Safety Pharmacology Assays Research Articles

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

IntroductionDSM421, 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. MethodsPotential testing errors, differences in types of plethysmography testing chambers, effects on stress indicators, and off-target activity were investigated. ResultsRespiratory 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). DiscussionThe 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.

15th Annual Meeting of the Safety Pharmacology Society: Focus on traditional sensory systems

IntroductionThis report summarizes and comments key talks on the five traditional senses (ear, vestibular system, vision, taste, olfaction, and touch) which were delivered during the 2015 Annual Meeting of the Safety Pharmacology (SP) Society. Areas coveredThe functional observational battery (FOB) can detect major candidate drug liabilities only on ear, touch and vision. Anatomy, physiology, pharmacology, and pathology notions on each sensory system introduce speaker talks. Techniques for evaluating drug effects on hearing functions are reviewed. Nonclinical approaches to assess vestibular toxicity leading to balance deficits are presented. Retinal explants studied with multielectrode arrays allow the identification of drug liability sites on the retina. Routinely performed Safety Pharmacology assays are not powered to address candidate drug-induced disturbances on taste and smell. This weakness needs correction since unintended pharmacological impairment of these sensorial functions may have serious health consequences. Neuropathy produced by chemotherapeutic agents may cause multiple sensorial perception distortions. ConclusionsSafety Pharmacology studies should ensure the safety of any candidate drug on the five sensorial systems.

ADME studies and preliminary safety pharmacology of LDT5, a lead compound for the treatment of benign prostatic hyperplasia.

This study aimed to estimate the absorption, distribution, metabolism and excretion (ADME) properties and safety of LDT5, a lead compound for oral treatment of benign prostatic hyperplasia that has previously been characterized as a multi-target antagonist of α1A-, α1D-adrenoceptors and 5-HT1A receptors. The preclinical characterization of this compound comprised the evaluation of its in vitro properties, including plasma, microsomal and hepatocytes stability, cytochrome P450 metabolism and inhibition, plasma protein binding, and permeability using MDCK-MDR1 cells. De-risking and preliminary safety pharmacology assays were performed through screening of 44 off-target receptors and in vivo tests in mice (rota-rod and single dose toxicity). LDT5 is stable in rat and human plasma, human liver microsomes and hepatocytes, but unstable in rat liver microsomes and hepatocytes (half-life of 11 min). LDT5 is highly permeable across the MDCK-MDR1 monolayer (Papp ∼32×10-6 cm/s), indicating good intestinal absorption and putative brain penetration. LDT5 is not extensively protein-bound and is a substrate of human CYP2D6 and CYP2C19 but not of CYP3A4 (half-life >60 min), and did not significantly influence the activities of any of the human cytochrome P450 isoforms screened. LDT5 was considered safe albeit new studies are necessary to rule out putative central adverse effects through D2, 5-HT1A and 5-HT2B receptors, after chronic use. This work highlights the drug-likeness properties of LDT5 and supports its further preclinical development.

Safety Pharmacology in Drug Discovery and Development.

Safety pharmacology is essential throughout the spectrum of drug discovery and development. Prior to first-in-human studies, safety pharmacology assays, tests, and models predict the clinical risk profile of a potential new drug. During clinical development, safety pharmacology can be used to explore--and potentially explain--both predicted and unpredicted side effects (e.g., adverse events, changes in vital signs, abnormal laboratory values) in order to refine the original clinical risk profile. This chapter will introduce the reader to safety pharmacology's role in translational medicine: the science of translating potential drugs' on- and off-target nonclinical properties to clinical consequences in order to select the best drug candidates to move into early clinical testing. Case studies will be used to illustrate the importance of safety pharmacology testing throughout all phases of drug development.

Drug safety is a barrier to the discovery and development of new androgen receptor antagonists

Androgen receptor (AR) antagonists are part of the standard of care for prostate cancer. Despite the almost inevitable development of resistance in prostate tumors to AR antagonists, no new AR antagonists have been approved for over a decade. Treatment failure is due in part to mutations that increase activity of AR in response to lower ligand concentrations as well as to mutations that result in AR response to a broader range of ligands. The failure to discover new AR antagonists has occurred in the face of continued research; to enable progress, a clear understanding of the reasons for failure is required. Non-clinical drug safety studies and safety pharmacology assays were performed on previously approved AR antagonists (bicalutamide, flutamide, nilutamide), next generation antagonists in clinical testing (MDV3100, BMS-641988), and a pre-clinical drug candidate (BMS-501949). In addition, non-clinical studies with AR mutant mice, and EEG recordings in rats were performed. Non-clinical findings are compared to disclosures of clinical trial results. As a drug class, AR antagonists cause seizure in animals by an off-target mechanism and are found in vitro to inhibit GABA-A currents. Clinical trials of candidate next generation AR antagonists identify seizure as a clinical safety risk. Non-clinical drug safety profiles of the AR antagonist drug class create a significant barrier to the identification of next generation AR antagonists. GABA-A inhibition is a common off-target activity of approved and next generation AR antagonists potentially explaining some side effects and safety hazards of this class of drugs.

