Evobrutinib pathway to its major metabolite M463-2 and insights from a biotransformation and DDI perspective.

Evobrutinib is a highly selective, covalent, central nervous system-penetrant Bruton's tyrosine kinase (BTK) inhibitor, currently in Phase III trials for the treatment of relapsing multiple sclerosis. One major circulating metabolite of evobrutinib has been previously identified as the racemic dihydro-diol M463-2 (MSC2430422) in a Phase I human mass balance study.Phenotyping experiments were conducted to confirm the metabolic pathway of evobrutinib to M463-2. Ratio of the enantiomers was determined by enantioselective liquid chromatography with tandem mass spectrometry analysis of plasma samples from humans and preclinical species. Drug-drug interaction (DDI) characterisation, evaluation of pharmacological activity on BTK, and off-target screening experiments followed assessing safety of the metabolite.The biotransformation of evobrutinib to M463-2 was determined to be a two-step process with a CYP-mediated oxidation acting to form an epoxide intermediate, which was further hydrolysed by soluble and mitochondrial epoxide hydrolase. Only the (S)-enantiomer was determined to be a major metabolite, the (R)-enantiomer was minor. In vitro studies demonstrated the (S)-enantiomer lacked clinically relevant pharmacological activity, off-target effects and DDIs.The biotransformation of evobrutinib to its major metabolite has been elucidated, with the major (S)-enantiomer being shown to pose no on/off target or DDI risks.

Examination of the Human Cytochrome P4503A4 Induction Potential of PF-06282999, an Irreversible Myeloperoxidase Inactivator: Integration of Preclinical, In Silico, and Biomarker Methodologies in the Prediction of the Clinical Outcome.

The propensity for CYP3A4 induction by 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999), an irreversible inactivator of myeloperoxidase, was examined in the present study. Studies using human hepatocytes revealed moderate increases in CYP3A4 mRNA and midazolam-1'-hydroxylase activity in a PF-06282999 dose-dependent fashion. At the highest tested concentration of 300 µM, PF-06282999 caused maximal induction in CYP3A4 mRNA and enzyme activity ranging from 56% to 86% and 47% t0 72%, respectively, of rifampicin response across the three hepatocyte donor pools. In a clinical drug-drug interaction (DDI) study, the mean midazolam Cmax and area under the curve (AUC) values following 14-day treatment with PF-06282999 decreased in a dose-dependent fashion with a maximum decrease in midazolam AUC0-inf and Cmax of ∼57.2% and 41.1% observed at the 500 mg twice daily dose. The moderate impact on midazolam pharmacokinetics at the 500 mg twice daily dose of PF-06282999 was also reflected in statistically significant changes in plasma 4ß-hydroxycholesterol/cholesterol and urinary 6ß-hydroxycortisol/cortisol ratios. Changes in plasma and urinary CYP3A4 biomarkers did not reach statistical significance at the 125 mg three times daily dose of PF-06282999, despite a modest decrease in midazolam systemic exposure. Predicted DDI magnitude based on the in vitro induction parameters and simulated pharmacokinetics of perpetrator (PF-06282999) and victim (midazolam) using the Simcyp (Simcyp Ltd., Sheffield, United Kingdom) population-based simulator were in reasonable agreement with the observed clinical data. Since the magnitude of the 4ß-hydroxycholesterol or 6ß-hydroxycortisol ratio change was generally smaller than the magnitude of the midazolam AUC change with PF-06282999, a pharmacokinetic interaction study with midazolam ultimately proved important for assessment of DDI via CYP3A4 induction.

Once-weekly administration of a long-acting fibroblast growth factor 21 analogue modulates lipids, bone turnover markers, blood pressure and body weight differently in obese people with hypertriglyceridaemia and in non-human primates.

