Survey of Pharmaceutical Industry's Best Practices around In Vitro Transporter Assessment and Implications for Drug Development: Considerations from the International Consortium for Innovation and Quality for Pharmaceutical Development Transporter Working Group.

The International Consortium for Innovation and Quality in Pharmaceutical Development Transporter Working Group had a rare opportunity to analyze a crosspharma collation of in vitro data and assay methods for the evaluation of drug transporter substrate and inhibitor potential. Experiments were generally performed in accordance with regulatory guidelines. Discrepancies, such as not considering the impact of preincubation for inhibition and free or measured in vitro drug concentrations, may be due to the retrospective nature of the dataset and analysis. Lipophilicity was a frequent indicator of crosstransport inhibition (P-gp, BCRP, OATP1B, and OCT1), with high molecular weight (MW ≥500 Da) also common for OATP1B and BCRP inhibitors. A high level of overlap in in vitro inhibition across transporters was identified for BCRP, OATP1B1, and MATE1, suggesting that prediction of DDIs for these transporters will be common. In contrast, inhibition of OAT1 did not coincide with inhibition of any other transporter. Neutrals, bases, and compounds with intermediate-high lipophilicity tended to be P-gp and/or BCRP substrates, whereas compounds with MW <500 Da tended to be OAT3 substrates. Interestingly, the majority of in vitro inhibitors were not reported to be followed up with a clinical study by the submitting company, whereas those compounds identified as substrates generally were. Approaches to metabolite testing were generally found to be similar to parent testing, with metabolites generally being equally or less potent than parent compounds. However, examples where metabolites inhibited transporters in vitro were identified, supporting the regulatory requirement for in vitro testing of metabolites to enable integrated clinical DDI risk assessment. SIGNIFICANCE STATEMENT: A diverse dataset showed that transporter inhibition often correlated with lipophilicity and molecular weight (>500 Da). Overlapping transporter inhibition was identified, particularly that inhibition of BCRP, OATP1B1, and MATE1 was frequent if the compound inhibited other transporters. In contrast, inhibition of OAT1 did not correlate with the other drug transporters tested.

Prediction of the Renal Organic Anion Transporter 1 (OAT1)- Mediated Drug Interactions for LY404039, the Active Metabolite of Pomaglumetad Methionil.

PURPOSE: The objective of this work was to demonstrate that clinical OAT1-mediated DDIs can be predicted using physiologically based pharmacokinetic (PBPK) modeling. METHODS: LY404039 is a metabotropic glutamate receptor 2/3 agonist and the active moiety of the prodrug pomaglumetad methionil (LY2140023). After oral administration, pomaglumetad methionil is rapidly taken up by enterocytes via PEPT1 and once absorbed, converted to LY404039 via membrane dehydropeptidase 1 (DPEP1). LY404039 is renally excreted by both glomerular filtration and active secretion and in vitro studies showed that the active secretion of LY404039 was mediated by the organic anion transporter 1 (OAT1). Both clinical and in vitro data were used to build a PBPK model to predict OAT1-mediated DDIs. RESULTS: In vitro inhibitory potencies (IC50) of the known OAT inhibitors, probenecid and ibuprofen, were determined to be 4.00 and 2.63 µM, respectively. Subsequently, clinical drug-drug interaction (DDI) study showed probenecid reduced the renal clearance of LY404039 by 30 to 40%. The PBPK bottom-up model, predicted a renal clearance that was approximately 20% lower than the observed one. The middle-out model, using an OAT1 relative activity factor (RAF) of 3, accurately reproduced the renal clearance of LY404039 and pharmacokinetic (PK) changes of LY404039 in the presence of probenecid. CONCLUSIONS: OAT1- mediated DDIs can be predicted using in vitro measured IC50 and PBPK modeling. The effect of ibuprofen was predicted to be minimal (AUC ratio of 1.15) and not clinically relevant.

Pharmacokinetics of Organic Cation Transporter 1 (OCT1) Substrates in Oct1/2 Knockout Mice and Species Difference in Hepatic OCT1-Mediated Uptake.

