Analysis of secondary pharmacology assays received by the US Food and Drug Administration.

Secondary pharmacology studies are a time-efficient and cost-effective method for determining the safety profile of a potential new drug before it enters human trials. The results of these multi-target screens are commonly submitted with Investigational New Drug (IND) applications, but there currently is little guidance on how such information is presented and which targets are chosen for testing. In this study, we expand on our previous analysis of secondary pharmacology reports by manually curating and analyzing all secondary pharmacology results received by the FDA received as part of an IND submission. A total of 1120 INDs submitted by 480 sponsors between 1999 and October 2020 were included in this study. The overall results were largely consistent with previous internal and external studies, showing that the most tested target in our set was the histamine 1 receptor (tested 938 times), the most hit target was sodium channel site 2 (hit 141 times), and the target with the highest hit percentage was the vesicular monoamine transporter 2 (hit 42.2% of the time). Additionally, this study demonstrated that improvements in the secondary pharmacology submission process, such as changes in formatting and nomenclature, could enhance the utility of these assays for regulatory review, including assisting with identifying the safety liabilities of a drug candidate early in development. This updated data set will allow FDA-industry collaborative working groups to continue developing the best methods for regulatory submission of secondary pharmacology data and evaluate the need for a standard target panel.

Aggregation and analysis of secondary pharmacology data from investigational new drug submissions at the US Food and Drug Administration.

Secondary pharmacology studies are utilized by the pharmaceutical industry as a cost-efficient tool to identify potential safety liabilities of drugs before entering Phase 1 clinical trials. These studies are recommended by the Food and Drug Administration (FDA) as a part of the Investigational New Drug (IND) application. However, despite the utility of these assays, there is little guidance on which targets should be screened and which format should be used. Here, we evaluated 226 secondary pharmacology profiles obtained from close to 90 unique sponsors. The results indicated that the most tested target in our set was the GABA benzodiazepine receptor (tested 168 times), the most hit target was adenosine 3 (hit 24 times), and the target with the highest hit percentage was the quinone reductase 2 (NQO2) receptor (hit 29% of the time). The overall results were largely consistent with those observed in previous publications. However, this study also identified the need for improvement in the submission process of secondary pharmacology studies by industry, which could enhance their utility for regulatory purpose. FDA-industry collaborative working groups will utilize this data to determine the best methods for regulatory submission of these studies and evaluate the need for a standard target panel.

An FDA/CDER perspective on nonclinical testing strategies: Classical toxicology approaches and new approach methodologies (NAMs).

Nonclinical testing of human pharmaceuticals is conducted to assess the safety of compounds to be studied in human clinical trials and for marketing of new drugs. Although there is no exact number and type of nonclinical studies required for safety assessments, as there is inherent flexibility for each new compound, the traditional approach is outlined in various FDA and ICH guidance documents and involves a combination of in vitro assays and whole animal testing methods. Recent advances in science have led to the emergence of numerous new approach methodologies (NAMs) for nonclinical testing that are currently being used in various aspects of drug development. Traditional nonclinical testing methods can predict clinical outcomes, although improvements in these methods that can increase predictivity of clinical outcomes are encouraged and needed. This paper discusses FDA/CDER's view on the opportunities and challenges of using NAMs in drug development especially for regulatory purposes, and also includes examples where NAMs are currently being used in nonclinical safety assessments and where they may supplement and/or enhance current testing methods. FDA/CDER also encourages communication with stakeholders regarding NAMs and is committed to exploring the use of NAMs to improve regulatory efficiency and potentially expedite drug development.

Drug-induced cardiac abnormalities in premature infants and neonates.

