Xenografted Tumors Share Comparable Fraction Unbound and Can Be Surrogated by Mouse Lung Tissue.

Free (unbound) drug concentration at the site of action is the key determinant of biologic activity since only unbound drugs can exert pharmacological and toxicological effects. Unbound drug concentration in tumors for solid cancers is needed to understand/explain/predict pharmacokinetics, pharmacodynamics, and efficacy relations. Fraction unbound (fu ) in tumors is usually determined across several xenografted tumors derived from various cell lines in the drug discovery stage, which is time consuming and a resource burden. In this study, we determined the fu values for a set of diverse compounds (comprising acid, base, neutral, zwitterion, and covalent drugs) across five different xenografted tumors and five commercially available mouse tissues to explore the correlation of fu between tumors and the possibility of surrogate tissue(s) for tumor fu (fu,tumor) determination. The crosstumor comparison showed that fu,tumor values across tumors are largely comparable, and systematic tissue versus tumor comparison demonstrated that only lung tissue had comparable fu to all five tumors (fu values within twofold change for >80% compounds in both comparisons). These results indicated that mouse lung tissue can be used as a surrogate matrix for a fu,tumor assay. This study will increase efficiency in fu,tumor assessment and reduce animal use (adapting the replace, reduce, and refine principle) in drug discovery. SIGNIFICANCE STATEMENT: The free drug concept is a well accepted principle in drug discovery research. Currently, tumor fraction unbound (fu,tumor) is determined in several tumors derived from different cell lines to estimate free drug concentrations of a compound. The results from this study indicated that fu,tumor across xenografted tumors is comparable, and fu,tumor can be estimated using a surrogate tissue, mouse lung. The results will increase efficiency in fu,tumor assessment and reduce animal use in drug discovery.

Absorption, Distribution, Metabolism, and Excretion of [14C]-Sotorasib in Rats and Dogs: Interspecies Differences in Absorption, Protein Conjugation and Metabolism.

Sotorasib is a first-in-class, targeted covalent inhibitor of Kirsten rat sarcoma viral oncogene homolog (KRAS)G12C approved by the FDA to treat patients with locally advanced or metastatic non-small cell lung cancer with the KRASG12C mutation. The mass balance, excretion, and metabolism of [14C]-sotorasib was characterized in rats and dogs after a single dose of 60 or 500 mg/kg, respectively. Mean recovery was >90% for both species. Excretion of unchanged sotorasib was a minor pathway in rats, accounting for <4% of administered dose in urine and <7% of administered dose in feces. Approximately 66% of administered dose was recovered in the bile from bile duct cannulated rats as metabolites. Excretion of unchanged sotorasib was the major excretion pathway in dogs, likely caused by solubility-limited absorption. Major pathways of sotorasib biotransformation included glutathione conjugation and oxidative metabolism. In vitro experiments demonstrated that nonenzymatic conjugation (Michael addition) was the primary mechanism of the reaction with glutathione. Extended radioactivity profiles in blood and plasma were observed in rats, but not dogs, after dosing with [14C]-sotorasib. In vitro experiments demonstrated that sotorasib-protein adducts were observed with both rat hemoglobin and serum albumin, explaining the extended radioactivity profile. SIGNIFICANCE STATEMENT: This study characterized the mass balance, excretion, and metabolism of [14C]-sotorasib, a covalent Kirsten rat sarcoma viral oncogene homolog G12C inhibitor, in rats and dogs. Rapid absorption and extensive metabolism of sotorasib was observed in rats, while sotorasib was primarily excreted unchanged in dog feces, likely due to solubility-limited absorption. Protein adducts with rat hemoglobin and serum albumin were characterized, explaining observed extended blood and plasma radioactivity profiles. The primary biotransformation pathway, glutathione conjugation, was mediated through nonenzymatic conjugation.

Pharmacokinetics, Disposition, and Biotransformation of [14C]Omecamtiv Mecarbil in Healthy Male Subjects after a Single Intravenous or Oral Dose.