Organic cation transporter 1 (OCT1) plays a role in hepatic uptake of drugs, affecting in vivo exposure, distinguished primarily through pharmacogenetics of the SLC22A1 gene. The role of OCT1 in vivo has not been confirmed, however, via drug-drug interactions that similarly affect exposure. In the current research, we used Oct1/2 knockout mice to assess the role of Oct1 in hepatic clearance and liver partitioning of clinical substrates and assess the model for predicting an effect of OCT1 function on pharmacokinetics in humans. Four OCT1 substrates (sumatriptan, fenoterol, ondansetron, and tropisetron) were administered to wild-type and knockout mice, and plasma, tissue, and urine were collected. Tissue transporter expression was evaluated using liquid chromatography-mass spectrometry. In vitro, uptake of all compounds in human and mouse hepatocytes and human OCT1- and OCT2-expressing cells was evaluated. The largest effect of knockout was on hepatic clearance and liver partitioning of sumatriptan (2- to 5-fold change), followed by fenoterol, whereas minimal changes in the pharmacokinetics of ondansetron and tropisetron were observed. This aligned with uptake in mouse hepatocytes, in which inhibition of uptake of sumatriptan and fenoterol into mouse hepatocytes by an OCT1 inhibitor was much greater compared with ondansetron and tropisetron. Conversely, inhibition of all four substrates was evident in human hepatocytes, in line with reported clinical pharmacogenetic data. These data confirm the role of Oct1 in the hepatic uptake of the four OCT1 substrates and elucidate species differences in OCT1-mediated hepatocyte uptake that should be considered when utilizing the model to predict effects in humans. SIGNIFICANCE STATEMENT: Studies in carriers of SLC22A1 null variants indicate a role of organic cation transporter 1 (OCT1) in the hepatic uptake of therapeutic agents, although OCT1-mediated drug-drug interactions have not been reported. This work used Oct1/2 knockout mice to confirm the role of Oct1 in the hepatic clearance and liver partitioning in mice for OCT1 substrates with reported pharmacogenetic effects. Species differences observed in mouse and human hepatocyte uptake clarify limitations of the knockout model for predicting exposure changes in humans for some OCT1 substrates.

Hepatic Organic Anion Transporting Polypeptide-Mediated Clearance in the Beagle Dog: Assessing In Vitro-In Vivo Relationships and Applying Cross-Species Empirical Scaling Factors to Improve Prediction of Human Clearance.

In the present study, the beagle dog was evaluated as a preclinical model to investigate organic anion transporting polypeptide (OATP)-mediated hepatic clearance. In vitro studies were performed with nine OATP substrates in three lots of plated male dog hepatocytes ± OATP inhibitor cocktail to determine total uptake clearance (CLuptake) and total and unbound cell-to-medium concentration ratio (Kpuu). In vivo intrinsic hepatic clearances (CLint,H) were determined following intravenous drug administration (0.1 mg/kg) in male beagle dogs. The in vitro parameters were compared with those previously reported in plated human, monkey, and rat hepatocytes; the ability of cross-species scaling factors to improve prediction of human in vivo clearance was assessed. CLuptake in dog hepatocytes ranged from 9.4 to 135 µl/min/106 cells for fexofenadine and telmisartan, respectively. Active process contributed >75% to CLuptake for 5/9 drugs. Rosuvastatin and valsartan showed Kpuu > 10, whereas cerivastatin, pitavastatin, repaglinide, and telmisartan had Kpuu < 5. The extent of hepatocellular binding in dog was consistent with other preclinical species and humans. The bias (2.73-fold) obtained from comparison of predicted versus in vivo dog CLint,H was applied as an average empirical scaling factor (ESFav) for in vitro-in vivo extrapolation of human CLint,H The ESFav based on dog reduced underprediction of human CLint,H for the same data set (geometric mean fold error = 2.1), highlighting its utility as a preclinical model to investigate OATP-mediated uptake. The ESFav from all preclinical species resulted in comparable improvement of human clearance prediction, in contrast to drug-specific empirical scalars, rationalized by species differences in expression and/or relative contribution of particular transporters to drug hepatic uptake.

