugs. However, using REF and TEC, we successfully predicted the magnitude of PET-imaged inhibition of RSV CL uptake,in vivo by cyclosporine A. SIGNIFICANCE STATEMENT: We showed that the in vivo transporter-mediated hepatic uptake CL of rosuvastatin, determined by PET imaging, can be predicted (within 2-fold) from in vitro studies in transporter-expressing cells (TECs) (scaled using REF), but only when plasma proteins were included in the in vitro studies. This conclusion did not hold when plasma proteins were absent in the TEC or human hepatocyte studies. Thus, additional studies are needed to improve in vitro to in vivo extrapolation of transporter-mediated drug CL.

Design, synthesis and identification of novel, orally bioavailable non-covalent Nrf2 activators.

Nrf2 is a transcription factor regulating expression of the Phase II Antioxidant Response and plays an important role in neuroprotection and detoxification. Nrf2 activation is inhibited by interaction with Keap1. Covalent Keap1 inhibitors such as dimethyl fumarate (DMF) and RTA-408 are either on the market or in late stage clinical trials which implies potential benefit of Nrf2 activation. Activation of Nrf2 by disrupting Nrf2-Keap1 interaction through a non-covalent small molecule is an attractive approach with the promise of greater selectivity. However, there are no known non-covalent Nrf2 activators with acceptable pharmacokinetic properties to test the hypothesis in vivo. Based on our early reported work, using structural-based design, followed by extensive SAR exploration, we have identified a novel series of non-covalent Nrf2 activators, with sub-nanomolar binding affinity on Keap1 and single digit nanomolar activity in an astrocyte assay. A representative analog shows excellent oral PK and good Nrf2-dependent gene inductions in kidney. These results provide a peripheral in vivo tool compound to validate the biology of non-covalent activation of Nrf2.

Effects of organizational commitment, job satisfaction and workplace violence on turnover intention of emergency nurses: A cross-sectional study.

AIMS: To describe turnover intention of emergency nurses and clarify the effects of organizational commitment, job satisfaction and workplace violence on turnover intention. BACKGROUND: Research has showed the predictors of turnover intention differed among nurses of different specialties. However, research on turnover intention has mostly focused on general nurses rather than emergency nurses. DESIGN: A descriptive, cross-sectional study was conducted. METHODS: A self-administered questionnaire was used to collect data from 415 emergency nurses in Beijing, China, using convenience sampling. Path analysis was used to test the relationships between organizational commitment, job satisfaction, workplace violence and turnover intention. RESULTS: Most emergency nurses (90.2%) had a high level or very high level of turnover intention. Contrary to previous studies, organizational commitment had a significant direct positive effect on workplace violence. It also had a direct positive effect on job satisfaction and a negative effect on turnover intention. Workplace violence had a negative effect on job satisfaction and a positive effect on turnover intention. Job satisfaction had a direct negative effect on turnover intention. CONCLUSION: To reduce turnover intention in the emergency department, measures should be taken to reduce workplace violence and increase nurses' job satisfaction, especially those with high organizational commitment.

A Comparison of Total and Plasma Membrane Abundance of Transporters in Suspended, Plated, Sandwich-Cultured Human Hepatocytes Versus Human Liver Tissue Using Quantitative Targeted Proteomics and Cell Surface Biotinylation.

Suspended (SH), plated (PH), and sandwich-cultured hepatocytes (SCH) are commonly used models to predict in vivo transporter-mediated hepatic uptake (SH or PH) or biliary (SCH) clearance of drugs. When doing so, the total and the plasma membrane abundance (PMA) of transporter are assumed not to differ between hepatocytes and liver tissue (LT). This assumption has never been tested. In this study, we tested this assumption by measuring the total and PMA of the transporters in human hepatocyte models versus LT (total only) from which they were isolated. Total abundance of OATP1B1/2B1/1B3, OCT1, and OAT2 was not significantly different between the hepatocytes and LT. The same was true for the PMA of these transporters across the hepatocyte models. In contrast, total abundance of the sinusoidal efflux transporter, MRP3, and the canalicular efflux transporters, MRP2 and P-gp, was significantly greater (P < 0.05) in SCH versus LT. Of the transporters tested, only the percentage of PMA of OATP1B1, P-gp, and MRP3, in SCH (82.8% ± 7.3%, 57.5% ± 10.9%, 69.3% ± 5.7%) was significantly greater (P < 0.05) than in SH (73.3% ± 6.4%, 27.4% ± 6.4%, 53.6% ± 4.1%). If the transporters measured in the plasma membrane are functional and the PMA in SH is representative of that in LT, these data suggest that SH, PH, and SCH will result in equal prediction of hepatic uptake clearance of drugs mediated by the transporters tested above. However, SCH will predict higher sinusoidal efflux and biliary clearance of drugs if the change in PMA of these transporters is not taken into consideration.