The Cardiac Safety Research Consortium (CSRC) is a transparent, public-private partnership that was established in 2005 as a Critical Path Program and formalized in 2006 under a Memorandum of Understanding between the United States Food and Drug Administration and Duke University. Our continuing goal is to advance paradigms for more efficient regulatory science related to the cardiovascular safety of new therapeutics, both in the United States and globally, particularly where such safety questions add burden to innovative research and development. This White Paper provides a summary of discussions by a cardiovascular committee cosponsored by the CSRC and the US Food and Drug Administration (FDA) that initially met in December 2014, and periodically convened at FDA's White Oak headquarters from March 2015 to September 2016. The committee focused on the lack of information concerning the cardiac effects of medications in the premature infant and neonate population compared with that of the older pediatric and adult populations. Key objectives of this paper are as follows: Provide an overview of human developmental cardiac electrophysiology, as well as the electrophysiology of premature infants and neonates; summarize all published juvenile animal models relevant to drug-induced cardiac toxicity; provide a consolidated source for all reported drug-induced cardiac toxicities by therapeutic area as a resource for neonatologists; present drugs that have a known cardiac effect in an adult population, but no reported toxicity in the premature infant and neonate populations; and summarize what is not currently known about drug-induced cardiac toxicity in premature infants and neonates, and what could be done to address this lack of knowledge. This paper presents the views of the authors and should not be construed to represent the views or policies of the FDA or Health Canada.

Regulatory Forum Review*: Utility of in Vitro Secondary Pharmacology Data to Assess Risk of Drug-induced Valvular Heart Disease in Humans: Regulatory Considerations.

Drug-induced valvular heart disease (VHD) is a serious side effect linked to long-term treatment with 5-hydroxytryptamine (serotonin) receptor 2B (5-HT2B) agonists. Safety assessment for off-target pharmacodynamic activity is a common approach used to screen drugs for this undesired property. Such studies include in vitro assays to determine whether the drug is a 5-HT2B agonist, a necessary pharmacological property for development of VHD. Measures of in vitro binding affinity (IC50, Ki) or cellular functional activity (EC50) are often compared to maximum therapeutic free plasma drug levels ( fCmax) from which safety margins (SMs) can be derived. However, there is no clear consensus on what constitutes an appropriate SM under various therapeutic conditions of use. The strengths and limitations of SM determinations and current risk assessment methodology are reviewed and evaluated. It is concluded that the use of SMs based on Ki values, or those relative to serotonin (5-HT), appears to be a better predictor than the use of EC50 or EC50/human fCmax values for determining whether known 5-HT2B agonists have resulted in VHD. It is hoped that such a discussion will improve efforts to reduce this preventable serious drug-induced toxicity from occurring and lead to more informed risk assessment strategies.

Preclinical QT safety assessment: cross-species comparisons and human translation from an industry consortium.

INTRODUCTION: In vivo models have been required to demonstrate relative cardiac safety, but model sensitivity has not been systematically investigated. Cross-species and human translation of repolarization delay, assessed as QT/QTc prolongation, has not been compared employing common methodologies across multiple species and sites. Therefore, the accurate translation of repolarization results within and between preclinical species, and to man, remains problematic. METHODS: Six pharmaceutical companies entered into an informal consortium designed to collect high-resolution telemetered data in multiple species (dog; n=34, cynomolgus; n=37, minipig; n=12, marmoset; n=14, guinea pig; n=5, and man; n=57). All animals received vehicle and varying doses of moxifloxacin (3-100 mg/kg, p.o.) with telemetered ECGs (≥500 Hz) obtained for 20-24h post-dose. Individual probabilistic QT-RR relationships were derived for each subject. The rate-correction efficacies of the individual (QTca) and generic correction formulae (Bazett, Fridericia, and Van de Water) were objectively assessed as the mean squared slopes of the QTc-RR relationships. Normalized moxifloxacin QTca responses (Veh Δ%/µM) were derived for 1h centered on the moxifloxacin Tmax. RESULTS: All QT-RR ranges demonstrated probabilistic uncertainty; slopes varied distinctly by species where dog and human exhibited the lowest QT rate-dependence, which was much steeper in the cynomolgus and guinea pig. Incorporating probabilistic uncertainty, the normalized QTca-moxifloxacin responses were similarly conserved across all species, including man. DISCUSSION: The current results provide the first unambiguous evidence that all preclinical in vivo repolarization assays, when accurately modeled and evaluated, yield results that are consistent with the conservation of moxifloxacin-induced QT prolongation across all common preclinical species. Furthermore, these outcomes are directly transferable across all species including man. The consortium results indicate that the implementation of standardized QTc data presentation, QTc reference cycle lengths, and rate-correction coefficients can markedly improve the concordance of preclinical and clinical outcomes in most preclinical species.