In Vitro and Clinical Evaluations of the Drug-Drug Interaction Potential of a Metabotropic Glutamate 2/3 Receptor Agonist Prodrug with Intestinal Peptide Transporter 1.

Despite peptide transporter 1 (PEPT1) being responsible for the bioavailability for a variety of drugs, there has been little study of its potential involvement in drug-drug interactions. Pomaglumetad methionil, a metabotropic glutamate 2/3 receptor agonist prodrug, utilizes PEPT1 to enhance absorption and bioavailability. In vitro studies were conducted to guide the decision to conduct a clinical drug interaction study and to inform the clinical study design. In vitro investigations determined the prodrug (LY2140023 monohydrate) is a substrate of PEPT1 with Km value of approximately 30 µM, whereas the active moiety (LY404039) is not a PEPT1 substrate. In addition, among the eight known PEPT1 substrates evaluated in vitro, valacyclovir was the most potent inhibitor (IC50 = 0.46 mM) of PEPT1-mediated uptake of the prodrug. Therefore, a clinical drug interaction study was conducted to evaluate the potential interaction between the prodrug and valacyclovir in healthy subjects. No effect of coadministration was observed on the pharmacokinetics of the prodrug, valacyclovir, or either of their active moieties. Although in vitro studies showed potential for the prodrug and valacyclovir interaction via PEPT1, an in vivo study showed no interaction between these two drugs. PEPT1 does not appear to easily saturate because of its high capacity and expression in the intestine. Thus, a clinical interaction at PEPT1 is unlikely even with a compound with high affinity for the transporter.

Chimeric TK-NOG mice: a predictive model for cholestatic human liver toxicity.

Due to the substantial interspecies differences in drug metabolism and disposition, drug-induced liver injury (DILI) in humans is often not predicted by studies performed in animal species. For example, a drug (bosentan) used to treat pulmonary artery hypertension caused unexpected cholestatic liver toxicity in humans, which was not predicted by preclinical toxicology studies in multiple animal species. In this study, we demonstrate that NOG mice expressing a thymidine kinase transgene (TK-NOG) with humanized livers have a humanized profile of biliary excretion of a test (cefmetazole) drug, which was shown by an in situ perfusion study to result from interspecies differences in the rate of biliary transport and in liver retention of this drug. We also found that readily detectable cholestatic liver injury develops in TK-NOG mice with humanized livers after 1 week of treatment with bosentan (160, 32, or 6 mg/kg per day by mouth), whereas liver toxicity did not develop in control mice after 1 month of treatment. The laboratory and histologic features of bosentan-induced liver toxicity in humanized mice mirrored that of human subjects. Because DILI has become a significant public health problem, drug safety could be improved if preclinical toxicology studies were performed using humanized TK-NOG.

Prediction of renal transporter mediated drug-drug interactions for pemetrexed using physiologically based pharmacokinetic modeling.

Pemetrexed, an anionic anticancer drug with a narrow therapeutic index, is eliminated mainly by active renal tubular secretion. The in vitro to in vivo extrapolation approach used in this work was developed to predict possible drug-drug interactions (DDIs) that may occur after coadministration of pemetrexed and nonsteroidal anti-inflammatory drugs (NSAIDs), and it included in vitro assays, risk assessment models, and physiologically based pharmacokinetic (PBPK) models. The pemetrexed transport and its inhibition parameters by several NSAIDs were quantified using HEK-PEAK cells expressing organic anion transporter (OAT) 3 or OAT4. The NSAIDs were ranked according to their DDI index, calculated as the ratio of their maximum unbound concentration in plasma over the concentration inhibiting 50% (IC50) of active pemetrexed transport. A PBPK model for ibuprofen, the NSAID with the highest DDI index, was built incorporating active renal secretion in Simcyp Simulator. The bottom-up model for pemetrexed underpredicted the clearance by 2-fold. The model we built using a scaling factor of 5.3 for the maximal uptake rate (Vmax) of OAT3, which estimated using plasma concentration profiles from patients given a 10-minute infusion of 500 mg/m(2) of pemetrexed supplemented with folic acid and vitamin B12, recovered the clinical data adequately. The observed/predicted increases in Cmax and the area under the plasma-concentration time curve (AUC0-inf) of pemetrexed when ibuprofen was coadministered were 1.1 and 1.0, respectively. The coadministration of all other NSAIDs was predicted to have no significant impact on the AUC0-inf based on their DDI indexes. The PBPK model reasonably reproduced pemetrexed concentration time profiles in cancer patients and its interaction with ibuprofen.

