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There are many factors which are known to cause variability in human in vitro enzyme kinetic data. Factors such as the source of enzyme and how it was prepared, the genetics and background of the donor, how the in vitro studies are designed, and how the data are analyzed contribute to variability in the resulting kinetic parameters. It is important to consider not only the factors which cause variability within an experiment, such as selection of a probe substrate, but also those that cause variability when comparing kinetic data across studies and laboratories. For example, the artificial nature of the microsomal lipid membrane and microenvironment in some recombinantly expressed enzymes, relative to those found in native tissue microsomes, has been shown to influence enzyme activity and thus can be a source of variability when comparing across the two different systems. All of these factors, and several others, are discussed in detail in the chapter below. In addition, approaches which can be used to visualize the uncertainty arising from the use of enzyme kinetic data within the context of predicting human pharmacokinetics are discussed.

Purpose This work describes a staged approach to the application of pharmacokinetic-pharmacodynamic (PK-PD) modeling in the voltage-gated sodium ion channel (NaV1.7) inhibitor drug discovery effort to address strategic questions regarding in vitro to in vivo translation of target modulation. Methods PK-PD analysis was applied to data from a functional magnetic resonance imaging (fMRI) technique to non-invasively measure treatment mediated inhibition of olfaction signaling in non-human primates (NHPs). Initial exposure-response was evaluated using single time point data pooled across 27 compounds to inform on in vitro to in vivo correlation (IVIVC). More robust effect compartment PK-PD modeling was conducted for a subset of 10 compounds with additional PD and PK data to characterize hysteresis. Results The pooled compound exposure-response facilitated an early exploration of IVIVC with a limited dataset for each individual compound, and it suggested a 2.4-fold in vitro to in vivo scaling factor for the NaV1.7 target. Accounting for hysteresis with an effect compartment PK-PD model as compounds advanced towards preclinical development provided a more robust determination of in vivo potency values, which resulted in a statistically significant positive IVIVC with a slope of 1.057 ± 0.210, R-squared of 0.7831, and p value of 0.006. Subsequent simulations with the PK-PD model informed the design of anti-nociception efficacy studies in NHPs. Conclusions A staged approach to PK-PD modeling and simulation enabled integration of in vitro NaV1.7 potency, plasma protein binding, and pharmacokinetics to describe the exposure-response profile and inform future study design as the NaV1.7 inhibitor effort progressed through drug discovery.

Pharmacokinetics (PKs) in Japanese healthy subjects were simulated for nine compounds using physiologically based PK (PBPK) models parameterized with physicochemical properties, preclinical absorption, distribution, metabolism, and excretion (ADME) data, and clinical PK data from non‐Japanese subjects. For each dosing regimen, 100 virtual trials were simulated and predicted/observed ratios for peak plasma concentration (C max) and area under the curve (AUC) were calculated. As qualification criteria, it was prespecified that >80% of simulated trials should demonstrate ratios to observed data ranging from 0.5–2.0. Across all compounds and dose regimens studied, 93% of simulated C max values in Japanese subjects fulfilled the criteria. Similarly, for AUC, 77% of single‐dosing regimens and 100% of multiple‐dosing regimens fulfilled the criteria. In summary, mechanistically incorporating the appropriate ADME properties into PBPK models, followed by qualification using non‐Japanese clinical data, can predict PKs in the Japanese population and lead to efficient trial design and conduct of Japanese phase I studies.

This open-label 2-period study assessed the effect of multiple-dose administration of rifampin, a strong cytochrome P450 3A (CYP3A) and P-glycoprotein inducer, on the pharmacokinetics of odanacatib, a cathepsin K inhibitor. In period 1, 12 healthy male subjects (mean age, 30 years) received a single dose of odanacatib 50 mg on day 1, followed by a 28-day washout. In period 2, subjects received rifampin 600 mg/day for 28 days; odanacatib 50 mg was coadministered on day 14. Blood samples for odanacatib pharmacokinetics were collected at predose and on day 1 of period 1 and day 14 of period 2. Coadministration of odanacatib and rifampin significantly reduced odanacatib exposure. The odanacatib AUC0-∞ geometric mean ratio (90% confidence interval) of odanacatib + rifampin/odanacatib alone was 0.13 (0.11-0.16). The harmonic mean ± jackknife standard deviation apparent terminal half-life (t½ ) was 71.6 ± 10.2 hours for odanacatib alone and 16.0 ± 3.4 hours for odanacatib + rifampin, indicating greater odanacatib clearance following coadministration with rifampin. Samples were collected in period 2 during rifampin dosing (days 1, 14, and 28) and after rifampin discontinuation (days 35, 42, and 56) to evaluate the ratio of plasma 4β-hydroxycholesterol to total serum cholesterol as a CYP3A4 induction biomarker; the ratio increased ∼5-fold over 28 days of daily dosing with 600 mg rifampin, demonstrating sensitivity to CYP3A4 induction.

The application of modeling and simulation techniques is increasingly common in preclinical stages of the drug discovery and development process. A survey focusing on preclinical pharmacokinetic/pharmacodynamics (PK/PD) analysis was conducted across pharmaceutical companies that are members of the International Consortium for Quality and Innovation in Pharmaceutical Development. Based on survey responses, ~68% of companies use preclinical PK/PD analysis in all therapeutic areas indicating its broad application. An important goal of preclinical PK/PD analysis in all pharmaceutical companies is for the selection/optimization of doses and/or dose regimens, including prediction of human efficacious doses. Oncology was the therapeutic area with the most PK/PD analysis support and where it showed the most impact. Consistent use of more complex systems pharmacology models and hybrid physiologically based pharmacokinetic models with PK/PD components was less common compared to traditional PK/PD models. Preclinical PK/PD analysis is increasingly being included in regulatory submissions with ~73% of companies including these data to some degree. Most companies (~86%) have seen impact of preclinical PK/PD analyses in drug development. Finally, ~59% of pharmaceutical companies have plans to expand their PK/PD modeling groups over the next 2 years indicating continued growth. The growth of preclinical PK/PD modeling groups in pharmaceutical industry is necessary to establish required resources and skills to further expand use of preclinical PK/PD modeling in a meaningful and impactful manner.