AIMS: To assess the safety, tolerability, pharmacokinetics and pharmacodynamics of PF-05231023, a long-acting fibroblast growth factor 21 (FGF21) analogue, in obese people with hypertriglyceridaemia on atorvastatin, with or without type 2 diabetes. METHODS: Participants received PF-05231023 or placebo intravenously once weekly for 4 weeks. Safety (12-lead ECGs, vital signs, adverse events [AEs], laboratory tests) and longitudinal weight assessments were performed. Blood samples were collected for pharmacokinetic and pharmacodynamic analyses. Cardiovascular safety studies were also conducted in telemetered rats and monkeys. Blood pressure (BP; mean, systolic and diastolic) and ECGs were monitored. RESULTS: A total of 107 people were randomized. PF-05231023 significantly decreased mean placebo-adjusted fasting triglycerides (day 25, 33%-43%) and increased HDL cholesterol (day 25, 15.7%-28.6%) and adiponectin (day 25, 1574 to 3272 ng/mL) across all doses, without significant changes in body weight (day 25, -0.45% to -1.21%). Modest decreases from baseline were observed for N-terminal propeptides of type 1 collagen (P1NP) on day 25, although C-telopeptide cross-linking of type 1 collagen (CTX-1) increased minimally. Systolic, diastolic BP, and pulse rate increased in a dose- and time-related manner. There were 5 serious AEs (one treatment-related) and no deaths. Three participants discontinued because of AEs. The majority of AEs were gastrointestinal. PF-05231023 increased BP and heart rate in rats, but not in monkeys. CONCLUSIONS: Once-weekly PF-05231023 lowered triglycerides markedly in the absence of weight loss, with modest changes in markers of bone homeostasis. This is the first report showing increases in BP and pulse rate in humans and rats after pharmacological administration of a long-acting FGF21 molecule.

Non-canonical inflammasome activation targets caspase-11.

Caspase-1 activation by inflammasome scaffolds comprised of intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and the adaptor ASC is believed to be essential for production of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 during the innate immune response. Here we show, with C57BL/6 Casp11 gene-targeted mice, that caspase-11 (also known as caspase-4) is critical for caspase-1 activation and IL-1ß production in macrophages infected with Escherichia coli, Citrobacter rodentium or Vibrio cholerae. Strain 129 mice, like Casp11(-/-) mice, exhibited defects in IL-1ß production and harboured a mutation in the Casp11 locus that attenuated caspase-11 expression. This finding is important because published targeting of the Casp1 gene was done using strain 129 embryonic stem cells. Casp1 and Casp11 are too close in the genome to be segregated by recombination; consequently, the published Casp1(-/-) mice lack both caspase-11 and caspase-1. Interestingly, Casp11(-/-) macrophages secreted IL-1ß normally in response to ATP and monosodium urate, indicating that caspase-11 is engaged by a non-canonical inflammasome. Casp1(-/-)Casp11(129mt/129mt) macrophages expressing caspase-11 from a C57BL/6 bacterial artificial chromosome transgene failed to secrete IL-1ß regardless of stimulus, confirming an essential role for caspase-1 in IL-1ß production. Caspase-11 rather than caspase-1, however, was required for non-canonical inflammasome-triggered macrophage cell death, indicating that caspase-11 orchestrates both caspase-1-dependent and -independent outputs. Caspase-1 activation by non-canonical stimuli required NLRP3 and ASC, but caspase-11 processing and cell death did not, implying that there is a distinct activator of caspase-11. Lastly, loss of caspase-11 rather than caspase-1 protected mice from a lethal dose of lipopolysaccharide. These data highlight a unique pro-inflammatory role for caspase-11 in the innate immune response to clinically significant bacterial infections.

Pharmacokinetics and Disposition of the Thiouracil Derivative PF-06282999, an Orally Bioavailable, Irreversible Inactivator of Myeloperoxidase Enzyme, Across Animals and Humans.

The thiouracil derivative PF-06282999 [2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide] is an irreversible inactivator of myeloperoxidase and is currently in clinical trials for the potential treatment of cardiovascular diseases. Concerns over idiosyncratic toxicity arising from bioactivation of the thiouracil motif to reactive species in the liver have been largely mitigated through the physicochemical (molecular weight, lipophilicity, and topological polar surface area) characteristics of PF-06282999, which generally favor elimination via nonmetabolic routes. To test this hypothesis, pharmacokinetics and disposition studies were initiated with PF-06282999 using animals and in vitro assays, with the ultimate goal of predicting human pharmacokinetics and elimination mechanisms. Consistent with its physicochemical properties, PF-06282999 was resistant to metabolic turnover from liver microsomes and hepatocytes from animals and humans and was devoid of cytochrome P450 inhibition. In vitro transport studies suggested moderate intestinal permeability and minimal transporter-mediated hepatobiliary disposition. PF-06282999 demonstrated moderate plasma protein binding across all of the species. Pharmacokinetics in preclinical species characterized by low to moderate plasma clearances, good oral bioavailability at 3- to 5-mg/kg doses, and renal clearance as the projected major clearance mechanism in humans. Human pharmacokinetic predictions using single-species scaling of dog and/or monkey pharmacokinetics were consistent with the parameters observed in the first-in-human study, conducted in healthy volunteers at a dose range of 20-200 mg PF-06282999. In summary, disposition characteristics of PF-06282999 were relatively similar across preclinical species and humans, with renal excretion of the unchanged parent emerging as the principal clearance mechanism in humans, which was anticipated based on its physicochemical properties and supported by preclinical studies.