Deciphering ADME genetic data using an automated haplotype approach.

OBJECTIVE: To investigate the utility of statistical tools in translating Affymetrix Drug Metabolizing Enzyme and Transporter (DMET) Assay single-nucleotide polymorphisms (SNPs) into common consensus star alleles. METHODS: DMET SNP data from clinical trials in different ethnicities were pooled for analyses. Three different statistical methods, PHASE, Bayesian, and expectation-maximization (EM), were first assessed by comparing the consistency of calling CYP2D6 alleles among 1108 Asians and 55 Caucasians. Subsequently, the performance of EM in deriving haplotype calls was evaluated against the Affymetrix Translation Table for CYPs 2B6, 2C19, 2C9, and 3A4/5 in 582 Asians, 296 Caucasians, and 369 Africans. Selected DNA samples were sequenced to verify the EM-predicted haplotype calls. RESULTS: PHASE, Bayesian, and EM methods showed a similar CYP2D6 star allele call rate. The EM method, with a 0.99 posterior probability cutoff, was chosen for further evaluation because of its low false-positive call rate. Haplotype calls obtained with the EM method were consistent with the Affymetrix Translation Table more than 95% of the time for all five CYPs, except for the CYP2B6 calls in the African descents (83%). In addition, the EM method was superior to the Translation Table-only approach in resolving complex haplotype patterns, identifying novel haplotypes in CYP2B6 and CYP3A5, and determining genotype calls in the presence of missing SNP data. CONCLUSION: A statistical method such as EM could be used to augment the translation of DMET assay SNP data into star alleles, especially for complex genes, to facilitate full utilization and interpretation of clinical pharmacogenetics data.

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.

Perspectives on the evaluation and adoption of complex in vitro models in drug development: Workshop with the FDA and the pharmaceutical industry (IQ MPS Affiliate).

Complex in vitro models (CIVM) offer the potential to improve pharmaceutical clinical drug attrition due to safety and/ or efficacy concerns. For this technology to have an impact, the establishment of robust characterization and qualifi­cation plans constructed around specific contexts of use (COU) is required. This article covers the output from a workshop between the Food and Drug Administration (FDA) and Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate. The intent of the workshop was to understand how CIVM technologies are currently being applied by pharma­ceutical companies during drug development and are being tested at the FDA through various case studies in order to identify hurdles (real or perceived) to the adoption of microphysiological systems (MPS) technologies, and to address evaluation/qualification pathways for these technologies. Output from the workshop includes the alignment on a working definition of MPS, a detailed description of the eleven CIVM case studies presented at the workshop, in-depth analysis, and key take aways from breakout sessions on ADME (absorption, distribution, metabolism, and excretion), pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of CIVM data. In conclusion, IQ MPS Affiliate and FDA scientists were able to build a general consensus on the need for animal CIVMs for preclinical species to better determine species concordance. Furthermore, there was acceptance that CIVM technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific COU.

Efflux transport is an important determinant of ethinylestradiol glucuronide and ethinylestradiol sulfate pharmacokinetics.