Omecamtiv mecarbil (OM) is a novel cardiac myosin activator that is currently in clinical development for the treatment of heart failure. The absorption and disposition of [14C]OM (60 µCi) were studied after a single intravenous infusion (35 mg over 1 hour) or oral solution dose (35 mg) in 14 healthy male subjects. Mean recovery of the administered [14C]OM dose was 85.1% and 86.5% over 336 hours for the intravenous and oral routes, respectively. After intravenous dosing, 47.8% and 37.3% of the dose was recovered in urine and feces, respectively; after oral dosing, 48.6% and 38.0% was recovered in urine and feces, respectively. Unchanged OM accounted for a minor percentage of radioactivity in urine (mean 7.7% of dose) and feces (mean 4.1% of dose) across all subjects. The major metabolites recovered in urine and feces were M3 (decarbamoylation product) and sequential metabolite M4 (lactam of M3), which accounted for means of 26.5% and 11.6% of the administered dose, respectively. The CYP4 family of enzymes was primarily responsible for the formation of M3 based on in vitro studies. Other metabolic pathways accounted for 14.9% of the administered dose. In pooled plasma, OM, M3, and M4 accounted for 83.8%, 6.0%, and 3.3% of the total [14C]OM-related materials. No other plasma metabolites constituted more than 3% of the administered dose. The bioavailability for OM solution was 93.5% after rapid and extensive absorption. SIGNIFICANCE STATEMENT: This study characterized the absorption and disposition of OM, a novel small molecule being developed for the treatment of heart failure. OM was primarily cleared through metabolism by the CYP4 family through oxidative cleavage of a terminal carbamate moiety that resembles hydrolysis.

Mercapturate pathway metabolites of sotorasib, a covalent inhibitor of KRASG12C, are associated with renal toxicity in the Sprague Dawley rat.

Sotorasib is a first-in class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. In the nonclinical toxicology studies of sotorasib, the kidney was identified as a target organ of toxicity in the rat but not the dog. Renal toxicity was characterized by degeneration and necrosis of the proximal tubular epithelium localized to the outer stripe of the outer medulla (OSOM), which suggested that renal metabolism was involved. Here, we describe an in vivo mechanistic rat study designed to investigate the time course of the renal toxicity and sotorasib metabolites. Renal toxicity was dose- and time-dependent, restricted to the OSOM, and the morphologic features progressed from vacuolation and necrosis to regeneration of tubular epithelium. The renal toxicity correlated with increases in renal biomarkers of tubular injury. Using mass spectrometry and matrix-assisted laser desorption/ionization, a strong temporal and spatial association between renal toxicity and mercapturate pathway metabolites was observed. The rat is reported to be particularly susceptible to the formation of nephrotoxic metabolites via this pathway. Taken together, the data presented here and the literature support the hypothesis that sotorasib-related renal toxicity is mediated by a toxic metabolite derived from the mercapturate and ß-lyase pathway. Our understanding of the etiology of the rat specific renal toxicity informs the translational risk assessment for patients.

Methylome and transcriptome signature of bronchoalveolar cells from multiple sclerosis patients in relation to smoking.

BACKGROUND: Despite compelling evidence that cigarette smoking impacts the risk of developing multiple sclerosis (MS), little is known about smoking-associated changes in the primary exposed lung cells of patients. OBJECTIVES: We aimed to examine molecular changes occurring in bronchoalveolar lavage (BAL) cells from MS patients in relation to smoking and in comparison to healthy controls (HCs). METHODS: We profiled DNA methylation in BAL cells from female MS (n = 17) and HC (n = 22) individuals, using Illumina Infinium EPIC and performed RNA-sequencing in non-smokers. RESULTS: The most prominent changes were found in relation to smoking, with 1376 CpG sites (adjusted P < 0.05) differing between MS smokers and non-smokers. Approximately 30% of the affected genes overlapped with smoking-associated changes in HC, leading to a strong common smoking signature in both MS and HC after gene ontology analysis. Smoking in MS patients resulted in additional discrete changes related to neuronal processes. Methylome and transcriptome analyses in non-smokers suggest that BAL cells from MS patients display very subtle (not reaching adjusted P < 0.05) but concordant changes in genes connected to reduced transcriptional/translational processes and enhanced cellular motility. CONCLUSIONS: Our study provides insights into the impact of smoking on lung inflammation and immunopathogenesis of MS.