Comparison of uptake transporter functions in hepatocytes in different species to determine the optimal model for evaluating drug transporter activities in humans.

A thorough understanding of species-dependent differences in hepatic uptake transporters is critical for predicting human pharmacokinetics (PKs) from preclinical data. In this study, the activities of organic anion transporting polypeptide (OATP/Oatp), organic cation transporter 1 (OCT1/Oct1), and sodium-taurocholate cotransporting polypeptide (NTCP/Ntcp) in cultured rat, dog, monkey and human hepatocytes were compared. The activities of hepatic uptake transporters were evaluated with respect to culture duration, substrate and species-dependent differences in hepatocytes. Longer culture duration reduced hepatic uptake transporter activities across species except for Oatp and Ntcp in rats. Comparable apparent Michaelis-Menten constant (Km,app) values in hepatocytes were observed across species for atorvastatin, estradiol-17ß-glucuronide and metformin. The Km,app values for rosuvastatin and taurocholate were significantly different across species. Rat hepatocytes exhibited the highest Oatp percentage of uptake transporter-mediated permeation clearance (PSinf,act) while no difference in %PSinf,act of probe substrates were observed across species. The in vitro hepatocyte inhibition data in rats, monkeys and humans provided reasonable predictions of in vivo drug-drug interaction (DDIs) between atorvastatin/rosuvastatin and rifampin. These findings suggested that using human hepatocytes with a short culture time is the most robust preclinical model for predicting DDIs for compounds exhibiting active hepatic uptake in humans.

Emerging Kidney Models to Investigate Metabolism, Transport, and Toxicity of Drugs and Xenobiotics.

The kidney is a major clearance organ of the body and is responsible for the elimination of many xenobiotics and prescription drugs. With its multitude of uptake and efflux transporters and metabolizing enzymes, the proximal tubule cell (PTC) in the nephron plays a key role in the disposition of xenobiotics and is also a primary site for toxicity. In this minireview, we first provide an overview of the major transporters and metabolizing enzymes in the PTCs responsible for biotransformation and disposition of drugs. Next, we discuss different cell sources that have been used to model PTCs in vitro, their pros and cons, and their characterization. As current technology is inadequate to evaluate reliably drug disposition and toxicity in the kidney, we then discuss recent advancements in kidney microphysiological systems (MPS) and the need to develop robust in vitro platforms that could be routinely used by pharmaceutical companies to screen compounds. Finally, we discuss the new and exciting field of stem cell-derived kidney models as potential cell sources for future kidney MPS. Given the push from both regulatory agencies and pharmaceutical companies to use more predictive "human-like" in vitro systems in the early stages of drug development to reduce attrition, these emerging models have the potential to be a game changer and may revolutionize how renal disposition and kidney toxicity in drug discovery are evaluated in the future.

Abundance of Phase 1 and 2 Drug-Metabolizing Enzymes in Alcoholic and Hepatitis C Cirrhotic Livers: A Quantitative Targeted Proteomics Study.

To predict the impact of liver cirrhosis on hepatic drug clearance using physiologically based pharmacokinetic (PBPK) modeling, we compared the protein abundance of various phase 1 and phase 2 drug-metabolizing enzymes (DMEs) in S9 fractions of alcoholic (n = 27) or hepatitis C (HCV, n = 30) cirrhotic versus noncirrhotic (control) livers (n = 25). The S9 total protein content was significantly lower in alcoholic or HCV cirrhotic versus control livers (i.e., 38.3 ± 8.3, 32.3 ± 12.8, vs. 51.1 ± 20.7 mg/g liver, respectively). In general, alcoholic cirrhosis was associated with a larger decrease in the DME abundance than HCV cirrhosis; however, only the abundance of UGT1A4, alcohol dehydrogenase (ADH)1A, and ADH1B was significantly lower in alcoholic versus HCV cirrhotic livers. When normalized to per gram of tissue, the abundance of nine DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, CYP1A2, ADH1A, ADH1B, aldehyde oxidase (AOX)1, and carboxylesterase (CES)1) in alcoholic cirrhosis and five DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, and CYP1A2) in HCV cirrhosis was <25% of that in control livers. The abundance of most DMEs in cirrhotic livers was 25% to 50% of control livers. CES2 abundance was not affected by cirrhosis. Integration of UGT2B7 abundance in cirrhotic livers into the liver cirrhosis (Child Pugh C) model of Simcyp improved the prediction of zidovudine and morphine PK in subjects with Child Pugh C liver cirrhosis. These data demonstrate that protein abundance data, combined with PBPK modeling and simulation, can be a powerful tool to predict drug disposition in special populations.