17α-ethinylestradiol (EE) undergoes extensive conjugation to 17α-ethinylestradiol-3-O-glucuronide (EEG) and 17α-ethinylestradiol-3-O-sulfate (EES). Thus, oral contraceptive drug-drug interaction (DDI) studies usually characterize metabolite pharmacokinetics, with changes typically attributed to modulation of metabolism. EE passively diffuses through plasma membranes, but its conjugates are hydrophilic and require active transport. Unlike EE metabolism, EEG and EES transport has not been explored in vivo as a potential mechanism of DDIs. Recent in vitro studies demonstrated that EEG is transported by multidrug resistance-associated protein (MRP) 2 and MRP3 and EES is a breast cancer resistance protein (BCRP) substrate. In the study presented here, pharmacokinetics of EE and conjugates were studied in TR⁻ rats, which lack Mrp2, have marginal hepatic Bcrp expression, and overexpress hepatic Mrp3. EE pharmacokinetics in TR⁻ rats were comparable to wild type; however, EEG and EES systemic exposures were altered markedly. EEG exposure was greatly increased: 20-fold and >100-fold after intravenous and oral EE administration, respectively. In contrast, EES exposure was lower in TR⁻ rats: 65% decreased (intravenously) and 83% decreased (orally). In intestinal and liver perfusions, EE intestinal permeability and metabolism and hepatic clearance were unchanged in TR⁻ rats; however, secretion of EEG into intestinal lumen was halved, EEG was not detected in TR⁻ bile, and EES biliary excretion was 98% decreased. After oral EE administration to Mrp2- and Bcrp-knockout mice, EEG exposure increased 46- and 2-fold, respectively, whereas EES concentrations were decreased modestly. In conclusion, altered efflux transport resulted in major alterations of EEG and EES pharmacokinetics, highlighting transport as a potential site of DDIs with EE conjugates.

Comparison of Hepatic Transporter Tissue Expression in Rodents and Interspecies Hepatic OCT1 Activity.

Hepatic clearance may be uptake rate limited by organic anion transporting polypeptides (OATPs) and organic cation transporter 1 (OCT1). While comparison of OATP activity has been investigated across species, little has been reported for OCT1. Additionally, while data on interspecies transporter expression in the liver exist, quantitative comparison of these transporters in multiple tissues is lacking. In the current research, the pharmacokinetics of OCT1 substrates (sumatriptan and metformin) were assessed in Oct knockout rats for comparison with previous Oct1/2-/- mice data and OCT1 pharmacogenetics in humans. Effect of OCT1 inhibitors verapamil and erlotinib on OCT1 substrate liver partitioning was also evaluated in rats. Expression of 18 transporters, including Oatps and Octs, in 9 tissues from mice and rats was quantitated using nanoLC/MS-MS, along with uptake transporters in hepatocytes from 5 species. Interspecies differences in OCT1 activity were further evaluated via uptake of OCT1 substrates in hepatocytes with corresponding in vivo liver partitioning in rodents and monkey. In Oct1-/- rats, sumatriptan hepatic clearance and liver partitioning decreased; however, metformin pharmacokinetics were unaffected. OCT1 inhibitor coadministration decreased sumatriptan liver partitioning. In rodents, Oatp expression was highest in the liver, although comparable expression of Oatps in other tissues was determined. Expression of Octs was highest in the kidney, with liver Oct1 expression comparably lower than Oatps. Liver partitioning of OCT1 substrates was lower in rodents than in monkey, in agreement with the highest OCT1 expression and uptake of OCT1 substrates in monkey hepatocytes. Species-dependent OCT1 activity requires consideration when translating preclinical data to the clinic.

Expansion of Knowledge on OCT1 Variant Activity In Vitro and In Vivo Using Oct1/2-/- Mice.