Nonclinical Safety Profile of Sotorasib, a KRASG12C-Specific Covalent Inhibitor for the Treatment of KRAS p.G12C-Mutated Cancer.

Sotorasib is a first-in-class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. A comprehensive nonclinical safety assessment package, including secondary/safety pharmacology and toxicology studies, was conducted to support the marketing application for sotorasib. Sotorasib was negative in a battery of genotoxicity assays and negative in an in vitro phototoxicity assay. Based on in vitro assays, sotorasib had no off-target effects against various receptors, enzymes (including numerous kinases), ion channels, or transporters. Consistent with the tumor-specific target distribution (ie, KRASG12C), there were no primary pharmacology-related on-target effects identified. The kidney was identified as a target organ in the rat but not the dog. Renal toxicity in the rat was characterized by tubular degeneration and necrosis restricted to a specific region suggesting that the toxicity was attributed to the local formation of a putative toxic reactive metabolite. In the 3-month dog study, adaptive changes of hepatocellular hypertrophy due to drug metabolizing enzyme induction were observed in the liver that was associated with secondary effects in the pituitary and thyroid gland. Sotorasib was not teratogenic and had no direct effect on embryo-fetal development in the rat or rabbit. Human, dog, and rat circulating metabolites, M24, M10, and M18, raised no clinically relevant safety concerns based on the general toxicology studies, primary/secondary pharmacology screening, an in vitro human ether-à-go-go-related gene assay, or mutagenicity assessment. Overall, the results of the nonclinical safety program support a high benefit/risk ratio of sotorasib for the treatment of patients with KRAS p.G12C-mutated tumors.

Strain-Dependent Variability of Early Discovery Small Molecule Pharmacokinetics in Mice: Does Strain Matter?

Drug discovery programs routinely perform pharmacokinetic (PK) studies in mice to prioritize lead compounds based on anticipated exposure-efficacy and exposure-toxicity relationships. Because of logistical and/or technical issues, the strain of mouse in early discovery PK studies may not always match the strain in toxicity or efficacy studies. This elicits the question do appreciable strain-dependent differences in PK parameters exist to an extent that would warrant conducting PK studies in a strain that matches efficacy and toxicity models? To understand the impact that strain may have on PK parameters, we selected eight marketed drugs with well characterized absorption, distribution, metabolism, and excretion properties and diverse structures to perform PK studies in three common mouse strains (Bagg Albino c, C57BL/6, and CD-1). Some statistical strain-dependent differences were observed; however, we found good general agreement of PK parameters between strains: 88%, 100%, 75%, 76%, 94%, and 88% of compounds were within twofold across strains for clearance, volume of distribution at steady state, t 1/2, C max, T max, and oral bioavailability, respectively. Overall, we recommend that an approach using a single strain of mouse is appropriate for discovery screening PK studies, provided that proper caution is exercised. SIGNIFICANCE STATEMENT: The mouse strain in discovery pharmacokinetic (PK) studies may not match the strain in efficacy and toxicology studies. Currently, there is a gap in the literature addressing whether differences in PK parameters across mouse strains exist such that multiple PK studies are warranted. The results from this study indicated that the PK properties of clinically used drugs between mouse strains are within an acceptable range such that single strain PK is appropriate.

Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples.

Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides.Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 10(6) bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10-15 × 10(6) cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls.In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 10(6) cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients and healthy controls in order to identify disease-associated peptides.