Pharmacokinetics and pharmacodynamics of PF-05231023, a novel long-acting FGF21 mimetic, in a first-in-human study.

AIMS: The aim of the present study was to evaluate the pharmacokinetics/pharmacodynamics (PK/PD), safety and tolerability of single intravenous (IV) doses of PF-05231023, a long acting fibroblast growth factor 21 (FGF21) analogue being developed for the treatment of type 2 diabetes mellitus (T2DM). METHODS: T2DM subjects (glycosylated haemoglobin: 7.0-10.5%; on stable metformin therapy and/or diet and exercise) were randomized to receive a single dose of placebo or PF-05231023 (0.5-200 mg). Safety evaluations were performed up to 14 days after dosing. PK and PD endpoints were measured and a PK/PD model was developed for triglyceride - an early marker of drug activity. RESULTS: No antidrug antibody or serious adverse events (AEs) were observed. The most frequent AEs were gastrointestinal but were generally mild. Plasma PF-05231023 levels peaked immediately post-IV dosing, with mean terminal half-lives of 6.5-7.7 h and 66.5- 96.6 h for intact C- and N-termini, respectively. Intact C-terminus exposures increased proportionally with increasing dose, whereas N-terminus exposures appeared to trend higher than dose-proportionally. Although no apparent effect on plasma glucose was seen, dose-dependent decreases in triglyceride were observed, with a maximum reduction of 48.5 ± 10.0% (mean ± standard deviation) for the 200 mg dose compared with a reduction of 19.1 ± 26.4% for placebo, demonstrating proof of pharmacology. Moreover, a reduction in total cholesterol and low-density lipoprotein cholesterol and an increase in high-density lipoprotein cholesterol were observed in the high-dose groups. CONCLUSIONS: Single IV doses of PF-05231023 up to 200 mg were generally safe and well tolerated by subjects with T2DM. The observed early sign of pharmacology supports further clinical testing of PF-05231023 upon repeated administration.

Mesodermal Fgf10b cooperates with other fibroblast growth factors during induction of otic and epibranchial placodes in zebrafish.

BACKGROUND: Vertebrate otic and epibranchial placodes develop in close proximity in response to localized fibroblast growth factor (Fgf) signaling. Although less is known about epibranchial induction, the process of otic induction in highly conserved, with important roles for Fgf3 and Fgf8 reported in all species examined. Fgf10 is also critical for otic induction in mouse, but the only zebrafish ortholog examined to date, fgf10a, is not expressed early enough to play such a role. A second zebrafish ortholog, fgf10b, has not been previously examined. RESULTS: We find that zebrafish fgf10b is expressed at tailbud stage in paraxial cephalic mesoderm beneath prospective epibranchial tissue, lateral to the developing otic placode. Knockdown of fgf10b does not affect initial otic induction but impairs subsequent accumulation of otic cells. Formation of epibranchial placodes and ganglia are also moderately impaired. Combinatorial disruption of fgf10b and fgf3 exacerbates the deficiency of otic cells and eliminates epibranchial induction entirely. Disruption of fgf10b and fgf24 also strongly reduces, but does not eliminate, epibranchial induction. CONCLUSIONS: fgf10b participates in a late phase of otic induction and, in combination with fgf3, is especially critical for epibranchial induction.

Implementation of whole-exome sequencing for pharmacogenomics profiling and exploring its potential clinical utilities.

Whole-exome sequencing (WES) is widely used in clinical settings; however, the exploration of its use in pharmacogenomic analysis remains limited. Our study compared the variant callings for 28 core absorption, distribution, metabolism and elimination genes by WES and array-based technology using clinical trials samples. The results revealed that WES had a positive predictive value of 0.71-0.92 and a sensitivity of single-nucleotide variants between 0.68 and 0.95, compared with array-based technology, for the variants in the commonly targeted regions of the WES and PhamacoScan™ assay. Besides the common variants detected by both assays, WES identified 200-300 exclusive variants per sample, totalling 55 annotated exclusive variants, including important modulators of metabolism such as rs2032582 (ABCB1) and rs72547527 (SULT1A1). This study highlights the potential clinical advantages of using WES to identify a wider range of genetic variations and enabling precision medicine.

