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RO7297089, an anti-B-cell maturation antigen (BCMA)/CD16A bispecific tetravalent antibody, is being developed as a multiple myeloma (MM) therapeutic. This study characterized nonclinical pharmacokinetics (PK), pharmacodynamics (PD), soluble BCMA (sBCMA), and soluble CD16 (sCD16) changes following administration of RO7297089 to support clinical trials. Unbound and total RO7297089 concentrations were measured in cynomolgus monkeys. RO7297089 exhibited a bi-phasic systemic concentration-time profile, similar to a typical human immunoglobulin 1 antibody. Target engagement by RO7297089 led to a robust increase (~100-fold) in total systemic sBCMA levels and relatively mild increase (~2-fold) in total sCD16 levels. To describe the relationship of nonclinical PK/PD data, we developed a target-mediated drug disposition (TMDD) model that includes the systemic target engagement of membrane BCMA (mBCMA), sBCMA, membrane CD16 (mCD16), and sCD16. We then used this model to simulate the PK/PD relationship of RO7297089 in MM patients by translating relevant PK parameters and target levels, based on the literature and newly generated data such as baseline sCD16A levels. Our model suggested that the impact of TMDD on RO7297089 exposure may be more significant in MM patients due to significantly higher expression levels of both mBCMA and sBCMA compared to healthy cynomolgus monkeys. Based on model simulations, we propose more frequent dosing of RO7297089 compared to regular monthly frequency in the clinic at the beginning of treatment to ensure sustained target engagement. This study demonstrates a translational research strategy for collecting relevant nonclinical data, establishing a TMDD model, and using simulations from this model to inform clinical dose regimens. Graphical Abstract

[Image: see text] Bruton’s tyrosine kinase (Btk) is thought to play a pathogenic role in chronic immune diseases such as rheumatoid arthritis and lupus. While covalent, irreversible Btk inhibitors are approved for treatment of hematologic malignancies, they are not approved for autoimmune indications. In efforts to develop additional series of reversible Btk inhibitors for chronic immune diseases, we sought to differentiate from our clinical stage inhibitor fenebrutinib using cyclopropyl amide isosteres of the 2-aminopyridyl group to occupy the flat, lipophilic H2 pocket. While drug-like properties were retained—and in some cases improved—a safety liability in the form of hERG inhibition was observed. When a fluorocyclopropyl amide was incorporated, Btk and off-target activity was found to be stereodependent and a lead compound was identified in the form of the (R,R)- stereoisomer.

The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent and non-covalent agonists elicit the same physiological responses are not understood. Here, we report the discovery of a non-covalent agonist, GNE551, and determine a cryo-EM structure of the TRPA1-GNE551 complex, revealing a distinct binding pocket and ligand-interaction mechanism. Unlike the covalent agonist allyl isothiocyanate, which elicits channel desensitization, tachyphylaxis, and transient pain, GNE551 activates TRPA1 into a distinct conducting state without desensitization and induces persistent pain. Furthermore, GNE551-evoked pain is relatively insensitive to antagonist treatment. Thus, we demonstrate the biased agonism of TRPA1, a finding that has important implications for the discovery of effective drugs tailored to different disease etiologies.

A thorough knowledge of the newborn (age, birth to 1 month postpartum) infant's gastrointestinal tract (GIT) is critical to the evaluation of the absorption, distribution, metabolism, and excretion (ADME) of orally administered drugs in this population. Developmental changes in the GIT during the newborn period are important for nutrient uptake as well as the disposition of orally administered medications. Some aspects of gastrointestinal function do not mature until driven by increased dietary complexity and nutritional demands later in the postnatal period. The functionalities present at birth, and subsequent maturation, can also impact the ADME parameters of orally administered compounds. This review will examine some specific contributors to the ADME processes in human neonates, as well as what is currently understood about the drivers for their maturation. Key species differences will be highlighted, with a focus on laboratory animals used in juvenile toxicity studies. Because of the gaps and inconsistencies in our knowledge, we will also highlight areas where additional study is warranted to better inform the appropriate use of medicines specifically intended for neonates.