The role of organic cation transporter 1 (OCT1) in humans is gaining attention as data emerges regarding its role in physiology, drug exposure, and drug response. OCT1 variants with decreased in vitro function correlate well with altered exposure of multiple OCT1 substrates in variant carriers. In the current research, we investigate mechanisms behind activity of OCT1 variants in vitro by generating cell lines expressing known OCT1 variants and quantifying membrane OCT1 protein expression with corresponding OCT1 activity and kinetics. Oct knockout mice have provided additional insight into the role of Oct1 in the liver and have reproduced effects of altered OCT1 activity observed in the clinic. To assess the complex effect of Oct1 depletion on pharmacokinetics of prodrug proguanil and its active moiety cycloguanil, both of which are OCT1 substrates, Oct1/2-/- mice were used. Decreased membrane expression of OCT1 was demonstrated for all variant cell lines, although activity was substrate-dependent, as reported previously. Lack of change in activity for OCT1*2 resulted in increased intrinsic activity per pmol of OCT1 protein, particularly for sumatriptan but also for proguanil and cycloguanil. Similar to that reported in humans with decreased OCT1 function, systemic exposure of proguanil was minimally affected in Oct1/2-/- mice. However, proguanil liver partitioning and exposure decreased. Cycloguanil exposure decreased following proguanil administration in Oct1/2-/- mice, as did the systemic metabolite:parent ratio. When administered directly, systemic exposure of cycloguanil decreased slightly; however liver partitioning and exposure were decreased in Oct1/2-/- mice. Unexpectedly, following proguanil administration, the metabolite ratio in the liver changed only minimally, and liver partitioning of cycloguanil was affected in Oct1/2-/- mice to a lesser extent following proguanil administration than direct administration of cycloguanil. In conclusion, these in vitro and in vivo data offer additional complexity in understanding mechanisms of OCT1 variant activity as well as the effects of these variants in vivo. From cell lines, it is apparent that intrinsic activity is not directly related to OCT1 membrane expression. Additionally, in situations with a more complicated role of OCT1 in drug pharmacokinetics there is difficulty translating in vivo impact simply from intrinsic activity from cellular data.

Assessment of Transporter Polymorphisms as a Factor in a BCRP Drug Interaction Study With Lanabecestat.

Lanabecestat is a human ß-site amyloid precursor protein-cleaving enzyme 1 inhibitor in development to slow disease progression in patients with early Alzheimer's disease. The study evaluated the breast cancer resistance protein (BCRP) inhibition potential of lanabecestat on the pharmacokinetics (PK) of rosuvastatin, a probe for BCRP activity, in healthy white subjects who were not carriers of SLCO1B1 (c.521T>C), not homozygotes for ABCG2 (c.421C>A or c.34G>A), and not heterozygotes of ABCG2 (c.421C>A and c.34G>A). The safety of lanabecestat + rosuvastatin, the effects of rosuvastatin on the PK of lanabecestat, and the effects of multiple genetic polymorphisms on rosuvastatin exposure were assessed. Geometric mean ratios of the maximum observed rosuvastatin concentration (Cmax ), area under the rosuvastatin concentration-versus-time curve (AUC) from time 0 to infinity, and time of maximum observed drug concentration (tmax ) when rosuvastatin was administered alone and with lanabecestat were contained within 0.8-1.25, as were lanabecestat AUC at steady state and tmax at steady state when lanabecestat was administered alone or with rosuvastatin. Lanabecestat Cmax at steady state increased 8% in the presence of rosuvastatin. Except for an approximately 80% increase of rosuvastatin AUC (P < .05) in the heterozygotes of ABCG2 c.421C>A relative to the CC genotype, there were no statistically significant associations between rosuvastatin exposure and polymorphisms assessed. Lanabecestat + rosuvastatin was associated with few treatment-emergent adverse events, all of which resolved and were mild. Lanabecestat does not meaningfully impact BCRP activity; therefore, restriction of concomitant administration with BCRP substrates, such as rosuvastatin, may be unnecessary.

Advances in blood-brain barrier modeling in microphysiological systems highlight critical differences in opioid transport due to cortisol exposure.