To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs.siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored. The rat livers were harvested to produce microsomes to determine the impact of POR knockdown on the metabolism of several probe substrates. Midazolam (a CYP3A substrate with high intrinsic clearance) was administered into LNP-treated rats to determine the impact of POR knockdown on midazolam pharmacokinetics.Hepatic POR mRNA and protein levels were significantly reduced by administering siRNA and the maximum POR enzyme activity reduction (~85%) occurred 2 weeks post-dose. In vitro analysis showed significant reductions in metabolism of probe substrates due to POR knockdown in liver, and in vivo POR knockdown resulted in greater than 10-fold increases in midazolam plasma concentrations following oral dosing.Anti-POR siRNA can be used to significantly reduce hepatic metabolism by various CYPs as well as greatly increase the bioavailability of high clearance compounds following an oral dose, thus enabling it to be used as a tool to increase drug exposure in vivo.

Recently, the US Food and Drug Administration and European Medicines Agency have issued new guidance for industry on drug interaction studies, which outline comprehensive recommendations on a broad range of in vitro and in vivo studies to evaluate drug–drug interaction (DDI) potential. This paper aims to provide an overview of these new recommendations and an in-depth scientifically based perspective on issues surrounding some of the recommended approaches in emerging areas, particularly, transporters and complex DDIs. We present a number of theoretical considerations and several case examples to demonstrate complexities in applying (1) the proposed transporter decision trees and associated criteria for studying a broad spectrum of transporters to derive actionable information and (2) the recommended model-based approaches at an early stage of drug development to prospectively predict DDIs involving time-dependent inhibition and mixed inhibition/induction of drug metabolizing enzymes. We hope to convey the need for conducting DDI studies on a case-by-case basis using a holistic scientifically based interrogative approach and to communicate the need for additional research to fill in knowledge gaps in these areas where the science is rapidly evolving to better ensure the safety and efficacy of new therapeutic agents.

With inadequate efficacy being the primary cause for the attrition of drug candidates in clinical development, the need to better predict clinical efficacy earlier in the drug development process has increased in importance in the pharmaceutical industry. Here, we review current applications of translational pharmacokinetic-pharmacodynamic (PK-PD) modeling of preclinical data in the pharmaceutical industry, including best practices. Preclinical translational PK-PD modeling has been used in many therapeutic areas and has been impactful to drug development. The role of preclinical translational PK-PD modeling in drug discovery and development will continue to evolve and broaden, given that its broad implementation in the pharmaceutical industry is relatively recent and many opportunities still exist for its further application.

A series of N -heterocyclic pyridinone catechol- O -methyltransferase (COMT) inhibitors were synthesized. Physicochemical properties, including ligand lipophilic efficiency (LLE) and c log P , were used to guide compound design and attempt to improve inhibitor pharmacokinetics. Incorporation of heterocyclic central rings provided improvements in physicochemical parameters but did not significantly reduce in vitro or in vivo clearance. Nevertheless, compound 11 was identified as a potent inhibitor with sufficient in vivo exposure to significantly affect the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), and indicate central COMT inhibition.

MK-7246, an antagonist of the chemoattractant receptor on T helper type 2 (Th2) cells, is being developed for the treatment of respiratory diseases. In a first-in-human study, we investigated whether genetic polymorphisms contributed to the marked intersubject variability in the pharmacokinetics of MK-7246 and its glucuronide metabolite M3. Results from in vitro enzyme kinetic studies suggested that UGT2B17 is probably the major enzyme responsible for MK-7246 metabolism in both the liver and the intestine. As compared with those with the UGT2B17*1/*1 wild-type genotype, UGT2B17*2/*2 carriers, who possess no UGT2B17 protein, had 25- and 82-fold greater mean dose-normalized values of area under the plasma concentration–time curve (AUC) and peak concentration of MK-7246, respectively, and a 24-fold lower M3-to-MK-7246 AUC ratio. The apparent half-life of MK-7246 was not as variable between these two genotypes. Therefore, the highly variable pharmacokinetics of MK-7246 is attributable primarily to the impact of UGT2B17 genetic polymorphisms and extensive first-pass metabolism of MK-7246.

Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.

The objective of this study is to assess the effectiveness of physiologically based pharmacokinetic (PBPK) models for simulating human plasma concentration-time profiles for the unique drug dataset of blinded data that has been assembled as part of a Pharmaceutical Research and Manufacturers of America initiative. Combinations of absorption, distribution, and clearance models were tested with a PBPK approach that has been developed from published equations. An assessment of the quality of the model predictions was made on the basis of the shape of the plasma time courses and related parameters. Up to 69% of the simulations of plasma time courses made in human demonstrated a medium to high degree of accuracy for intravenous pharmacokinetics, whereas this number decreased to 23% after oral administration based on the selected criteria. The simulations resulted in a general underestimation of drug exposure (Cmax and AUC0- t ). The explanations for this underestimation are diverse. Therefore, in general it may be due to underprediction of absorption parameters and/or overprediction of distribution or oral first-pass. The implications of compound properties are demonstrated. The PBPK approach based on in vitro-input data was as accurate as the approach based on in vivo data. Overall, the scientific benefit of this modeling study was to obtain more extensive characterization of predictions of human PK from PBPK methods.