Motivation Kinases play a significant role in diverse disease signaling pathways and understanding kinase inhibitor selectivity, the tendency of drugs to bind to off-targets, remains a top priority for kinase inhibitor design and clinical safety assessment. Traditional approaches for kinase selectivity analysis using biochemical activity and binding assays are useful but can be costly and are often limited by the kinases that are available. On the other hand, current computational kinase selectivity prediction methods are computational intensive and can rarely achieve sufficient accuracy for large-scale kinome wide inhibitor selectivity profiling. Results Here, we present a KinomeFEATURE database for kinase binding site similarity search by comparing protein microenvironments characterized using diverse physiochemical descriptors. Initial selectivity prediction of 15 known kinase inhibitors achieved an >90% accuracy and demonstrated improved performance in comparison to commonly used kinase inhibitor selectivity prediction methods. Additional kinase ATP binding site similarity assessment (120 binding sites) identified 55 kinases with significant promiscuity and revealed unexpected inhibitor cross-activities between PKR and FGFR2 kinases. Kinome-wide selectivity profiling of 11 kinase drug candidates predicted novel as well as experimentally validated off-targets and suggested structural mechanisms of kinase cross-activities. Our study demonstrated potential utilities of our approach for large-scale kinase inhibitor selectivity profiling that could contribute to kinase drug development and safety assessment. Availability and implementation The KinomeFEATURE database and the associated scripts for performing kinase pocket similarity search can be downloaded from the Stanford SimTK website (https://simtk.org/projects/kdb). Supplementary information Supplementary data are available at Bioinformatics online.

Several chemicals and pharmaceuticals increase the incidence of hemangiosarcomas (HSAs) in mice, but the relevance to humans is uncertain. Recently, canine HSAs were identified as a powerful tool for investigating the pathogenesis of human HSAs. To characterize the cellular phenotype of canine HSAs, we evaluated immunoreactivity and/or messenger RNA (mRNA) expression of markers for hematopoietic stem cells (HSCs), endothelial cells (ECs), a tumor suppressor protein, and a myeloid marker in canine HSAs. Neoplastic canine cells expressed EC markers and a myeloid marker, but expressed HSC markers less consistently. The canine tumor expression results were then compared to previously published immunoreactivity results for these markers in human and mouse HSAs. There are 2 noteworthy differences across species: (1) most human HSAs had HSC marker expression, indicating that they were comprised of tumor cells that were less differentiated than those in canine and mouse tumors; and (2) human and canine HSAs expressed a late-stage EC maturation marker, whereas mouse HSAs were negative, suggesting that human and canine tumors may retain greater differentiation potential than mouse tumors. These results indicate that HSA development is variable across species and that caution is necessary when discussing translation of carcinogenic risk from animal models to humans.

The Hippo pathway is a critical regulator of cell and organ growth and has emerged as a target for therapeutic intervention in cancers. Its signaling is thought to play an important role in various physiological processes including homeostasis and tissue regeneration. To date there has been limited information about potential pharmacology-related (on-target) safety liabilities of Hippo pathway inhibitors in the context of cancer indications. Herein, we review data from human genetic disorders and genetically engineered rodent models to gain insight into safety liabilities that may emerge from the inhibition of Hippo pathway. Germline systemic deletion of murine Hippo pathway effectors (Yap, Taz, and Teads) resulted in embryonic lethality or developmental phenotypes. Mouse models with tissue-specific deletion (or mutant overexpression) of the key effectors in Hippo pathways have indicated that, at least in some tissues, Hippo signaling may be dispensable for physiological homeostasis; and appears to be critical for regeneration upon tissue damage, indicating that patients with underlying comorbidities and/or insults caused by therapeutic agents and/or comedications may have a higher risk. Caution should be taken in interpreting phenotypes from tissue-specific transgenic animal models since some tissue-specific promoters are turned on during development. In addition, therapeutic agents may result in systemic effects not well-predicted by animal models with tissue-specific gene deletion. Therefore, the development of models that allows for systemic deletion of Yap and/or Taz in adult animals will be key in evaluating the potential safety liabilities of Hippo pathway modulation. In this review, we focus on potential challenges and strategies for targeting the Hippo pathway in cancers.

