Points to consider regarding the use and implementation of virtual controls in nonclinical general toxicology studies.

The replacement of a proportion of concurrent controls by virtual controls in nonclinical safety studies has gained traction over the last few years. This is supported by foundational work, encouraged by regulators, and aligned with societal expectations regarding the use of animals in research. This paper provides an overview of the points to consider for any institution on the verge of implementing this concept, with emphasis given on database creation, risks, and discipline-specific perspectives.

Development of BET Inhibitors as Potential Treatments for Cancer: Optimization of Pharmacokinetic Properties.

We describe the synthesis of triazole-containing carboline derivatives and their utility as bromodomain and extra-terminal (BET) inhibitors. A convergent synthetic route permitted the detailed investigation of deuteration and fluorination strategies to reduce clearance while maintaining a favorable in vitro profile. This work led to the identification of a potent BET inhibitor, 2-{8-fluoro-3-[4-(2H3)methyl-1-methyl-1H-1,2,3-triazol-5-yl]-5-[(S)-(oxan-4-yl)(phenyl)methyl]-5H-pyrido[3,2-b]indol-7-yl}propan-2-ol (15), which demonstrated reduced clearance and an improved pharmacokinetic (PK) profile across preclinical species. Importantly, no major metabolite was observed when 15 was incubated with human hepatocytes (hHEP) for 2 h. This study culminated with the evaluation of 15 in a mouse triple-negative breast cancer (TNBC) tumor model where it demonstrated robust efficacy at low doses.

Development of BET inhibitors as potential treatments for cancer: A new carboline chemotype.

We describe our efforts to introduce structural diversity to a previously described triazole-containing N1-carboline series of bromodomain and extra-terminal (BET) inhibitors. N9 carbolines were designed to retain favorable binding interactions that the N1-carbolines possess. A convergent synthetic route enabled modifications to reduce clearance, enhance physicochemical properties, and improve the overall in vitro profile. This work led to the identification of a potent BET inhibitor, (S)-2-{8-fluoro-5-[(3-fluoropyridin-2-yl)(oxan-4-yl)methyl]-7-[4-(2H3)methyl-1-methyl-1H-1,2,3-triazol-5-yl]-5H-pyrido[3,2-b]indol-3-yl}propan-2-ol (10), a compound with enhanced oral exposure in mice. Subsequent evaluation in a mouse triple-negative breast cancer tumor model revealed efficacy at 4 mg/kg of N9-carboline 10.

Discovery and Preclinical Pharmacology of an Oral Bromodomain and Extra-Terminal (BET) Inhibitor Using Scaffold-Hopping and Structure-Guided Drug Design.

Inhibition of the bromodomain and extra-terminal (BET) family of adaptor proteins is an attractive strategy for targeting transcriptional regulation of key oncogenes, such as c-MYC. Starting with the screening hit 1, a combination of structure-activity relationship and protein structure-guided drug design led to the discovery of a differently oriented carbazole 9 with favorable binding to the tryptophan, proline, and phenylalanine (WPF) shelf conserved in the BET family. Identification of an additional lipophilic pocket and functional group optimization to optimize pharmacokinetic (PK) properties culminated in the discovery of 18 (BMS-986158) with excellent potency in binding and functional assays. On the basis of its favorable PK profile and robust in vivo activity in a panel of hematologic and solid tumor models, BMS-986158 was selected as a candidate for clinical evaluation.

Development of BET inhibitors as potential treatments for cancer: A search for structural diversity.

We describe our efforts to identify structurally diverse leads in the triazole-containing N1-carboline series of bromodomain and extra-terminal inhibitors. Replacement of the N5 "cap" phenyl moiety with various heteroaryls, coupled with additional modifications to the carboline core, provided analogs with similar potency, improved pharmacokinetic properties, and increased solubility compared to our backup lead, BMS-986225 (2). Rapid SAR exploration was enabled by a convergent, synthetic route. These efforts provided a potent BET inhibitor, 3-fluoropyridyl 12, that demonstrated robust efficacy in a multiple myeloma mouse tumor model at 1 mg/kg.

TGFß2 and TGFß3 mediate appropriate context-dependent phenotype of rat valvular interstitial cells.

This study focused on characterizing the potential mechanism of valvular toxicity caused by TGFß receptor inhibitors (TGFßRis) using rat valvular interstitial cells (VICs) to evaluate early biological responses to TGFßR inhibition. Three TGFßRis that achieved similar exposures in the rat were assessed. Two dual TGFßRI/-RII inhibitors caused valvulopathy, whereas a selective TGFßRI inhibitor did not, leading to a hypothesis that TGFß receptor selectivity may influence the potency of valvular toxicity. The dual valvular toxic inhibitors had the most profound effect on altering VIC phenotype including altered morphology, migration, and extracellular matrix production. Reduction of TGFß expression demonstrated that combined TGFß2/ß3 inhibition by small interfering RNA or neutralizing antibodies caused similar alterations as TGFßRis. Inhibition of TGFß3 transcription was only associated with the dual TGFßRis, suggesting that TGFßRII inhibition impacts TGFß3 transcriptional regulation, and that the potency of valvular toxicity may relate to alteration of TGFß2/ß3-mediated processes involved in maintaining proper balance of VIC phenotypes in the heart valve.