Transporter Expression in Noncancerous and Cancerous Liver Tissue from Donors with Hepatocellular Carcinoma and Chronic Hepatitis C Infection Quantified by LC-MS/MS Proteomics.

Protein expression of major hepatobiliary drug transporters (NTCP, OATPs, OCT1, BSEP, BCRP, MATE1, MRPs, and P-gp) in cancerous (C, n = 8) and adjacent noncancerous (NC, n = 33) liver tissues obtained from patients with chronic hepatitis C with hepatocellular carcinoma (HCV-HCC) were quantified by LC-MS/MS proteomics. Herein, we compare our results with our previous data from noninfected, noncirrhotic (control, n = 36) and HCV-cirrhotic (n = 30) livers. The amount of membrane protein yielded from NC and C HCV-HCC tissues decreased (31%, 67%) relative to control livers. In comparison with control livers, with the exception of NTCP, MRP2, and MATE1, transporter expression decreased in NC (38%-76%) and C (56%-96%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP expression increased (113%), MATE1 expression decreased (58%), and MRP2 expression was unchanged relative to control livers. In C HCV-HCC tissues, NTCP and MRP2 expression decreased (63%, 56%) and MATE1 expression was unchanged relative to control livers. Compared with HCV-cirrhotic livers, aside from NTCP, OCT1, BSEP, and MRP2, transporter expression decreased in NC (41%-71%) and C (54%-89%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP and MRP2 expression increased (362%, 142%), whereas OCT1 and BSEP expression was unchanged. In C HCV-HCC tissues, OCT1 and BSEP expression decreased (90%, 80%) relative to HCV-cirrhotic livers, whereas NTCP and MRP2 expression was unchanged. Expression of OATP2B1, BSEP, MRP2, and MRP3 decreased (56%-72%) in C HCV-HCC tissues in comparison with matched NC tissues (n = 8), but the expression of other transporters was unchanged. These data will be helpful in the future to predict transporter-mediated hepatocellular drug concentrations in patients with HCV-HCC.

Fampridine is a Substrate and Inhibitor of Human OCT2, but not of Human MATE1, or MATE2K.

PURPOSE: The renal clearance of fampridine (Fampyra®, or Ampyra®) significantly exceeds the glomerular filtration rate, suggesting active renal secretion is likely the major elimination pathway. The goal of this study was to identify the renal transporters that are involved in the renal active secretion, and elucidate the active renal secretion mechanism of fampridine. METHODS: The uptake of fampridine to HEK-293 cells overexpressing human OCT2, MATE1 or MATE2K was determined in the absence and presence of Cimetidine, the prototypical inhibitor of the transporters. The inhibition potential of fampridine on the renal transporters was evaluated by determining the uptake of TEA and Metformin, the probe substrates of the transporters of OCT2 and MATEs, respectively, in the absence or presence of fampridine. RESULTS: Significant time- and concentration-dependent uptake of fampridine by human OCT2 was observed. The Km and Vmax were determined as 51.0 ± 17.1 µM and 1107 ± 136 pmole/min/106 cells, respectively. Fampridine also inhibited OCT2 mediated uptake of Metformin with estimated IC50 of 66.8 µM. In contrast, there was not significant uptake of fampridine by human MATE1 or MATE2K, and fampridine did not inhibit MATE1 or MATE2K mediated uptake of TEA. CONCLUSION: The studies indicated fampridine is a substrate and inhibitor of OCT2, but not MATE1 or MATE2K. Results from the study suggested the active renal secretion of fampridine is mediated by human OCT2 but not MATE1 or MATE2K. To our knowledge, fampridine is the first reported substrate specific to OCT2 but not to MATE1 or MATE2K.

A pre-clinical quantitative model predicts the pharmacokinetics/pharmacodynamics of an anti-BDCA2 monoclonal antibody in humans.