Immunogenicity of avelumab in patients with metastatic Merkel cell carcinoma or advanced urothelial carcinoma.

Like other monoclonal antibodies, immune checkpoint inhibitors may be immunogenic in some patients, potentially affecting pharmacokinetics (PKs) and clinical outcomes. In post hoc analyses, we characterized antidrug antibody (ADA) development with avelumab monotherapy in patients with metastatic Merkel cell carcinoma (mMCC) from the JAVELIN Merkel 200 trial (first-line [1L; N = 116] and second-line or later [≥2L; N = 88] cohorts) or with advanced urothelial carcinoma (aUC) from the JAVELIN Bladder 100 (1L maintenance [N = 350]) and JAVELIN Solid Tumor (≥2L [N = 249]) trials. Treatment-emergent ADAs developed in a numerically higher proportion of patients with aUC (1L maintenance, 19.1%; ≥2L, 18.1%) versus mMCC (1L, 8.2%; ≥2L, 8.9%); incidences within tumor types were similar by line of therapy. In PK analyses, numerically lower avelumab trough concentration and higher baseline clearance were observed in treatment-emergent ADA+ versus ADA- subgroups; however, differences were not clinically relevant. Numerical differences in overall survival, progression-free survival, or objective response rate by ADA status were observed; however, no clinically meaningful trends were identified. Proportions of patients with treatment-emergent adverse events (TEAEs; any grade or grade 3/4), serious TEAEs, TEAEs leading to treatment discontinuation, or infusion-related reactions were similar, with overlapping 80% confidence intervals between ADA subgroups. Efficacy and safety observations were similar in subgroups defined by early development of ADA+ status during treatment. In conclusion, no meaningful differences in PKs, efficacy, and safety were observed between subgroups of avelumab-treated patients with different ADA status. Overall, these data suggest that ADAs are not relevant for treatment decisions with avelumab.

Asia-Inclusive Global Development of Enpatoran: Results of an Ethno-Bridging Study, Intrinsic/Extrinsic Factor Assessments and Disease Trajectory Modeling to Inform Design of a Phase II Multiregional Clinical Trial.

Enpatoran is a novel, highly selective, and potent dual toll-like receptor (TLR)7 and TLR8 inhibitor currently under development for the treatment of autoimmune disorders including systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), and myositis. The ongoing phase II study (WILLOW; NCT05162586) is evaluating enpatoran for 24 weeks in patients with active SLE or CLE and is currently recruiting. To support development of WILLOW as an Asia-inclusive multiregional clinical trial (MRCT) according to International Conference on Harmonisation E5 and E17 principles, we have evaluated ethnic sensitivity to enpatoran based on clinical pharmacokinetic (PK), pharmacodynamic (PD), and safety data from an ethno-bridging study (NCT04880213), supplemented by relevant quantitative PK, PD, and disease trajectory modeling (DTM) results, and drug metabolism/disease knowledge. A single-center, open-label, sequential dose group study in White and Japanese subjects matched by body weight, height, and sex demonstrated comparable PK and PD properties for enpatoran in Asian vs. non-Asian (White and other) subjects across single 100, 200, and 300 mg orally administered doses. DTM suggested no significant differences in SLE disease trajectory for Asian vs. non-Asian individuals. Aldehyde oxidase (AOX) is considered to be a key contributor to enpatoran metabolism, and a literature review indicated no relevant ethnic differences in AOX function based on in vitro and clinical PK data from marketed drugs metabolized by AOX, supporting the conclusion of low ethnic sensitivity for enpatoran. Taken together, the inclusion of Asian patients in MRCTs including WILLOW was informed based on a Totality of Evidence approach.

Multi-platform next-generation sequencing of the domestic turkey (Meleagris gallopavo): genome assembly and analysis.

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.

Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis.