Discovery of Branebrutinib (BMS-986195): A Strategy for Identifying a Highly Potent and Selective Covalent Inhibitor Providing Rapid in Vivo Inactivation of Bruton's Tyrosine Kinase (BTK).

Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase, is a member of the Tec family of kinases and is essential for B cell receptor (BCR) mediated signaling. BTK also plays a critical role in the downstream signaling pathways for the Fcγ receptor in monocytes, the Fcε receptor in granulocytes, and the RANK receptor in osteoclasts. As a result, pharmacological inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as rheumatoid arthritis and lupus. This article will outline the evolution of our strategy to identify a covalent, irreversible inhibitor of BTK that has the intrinsic potency, selectivity, and pharmacokinetic properties necessary to provide a rapid rate of inactivation systemically following a very low dose. With excellent in vivo efficacy and a very desirable tolerability profile, 5a (branebrutinib, BMS-986195) has advanced into clinical studies.

A novel function of CXCL10 in mediating monocyte production of proinflammatory cytokines.

IFN-γ-inducible protein 10 (CXCL10), a chemokine that is abundantly secreted in response to inflammatory stimuli, has been implicated in the pathogenesis of multiple inflammatory diseases, such as inflammatory bowel disease. Whereas CXCL10 is traditionally recognized for recruiting pathogenic T cells to inflamed sites, its nonchemotactic role during inflammation remains poorly defined. In this report, we identified a novel function of CXCL10 in the regulation of the inflammatory potential of human monocytes to produce cytokines. We found that CXCL10 was necessary and sufficient for IFN-γ-primed human monocytes to induce a robust production of proinflammatory cytokines, such as IL-12 and IL-23. CXCL10-induced monocyte production of these cytokines depended on CXCR3 receptor engagement as well as on the Iκ B kinase and p38 MAPK signaling pathways. By using an innate-mediated murine colitis model, we demonstrated that anti-CXCL10 Ab treatment robustly suppressed the local production of myeloid-derived inflammatory cytokines and intestinal tissue damage. Together, our data unravel a previously unappreciated role of CXCL10 in the amplification of myeloid cell-mediated inflammatory responses. Targeting CXCL10 is therefore an attractive approach to treating inflammatory diseases that are driven by innate and adaptive immunity.

Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors.

Structure-activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.

BMS-871: a novel orally active pan-Notch inhibitor as an anticancer agent.

This Letter describes synthesis, SAR, and biological activity of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides as inhibitors of γ-secretase mediated signaling of Notch receptors. Optimization of this series led to the identification of BMS-871 (compound 30) which displayed robust in vivo efficacy in Notch-dependent leukemia and solid tumor xenograft models.

A bioanalytical strategy utilizing dried blood spot sampling and LC-MS/MS in discovery toxicology studies.

BACKGROUND & METHOD: The small sample volumes characteristic to dried blood spot (DBS) sampling enabled us to right-shift the linear dynamic range of an LC-MS/MS plasma assay tenfold and eliminate the need for extensive sample dilution in support of three discovery toxicology studies in which both plasma and DBS samples were collected. With the right-shifted DBS assay range, no DBS study samples required dilution, while all of the plasma samples were diluted 5-50-fold. RESULTS: DBS standard curves from 78-80,000 nM were linear, the performance of the curve and QC samples was within acceptable discovery-assay criteria and individual plasma and DBS data were comparable. Linear correlations of C(max) and AUC derived from DBS and plasma data resulted in R(2) > 0.9. CONCLUSION: This bioanalytical strategy represents a benefit to the bioanalyst that can expedite the return of data and minimize the potential for error and variability that can result from extensive dilutions of study samples.

Cytochrome P450 11A1 bioactivation of a kinase inhibitor in rats: use of radioprofiling, modulation of metabolism, and adrenocortical cell lines to evaluate adrenal toxicity.