BACKGROUND: The United States faces a national crisis involving opioid medications, where currently more than 130 people die every day. To combat this epidemic, a better understanding is needed of how opioids penetrate into the central nervous system (CNS) to facilitate pain relief and, potentially, result in addiction and/or misuse. Animal models, however, are a poor predictor of blood-brain barrier (BBB) transport and CNS drug penetration in humans, and many traditional 2D cell culture models of the BBB and neurovascular unit have inadequate barrier function and weak or inappropriate efflux transporter expression. Here, we sought to better understand opioid transport mechanisms using a simplified microfluidic neurovascular unit (NVU) model consisting of human brain microvascular endothelial cells (BMECs) co-cultured with astrocytes. METHODS: Human primary and induced pluripotent stem cell (iPSC)-derived BMECs were incorporated into a microfluidic NVU model with several technical improvements over our previous design. Passive barrier function was assessed by permeability of fluorescent dextrans with varying sizes, and P-glycoprotein function was assessed by rhodamine permeability in the presence or absence of inhibitors; quantification was performed with a fluorescent plate reader. Loperamide, morphine, and oxycodone permeability was assessed in the presence or absence of P-glycoprotein inhibitors and cortisol; quantification was performed with mass spectrometry. RESULTS: We first report technical and methodological optimizations to our previously described microfluidic model using primary human BMECs, which results in accelerated barrier formation, decreased variability, and reduced passive permeability relative to Transwell models. We then demonstrate proper transport and efflux of loperamide, morphine, and oxycodone in the microfluidic NVU containing BMECs derived from human iPSCs. We further demonstrate that cortisol can alter permeability of loperamide and morphine in a divergent manner. CONCLUSIONS: We reveal a novel role for the stress hormone cortisol in modulating the transport of opioids across the BBB, which could contribute to their abuse or overdose. Our updated BBB model represents a powerful tool available to researchers, clinicians, and drug manufacturers for understanding the mechanisms by which opioids access the CNS.

Physiologically-Based Pharmacokinetic Modeling of Atorvastatin Incorporating Delayed Gastric Emptying and Acid-to-Lactone Conversion.

The drug-drug interaction profile of atorvastatin confirms that disposition is determined by cytochrome P450 (CYP) 3A4 and organic anion transporting polypeptides (OATPs). Drugs that affect gastric emptying, including dulaglutide, also affect atorvastatin pharmacokinetics (PK). Atorvastatin is a carboxylic acid that exists in equilibrium with a lactone form in vivo. The purpose of this work was to assess gastric acid-mediated lactone equilibration of atorvastatin and incorporate this into a physiologically-based PK (PBPK) model to describe atorvastatin acid, lactone, and their major metabolites. In vitro acid-to-lactone conversion was assessed in simulated gastric fluid and included in the model. The PBPK model was verified with in vivo data including CYP3A4 and OATP inhibition studies. Altering the gastric acid-lactone equilibrium reproduced the change in atorvastatin PK observed with dulaglutide. The model emphasizes the need to include gastric acid-lactone conversion and all major atorvastatin-related species for the prediction of atorvastatin PK.

PhRMA white paper on ADME pharmacogenomics.

Pharmacogenomic (PGx) research on the absorption, distribution, metabolism, and excretion (ADME) properties of drugs has begun to have impact for both drug development and utilization. To provide a cross-industry perspective on the utility of ADME PGx, the Pharmaceutical Research and Manufacturers of America (PhRMA) conducted a survey of major pharmaceutical companies on their PGx practices and applications during 2003-2005. This white paper summarizes and interprets the results of the survey, highlights the contributions and applications of PGx by industrial scientists as reflected by original research publications, and discusses changes in drug labels that improve drug utilization by inclusion of PGx information. In addition, the paper includes a brief review on the clinically relevant genetic variants of drug-metabolizing enzymes and transporters most relevant to the pharmaceutical industry.

Post-translational modifications of transporters.