Macrophages respond to cytosolic nucleic acids by activating cysteine protease caspase-1 within a complex called the inflammasome. Subsequent cleavage and secretion of proinflammatory cytokines IL-1beta and IL-18 are critical for innate immunity. Here, we show that macrophages from mice lacking absent in melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger inflammasome assembly. Caspase-1 activation in response to intracellular pathogen Francisella tularensis also required AIM2. Immunofluorescence microscopy of macrophages infected with F. tularensis revealed striking colocalization of bacterial DNA with endogenous AIM2 and inflammasome adaptor ASC. By contrast, type I IFN (IFN-alpha and -beta) secretion in response to F. tularensis did not require AIM2. IFN-I did, however, boost AIM2-dependent caspase-1 activation by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in infected macrophages lacking either the IFN-I receptor or stimulator of interferon genes (STING). Finally, AIM2-deficient mice displayed increased susceptibility to F. tularensis infection compared with wild-type mice. Their increased bacterial burden in vivo confirmed that AIM2 is essential for an effective innate immune response.

Alternatives to rifampicin: A review and perspectives on the choice of strong CYP3A inducers for clinical drug-drug interaction studies.

N-Nitrosamine (NA) impurities are considered genotoxic and have gained attention due to the recall of several marketed drug products associated with higher-than-permitted limits of these impurities. Rifampicin is an index inducer of multiple cytochrome P450s (CYPs) including CYP2B6, 2C8, 2C9, 2C19, and 3A4/5 and an inhibitor of OATP1B transporters (single dose). Hence, rifampicin is used extensively in clinical studies to assess drug-drug interactions (DDIs). Despite NA impurities being reported in rifampicin and rifapentine above the acceptable limits, these critical anti-infective drugs are available for therapeutic use considering their benefit-risk profile. Reports of NA impurities in rifampicin products have created uncertainty around using rifampicin in clinical DDI studies, especially in healthy volunteers. Hence, a systematic investigation through a literature search was performed to determine possible alternative index inducer(s) to rifampicin. The available strong CYP3A inducers were selected from the University of Washington DDI Database and their in vivo DDI potential assessed using the data from clinical DDI studies with sensitive CYP3A substrates. To propose potential alternative CYP3A inducers, factors including lack of genotoxic potential, adequate safety, feasibility of multiple dose administration to healthy volunteers, and robust in vivo evidence of induction of CYP3A were considered. Based on the qualifying criteria, carbamazepine, phenytoin, and lumacaftor were identified to be the most promising alternatives to rifampicin for conducting CYP3A induction DDI studies. Strengths and limitations of the proposed alternative CYP3A inducers, the magnitude of in vivo CYP3A induction, appropriate study designs for each alternative inducer, and future perspectives are presented in this paper.

Applying Modeling and Simulations for Rational Dose Selection of Novel Toll-Like Receptor 7/8 Inhibitor Enpatoran for Indications of High Medical Need.

Dual toll-like receptor (TLR) 7 and TLR8 inhibitor enpatoran is under investigation as a treatment for lupus and coronavirus disease 2019 (COVID-19) pneumonia. Population pharmacokinetic/pharmacodynamic (PopPK/PD) model-based simulations, using PK and PD (inhibition of ex vivo-stimulated interleukin-6 (IL-6) and interferon-α (IFN-α) secretion) data from a phase I study of enpatoran in healthy participants, were leveraged to inform dose selection for lupus and repurposed for accelerated development in COVID-19. A two-compartment PK model was linked to sigmoidal maximum effect (Emax ) models with proportional decrease from baseline characterizing the PD responses across the investigated single and multiple doses, up to 200 mg daily for 14 days (n = 72). Concentrations that maintain 50/60/90% inhibition (IC50/60/90 ) of cytokine secretion (IL-6/IFN-α) over 24 hours were estimated and stochastic simulations performed to assess target coverage under different dosing regimens. Simulations suggested investigating 25, 50, and 100 mg enpatoran twice daily (b.i.d.) to explore the anticipated therapeutic dose range for lupus. With 25 mg b.i.d., > 50% of subjects are expected to achieve 60% inhibition of IL-6. With 100 mg b.i.d., most subjects are expected to maintain almost complete target coverage for 24 hours (> 80% subjects IC90,IL-6  = 15.5 ng/mL; > 60% subjects IC90,IFN-α  = 22.1 ng/mL). For COVID-19, 50 and 100 mg enpatoran b.i.d. were recommended; 50 mg b.i.d. provides shorter IFN-α inhibition (median time above IC90  = 13 hours/day), which may be beneficial to avoid interference with the antiviral immune response. Utilization of PopPK/PD models initially developed for lupus enabled informed dose selection for the accelerated development of enpatoran in COVID-19.