T-cell activation and HLA-regulated response to smoking in the deep airways of patients with multiple sclerosis.

Cigarette smoking is a risk factor for multiple sclerosis (MS), and the risk is further multiplied for HLA-DRB1*15(+) smokers. To define the smoke-induced immune responses in the lung we performed bronchoscopy with bronchoalveolar lavage (BAL) on smokers and non-smokers, both MS-patients and healthy volunteers. In the BAL, non-smokers with MS showed an increased preformed CD40L expression in CD4(+) T-cells while smokers displayed an increase in proliferating (Ki-67(+)) T-cells. In addition, our results confirm that smoking induces an increase of alveolar macrophages in BAL, and further defined a significant attenuation of this response in carriers of the HLA-DRB1*15 allele, in both MS patients and healthy controls. This first systematic investigation of the immune response in the lungs of smokers and non-smokers diagnosed with MS, thus suggests an MS-associated lung T-cell phenotype, involvement of a specific T-cell response to smoke, and a genetic regulation of the macrophage response.

T-cell receptor-HLA-DRB1 associations suggest specific antigens in pulmonary sarcoidosis.

In pulmonary sarcoidosis, CD4(+) T-cells expressing T-cell receptor Vα2.3 accumulate in the lungs of HLA-DRB1*03(+) patients. To investigate T-cell receptor-HLA-DRB1*03 interactions underlying recognition of hitherto unknown antigens, we performed detailed analyses of T-cell receptor expression on bronchoalveolar lavage fluid CD4(+) T-cells from sarcoidosis patients.Pulmonary sarcoidosis patients (n=43) underwent bronchoscopy with bronchoalveolar lavage. T-cell receptor α and ß chains of CD4(+) T-cells were analysed by flow cytometry, DNA-sequenced, and three-dimensional molecular models of T-cell receptor-HLA-DRB1*03 complexes generated.Simultaneous expression of Vα2.3 with the Vß22 chain was identified in the lungs of all HLA-DRB1*03(+) patients. Accumulated Vα2.3/Vß22-expressing T-cells were highly clonal, with identical or near-identical Vα2.3 chain sequences and inter-patient similarities in Vß22 chain amino acid distribution. Molecular modelling revealed specific T-cell receptor-HLA-DRB1*03-peptide interactions, with a previously identified, sarcoidosis-associated vimentin peptide, (Vim)429-443 DSLPLVDTHSKRTLL, matching both the HLA peptide-binding cleft and distinct T-cell receptor features perfectly.We demonstrate, for the first time, the accumulation of large clonal populations of specific Vα2.3/Vß22 T-cell receptor-expressing CD4(+) T-cells in the lungs of HLA-DRB1*03(+) sarcoidosis patients. Several distinct contact points between Vα2.3/Vß22 receptors and HLA-DRB1*03 molecules suggest presentation of prototypic vimentin-derived peptides.

Evaluation of a New Molecular Entity as a Victim of Metabolic Drug-Drug Interactions-an Industry Perspective.

Under the guidance of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ), scientists from 20 pharmaceutical companies formed a Victim Drug-Drug Interactions Working Group. This working group has conducted a review of the literature and the practices of each company on the approaches to clearance pathway identification (fCL), estimation of fractional contribution of metabolizing enzyme toward metabolism (fm), along with modeling and simulation-aided strategy in predicting the victim drug-drug interaction (DDI) liability due to modulation of drug metabolizing enzymes. Presented in this perspective are the recommendations from this working group on: 1) strategic and experimental approaches to identify fCL and fm, 2) whether those assessments may be quantitative for certain enzymes (e.g., cytochrome P450, P450, and limited uridine diphosphoglucuronosyltransferase, UGT enzymes) or qualitative (for most of other drug metabolism enzymes), and the impact due to the lack of quantitative information on the latter. Multiple decision trees are presented with stepwise approaches to identify specific enzymes that are involved in the metabolism of a given drug and to aid the prediction and risk assessment of drug as a victim in DDI. Modeling and simulation approaches are also discussed to better predict DDI risk in humans. Variability and parameter sensitivity analysis were emphasized when applying modeling and simulation to capture the differences within the population used and to characterize the parameters that have the most influence on the prediction outcome.