We studied the time course for the reversal of rifampin's effect on the pharmacokinetics of oral midazolam (a cytochrome P450 (CYP) 3A4 substrate) and digoxin (a P-glycoprotein (P-gp) substrate). Rifampin increased midazolam metabolism, greatly reducing the area under the concentration-time curve (AUC(0-∞)). The midazolam AUC(0-∞) returned to baseline with a half-life of ~8 days. Rifampin's effect on the AUC(0-3 h) of digoxin was biphasic: the AUC(0-3 h) increased with concomitant dosing of the two drugs but decreased when digoxin was administered after rifampin. Digoxin was found to be a weak substrate of organic anion-transporting polypeptide (OATP) 1B3 in transfected cells. Although the drug was transported into isolated hepatocytes, it is not likely that this transport was through OATP1B3 because the transport was not inhibited by rifampin. However, rifampin did inhibit the P-gp-mediated transport of digoxin with a half-maximal inhibitory concentration (IC(50)) below anticipated gut lumen concentrations, suggesting that rifampin inhibits digoxin efflux from the enterocyte to the intestinal lumen. Pharmacokinetic modeling suggested that the effects on digoxin are consistent with a combination of inhibitory and inductive effects on gut P-gp. These results suggest modifications to drug-drug interaction (DDI) trial designs.

The Fc region of an antibody mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and plays a key role in the in vivo half-life of an antibody. In designing antibody therapeutics, it is sometimes desirable that the antibody has altered Fc-mediated properties. In the case of a "benign blocker" antibody, it is often desirable to diminish or abolish the ADCC and CDC functions while retaining its PK profile. Here, we report a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S). An IgG2m4 antibody has an overall reduction in complement and Fc gamma receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus.

1. Ten compounds from the Merck Research Laboratories pipeline were selected to evaluate the utility of using intrinsic clearance derived from recombinantly expressed cytochromes P450 (CYP) and physiologically based pharmacokinetic modelling to predict Phase I pharmacokinetics using simCYP. The compounds selected were anticipated to be eliminated predominantly by P450 metabolism. 2. There was a reasonable agreement between the predicted and actual clinical exposure with 80% of the predicted exposures being within three-fold of the observed values. Furthermore, prediction of C(t) (plasma concentration at a specified time point) and T(max) were acceptable with greater than or equal to 70% of the predicted data being within three-fold of the observed values. However, prediction of C(max) was unreliable and may have been due to error in predicting the time-dependent change in volume of distribution and/or error in estimating absorption rate. 3. Although it is acknowledged that research is needed to improve predictive performance, the data presented are supportive of using recombinant P450 intrinsic clearance and physiologically based pharmacokinetic modelling to predict Phase I pharmacokinetics for compounds eliminated by P450 metabolism.

Glycine transporter 1 (GlyT1) represents a novel target for the treatment of schizophrenia via the potentiation of glutamatergic NMDA receptors. The discovery of 4,4-disubstituted piperidine inhibitors of GlyT1 which exhibit improved pharmacokinetic properties, including oral bioavailability, is discussed.

Background Characterization of PD-L1 expression within clinically/radiologically negative but microscopically tumor positive sentinel lymph nodes (SLN) is important to our understanding of the relevance of this immune checkpoint pathway for adjuvant therapy. Methods Patients included had primary cutaneous melanoma, Breslow thickness of 2.01–4.0 or >4 mm with or without tumor ulceration (T3a, T3b, T4a, T4b). All patients had microscopically tumor positive SLN. Hematoxylin and eosin (H&E) staining was performed, followed by PD-L1 immunohistochemical (IHC) staining using a preliminary IHC assay with anti-PD-L1 antibody clone 22C3. The slides were separately evaluated by two pathologists (JY and CG). Samples containing metastatic melanoma lesions were scored separately for PD-L1 expression in intratumoral and peritumoral locations, by utilizing two scoring methods. Results Twenty-four patients where metastatic melanoma presence in the SLN was confirmed by H&E review of the cut sections were included in the final analysis of PD-L1 expression. SLN tumor size ranged from 1 to 2 mm. For three patients, the melanin content was too high to confidently assign a PD-L1 score. For the remaining 21 patients, all had some evidence of either intratumoral or peritumoral PD-L1 expression. The frequency of intratumoral tumor-associated PD-L1 expression was: 0 % of tumor cells (3 pts, 14 %); 10 % (7 pts, 33 %). Conclusions Tumor-associated PD-L1 expression is readily detectable within melanoma micrometastases in the SLN of the majority of patients. These results support the testing of a therapeutic role for PD1/PD-L1 inhibition in the adjuvant setting, targeting melanoma micrometastases.