RO7297089 is a bispecific antibody (IgG-scFv) targeting Bcell maturation antigen (BCMA) and CD16a (FcγRIIIA) that is being developed for the treatment of multiple myeloma (MM). BCMA is exclusively expressed on plasmablasts and differentiated plasma cells (PCs), and is overexpressed on malignant PCs in MM patients. CD16a is expressed on natural killer (NK) cells, monocytes, mast cells, and macrophages. Herein, we characterized the mode of action and safety profile of RO7297089 in vitro and in vivo. RO7297089 showed potent cell killing when using BCMA+ MM tumor cell lines as target cells and human peripheral blood mononuclear cells, NK cells or macrophages as effector cells. Minimal increases in TNFα (2x) and IFNγ (4x), but not other cytokines/chemokines, were observed compared to the vehicle control treatment only in the presence of the BCMA+ MM tumor cell line up to the concentration tested. This suggests that, unlike T-cell engagers, the risk of cytokine release syndrome in patients receiving RO7297089 is low. Cynomolgus monkey is the only relevant nonclinical species for RO7297089 as it showed binding to both recombinant CD16 and BCMA with comparable affinity to human antigens. Following five weekly intravenous administrations to monkeys at 0, 15, and 50 mg/kg, RO7297089 was well tolerated. In line with the mechanism of action, there were no test article-related cytokine increases or adverse findings observed in both dose levels. Systemic exposure of RO7297089 was approximately dose proportional from 15 to 50 mg/kg. Anti-drug antibodies (ADA) were observed in some animals at both dose levels, and ADA-related decreases in concentrations were observed at only 15 mg/kg. To evaluate in vivo activity, RO7297089-related effects on total plasma sBCMA and PCs were assessed. Elevations of sBCMA levels (100x) were observed post dose at both dose levels, and these effects returned to predose levels in animals that did not maintain concentrations at 15 mg/kg, suggesting that RO7297089 bound to and stabilized circulating cynomolgus sBCMA. Time- and dose-dependent reductions in serum IgM levels were observed at both dose levels. Changes in PC numbers were not detected by immunophenotyping; however, gene expression analysis of PC markers was included and demonstrated clear reductions in mRNA expression levels of PC markers including BCMA and J-chain in blood at both dose levels, suggesting reductions in BCMA+ cells. Collectively, these studies suggest that RO7297089 selectively kills BCMA+ cells by engaging CD16a-positive immune cells and has a favorable safety profile. Citation Format: Satoko Kakiuchi-Kiyota, Melissa M. Schutten, Adeyemi O. Adedeji, Hao Cai, Robert Hendricks, Luna Liu, Sivan Cohen, Aaron M. Fullerton, Nicholas Corr, Lanlan Yu, Denise de Almeida Nagata, Shelly Zhong, Michael Dillon, Christoph Spiess, Steve R. Leong, Bing Zheng, Susanne Wingert, Uwe Reusch, Stefan Knackmuss, Thorsten Ross, Andrew Polson, Ayse M. Ovacik. Preclinical pharmacology and safety of RO7297089, a novel anti-BCMA/CD16a bispecific antibody for the treatment of multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4556.