T cell receptor-Vß repertoires in lung and blood CD4+ and CD8+ T cells of pulmonary sarcoidosis patients.

BACKGROUND: Sarcoidosis patients have accumulations of activated CD4+ T cells in affected organs, such as the lungs. T cell receptor (TCR) Vß-chain usage has been incompletely characterized in these patients. METHODS: We surveyed the TCR Vß usage in CD4+ and CD8+ T cells in bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMC) from 15 HLA-typed Scandinavian sarcoidosis patients. In addition, PBMC from 9 healthy volunteers and BAL cells from three of them were examined. Using 21 Vß family-specific antibodies, we covered approximately 70% of all Vß chains. RESULTS: In BAL T cells from sarcoidosis patients, we identified 16 CD4+ T cell expansions in 271 analyses (5.9%) and 21 CD8+ expansions in 240 analyses (8.7%). In PBMC we found 9 CD4+ expansions in 276 analyses (3.3%) and 12 CD8+ expansions out of 263 analyses (4.6%). Consistent with previous studies we found Vß8 and Vß16 expansions in sarcoidosis patients' lungs. In addition, we found lung restricted Vß22 expansions in three HLA DRB1 03+ patients. However, we found no statistically significant difference in frequency of expansions between patients and healthy controls. CONCLUSIONS: The identified T cell expansions in present study indicate specific antigen recognition in the lungs of sarcoidosis patients.

Tolerability and efficacy of the monoaminergic stabilizer (-)-OSU6162 (PNU-96391A) in Huntington's disease: a double-blind cross-over study.

OBJECTIVE: To evaluate the safety (primary objective) and efficacy (secondary objective) of (-)-OSU6162 in Huntington's disease (HD). METHODS: In a double-blind, cross-over trial, patients with HD were randomly assigned to start treatment on either (-)-OSU6162 or placebo. After 4 weeks, those patients who initially received active drug were switched to placebo for another 4 weeks, and vice versa. During the first week the (-)-OSU6162 dose was 15 mg twice daily, during the second week 30 mg twice daily, and during the last 2 weeks 45 mg twice daily. Motor, cognitive, mental and social functions were rated by the clinical investigator or by self-assessment, using established rating scales. RESULTS: Fifteen patients fulfilling inclusion and exclusion criteria completed the study. (-)-OSU6162 was well tolerated by all patients and no adverse effects were observed. (-)-OSU6162 treatment significantly improved the Short Form 36 Vitality score, mainly due to an improvement of the individual item 'worn-out' (VT3). In addition, an improvement of depressive symptoms was found using Beck Depression Inventory. In contrast to a general trend of improvement in several non-motor variables only small and non-significant differences between (-)-OSU6162 and placebo were found regarding motor functions. CONCLUSIONS: (-)-OSU6162 offers promise for the treatment of HD, as a drug with good tolerability, capable of improving the patients' experienced non-motor functions such as energy and mood and thus alleviating symptoms of great importance for their quality of life.

Development and Evaluation of Ontogeny Functions of the Major UDP-Glucuronosyltransferase Enzymes to Underwrite Physiologically Based Pharmacokinetic Modeling in Pediatric Populations.

Uridine 5'-diphospho-glucuronosyltransferases (UGTs) demonstrate variable expression in the pediatric population. Thus, understanding of age-dependent maturation of UGTs is critical for accurate pediatric pharmacokinetics (PK) prediction of drugs that are susceptible for glucuronidation. Ontogeny functions of major UGTs have been previously developed and reported. However, those ontogeny functions are based on in vitro data (i.e., enzyme abundance, in vitro substrate activity, and so on) and therefore, may not translate to in vivo maturation of UGTs in the clinical setting. This report describes meta-analysis of the literature to develop and compare ontogeny functions for 8 primary UGTs (UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B10, UGT2B15, and UGT2B17) based on published in vitro and in vivo studies. Once integrated with physiologically based pharmacokinetics modeling models, in vivo activity-based ontogeny functions demonstrated somewhat greater prediction accuracy (mean squared error, MSE: 0.05) compared to in vitro activity (MSE: 0.104) and in vitro abundance-based ontogeny functions (MSE: 0.129).