Compound I [3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one] is a potent inhibitor of human kinase insert domain-containing receptor (KDR kinase), which is under investigation for the treatment of cancer. Bile duct-cannulated male beagle dogs were administered 6 mg/kg compound I q.d. for 14 days. There was an approximately 2.5-fold decrease in the mean plasma area under the curve of I on days 7 and 14 (approximately 11.3 microM . h), relative to day 1 (28.2 microM . h). In the dog, compound I was eliminated by metabolism, with a major pathway being aromatic hydroxylation and subsequent sulfation to form the metabolite M3. Metabolic profiling suggested that the pathway leading to the formation of the sulfated conjugate M3 was induced upon multiple dosing of I. Studies conducted in vitro suggested that CYP1A1/2 was responsible for the formation of the hydroxylated metabolite, which is sulfated to yield M3. Additional studies confirmed induction of CYP1A protein and activity in the livers of dogs treated with I. However, studies in a dog hepatocyte model of induction showed a surprising decrease both in CYP1A mRNA and enzymatic activity in the presence of I, emphasizing the need to consider the results from a variety of in vitro and in vivo studies in deriving an understanding of the metabolic fate of a drug candidate. It is concluded that the autoinduction observed after multiple treatments with compound I occurs since compound I is both an inducer and a substrate for dog CYP1A.

We have developed a quantitative assay using electrospray ionization mass spectrometry coupled to reversed-phase ion-pair liquid chromatography (LC/MS) for quantitation of sodium borocaptate (BSH) in human plasma. The assay was developed using a Micromass Q-TOF II mass spectrometer equipped with an orthogonal electrospray source. The mobile phase was a 1:1 solution of methanol and 5 mM aqueous tetrabutylammonium acetate flowing at 0.2 mL/min, and the chromatography was performed using a Machery-Nagel Nucleosil C18 column. Plasma samples from patients who had received an intravenous infusion of sodium borocaptate (Na2B12H11SH), frequently referred to as BSH, were prepared for analysis by precipitation with acetonitrile. Following this, the supernatants were collected, and 40 microL was injected onto the column for analysis. The LC/MS assay was linear over a BSH plasma concentration range of 20-0.5 microg/mL with acceptable variability for both intra- and interassay precision. The LC/MS assay was used to generate pilot pharmacokinetic data for the plasma disposition of BSH in humans. The disposition of BSH was found to be consistent with a two-compartment model with first-order elimination from the central compartment. The mean total body plasma clearance was 95.7 +/- 30.8 m/min and the harmonic mean terminal half-life was 3.6 h.

The application of physiologically based pharmacokinetic (PBPK) modeling has developed rapidly within the pharmaceutical industry and is becoming an integral part of drug discovery and development. In this study, we provide a cross pharmaceutical industry position on "how PBPK modeling can be applied in industry" focusing on the strategies for application of PBPK at different stages, an associated perspective on the confidence and challenges, as well as guidance on interacting with regulatory agencies and internal best practices.

We evaluated the effect of prednisone on midazolam and odanacatib pharmacokinetics. In this open-label, 2-period crossover study in healthy male subjects, midazolam 2 mg was administered (Day -1) followed by odanacatib 50 mg (Day 1) during Part 1. In Period 2, prednisone 10 mg once daily (qd) was administered on Days 1-28; odanacatib was co-administered on Day 14 and midazolam 2 mg was co-administered on Days 1 and 28. Subjects were administered midazolam 2 mg on Days 42 and 56. Safety and tolerability were assessed throughout the study. A physiologically-based pharmacokinetic (PBPK) model was also built. There were 15 subjects enrolled; mean age was 31 years. The odanacatib AUC(0- ∞) GMR (90% CI) [odanacatib + prednisone (Day 14, Period 2)/odanacatib alone (Day 1, Period 1] was 1.06 (0.96, 1.17). AUC(0-∞) GMR (90%CI) [midazolam + prednisone (Day 28, Period 2)/midazolam alone (Day -1, Period 1] was 1.08 (0.93,1.26). There were no serious AEs or AEs leading to discontinuation. PBPK modeling showed that prednisone does not cause significant effects on the exposure of sensitive CYP3A4 substrates in vivo at therapeutic doses. Co-administration of prednisone 10 mg qd had no effect on pharmacokinetics of either odanacatib 10 mg or midazolam 2 mg.

There are many factors which are known to cause variability in human in vitro enzyme kinetic data. Factors such as the source of enzyme and how it was prepared, the genetics and background of the donor, how the in vitro studies are designed, and how the data are analyzed contribute to variability in the resulting kinetic parameters. It is important to consider not only the factors which cause variability within an experiment, such as selection of a probe substrate, but also those that cause variability when comparing kinetic data across studies and laboratories. For example, the artificial nature of the microsomal lipid membrane and microenvironment in some recombinantly expressed enzymes, relative to those found in native tissue microsomes, has been shown to influence enzyme activity and thus can be a source of variability when comparing across the two different systems. All of these factors, and several others, are discussed in detail in the chapter below.