Risk assessment of drug-drug interaction potential for bintrafusp alfa with cytochrome P4503A4 substrates: A totality of evidence approach.

Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of TGF-ßRII (a TGF-ß "trap") fused to a human IgG1 mAb blocking PD-L1, is being evaluated for efficacy and safety in solid tumor indications as monotherapy and in combination with small-molecule drugs. We evaluated the perpetrator drug-drug interaction (DDI) potential of bintrafusp alfa via cytochrome P4503A4 (CYP3A4) enzyme modulation, which is responsible for the metabolism of a majority of drugs. The holistic approach included (1) evaluation of longitudinal profiles of cytokines implicated in CYP3A4 modulation and serum 4ß-hydroxycholesterol, an endogenous marker of CYP3A4 activity, in a phase I clinical study, and (2) transcriptomics analysis of the CYP3A4 mRNA levels vs the TGFB gene expression signature in normal hepatic tissues. Bintrafusp alfa was confirmed not to cause relevant proinflammatory cytokine modulation or alterations in 4ß-hydroxycholesterol serum concentrations in phase I studies. Transcriptomics analyses revealed no meaningful correlations between TGFB gene expression and CYP3A4 mRNA expression, supporting the conclusion that the risk of CYP3A4 enzyme modulation due to TGF-ß neutralization by bintrafusp alfa is low. Thus, bintrafusp alfa is not expected to have DDI potential as a perpetrator with co-administered drugs metabolized by CYP3A4; this information is relevant to clinical evaluations of bintrafusp alfa in combination settings.

Exposure-response analyses for the MET inhibitor tepotinib including patients in the pivotal VISION trial: support for dosage recommendations.

PURPOSE: Tepotinib is a highly selective MET inhibitor approved for treatment of non-small cell lung cancer (NSCLC) harboring METex14 skipping alterations. Analyses presented herein evaluated the relationship between tepotinib exposure, and efficacy and safety outcomes. METHODS: Exposure-efficacy analyses included data from an ongoing phase 2 study (VISION) investigating 500 mg/day tepotinib in NSCLC harboring METex14 skipping alterations. Efficacy endpoints included objective response, duration of response, and progression-free survival. Exposure-safety analyses included data from VISION, plus four completed studies in advanced solid tumors/hepatocellular carcinoma (30-1400 mg). Safety endpoints included edema, serum albumin, creatinine, amylase, lipase, alanine aminotransferase, aspartate aminotransferase, and QT interval corrected using Fridericia's method (QTcF). RESULTS: Tepotinib exhibited flat exposure-efficacy relationships for all endpoints within the exposure range observed with 500 mg/day. Tepotinib also exhibited flat exposure-safety relationships for all endpoints within the exposure range observed with 30-1400 mg doses. Edema is the most frequently reported adverse event and the most frequent cause of tepotinib dose reductions and interruptions; however, the effect plateaued at low exposures. Concentration-QTc analyses using data from 30 to 1400 mg tepotinib resulted in the upper bounds of the 90% confidence interval being less than 10 ms for the mean exposures at the therapeutic (500 mg) and supratherapeutic (1000 mg) doses. CONCLUSIONS: These analyses provide important quantitative pharmacologic support for benefit/risk assessment of the 500 mg/day dosage of tepotinib as being appropriate for the treatment of NSCLC harboring METex14 skipping alterations. REGISTRATION NUMBERS: NCT01014936, NCT01832506, NCT01988493, NCT02115373, NCT02864992.

A phase 1b study evaluating the safety and preliminary efficacy of berzosertib in combination with gemcitabine in patients with advanced non-small cell lung cancer.