Impact of Sotorasib on the Pharmacokinetics and Pharmacodynamics of Metformin, a MATE1/2K Substrate, in Healthy Subjects.

BACKGROUND AND OBJECTIVE: The objectives of this study were to evaluate the effect of sotorasib on metformin pharmacokinetics and pharmacodynamics and the effect of metformin on sotorasib pharmacokinetics in healthy subjects. Sotorasib is an oral, small molecule inhibitor of the Kirsten rat sarcoma oncogene homolog (KRAS) G12C mutant protein (KRASG12C) protein approved by the U.S. Food and Drug Administration in 2021 for the treatment of KRASG12C-mutated locally advanced or metastatic non-small cell lung cancer (NSCLC) in adults who have received at least one prior systemic therapy METHODS: This was a phase I, single-center, open-label, three-period, fixed-sequence study. Subjects received single oral doses of metformin 850 mg, sotorasib 960 mg, and metformin 850 mg with sotorasib 960 mg. Urine and plasma were collected and assayed for metformin and sotorasib pharmacokinetics. Blood glucose was also measured for metformin pharmacodynamics. In addition, an in vitro study was conducted to determine whether sotorasib was an inhibitor of MATE1/2K or OCT2 transport. RESULTS: Geometric least-squares mean ratio of sotorasib area under the concentration-time curve from time 0 to infinity and peak plasma concentration were 0.910 and 0.812, respectively, when sotorasib was coadministered with metformin compared with administration of sotorasib alone. Geometric least-squares mean ratio of metformin area under the concentration-time curve from time 0 to infinity and peak plasma concentration were 0.99 and 1.00, respectively, when comparing metformin coadministered with sotorasib to metformin alone. Geometric mean estimates of serum glucose area under the concentration-time curve from time 0 to 2 h following metformin alone, sotorasib alone, and metformin with sotorasib were 179, 222, and 194, respectively. CONCLUSIONS: These results demonstrated that coadministration of metformin with sotorasib does not impact sotorasib exposure to a clinically significant extent. Coadministration of sotorasib with metformin does not affect metformin exposure or its antihyperglycemic effect, in contrast to the inhibitory effect observed in vitro. Doses of sotorasib 960 mg and metformin 850 mg were safe and well tolerated when coadministered to healthy subjects.

Evaluation of levocetirizine in beagle dog and cynomolgus monkey telemetry assays: Defining the no QTc effect profile by timepoint and concentration-QTc analysis.

In prior clinical studies, levocetirizine (LEVO) has demonstrated no effect on ventricular repolarization (QTc intervals), therefore it is a relevant negative control to assess in nonclinical assays to define low proarrhythmic risk. LEVO was tested in beagle dog and cynomolgus monkey (nonhuman primate [NHP]) telemetry models to understand the nonclinical-clinical translation of this negative control. One oral dose of vehicle, LEVO (10 mg/kg/species) or moxifloxacin (MOXI; 30 mg/kg/dog; 80 mg/kg/NHP) was administered to instrumented animals (N = 8/species) using a cross-over dosing design; MOXI was the in-study positive control. Corrected QT interval values (QTcI) were calculated using an individual animal correction factor. Blood samples were taken for drug exposure during telemetry and for pharmacokinetic (PK) analysis (same animals; different day) for exposure-response (C-QTc) modeling. Statistical analysis of QTc-by-timepoint data showed that LEVO treatment was consistent with vehicle, thus no effect on ventricular repolarization was observed over 24 h in both species. PK analysis indicated that LEVO-maximum concentration levels in dogs (range: 12,300-20,100 ng/ml) and NHPs (range: 4090-12,700 ng/ml) were ≥4-fold higher than supratherapeutic drug levels in clinical QTc studies. Slope analysis values in dogs (0.00019 ms/ng/ml) and NHPs (0.00016 ms/ng/ml) were similar to the human C-QTc relationship and indicated no relationship between QTc intervals and plasma levels of LEVO. MOXI treatment caused QTc interval prolongation (dog: 18 ms; NHP: 29 ms). The characterization of LEVO in these non-rodent telemetry studies further demonstrates the value and impact of the in vivo QTc assay to define a "no QTc effect" profile and support clinical safety assessment.