OBJECTIVE The purpose of this study was to obtain tumor and normal brain tissue biodistribution data and pharmacokinetic profiles for sodium borocaptate (Na2B12H11SH) (BSH), a drug that has been used clinically in Europe and Japan for boron neutron capture therapy of brain tumors. The study was performed with a group of 25 patients who had preoperative diagnoses of either glioblastoma multiforme (GBM) or anaplastic astrocytoma (AA) and were candidates for debulking surgery. Nineteen of these patients were subsequently shown to have histopathologically confirmed diagnoses of GBM or AA, and they constituted the study population. METHODS BSH (non-10 B-enriched) was infused intravenously, in a 1-hour period, at doses of 15, 25, and 50 mg boron/kg body weight (corresponding to 26.5, 44.1, and 88.2 mg BSH/kg body weight, respectively) to groups of 3, 3, and 13 patients, respectively. Multiple samples of tumor tissue, brain tissue around the tumors, and normal brain tissue were obtained at either 3 to 7 or 13 to 15 hours after infusion. Blood samples for pharmacokinetic studies were obtained at times up to 120 hours after termination of the infusion. Sixteen of the patients underwent surgery at the Beijing Neurosurgical Institute and three at The Ohio State University, where all tissue samples were subsequently analyzed for boron content by direct current plasma-atomic emission spectroscopy. RESULTS Blood boron values peaked at the end of the infusion and then decreased triexponentially during the 120-hour sampling period. At 6 hours after termination of the infusion, these values had decreased to 20.8, 29.1, and 62.6 μg/ml for boron doses of 15, 25, and 50 mg/kg body weight, respectively. For a boron dose of 50 mg/kg body weight, the maximum (mean ± standard deviation) solid tumor boron values at 3 to 7 hours after infusion were 17.1 ± 5.8 and 17.3 ± 10.1 μg/g for GBMs and AAs, respectively, and the mean tumor value averaged across all samples was 11.9 μg/g for both GBMs and AAs. In contrast, the mean normal brain tissue values, averaged across all samples, were 4.6 ± 5.1 and 5.5 ± 3.9 μg/g and the tumor/normal brain tissue ratios were 3.8 and 3.2 for patients with GBMs and AAs, respectively. The large standard deviations indicated significant heterogeneity in uptake in both tumor and normal brain tissue. Regions histopathologically classified either as a mixture of tumor and normal brain tissue or as infiltrating tumor exhibited slightly lower boron concentrations than those designated as solid tumor. After a dose of 50 mg/kg body weight, boron concentrations in blood decreased from 104 μg/ml at 2 hours to 63 μg/ml at 6 hours and concentrations in skin and muscle were 43.1 and 39.2 μg/g, respectively, during the 3- to 7-hour sampling period. CONCLUSION When tumor, blood, and normal tissue boron concentrations were taken into account, the most favorable tumor uptake data were obtained with a boron dose of 25 mg/kg body weight, 3 to 7 hours after termination of the infusion. Although blood boron levels were high, normal brain tissue boron levels were almost always lower than tumor levels. However, tumor boron concentrations were less than those necessary for boron neutron capture therapy, and there was significant intratumoral and interpatient variability in the uptake of BSH, which would make estimation of the radiation dose delivered to the tumor very difficult. It is unlikely that intravenous administration of a single dose of BSH would result in therapeutically useful levels of boron. However, combining BSH with boronophenylalanine, the other compound that has been used clinically, and optimizing their delivery could increase tumor boron uptake and potentially improve the efficacy of boron neutron capture therapy.

There are many factors which are known to cause variability in human in vitro enzyme kinetic data. Factors such as the source of enzyme and how it was prepared, the genetics and background of the donor, how the in vitro studies are designed, and how the data are analyzed contribute to variability in the resulting kinetic parameters. It is important to consider not only the factors which cause variability within an experiment, such as selection of a probe substrate, but also those that cause variability when comparing kinetic data across studies and laboratories. For example, the artificial nature of the microsomal lipid membrane and microenvironment in some recombinantly expressed enzymes, relative to those found in native tissue microsomes, has been shown to influence enzyme activity and thus can be a source of variability when comparing across the two different systems. All of these factors, and several others, are discussed in detail in the chapter below. In addition, approaches which can be used to visualize the uncertainty arising from the use of enzyme kinetic data within the context of predicting human pharmacokinetics are discussed.

A major pathway of elimination of the prostaglandin D2 receptor 1 antagonist laropiprant in humans is by uridine diphosphate-glucuronosyltransferase (UGT)-mediated biotransformation. In this study, liver and kidney relative activity factors were developed for UGT1A1, 1A9 and 2B7 to allow for in vitro-in vivo extrapolation of intrinsic clearance data to whole organ clearance using recombinant human UGT isoforms applying this to laropiprant as a model substrate. The total body metabolic clearance of laropiprant determined using this approach (5.0 L/hr) agreed well with the value determined in vivo following intravenous administration to healthy human volunteers (5.1 L/hr). The results suggest that approximately 36%, 36% and 28% of the hepatic metabolic clearance of laropiprant was mediated by UGT1A1, 1A9 and 2B7, respectively. Likewise, 80% and 20% of the renal metabolic clearance was mediated by UGT1A9 and 2B7, respectively. Furthermore, the data suggested that the contribution of the kidney to the overall total metabolic clearance was minor relative to the liver (≈ 12%).

Several drug–drug interaction (DDI) prediction models were evaluated for their ability to identify drugs with cytochrome P450 (CYP)3A induction liability based on in vitro mRNA data. The drug interaction magnitudes of CYP3A substrates from 28 clinical trials were predicted using (i) correlation approaches (ratio of the in vivo peak plasma concentration (Cmax) to in vitro half-maximal effective concentration (EC50); and relative induction score), (ii) a basic static model (calculated R3 value), (iii) a mechanistic static model (net effect), and (iv) mechanistic dynamic (physiologically based pharmacokinetic) modeling. All models performed with high fidelity and predicted few false negatives or false positives. The correlation approaches and basic static model resulted in no false negatives when total Cmax was incorporated; these models may be sufficient to conservatively identify clinical CYP3A induction liability. Mechanistic models that include CYP inactivation in addition to induction resulted in DDI predictions with less accuracy, likely due to an overprediction of the inactivation effect. Clinical Pharmacology & Therapeutics (2014); 95 2, 216–227. doi:10.1038/clpt.2013.170