Predicting safety toleration of pharmaceutical chemical leads: Cytotoxicity correlations to exploratory toxicity studies

The selection and application of appropriate safety screening paradigms could revolutionize the drug discovery process by reducing safety-related attrition. While mechanism specific genotoxicity and safety pharmacology assays are routinely used in screening, the overall value of employing nonspecific cytotoxicity assays remains controversial. A retrospective analysis of safety findings from rat exploratory toxicity studies (4–14 days) utilizing compounds that spanned broad therapeutic targets (protease, transport, G-protein-coupled receptors, and kinase inhibitors, cGMP modulators) demonstrated that safety toleration in vivo could be approximated using cytotoxicity values. A composite safety score was calculated for each compound dose based on findings in each of the following categories: systemic toleration (mortality, food consumption, and adverse clinical signs), clinical chemistry/hematology parameters (deviations from normal ranges), and multiorgan pathology (necrosis or incidence/severity of histologic change). Binning compounds into potent (LC 50 < 10 μM) and non-potent (LC 50 > 100 μM) cytotoxicants in vitro showed that compared to non-potent cytotoxicants the exposure to potent cytotoxicants in vivo resulted in higher overall severity scores at lower exposures. Correlating overall toleration for individual compounds was further refined when in vivo exposure was considered. When average plasma exposure (Cp ave) for a compound exceeded its mean lethal concentration (LC 50) in vitro (Cp ave/LC 50 > 1), higher overall severity scores were achieved compared to lower exposure margins (Cp ave/LC 50 <0.01). Based on this analysis, the ability to select lead series and individual compounds with better safety characteristics is presented. In summary, cytotoxicity screening can be used to approximate, not define, the safety characteristics of lead pharmaceutical series early in the drug discovery process.

PF-03475952: a potent and neutralizing fully human anti-CD44 antibody for therapeutic applications in inflammatory diseases

CD44 is a cell adhesion molecule believed to play a critical role in T cell and monocyte infiltration in the inflammatory process. The reduction of CD44 expression or its ability to properly interact with its key ligand, hyaluronic acid (HA), inhibits migration and subsequent activation of cells within sites of inflammation. CD44-deficient mice exhibit decreased disease in a mouse arthritis model. Accordingly, we developed PF-03475952, a fully human IgG2 anti-CD44 monoclonal antibody (mAb). Binding of PF-03475952 to CD44 inhibits binding of HA and induces loss of CD44 from the cell surface. PF-03475952 also passed a series of safety pharmacology assays designed to assess the risk of the mAb to bind Fc gamma receptors, stimulate cytokine release from human whole blood, and stimulate cytokine release from peripheral blood mononuclear cells (PBMC) using plate-bound antibodies. The latter assay was designed specifically to evaluate the risk of cytokine storm that had been observed with TGN1412 (immunostimulatory CD28 superagonist mAb). PF-003475952 exhibits high-affinity binding to both human and cynomolgus monkey CD44, but does not cross-react with rodent CD44. Thus, a rat anti-mouse CD44 mAb was used to demonstrate a dose-dependent decrease of disease in mouse collagen-induced arthritis. Importantly, efficacy was correlated with >50% loss of cell surface CD44 on circulating cells. Loss of CD44 expression on CD3+ lymphocytes was monitored following a single dose of PF-03475952 in cynomolgus monkeys as a pharmacodynamic marker. The recovery of CD44 expression was found to be dose-dependent. PF-03475952 doses of 1, 10, and 100 mg/kg reduced CD44 expression below 50% for 218, 373, and >504 hours, respectively. Targeting of CD44 is a unique mechanism of action in the treatment of inflammatory diseases and is expected to reduce joint damage induced by inflammatory mediators, resulting in disease modification in inflammatory diseases such as rheumatoid arthritis.

Prediction of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiomyocytes

Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. Block of the human Ether-a-go go Related Gene (hERG) ion channel in particular is associated with life-threatening arrhythmias, such as Torsade de Pointes (TdP). Here we investigated human cardiomyocytes derived from pluripotent (embryonic) stem cells (hESC) as a renewable, scalable, and reproducible system on which to base cardiac safety pharmacology assays. Analyses of extracellular field potentials in hESC-derived cardiomyocytes (hESC-CM) and generation of derivative field potential duration (FPD) values showed dose-dependent responses for 12 cardiac and noncardiac drugs. Serum levels in patients of drugs with known effects on QT interval overlapped with prolonged FPD values derived from hESC-CM, as predicted. We thus propose hESC-CM FPD prolongation as a safety criterion for preclinical evaluation of new drugs in development. This is the first study in which dose responses of such a wide range of compounds on hESC-CM have been generated and shown to be predictive of clinical effects. We propose that assays based on hESC-CM could complement or potentially replace some of the preclinical cardiac toxicity screening tests currently used for lead optimization and further development of new drugs.

Human embryonic stem cell-derived cardiomyocytes: drug discovery and safety pharmacology

Human embryonic stem cells (hESCs) can provide potentially unlimited quantities of a wide range of human cell types that can be used in drug discovery and development, basic research and regenerative medicine. In this review, the authors describe the differentiation of hESCs into cardiomyocytes and outline the properties of hESC-derived cardiomyocytes (hESC-CMs), including their cardiac-type action potentials and contractile characteristics. In vitro cellular assays using hESC-CMs, which can be genetically engineered to create target-specific reporters as well as human disease models, will have applications at multiple stages of the drug discovery process. Furthermore, cardiac safety pharmacology assays evaluating the risk of proarrhythmic side effects associated with QT prolongation may be enhanced in their predictive value with the use of hESC-CMs.