Antigen-specific multifunctional T-cells in sarcoidosis patients with Lofgren's syndrome.

Sarcoidosis is a granulomatous disease of unknown aetiology, mainly affecting the lungs. Recently, T-cell responses towards a specific mycobacterial protein, catalase-peroxidase (mKatG), were observed in sarcoidosis patients. Bronchoalveolar lavage (BAL) fluid and peripheral blood were obtained from a total of 23 sarcoidosis patients, of whom 13 had Löfgren's syndrome and lung accumulations of T-cell receptor AV2S3+ T-cells. Using six-colour flow cytometry in combination with intracellular cytokine staining, T-cell subsets were studied with regard to interferon (IFN)-γ, tumour necrosis factor (TNF) and interleukin-2 production, after stimulation with mKatG or Mycobacterium tuberculosis purified protein derivate (PPD). Stimulation with mKatG resulted in higher simultaneous IFN-γ and TNF production, but less single IFN-γ production, from total BAL fluid CD4+ T-cells of Löfgren's syndrome patients, when compared with non-Löfgren's patients. In contrast, PPD stimulation gave rise to largely similar cytokine responses in both patient subgroups. Furthermore, mKatG stimulated higher IFN-γ production in BAL fluid and blood AV2S3+ T-cells than AV2S3- T-cells, whereas the opposite was seen in BAL fluid with PPD stimulation. Our finding that patients with Löfgren's syndrome exhibited a more pronounced multifunctional cytokine profile (simultaneous IFN-γ and TNF production) towards the mycobacterial protein mKatG may help to explain the distinct disease presentation in this patient subgroup.

Prediction of CYP2D6 drug interactions from in vitro data: evidence for substrate-dependent inhibition.

Predicting the magnitude of potential drug-drug interactions is important for underwriting patient safety in the clinical setting. Substrate-dependent inhibition of cytochrome P450 enzymes may confound extrapolation of in vitro results to the in vivo situation. However, the potential for substrate-dependent inhibition with CYP2D6 has not been well characterized. The inhibition profiles of 20 known inhibitors of CYP2D6 were characterized in vitro against four clinically relevant CYP2D6 substrates (desipramine, dextromethorphan, metoprolol, and thioridazine) and bufuralol. Dextromethorphan exhibited the highest sensitivity to in vitro inhibition, whereas metoprolol was the least sensitive. In addition, when metoprolol was the substrate, inhibitors with structurally constrained amino moieties (clozapine, debrisoquine, harmine, quinidine, and yohimbine) exhibited at least a 5-fold decrease in inhibition potency when results were compared with those for dextromethorphan. Atypical inhibition kinetics were observed for these and other inhibitor-substrate pairings. In silico docking studies suggested that interactions with Glu216 and an adjacent hydrophobic binding pocket may influence substrate sensitivity and inhibition potency for CYP2D6. The in vivo sensitivities of the clinically relevant CYP2D6 substrates desipramine, dextromethorphan, and metoprolol were determined on the basis of literature drug-drug interaction (DDI) outcomes. Similar to the in vitro results, dextromethorphan exhibited the highest sensitivity to CYP2D6 inhibition in vivo. Finally, the magnitude of in vivo CYP2D6 DDIs caused by quinidine was predicted using desipramine, dextromethorphan, and metoprolol. Comparisons of the predictions with literature results indicated that the marked decrease in inhibition potency observed for the metoprolol-quinidine interaction in vitro translated to the in vivo situation.