Setileuton [4-(4-fluorophenyl)-7-[({5-[(1S)-1-hydroxy-1-(trifluoromethyl)propyl]-1,3,4-oxadiazol-2-yl}amino)methyl]-2H-1-benzopyran-2-one] is a selective inhibitor of the 5-lipoxygenase enzyme, which is under investigation for the treatment of asthma and atherosclerosis. During the development of setileuton, a metabolite (M5) was identified in incubations with rat, dog, and human liver microsomes that represented the addition of 18 Da to the 1,3,4-oxadiazole portion of the molecule. Based on mass spectral data, a ring opened structure was proposed and confirmed through comparison with a synthetic standard. The metabolic ring opening was examined in vitro in rat liver microsomes and was determined to be mediated by cytochrome P450s (P450s). Upon examination of the specific P450s involved using cDNA-expressed rat P450s, it was shown that CYP1A2 likely was the major isoform contributing to the formation of M5. Studies using stable labeled molecular oxygen and water demonstrated that the oxygen was incorporated from molecular oxygen, rather than water, and confirmed that the metabolic formation was oxidative. An alternative, comparatively slow pathway of chemical hydrolysis also was identified and described. Three potential mechanisms for the two-step metabolic ring opening of the 1,3,4-oxadizole are proposed.

The objective of this study was to evaluate the performance of the Wajima allometry (Css -MRT) approach published in the literature, which is used to predict the human plasma concentration-time profiles from a scaling of preclinical species data. A diverse and blinded dataset of 108 compounds from PhRMA member companies was used in this evaluation. The human intravenous (i.v.) and oral (p.o.) pharmacokinetics (PK) data were available for 18 and 107 drugs, respectively. Three different scenarios were adopted for prediction of human PK profiles. In the first scenario, human clearance (CL) and steady-state volume of distribution (Vss ) were predicted by unbound fraction corrected intercept method (FCIM) and Oie-Tozer (OT) approaches, respectively. Quantitative structure activity relationship (QSAR)-based approaches (TSrat-dog ) based on compound descriptors together with rat and dog data were utilized in the second scenario. Finally, in the third scenario, CL and Vss were predicted using the FCIM and Jansson approaches, respectively. For the prediction of oral pharmacokinetics, the human bioavailability and absorption rate constant were assumed as the average of preclinical species. Various statistical techniques were used for assessing the accuracy of the simulation scenarios. The human CL and Vss were predicted within a threefold error range for about 75% of the i.v. drugs. However, the accuracy in predicting key p.o. PK parameters appeared to be lower with only 58% of simulations falling within threefold of observed parameters. The overall ability of the Css -MRT approach to predict the curve shape of the profile was in general poor and ranged between low to medium level of confidence for most of the predictions based on the selected criteria.

This study is part of the Pharmaceutical Research and Manufacturers of America (PhRMA) initiative on predictive models of efficacy, safety, and compound properties. The overall goal of this part was to assess the predictability of human pharmacokinetics (PK) from preclinical data and to provide comparisons of available prediction methods from the literature, as appropriate, using a representative blinded dataset of drug candidates. The key objectives were to (i) appropriately assemble and blind a diverse dataset of in vitro, preclinical in vivo, and clinical data for multiple drug candidates, (ii) evaluate the dataset with empirical and physiological methodologies from the literature used to predict human PK properties and plasma concentration–time profiles, (iii) compare the predicted properties with the observed clinical data to assess the prediction accuracy using routine statistical techniques and to evaluate prediction method(s) based on the degree of accuracy of each prediction method, and (iv) compile and summarize results for publication. Another objective was to provide a mechanistic understanding as to why one methodology provided better predictions than another, after analyzing the poor predictions. A total of 108 clinical lead compounds were collected from 12 PhRMA member companies. This dataset contains intravenous (n = 19) and oral pharmacokinetic data (n = 107) in humans as well as the corresponding preclinical in vitro, in vivo, and physicochemical data. All data were blinded to protect the anonymity of both the data and the company submitting the data. This manuscript, which is the first of a series of manuscripts, summarizes the PhRMA initiative and the 108 compound dataset. More details on the predictability of each method are reported in companion manuscripts. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4050–4073, 2011

The objective of this study was to evaluate the performance of various allometric and in vitro-in vivo extrapolation (IVIVE) methodologies with and without plasma protein binding corrections for the prediction of human intravenous (i.v.) clearance (CL). The objective was also to evaluate the IVIVE prediction methods with animal data. Methodologies were selected from the literature. Pharmaceutical Research and Manufacturers of America member companies contributed blinded datasets from preclinical and clinical studies for 108 compounds, among which 19 drugs had i.v. clinical pharmacokinetics data and were used in the analysis. In vivo and in vitro preclinical data were used to predict CL by 29 different methods. For many compounds, in vivo data from only two species (generally rat and dog) were available and/or the required in vitro data were missing, which meant some methods could not be properly evaluated. In addition, 66 methods of predicting oral (p.o.) area under the curve (AUCp.o. ) were evaluated for 107 compounds using rational combinations of i.v. CL and bioavailability (F), and direct scaling of observed p.o. CL from preclinical species. Various statistical and outlier techniques were employed to assess the predictability of each method. Across methods, the maximum success rate in predicting human CL for the 19 drugs was 100%, 94%, and 78% of the compounds with predictions falling within 10-fold, threefold, and twofold error, respectively, of the observed CL. In general, in vivo methods performed slightly better than IVIVE methods (at least in terms of measures of correlation and global concordance), with the fu intercept method and two-species-based allometry (rat-dog) being the best performing methods. IVIVE methods using microsomes (incorporating both plasma and microsomal binding) and hepatocytes (not incorporating binding) resulted in 75% and 78%, respectively, of the predictions falling within twofold error. IVIVE methods using other combinations of binding assumptions were much less accurate. The results for prediction of AUCp.o. were consistent with i.v. CL. However, the greatest challenge to successful prediction of human p.o. CL is the estimate of F in human. Overall, the results of this initiative confirmed predictive performance of common methodologies used to predict human CL.

