Automated blood sampling in canine telemetry model: Enhanced assessment of immune liabilities.

INTRODUCTION: This manuscript presents a successful integration of multi-timepoint biomarker blood sampling (e.g., cytokines) in a conscious dog cardiovascular study using automated blood sampling via vascular access ports in telemetry instrumented dogs. In addition to determining plasma exposure of the test compound, the assessment of biomarkers of interest allows for more comprehensive preclinical evaluation on a traditional conscious dog cardiovascular (CV) telemetry study especially for immunology and immune-oncology molecules. This model system provides a rapid and efficient means to quickly gain understanding of potential effects on key cardiovascular parameters in large species that are commonly used for preclinical safety evaluations while collecting multiple blood samples for drug and cytokine analysis. METHODS: Male beagle dogs were chronically implanted with telemetry devices (PhysioTel™ model D70-PCTP) and vascular access ports (SPMID-GRIDAC-5NC). BASi Culex-L automated blood sampling (ABS) (Bioanalytical Systems, Inc) system was used to collect blood samples at multiple time points for cytokine analysis. Four beagles received low-dose lipopolysaccharide solution (LPS) (0.1 and 0.5 µg/mL). The following cytokines were measured by Milliplex® map Canine Cytokine Magnetic Bead Panel: Interleukin (IL) 2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, TNF-α, MCP-1, KC-like, GM-CSF, IFN gamma, and IP10. RESULTS: Low dose LPS administration induced a pronounced dose-dependent, transient release of key inflammatory cytokines (IL-2, IL-6, IL-10, TNF-α, MCP-1, and KC-like). Cytokine responses were similar to other canine and human endotoxin models. LPS administration led to an increase in body temperature, heart rate, and mean arterial pressure, as well as a decrease in QTcV interval. CONCLUSION: Successful incorporation of cytokine analysis in telemetry instrumented dogs with vascular access ports allows for translational PK/PD modeling of both efficacy and safety of compounds in the immunology as well as the immune-oncology therapeutic areas designed to modulate the immune system. Remote collection of blood samples simultaneously with CV endpoints is a significant enhancement for assessment of biomarkers that are sensitive to animal handling and excitement associated with room disturbances which are obligatory with manual blood collection. Furthermore, implementing this approach has also refined our animal welfare procedure by reducing the handling during a study and thereby reducing stress (positive refinement 3R impact).

Increased stress associated with head-out plethysmography testing can exacerbate respiratory effects and lead to mortality in rats.

INTRODUCTION: DSM421, a dihydroorotate dehydrogenase inhibitor, was in preclinical development as a potential treatment option for malaria. When tested in a core battery of safety pharmacology assays, DSM421 did not produce any effects at oral doses up to 750 mg/kg in an Irwin test in rats, but a respiratory study in rats using head-out plethysmography resulted in substantial changes in respiratory function as well as moribundity and mortality at that and lower doses. An investigation was performed to determine the source of this discrepancy. METHODS: Potential testing errors, differences in types of plethysmography testing chambers, effects on stress indicators, and off-target activity were investigated. RESULTS: Respiratory changes and toxicity (resulting in euthanasia in extremis) were confirmed in a repeat, head-out plethysmography test, but the effects of DSM421 were much less severe overall when the rats were tested in whole-body chambers. Additionally, at the end of the 5-h post-dosing respiratory monitoring periods, levels of stress-related hormones (particularly corticosterone) were higher overall in the head-out, than in the whole-body, tested rats. Furthermore, DSM421 was found to produce changes in cardiovascular function in unrestrained rats, and it was shown to have off-target binding affinity at the adenosine A3 receptor (which is associated with bronchoconstriction). DISCUSSION: The generalized stress inherent to head-out plethysmography testing exacerbated the respiratory effects of DSM421 and was possibly compounded by DSM421's cardiovascular effects, thus artifactually resulting in moribundity and mortality in rats. Care should be taken when choosing whether to use head-out versus whole-body plethysmography chambers during respiratory function testing in animals.

Novel Computational Approach to Predict Off-Target Interactions for Small Molecules.

Most small molecule drugs interact with unintended, often unknown, biological targets and these off-target interactions may lead to both preclinical and clinical toxic events. Undesired off-target interactions are often not detected using current drug discovery assays, such as experimental polypharmacological screens. Thus, improvement in the early identification of off-target interactions represents an opportunity to reduce safety-related attrition rates during preclinical and clinical development. In order to better identify potential off-target interactions that could be linked to predictable safety issues, a novel computational approach to predict safety-relevant interactions currently not covered was designed and evaluated. These analyses, termed Off-Target Safety Assessment (OTSA), cover more than 7,000 targets (~35% of the proteome) and > 2,46,704 preclinical and clinical alerts (as of January 20, 2019). The approach described herein exploits a highly curated training set of >1 million compounds (tracking >20 million compound-structure activity relationship/SAR data points) with known in vitro activities derived from patents, journals, and publicly available databases. This computational process was used to predict both the primary and secondary pharmacological activities for a selection of 857 diverse small molecule drugs for which extensive secondary pharmacology data are readily available (456 discontinued and 401 FDA approved). The OTSA process predicted a total of 7,990 interactions for these 857 molecules. Of these, 3,923 and 4,067 possible high-scoring interactions were predicted for the discontinued and approved drugs, respectively, translating to an average of 9.3 interactions per drug. The OTSA process correctly identified the known pharmacological targets for >70% of these drugs, but also predicted a significant number of off-targets that may provide additional insight into observed in vivo effects. About 51.5% (2,025) and 22% (900) of these predicted high-scoring interactions have not previously been reported for the discontinued and approved drugs, respectively, and these may have a potential for repurposing efforts. Moreover, for both drug categories, higher promiscuity was observed for compounds with a MW range of 300 to 500, TPSA of ~200, and clogP ≥7. This computation also revealed significantly lower promiscuity (i.e., number of confirmed off-targets) for compounds with MW > 700 and MW<200 for both categories. In addition, 15 internal small molecules with known off-target interactions were evaluated. For these compounds, the OTSA framework not only captured about 56.8% of in vitro confirmed off-target interactions, but also identified the right pharmacological targets for 14 compounds as one of the top scoring targets. In conclusion, the OTSA process demonstrates good predictive performance characteristics and represents an additional tool with utility during the lead optimization stage of the drug discovery process. Additionally, the computed physiochemical properties such as clogP (i.e., lipophilicity), molecular weight, pKa and logS (i.e., solubility) were found to be statistically different between the approved and discontinued drugs, but the internal compounds were close to the approved drugs space in most part.

Aminomethyl tetrahydronaphthalene biphenyl carboxamide MCH-R1 antagonists--Increasing selectivity over hERG.

Aminomethyl tetrahydronaphthalene biphenyl carboxamide MCH-R1 antagonists with greater selectivity over hERG were identified. SAR studies addressing two distinct alternatives for structural modifications leading to improve hERG selectivity are described.

Aminomethyl tetrahydronaphthalene ketopiperazine MCH-R1 antagonists--Increasing selectivity over hERG.

A direct correlation between hERG binding and QTc prolongation was established for a series of aminomethyl tetrahydronaphthalene ketopiperazine MCH-R1 antagonists. Compounds within this class with greater selectivity over hERG were developed. Compound 4h proved to have the best profile, with MCH-R1 Ki = 16 nm and hERG IC50 = 25 microM.