Drug transporter proteins are critical to the distribution of a wide range of endogenous compounds and xenobiotics such as hormones, bile acids, peptides, lipids, sugars, and drugs. There are two classes of drug transporters- the solute carrier (SLC) transporters and ATP-binding cassette (ABC) transporters -which predominantly differ in the energy source utilized to transport substrates across a membrane barrier. Despite their hydrophobic nature and residence in the membrane bilayer, drug transporters have dynamic structures and adopt many conformations during the translocation process. Whereas there is significant literature evidence for the substrate specificity and structure-function relationship for clinically relevant drug transporters proteins, there is less of an understanding in the regulatory mechanisms that contribute to the functional expression of these proteins. Post-translational modifications have been shown to modulate drug transporter functional expression via a wide range of molecular mechanisms. These modifications commonly occur through the addition of a functional group (e.g. phosphorylation), a small protein (e.g. ubiquitination), sugar chains (e.g. glycosylation), or lipids (e.g. palmitoylation) on solvent accessible amino acid residues. These covalent additions often occur as a result of a signaling cascade and may be reversible depending on the type of modification and the intended fate of the signaling event. Here, we review the significant role in which post-translational modifications contribute to the dynamic regulation and functional consequences of SLC and ABC drug transporters and highlight recent progress in understanding their roles in transporter structure, function, and regulation.

Prediction of Transporter-Mediated Drug-Drug Interactions for Baricitinib.

Baricitinib, an oral selective Janus kinase 1 and 2 inhibitor, undergoes active renal tubular secretion. Baricitinib was not predicted to inhibit hepatic and renal uptake and efflux drug transporters, based on the ratio of the unbound maximum eliminating-organ inlet concentration and the in vitro half-maximal inhibitory concentrations (IC50 ). In vitro, baricitinib was a substrate for organic anion transporter (OAT)3, multidrug and toxin extrusion protein (MATE)2-K, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP). Probenecid, a strong OAT3 inhibitor, increased the area under the concentration-time curve from time zero to infinity (AUC[0-∞] ) of baricitinib by twofold and decreased renal clearance to 69% of control in healthy subjects. Physiologically based pharmacokinetic (PBPK) modeling reproduced the renal clearance of baricitinib and the inhibitory effect of probenecid using the in vitro IC50 value of 4.4 µM. Using ibuprofen and diclofenac in vitro IC50 values of 4.4 and 3.8 µM toward OAT3, 1.2 and 1.0 AUC(0-∞) ratios of baricitinib were predicted. These predictions suggest clinically relevant drug-drug interactions (DDIs) with ibuprofen and diclofenac are unlikely.

Development and validation of a sensitive, specific, and rapid hybridization-ELISA assay for determination of concentrations of a ribozyme in biological matrices.

Ribozymes are RNA or modified RNA polymers capable of catalyzing cleavage reactions in target strands RNA, and are under development as human therapeutics. Previous methods used for quantitation of nucleic acid polymers in serum or plasma required extraction of the polymer followed by capillary electrophoresis, HPLC, or gel electrophoresis. These methods are time consuming and lack sensitivity. A bioanalytical method has been developed that does not require extraction of the ribozyme analyte from serum. This technique relies on hybridization of the ribozyme molecule to two complementary biotin and digoxigenin labeled oligonucleotide probes. Serum containing the ribozyme is mixed with the labeled probes, and the mixture is heated at 75 degrees C for 5 min to disrupt the ribozyme secondary structure. Samples are then cooled to permit probe annealing and are added to a streptavidin-coated 96-well plate. The bound complex is detected with an anti-digoxigenin alkaline phosphatase (AP) conjugate using PNPP (p-nitrophenyl phosphate) as a substrate. The amount of colored product is measured on a microtiter plate reader at a wavelength of 405 nm. Concentrations of unknown ribozyme samples are estimated based on a standard curve (0.37-270 ng/ml) prepared in serum. The validated lower and upper limits of quantification are 5.0 and 120 ng/ml, respectively. The assay can be completed in approximately 5h and does not require extraction procedures or electrophoretic/chromatographic separation. It is therefore a simple, sensitive and rapid technique. This assay has been validated and has been used for quantitation of serum levels of the HEPTAZYME ribozyme in mouse, monkey, and human pharmacokinetic studies.