The objective of this study was to evaluate the performance of various empirical, semimechanistic and mechanistic methodologies with and without protein binding corrections for the prediction of human volume of distribution at steady state (Vss ). PhRMA member companies contributed a set of blinded data from preclinical and clinical studies, and 18 drugs with intravenous clinical pharmacokinetics (PK) data were available for the analysis. In vivo and in vitro preclinical data were used to predict Vss by 24 different methods. Various statistical and outlier techniques were employed to assess the predictability of each method. There was not simply one method that predicts Vss accurately for all compounds. Across methods, the maximum success rate in predicting human Vss was 100%, 94%, and 78% of the compounds with predictions falling within tenfold, threefold, and twofold error, respectively, of the observed Vss . Generally, the methods that made use of in vivo preclinical data were more predictive than those methods that relied solely on in vitro data. However, for many compounds, in vivo data from only two species (generally rat and dog) were available and/or the required in vitro data were missing, which meant some methods could not be properly evaluated. It is recommended to initially use the in vitro tissue composition-based equations to predict Vss in preclinical species and humans, putting the assumptions and compound properties into context. As in vivo data become available, these predictions should be reassessed and rationalized to indicate the level of confidence (uncertainty) in the human Vss prediction. The top three methods that perform strongly at integrating in vivo data in this way were the Oie-Tozer, the rat -dog-human proportionality equation, and the lumped-PBPK approach. Overall, the scientific benefit of this study was to obtain greater characterization of predictions of human Vss from several methods available in the literature.

Background: Anti-PD1/PDL1 immunotherapy has demonstrated unprecedented clinical antitumor activity in patients with metastatic melanoma supporting planned testing as adjuvant therapy in patients with operable disease at high risk of recurrence and death. Characterization of PDL1 expression within clinically/radiologically negative but microscopically tumor positive sentinel lymph nodes is critical to our understanding of the relevance of this immune checkpoint pathway for adjuvant therapy. Methods: Cut sections (5 µm) of sentinel lymph node from 25 patients reported positive for melanoma micrometastasis were included. H&E staining was performed, followed by PD-L1 immunohistochemical (IHC) staining using a preliminary IHC assay with anti-PD-L1 antibody clone 22C3. Slides from 2 patients were also stained using an anti-HMB45/MelA protocol to better ascertain the presence and/or localization of melanoma lesions in the tissue in order to facilitate interpretation of the PD-L1 staining in those samples. The slides were separately evaluated by 2 pathologists. Samples containing metastatic melanoma lesions were scored separately for PD-L1 expression in intratumoral (including along tumor periphery but with clear tumor cell labeling) and peritumoral (expression external to tumor nodule in immediately surrounding tissue) locations. For intratumoral signal, attempts were made to classify the expression as tumor cell associated (T), non-tumor cell associated (NT), or both (T/NT). Scores were assigned using a 0-5 semiquantitative scale assessing prevalence of positive cells where 0 = negative, 1 = minimal or rare, 2 = low, 3 = moderate, 4 = high, and 5 = very high. Samples where melanin content was too high to confidently assign a PD-L1 score were specifically noted. Results: Twenty patients where metastatic melanoma presence in regional sentinel nodes was confirmed by H&E review of the cut sections were included in the final analysis of PDL1 expression in the target population. Table 1 summarizes the study results. Except for samples where melanin content was too high to confidently assign a PD-L1 score, all SLNs examined demonstrated evidence of intratumoral and/or peritumoral PD-L1 expression. Conclusions: PD-L1 expression is readily detectable intratumorally and/or peritumorally within melanoma micrometastases in the SLN. These results support the testing of a therapeutic role for PD1/PDL1 inhibition in the adjuvant therapy setting, targeting melanoma micrometastases. Patient with SLN+ Tumor Associated Expression PD-L1 Score / Tumor ---- PD-L1 Score / Peritumoral 1. 1 T, very weak ---- 1 2. 4, predominantly T, peripheral ---- 1 3. melanin confounds ---- 2 4. 0 ---- 2 (adjacent subcapsular sinus) 5. 4 T/NT---- 2 6. 2, predominantly NT ---- 3 7. 0 ---- 3 (adjacent subcapsular sinus) 8. 2.5, predominantly NT ---- 3 9. 4.5, predominantly T, weak ---- 2 (adjacent sinus) 10. melanin confounds ---- 0 11. 1 T ---- 2.5 12. 0* ---- 2 13. 1 T ---- 1 14. 2.5 NT ---- 2 15. 2 predominantly NT ---- 3 16. 1 T ---- 2 17. melanin confounds ---- 0 18. 1 ---- 1 19. 1 NT ---- 2.5 20. 2 T, melanin confounds ---- 2 *Very few tumor cells. In one region where a few tumor cells were present, a dense area of PD-L1 positivity was present directly adjacent but was impossible to say if all were non-tumor; this is interpreted as most likely. Citation Format: Ahmad A. Tarhini, Jennifer A. Yearley, Christopher Gibson, Uma N. M. Rao, Cindy Sander, John M. Kirkwood. Tumor associated PDL1 expression pattern in microsopically tumor positive sentinel lymph nodes (SLN) in patients with melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A90.