BIIB059 is a novel humanized monoclonal antibody (mAb) that is currently under development for the treatment of Systemic Lupus Erythematosus and Cutaneous Lupus Erythematosus. BIIB059 is targeted against the blood dendritic cell antigen 2 (BDCA2), a receptor exclusively expressed on the surface of plasmacytoid dendritic cells (pDCs). Herein, we utilized pre-clinical pharmacokinetic (PK) and pharmacodynamic (PD) data to develop a non-human primate (NHP) model and to address whether the NHP model can be successfully scaled to predict the human PK/PD. In particular, PK data from 17 cynomolgus monkeys were utilized for PK model development, wherein BIIB059 was administered intravenously (1 and 10 mg/kg single-dosing and 5 mg/kg multiple-dosing) or subcutaneously (0.2 and 7.5 mg/kg single-dosing). Additionally, PD data (BDCA2 receptor density on pDCs) from 6 cynomolgus monkeys were used for the development of the PD model. The developed NHP two-compartment PK model, linked with an indirect response PD model, was subsequently scaled to humans by combining traditional allometric PK scaling with sensitivity-analysis-driven scaling of the PD. The scaled PK/PD model was then used to simulate the human PK/PD for different dose levels. When clinical data from the BIIB059 Phase I study became available, they were used to evaluate the predictability of the scaled PK/PD model and the model simulations were in agreement with the clinical data. Therefore, the presented approach is suggested to be employed in scaling pre-clinical mAb models to support the selection of safe first-in-human doses and, more broadly, the prediction of PK/PD in the clinic.

Current Approaches for Absorption, Distribution, Metabolism, and Excretion Characterization of Antibody-Drug Conjugates: An Industry White Paper.

An antibody-drug conjugate (ADC) is a unique therapeutic modality composed of a highly potent drug molecule conjugated to a monoclonal antibody. As the number of ADCs in various stages of nonclinical and clinical development has been increasing, pharmaceutical companies have been exploring diverse approaches to understanding the disposition of ADCs. To identify the key absorption, distribution, metabolism, and excretion (ADME) issues worth examining when developing an ADC and to find optimal scientifically based approaches to evaluate ADC ADME, the International Consortium for Innovation and Quality in Pharmaceutical Development launched an ADC ADME working group in early 2014. This white paper contains observations from the working group and provides an initial framework on issues and approaches to consider when evaluating the ADME of ADCs.

Transporter Expression in Liver Tissue from Subjects with Alcoholic or Hepatitis C Cirrhosis Quantified by Targeted Quantitative Proteomics.

Although data are available on the change of expression/activity of drug-metabolizing enzymes in liver cirrhosis patients, corresponding data on transporter protein expression are not available. Therefore, using quantitative targeted proteomics, we compared our previous data on noncirrhotic control livers (n = 36) with the protein expression of major hepatobiliary transporters, breast cancer resistance protein (BCRP), bile salt export pump (BSEP), multidrug and toxin extrusion protein 1 (MATE1), multidrug resistance-associated protein (MRP)2, MRP3, MRP4, sodium taurocholate-cotransporting polypeptide (NTCP), organic anion-transporting polypeptides (OATP)1B1, 1B3, 2B1, organic cation transporter 1 (OCT1), and P-glycoprotein (P-gp) in alcoholic (n = 27) and hepatitis C cirrhosis (n = 30) livers. Compared with control livers, the yield of membrane protein from alcoholic and hepatitis C cirrhosis livers was significantly reduced by 56 and 67%, respectively. The impact of liver cirrhosis on transporter protein expression was transporter-dependent. Generally, reduced protein expression (per gram of liver) was found in alcoholic cirrhosis livers versus control livers, with the exception that the expression of MRP3 was increased, whereas no change was observed for MATE1, MRP2, OATP2B1, and P-gp. In contrast, the impact of hepatitis C cirrhosis on protein expression of transporters (per gram of liver) was diverse, showing an increase (MATE1), decrease (BSEP, MRP2, NTCP, OATP1B3, OCT1, and P-gp), or no change (BCRP, MRP3, OATP1B1, and 2B1). The expression of hepatobiliary transporter protein differed in different diseases (alcoholic versus hepatitis C cirrhosis). Finally, incorporation of protein expression of OATP1B1 in alcoholic cirrhosis into the Simcyp physiologically based pharmacokinetics cirrhosis module improved prediction of the disposition of repaglinide in liver cirrhosis patients. These transporter expression data will be useful in the future to predict transporter-mediated drug disposition in liver cirrhosis patients.

[Quantitative structure characteristics and fractal dimension of Chinese medicine granules measured by synchrotron radiation X-ray computed micro tomography].