OBJECTIVES: Berzosertib (formerly M6620, VX-970) is an intravenous, highly potent and selective, first-in-class ataxia telangiectasia and Rad3-related (ATR) protein kinase inhibitor. We assessed the safety, tolerability, preliminary efficacy, and pharmacokinetics (PK) of berzosertib plus gemcitabine in an expansion cohort of patients with advanced non-small cell lung cancer (NSCLC). The association of efficacy with TP53 status and other tumor markers was also explored. MATERIALS AND METHODS: Adult patients with advanced histologically confirmed NSCLC received berzosertib 210 mg/m2 (days 2 and 9) and gemcitabine 1000 mg/m2 (days 1 and 8) at the recommended phase 2 dose established in the dose escalation part of the study. RESULTS: Thirty-eight patients received at least one dose of study treatment. The most common treatment-emergent adverse events were fatigue (55.3%), anemia (52.6%), and nausea (39.5%). Gemcitabine had no apparent effect on the PK of berzosertib. The objective response rate (ORR) was 10.5% (4/38, 90% confidence interval [CI]: 3.7-22.5%). In the exploratory analysis, the ORR was 30.0% (3/10, 90% CI: 9.0-61.0%) in patients with high loss of heterozygosity (LOH) and 11.0% (1/9, 90% CI: 1.0-43.0%) in patients with low LOH. The ORR was 33.0% (2/6, 90% CI: 6.0-73.0%) in patients with high tumor mutational burden (TMB), 12.5% (2/16, 90% CI: 2.0-34.0%) in patients with intermediate TMB, and 0% (0/3, 90% CI: 0.0-53.6%) in patients with low TMB. CONCLUSIONS: Berzosertib plus gemcitabine was well tolerated in patients with advanced, pre-treated NSCLC. Based on the observed clinical efficacy, future clinical trials should involve genomically selected patients.

Phase 1b study of berzosertib and cisplatin in patients with advanced triple-negative breast cancer.

Platinum derivatives are commonly used for the treatment of patients with metastatic triple-negative breast cancer (TNBC). However, resistance often develops, leading to treatment failure. This expansion cohort (part C2) of the previously reported phase 1b trial (NCT02157792) is based on the recommended phase 2 dose of the combination of the ataxia-telangiectasia and Rad3-related (ATR) inhibitor berzosertib and cisplatin observed in patients with advanced solid tumors, including TNBC. Forty-seven patients aged ≥18 years with advanced TNBC received cisplatin (75 mg/m2; day 1) and berzosertib (140 mg/m2; days 2 and 9), in 21-day cycles. Berzosertib was well tolerated, with a similar toxicity profile to that reported previously for this combination. The overall response rate (90% confidence interval) was 23.4% (13.7, 35.8). No relevant associations were observed between response and gene alterations. Further studies combining ATR inhibitors with platinum compounds may be warranted in highly selected patient populations.

A comparative physical map reveals the pattern of chromosomal evolution between the turkey (Meleagris gallopavo) and chicken (Gallus gallus) genomes.

BACKGROUND: A robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species. RESULTS: The turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes. CONCLUSION: The BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species.

Evobrutinib, a covalent Bruton's tyrosine kinase inhibitor: Mass balance, elimination route, and metabolism in healthy participants.

The highly selective, covalent Bruton's tyrosine kinase inhibitor evobrutinib is under investigation for treatment of patients with multiple sclerosis (MS). Early clinical studies in healthy participants and patients with relapsing MS indicated that evobrutinib is well-tolerated and effective. We undertook a mass balance study in six men who received a single 75-mg oral dose of evobrutinib containing ~ 3.6 MBq (100 µCi) 14 C-evobrutinib, to determine the absorption, metabolic pathways, and routes of excretion of evobrutinib. The primary objectives of this phase I study (NCT03725072) were to (1) determine the rates and routes of total radioactivity excretion, including the mass balance of total drug-related radioactivity in urine and feces, (2) assess the pharmacokinetics (PKs) of total radioactivity in blood and plasma, and (3) characterize the plasma PKs of evobrutinib. Exploratory end points included identifying and quantifying evobrutinib and its metabolites in plasma and excreta (urine and feces) and exploring key biotransformation pathways and clearance mechanisms. Evobrutinib was primarily eliminated in feces (arithmetic mean percentage, SD, 71.0, 2.1) and, to a lesser extent, in urine (20.6, 2.0), with most of the total radioactivity (85.3%) excreted in the first 72 h after administration. No unchanged evobrutinib was detected in excreta. Evobrutinib was rapidly absorbed and substantially metabolized upon absorption. Only one major metabolite M463-2 (MSC2430422) was identified in plasma above the 10% of total drug exposure threshold, which classifies M463-2 (MSC2430422) as a major metabolite according to the US Food and Drug Administration (FDA; metabolites in safety testing [MIST]) and the European Medicines Agency (EMA; International Conference on Harmonization [ICH] M3). These results support further development of evobrutinib and may help inform subsequent investigations.