Selective and nonselective cytochromes P450 (P450) chemical inhibitors and monoclonal antibodies (mAbs) are routinely used to determine the contribution of P450 enzymes involved in the biotransformation of a drug. A fluorometric assay has been established using fluorescein diacetate as a model substrate to determine the effect of some commonly used P450 inhibitors and mAbs on human liver microsomal esterase activity. Of those inhibitors studied, only alpha-naphthoflavone, clotrimazole, ketoconazole, miconazole, nicardipine, and verapamil significantly inhibited human liver microsomal esterase activity, with apparent IC50 values of 18.0, 20.5, 6.5, 15.0, 19.4, and 5.4 microM, respectively. All of these showed > or =20% inhibition of human liver microsomal esterase activity at concentrations typically used for P450 reaction phenotyping studies, with clotrimazole, miconazole, nicardipine, and verapamil showing >60% inhibition. Unlike the chemical inhibitors, no inhibition of human liver microsomal esterase activity was observed in the presence of mAb to CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. These results suggest that P450 chemical inhibitors are capable of inhibiting human liver microsomal esterase activity and should not be used to assess the role of P450 enzymes in the biotransformation of esters. The lack of inhibition of human liver microsomal esterase activity by P450-specific monoclonal antibodies suggests that they may be used to assess the role of P450 enzymes in the biotransformation of esters. Additional experiments to assess the contribution of oxidative enzymes in the metabolism of esters may include incubations in the presence and absence of beta-nicotinamide adenine dinucleotide 2'-phosphate reduced.

During the metabolic characterization of compound I, 2-{6-cyano-3-[(2,2-difluoro-2-pyridin-2-ylethyl)amino]-2-oxopyrazin-1(2H)-yl]-N-[(3-fluoropyridin-2-yl)methyl]acetamide, evidence was obtained for extensive oxidative bioactivation of the pyrazinone ring system and some of the resulting metabolites were apparently devoid of the cyano moiety. Two assays, a spectrophotometric and a high-pressure liquid chromatography (HPLC) pre-column derivatization method, were evaluated for their ability to detect and quantify cyanide that is metabolically generated from liver microsomal incubations. When I was incubated (45 microM) in the presence of NADPH-fortified human liver microsomes for 2h, 7.5 microM of cyanide was detected using the spectrophotometric assay and 8.9 microM was measured using the HPLC methodology. Overall, the results from the two assays appeared to agree reasonably well with each other. However, the HPLC assay was the preferred method for the evaluation of cyanide formation in vitro due to its sensitivity, reliability, and ease of use.

Thrombin is a serine protease that plays a key role in the blood coagulation cascade. Compound I [2-[6-chloro-3-[(2,2-difluoro-2-pyridin-2-ylethyl)amino]-2-oxopyrazin-1(2H)-yl]-N-[(3-fluoropyridin-2-yl)methyl]acetamide] is a potent, selective, and orally bioavailable thrombin inhibitor that is being studied as a possible anticoagulant. Biotransformation studies in rats revealed that 84% of an i.v. dose of I was excreted in the form of two metabolites. Both metabolites were formed by metabolic activation of the pyrazinone ring in I and subsequent rearrangement leading to two novel dihydro-imidazole and imidazolidine derivatives. The structures of these metabolites and their mechanism of formation were elucidated by additional use of two 13C single labels in the pyrazinone ring of I in combination with mass spectrometry and NMR techniques. The metabolite structures described here illustrate the rich metabolic chemistry of the amino-pyrazinone heterocycle.

The pharmacokinetics of sodium borocaptate (BSH), a drug that has been used clinically for boron neutron capture therapy (BNCT) of malignant brain tumors, have been characterized by measuring boron concentrations by direct current plasma-atomic emission spectroscopy (DCP-AES) in a group of 23 patients with high-grade gliomas. The disposition of BSH following intravenous (i.v.) infusion, which was determined by measuring plasma boron concentrations by DCP-AES, was consistent with a three-compartment open model with zero-order input and first-order elimination from the central compartment. Boron disposition was linear over the dose range of 26.5-88.2 mg BSH/kg body weight (b.w.), corresponding to 15-50 mg boron/kg b.w. Mean total body boron plasma clearance was 14.4 +/- 3.5 ml/min and the harmonic mean half-lives (range) were 0.6 (0.3-3.7), 6.5 (4.8-10.1) and 77.8 (49.6-172.0) h for the alpha, beta, and gamma disposition phases, respectively. Using an empirically determined plasma: blood boron concentration ratio of 1.3 +/- 0.2, the calculated total body boron blood clearance was 18.5 +/- 4.5 ml/min. In order to develop a model for selecting the optimum dosing paradigm, a pharmacokinetic correlation was established between the boron content of normal brain, solid tumor, and infiltrating tumor to the shallow tissue pharmacokinetic compartment (C2). Based on our model, it was concluded that although multiple i.v. infusions of BSH might increase absolute tumor boron concentrations, they will not improve the tumor: plasma boron concentration ratios over those attainable by a single i.v. infusion. The results from our study are confirmatory of those previously reported by others when blood sampling has been carried out for a sufficient period of time to adequately characterize the pharmacokinetics.

Tandem liquid chromatography-mass spectrometry coupled to online radioactive material detection (LC/RAM/MS/MS) is a technique that is used routinely for in vivo and in vitro drug metabolism studies and allows for a simultaneous correlation between radiochemical peaks and mass spectral data. The compound diethylene glycol monobutyl ether (DGBE), a component of a commercially available scintillation cocktail for RAM analysis, was identified as a source of overwhelming chemical noise in a mass spectrometer which was used in an LC/RAM/MS/MS configuration. In this report, we describe the identification of DGBE as the source of the chemical noise and the methods that were used to minimize the exposure of the mass spectrometer to volatile components of the scintillation cocktail.