The shape and structure of granules are controlled by the granulation process, which is one of the main factors to determine the nature of the solid dosage forms. In this article, three kinds of granules of a traditional Chinese medicine for improving appetite and promoting digestion, namely, Jianwei Granules, were prepared using granulation technologies as pendular granulation, high speed stirring granulation, and fluidized bed granulation and the powder properties of them were investigated. Meanwhile, synchrotron radiation X-ray computed micro tomography (SR-µCT) was applied to quantitatively determine the irregular internal structures of the granules. The three-dimensional (3D) structure models were obtained by 3D reconstruction, which were more accurately to characterize the three-dimensional structures of the particles through the quantitative data. The models were also used to quantitatively compare the structural differences of granules prepared by different granulation processes with the same formula, so as to characterize how the production process plays a role in the pharmaceutical behaviors of the granules. To focus on the irregularity of the particle structure, the box counting method was used to calculate the fractal dimensions of the granules. The results showed that the fractal dimension is more sensitive to reflect the minor differences in the structure features than the conventional parameters, and capable to specifically distinct granules in structure. It is proved that the fractal dimension could quantitatively characterize the structural information of irregular granules. It is the first time suggested by our research that the fractal dimension difference (Df,c) between two fractal dimension parameters, namely, the volume matrix fractal dimension and the surface matrix fractal dimension, is a new index to characterize granules with irregular structures and evaluate the effects of production processes on the structures of granules as a new indicator for the granulating process control and optimization.

Evaluation of OCT2-mediated drug-drug interactions between ulotaront and metformin in subjects with schizophrenia.

Ulotaront (SEP-363856) is a TAAR1 agonist, with 5-HT1A agonist activity, currently in clinical development for the treatment of schizophrenia. In vitro studies indicate ulotaront is an OCT2-specific inhibitor with IC50 of 1.27 µM. The primary objective of this study is to determine if a single dose of ulotaront affects the PK of metformin, an index substrate of OCT2, in subjects with schizophrenia. In a randomized, single-blind, 2-period crossover study, 25 adults with schizophrenia received a single dose of metformin-HCl 850 mg (approximately 663 mg metformin) with and without coadministration of 100 mg ulotaront. The plasma samples were analyzed by fully validated LC-MS/MS methods. The primary PK endpoints for metformin were AUCinf, AUClast, Cmax, and tmax. The highest-anticipated clinical dose of ulotaront (100 mg) had no statistically significant effect on the PK of a single dose of metformin based on Cmax and AUCinf. Geometric least squares mean ratios were 89.98% and 110.63%, respectively, with the 90% confidential interval (CI) for each parameter contained within 80%-125%. Median tmax was comparable across the treatments. Ulotaront does not act as a perpetrator of OCT2-mediated DDI against metformin. Co-administration of ulotaront is not expected to require dose adjustment of metformin or other drugs cleared by OCT2.

Variability in P-glycoprotein inhibitory potency (IC50) using various in vitro experimental systems: implications for universal digoxin drug-drug interaction risk assessment decision criteria.

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

Application of receiver operating characteristic analysis to refine the prediction of potential digoxin drug interactions.

In the 2012 Food and Drug Administration (FDA) draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits P-glycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when the maximum concentration of inhibitor at steady state divided by IC50 ([I1]/IC50) is ≥0.1 or concentration of inhibitor based on highest approved dose dissolved in 250 ml divide by IC50 ([I2]/IC50) is ≥10. In this article, refined criteria are presented, determined by receiver operating characteristic analysis, using IC50 values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for P-gp overexpressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this article take into account variability in IC50 values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of [I1]/IC50 ≥ 0.03 and [I2]/IC50 ≥ 45 and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, whereas the number of false positives (DDI predicted but not observed) substantially decreased from 51 to 40%, relative to the FDA criteria. On the basis of estimated overall variability in IC50 values, a theoretical 95% confidence interval calculation was developed for single laboratory IC50 values, translating into a range of [I1]/IC50 and [I2]/IC50 values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, attributable to variability in IC50 values.

A Phase I, Open-Label, Fixed Sequence Study to Investigate the Effect of Cytochrome P450 2D6 Inhibition on the Pharmacokinetics of Ulotaront in Healthy Subjects.