Inorganic arsenic (arsenite and arsenate) at high exposures is a known human carcinogen, inducing tumors of the urinary bladder, skin, and lungs. In two experiments, we examined the urothelial proliferative effects of treatment with 173 ppm sodium arsenite (100 ppm arsenic) in the drinking water for 6 and 24 hr, and 3, 7, and 14 days in female F344 rats and 43.3 ppm sodium arsenite (25 ppm arsenic) in female C57BL/6 wild-type and arsenic (+3 oxidation state) methyltransferase knockout (As3mt KO) mice that are unable to methylate arsenicals. In the rat and both mouse genotypes, scanning electron microscopy showed cytotoxic urothelial changes as early as 6 hr after the start of arsenic exposure. The severity of AsIII-induced cytotoxic urothelial changes increased over time in the rat and in the As3mt KO mouse. Light microscopy showed an increase in urothelial hyperplasia in the rat. No significant increases in bromodeoxyuridine-labeling index were observed. The data support the hypothesis that the sequence of events in the mode of action for urothelial effects of orally administered inorganic arsenic in the rat and mouse involves superficial cytotoxicity with consequent regenerative increased cell proliferation similar to the findings associated with the administration of dimethylarsinic acid (DMAV) in rats.

It is unclear whether the process of spontaneous and chemically induced hemangiosarcoma and hemangioma formation in mice involves the transformation of differentiated endothelial cells (ECs) or recruitment of multipotential bone marrow–derived hematopoietic stem cells or endothelial progenitor cells (EPCs), which show some degree of endothelial differentiation. In the present study, immunohistochemical staining for hematopoietic stem cell markers (CD45 and CD34), EC markers (vascular endothelial growth factor receptor 2 [VEGFR2], CD31, and factor VIII–related antigen), and a myeloid lineage marker (CD14) was employed to better define the origin of hemangiosarcomas and hemangiomas in mice. Staining was negative for CD45, factor VIII–related antigen, and CD14 and positive for CD34, VEGFR2, and CD31, indicating that mouse hemangiosarcomas and hemangiomas are composed of cells derived from EPCs expressing CD34, VEGFR2, and CD31 but not factor VIII–related antigen. The lack of CD45 expression suggests that mouse vascular tumors may arise from EPCs that are at a stage later than hematopoietic stem cells. Since factor VIII–related antigen expression is known to occur later than CD31 expression in EPCs, our observations may indicate that these tumor cells are arrested at a stage prior to complete differentiation. In addition, myeloid lineage cells do not appear to contribute to hemangiosarcoma and hemangioma formation in mice.

Transfluthrin, a pyrethroid insecticide, induced urinary bladder tumors in rats but not in mice in 2-year bioassays. We investigated the urothelial effects of transfluthrin in vivo in rats and the effects of its major metabolite tetrafluorobenzoic acid (TFBA) in vitro on rat (MYP3) and human (1T1) urothelial cell lines. Rats were fed diet containing 0, 2000 or 5000 (with and without 1.25% NH(4)Cl) ppm transfluthrin for 4 weeks or 0 or 2000 ppm transfluthrin for 13 weeks. After 4 weeks, there was no evidence of hyperplasia or increased proliferation in any treatment group. After 13 weeks treatment with 2000 ppm, cytotoxicity and necrosis of the rat urothelial superficial layer were detected by scanning electron microscopy. The urinary concentration of TFBA in rats fed 2000 ppm transfluthrin was 2.94±0.67 mM. The LC(50) of TFBA was 2.25 mM for MYP3 cells and 2.43 mM for 1T1 cells. These studies support cytotoxicity and regenerative proliferation as the mechanism for induction of bladder tumors with high oral doses of transfluthrin due to metabolism of transfluthrin to the weakly cytotoxic TFBA which is excreted at high concentrations in the urine of rats administered high doses of transfluthrin (≥2000 ppm) for an extended period.