A drug candidate, BMS-A ((N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl) 2-oxo-1,2-dihydropyridine- 3-carboxamide)), was associated with dose- and time-dependent vacuolar degeneration and necrosis of the adrenal cortex following oral administration to rats. Pretreatment with 1-aminobenzotriazole (ABT), a nonspecific P450 inhibitor, ameliorated the toxicity. In vivo and in vitro systems, including adrenal cortex-derived cell lines, were used to study the mechanism responsible for the observed toxicity. Following an oral dose of the C-14 labeled compound, two hydroxylated metabolites of the parent (M2 and M3) were identified as prominent species found only in adrenal glands and testes, two steroidogenic organs. In addition, a high level of radioactivity was covalently bound to adrenal tissue proteins, 40% of which was localized in the mitochondrial fraction. ABT pretreatment reduced localization of radioactivity in the adrenal gland. Low levels of radioactivity bound to proteins were also observed in testes. Both M3 and covalent binding to proteins were found in incubations with mitochondrial fraction isolated from adrenal tissue in the presence of NADPH. In vitro formation of M3 and covalent binding to proteins were not affected by addition of GSH or a CYP11B1/2 inhibitor, metyrapone (MTY), but were inhibited by ketoconazole (KTZ) and a CYP11A1 inhibitor, R-(+)-aminoglutethimide (R-AGT). BMS-A induced apoptosis in a mouse adrenocortical cell line (Y-1) but not in a human cell line (H295R). Metabolite M3 and covalent binding to proteins were also produced in Y-1 and to a lesser extent in H295R cells. The cell toxicity, formation of M3, and covalent binding to proteins were all diminished by R-AGT but not by MTY. These results are consistent with a CYP11A1-mediated bioactivation to generate a reactive species, covalent binding to proteins, and subsequently rat adrenal toxicity. The thorough understanding of the metabolism-dependent adrenal toxicity was useful to evaluate cross-species adrenal toxicity potential of this compound and related analogues.

Safety assessment considerations and strategies for targeted small molecule cancer therapeutics in drug discovery.

Less than 10% of all experimental drugs introduced into clinical trials ever achieve the approval of regulatory agencies for marketing. For experimental small molecule oncology therapeutics, the approval rate is even less (5%). Clinical safety and efficacy are the two main causes of failure for oncologic drugs in development. Because these failures of experimental drugs are tremendously expensive for pharmaceutical companies, strategies have been developed and refined for reducing this attrition. While these strategic activities can take place in drug development, more benefit may be gained by increasing efforts in drug discovery in the form of (1) target validation; (2) compound selectivity analysis from the perspective of balancing efficacy and toxicity; and (3) investigation of ancillary means to abrogate toxicity, especially with respect to undesirable target-related effects. Most pharmaceutical companies recognize the benefit of lead compound optimization, but the degree to which it is applied seems to vary greatly. This article presents concepts and strategies to reduce the attrition of small molecule oncology therapeutic drug candidates due to toxicity.

Tumor development by transgenic expression of a constitutively active insulin-like growth factor I receptor.

The insulin-like growth factor I receptor (IGF-IR) is a transmembrane tyrosine kinase that is essential to growth and development and also thought to provide a survival signal for the maintenance of the transformed phenotype. There has been increasing interest in further understanding the role of IGF-I signaling in cancer and in developing receptor antagonists for therapeutic application. We describe herein a novel animal model that involves transgenic expression of a fusion receptor that is constitutively activated by homodimerization. Transgenic mice that expressed the activated receptor showed aberrant development of the mammary glands and developed salivary and mammary adenocarcinomas as early as 8 weeks of age. Xenograft tumors and a cell line were derived from the transgenic animals and are sensitive to inhibition by a novel small-molecule inhibitor of the IGF-IR kinase. This new model should provide new opportunities for further understanding how aberrant IGF-IR signaling leads to tumorigenesis and for optimizing novel antagonists of the receptor kinase.

The north american control animal database: a resource based on standardized nomenclature and diagnostic criteria.

Historical control data have been shown to be valuable in the interpretation and evaluation of results from rodent carcinogenicity studies. Standardization of terminology and histopathology procedures is a prerequisite for meaningful comparison of control data across studies and analysis of potential carcinogenic effects. Standardization is particularly critical for the construction of a database that includes incidence data from different studies evaluated by pathologists in different laboratories. Standardized nomenclature and diagnostic criteria have been established for neoplasms and proliferative lesions. Efforts of the National Toxicology Program, the Society of Toxicologic Pathology (STP), and the Registry of Industrial Toxicology Animal-data (RITA) have led to a harmonized pathology nomenclature for the rat and the mouse. This nomenclature with detailed descriptions of lesions is available in publications by the STP and International Agency for Research on Cancer (IARC). A listing of these terms is available on the World Wide Web. Utilizing the model established by RITA and working with the International Life Sciences Institute (ILSI), companies with laboratories in North America formed a working group in 1994 to establish and maintain a database of neoplastic and proliferative lesions from control animals in carcinogenicity studies. The rationale for development of the North American Control Animal Database (NACAD), the factors that influence tumor incidence, operation of the database, and the benefits to be realized by using a standardized approach are discussed.