BACKGROUND: Ulotaront is a novel psychotropic agent with agonist activity at trace amine-associated receptor 1 (TAAR1) and 5-hydroxytryptamine type 1A (5-HT1A) receptors in phase III clinical development for the treatment of schizophrenia. OBJECTIVE: This study aimed to investigate the effect of paroxetine, a strong cytochrome P450 (CYP) 2D6 inhibitor, on ulotaront pharmacokinetics (PK) in healthy volunteers. METHODS: Subjects received a single oral dose of 25 mg ulotaront on Day 1 and an oral dose of 20 mg paroxetine once daily from Days 5 to 10 to achieve steady-state plasma paroxetine levels. On Day 11, subjects received another single oral dose of 25 mg ulotaront, with continued daily oral dosing of 20 mg paroxetine from Days 11 to 14. All 24 subjects were CYP2D6 normal metabolizers. RESULTS: Coadministration of paroxetine increased ulotaront maximum observed plasma concentration (Cmax) and area under the plasma concentration-time curve from time zero to infinity (AUC∞) by 31% and 72%, respectively, and decreased ulotaront apparent clearance (CL/F) by approximately 42%. While coadministration of paroxetine increased AUC∞ of active but minor metabolite SEP-363854 by 32%, it had no effect on SEP-363854 Cmax, or on SEP-363854 to the ulotaront AUC from time zero to the last quantifiable concentration (AUClast) ratio. Based on the acceptable adverse event profile of ulotaront across previous phase II studies, the increase in ulotaront exposure is unlikely to be clinically meaningful. CONCLUSIONS: Weak drug-drug interactions were observed between ulotaront and the strong CYP2D6 inhibitor paroxetine; however, dose adjustment as a precondition when ulotaront is coadministered with strong CYP2D6 inhibitors or administered to CYP2D6 poor metabolizers should not be necessary.

Cerebrospinal fluid can be used as a surrogate to assess brain exposures of breast cancer resistance protein and P-glycoprotein substrates.

The objectives of the study were to characterize the selectivity of dantrolene to breast cancer resistance protein (Bcrp) and to evaluate whether cerebrospinal fluid (CSF) can be used as a surrogate to assess brain exposures of BCRP and P-glycoprotein (Pgp) substrates. The impact of Bcrp and Pgp on dantrolene exposures in brain and CSF was examined in Bcrp and Mdr1a/1b knockout mice and was further investigated in wild-type mice in the presence of the Bcrp inhibitor (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester (Ko143), the Pgp inhibitor 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporine A (PSC833), and the dual inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). The effect of Bcrp and Pgp on digoxin exposures in brain and CSF was investigated in wild-type mice in the presence of the inhibitors. In vivo studies showed dantrolene exposures in brain and CSF, but not the blood, increased in Bcrp(-/-) and Mdr1a/1b(-/-)/Bcrp(-/-) mice, or in the presence of the Bcrp inhibitors Ko143 or GF120918. Inhibition of Pgp by GF120918 and PSC833 significantly increased digoxin exposures in brain, CSF, and blood to a lesser extent. Results from the present study demonstrated that inhibition of Bcrp and Pgp increased not only the exposures of dantrolene and digoxin in brain, but also the exposures in CSF. In addition, the change of exposures in CSF reflected the changes in brain. The present study strongly suggests that the dantrolene and digoxin exposures in CSF are primarily determined by the rapid transport from brain to CSF, and inhibition of Bcrp and Pgp exhibits little impact on using CSF as surrogates to assess brain exposures of Bcrp and Pgp substrates.

Current Approaches for Predicting Human PK for Small Molecule Development Candidates: Findings from the IQ Human PK Prediction Working Group Survey.

Accurate prediction of human clearance (CL) and volume of distribution at steady state (Vd,ss) for small molecule drug candidates is an essential component of assessing likely efficacious dose and clinical safety margins. In 2021, the IQ Consortium Human PK Prediction Working Group undertook a survey of IQ member companies to understand the current PK prediction methods being used to estimate these parameters across the pharmaceutical industry. The survey revealed a heterogeneity in approaches being used across the industry (e.g., the use of allometric approaches, differing incorporation of binding terms, and inconsistent use of empirical correction factors for in vitro-in vivo extrapolation, IVIVE), which could lead to different PK predictions with the same input data. Member companies expressed an interest in improving human PK predictions by identifying the most appropriate compound-class specific methods, as determined by physiochemical properties and knowledge of CL pathways. Furthermore, there was consensus that increased understanding of the uncertainty inherent to the compound class-dependent prediction would be invaluable in aiding communication of human PK and dose uncertainty at the time of candidate nomination for development. The human PK Prediction Working Group is utilizing these survey findings to help interrogate clinical IV datasets from across the IQ consortium member companies to understand PK prediction accuracy and uncertainty from preclinical datasets.