The enzyme arsenic (+3 oxidation state) methyltransferase (As3mt) catalyzes reactions converting inorganic arsenic to methylated metabolites, some of which are highly cytotoxic. In a previous study, female As3mt knockout (KO) mice treated with diet containing 100 or 150 ppm arsenic as arsenite showed systemic toxicity and significant effects on the urothelium. In the present study, we showed that the cytotoxic and proliferative effects of arsenite administration on the urothelium are dose dependent. Female wild-type C57BL/6 mice and As3mt KO mice were divided into five groups (n = 7) with free access to drinking water containing 0, 1, 10, 25, or 50 ppm arsenic as arsenite for 4 weeks. At sacrifice, urinary bladders of both As3mt KO and wild-type mice showed hyperplasia by light microscopy; however, the hyperplasia was more severe in the As3mt KO mice. Intracytoplasmic granules were detected in the urothelium of As3mt KO and wild-type mice at arsenic doses ≥ 10 ppm but were more numerous, more extensive, and larger in the KO mice. A no effect level for urothelial effects was identified at 1 ppm arsenic in the wild-type and As3mt KO mice. In As3mt KO mice, livers showed mild acute inflammation and kidneys showed hydronephrosis. The present study shows a dose-response for the effects of orally administered arsenite on the bladder urothelium of wild-type and As3mt KO mice, with greater effects in the KO strain but with a no effect level of 1 ppm for both.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, which belong to the nuclear receptor superfamily. Some PPARgamma agonists, such as pioglitazone, and dual PPARgamma/PPARalpha agonists, such as muraglitazar, induced urothelial bladder tumors in rats but not in mice. In this study, we investigated the early effects in the urine and bladder of rats treated with pioglitazone to evaluate the possible relation between urinary solids formation and urothelial cytotoxicity and regenerative proliferation. In a 4-week experiment, treatment of rats with 16 mg/kg pioglitazone induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation measured by bromodeoxyuridine labeling index and hyperplasia by histology. It also produced alterations in urinary solid formation, especially calcium-containing crystals and calculi. PPARgamma agonists (pioglitazone and troglitazone) in vitro reduced rat urothelial cell proliferation and induced uroplakin synthesis, a specific differentiation marker in urothelial cells. Our data support the hypothesis that the bladder tumors produced in rats by pioglitazone are related to the formation of urinary solids. This strongly supports the previous conclusion in studies with muraglitazar that this is a rat-specific phenomenon and does not pose a urinary bladder cancer risk to humans treated with these agents.

Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in the drinking water is carcinogenic to the urinary bladder of humans. Recently, models have been developed involving transplacental administration of inorganic arsenic and subsequent administration of another substance that produces a low incidence of urogenital neoplasms. Administration of arsenite or arsenate in the diet or drinking water to five-to eight-week-old mice or rats rapidly induces urothelial cytotoxicity and regenerative hyperplasia. In mice administered arsenite, we observed eosinophilic intracytoplasmic granules present in the urothelial cells. These granules were not present in urothelial cells of untreated mice or in treated or untreated rats. By transmission electron microscopy, the granules were located within the mitochondrial matrix, that is, mitochondrial inclusions. Arsenic, primarily as arsenite, was present in partially purified mitochondria containing these granules. Cells containing the granules were not usually associated with degenerative changes. Lack of these granules in rats suggests that they are not necessary for inorganic arsenic–induced urothelial cytotoxicity or hyperplasia. These granules have also been observed with exposures to other metals in other tissues and other species, suggesting that they represent a protective mechanism against metal-induced toxicity.

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists and PPARγ/α dual agonists are used in the treatment of type 2 diabetes mellitus and hyperlipidemias. In carcinogenicity studies, some of these agonists induced hemangiomas/hemangiosarcomas in mice, but not in rats. We hypothesized that increased endothelial cell (EC) proliferation may be involved in the mechanism of PPAR agonist–induced vascular tumors in mice. We previously showed that the sarcomagenic PPARγ agonist troglitazone (TG) increased EC proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses (400 and 800 mg/kg) after four weeks of treatment. In vitro, TG had a mitogenic effect on mouse microvascular mouse ECs by increasing cell proliferation and survival. The current studies showed that treatment of mouse ECs in vitro induced alterations in proliferation pathway gene expression, especially the expression of insulin-like growth factor-1, but had no effect on mouse oxidative stress pathways. In vivo, treatment with vitamin E did not inhibit TG-induced EC proliferation in liver and adipose tissue. In addition, no hypoxic effect was detected in adipose tissue of TG-treated mice; however, TG had a minor effect on hepatocellular hypoxia. These results provide additional evidence supporting a direct mitogenic effect in the mode of action of TG-induced hemangiosarcomas in mice.