Predicting the drug interaction potential of AMG 853, a dual antagonist of the D-prostanoid and chemoattractant receptor-homologous molecule expressed on T helper 2 cells receptors.

2-(4-(4-(tert-Butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenylsulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid (AMG 853) is an orally bioavailable and potent dual antagonist of the D-prostanoid and chemoattractant receptor-homologous molecule expressed on T helper 2 cells receptors. The drug interaction potential of AMG 853, both as a victim and a perpetrator, was investigated using in vitro, in silico, and in vivo methodologies. Experiments in human liver microsomes (HLM) and recombinant enzymes identified CYP2C8, CYP2J2, and CYP3A as well as multiple UDP-glucuronosyltransferase isoforms as being responsible for the metabolic clearance of AMG 853. With use of HLM and selective probe substrates, both AMG 853 and its acyl glucuronide metabolite (M1) were shown to be inhibitors of CYP2C8. AMG 853 and M1 did not inhibit any of the other cytochrome P450 isoforms tested, and AMG 853 exhibited minimal enzyme induction properties in human hepatocytes cultures. In light of the in vitro findings, modeling and simulation approaches were used to examine the potential for ketoconazole (a CYP3A inhibitor) to inhibit the metabolism of AMG 853 as well as for AMG 853 to inhibit the metabolism of paclitaxel, rosiglitazone, and montelukast, commonly used substrates of CYP2C8. A weak and clinically insignificant drug interaction (area under the drug concentration-time curve (AUC)(i)/AUC <2) was predicted between ketoconazole and AMG 853. No drug interactions were predicted for AMG 853 and paclitaxel, rosiglitazone, or montelukast. Finally, administration of AMG 853 to healthy human subjects in clinical trials in the presence or absence of ketoconazole confirmed that AMG 853 is unlikely to be involved in clinically significant drug interactions.

Absorption, metabolism and excretion of [14C]-sotorasib in healthy male subjects: characterization of metabolites and a minor albumin-sotorasib conjugate.

PURPOSE: The objectives of this study were to characterize the absorption, metabolism, and excretion of sotorasib and determine the metabolites present in plasma, urine, and feces in healthy male subjects following a single oral 720 mg dose containing approximately 1 µCi of [14C]-sotorasib. METHODS: Urine, feces, and plasma were collected post-dose and assayed for total radioactivity and profiled for sotorasib metabolites. Urine and plasma were also assayed for sotorasib pharmacokinetics. In addition, in vitro studies were performed to determine the enzymes responsible for formation of major circulating metabolites and protein adducts in human plasma. RESULTS: Sotorasib was rapidly absorbed, with a median time to peak concentration of 0.75 h. Mean t1/2,z of plasma sotorasib, whole blood total radioactivity, and plasma total radioactivity were 6.35, 174, and 128 h, respectively. The geometric mean cumulative recovery was 80.6%; the majority was excreted in feces (74.4%) with a low percentage excreted in urine (5.81%). M10, sotorasib, and M24 were present at 31.6%, 22.2%, and 13.7% of total radioactivity in plasma extracts, respectively. M10 and sotorasib were present at < 5% of administered radioactivity in urine, while only unchanged sotorasib, at 53% of administered radioactivity, was identified in feces. A sotorasib-albumin adduct was identified in plasma as a minor constituent, consistent with the observed radioactivity profile in plasma/blood. CONCLUSION: Sotorasib metabolism involves nonenzymatic glutathione conjugation, GGT-mediated hydrolysis of glutathione adduct, and direct CYP3A and CYP2C8-mediated oxidation. Elimination of sotorasib is predominantly fecal excretion, suggesting dose reduction is not necessary with renal impairment.