Anticipated classes of new medications and molecular targets for pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) remains a life-limiting condition with a major impact on the ability to lead a normal life. Although existing therapies may improve the outlook in some patients there remains a major unmet need to develop more effective therapies in this condition. There have been significant advances in our understanding of the genetic, cell and molecular basis of PAH over the last few years. This research has identified important new targets that could be explored as potential therapies for PAH. In this review we discuss whether further exploitation of vasoactive agents could bring additional benefits over existing approaches. Approaches to enhance smooth muscle cell apotosis and the potential of receptor tyrosine kinase inhibition are summarised. We evaluate the role of inflammation, epigenetic changes and altered glycolytic metabolism as potential targets for therapy, and whether inherited genetic mutations in PAH have revealed druggable targets. The potential of cell based therapies and gene therapy are also discussed. Potential candidate pathways that could be explored in the context of experimental medicine are identified.

VeloceGenomics: an accelerated in vivo drug discovery approach to rapidly predict the biologic, drug-like activity of compounds, proteins, or genes.

PURPOSE: The aim of this study is to test the predictive power of in vivo multiorgan RNA expression profiling in identifying the biologic activity of molecules. METHODS: Animals were treated with compound A or B. At the end of the treatment period, in vivo multiorgan microarray-based gene expression data were collected. Investigators masked to the identity of the compounds analyzed the transcriptome signatures to define the molecular pathways affected by treatment and to hypothesize the biologic activity and potential therapeutic indications of the blinded compounds. RESULTS: For compound A, G-protein-coupled receptors and factors associated with cell growth were affected-growth hormone/insulin-like growth factor-1, glucagon/insulin axes, and general somatomedin-like activity. Deblinding showed the compound to be a somatostatin analog, SOM230, confirming the accuracy of the predicted biologic activity. For compound B, components of the inflammatory cascade potentially mediated by lipopolysaccharide, tumor necrosis factor, or proinflammatory cytokines were affected. The gene expression signatures were most consistent with an interleukin-6 family activity. Deblinding revealed that compound B was leukemia inhibitory factor. CONCLUSIONS: VeloceGenomics is a strategy of coupling in vivo compound testing with genomic technologies. The process enables prediction of the mechanism of action and, coupled with other relevant data, prediction of the suitability of compounds for advancement in the drug development process.

Immunologic pathways in a quantitative model of immunosuppression based on rejection of an allogeneic or xenogeneic tumor graft.

BACKGROUND: A quantitative model of immunosuppression was previously developed based on the rejection of the allogeneic A/J murine tumor sarcoma 1 (Sa1) in immunocompetent mice. Here, the model is used to evaluate the immunologic mechanisms of graft rejection and to determine the potential of this model to detect synergistic effects of combined immunosuppressive therapies. METHODS: Wild-type, genetically-deficient, or drug-treated mice were used. Mice were challenged subcutaneously with the allogeneic murine tumor cell line, Sa1, or with the xenogenic human tumor, MDA435. Tumor growth was monitored with time, with increasing tumor size reflecting greater immunosuppression. In some cases, the mice were presensitized with either Sa1 or with A/J splenocytes. RESULTS: In naïve recipient mice, studies in major histocompatibility complex (MHC)-I-deficient mice and with depleting anti-CD8 monoclonal antibody (mAb) demonstrate that CD8 T cells are important for Sa1 rejection. A modest role for perforin but not for Fas/Fas ligand could be demonstrated. Blockade of CD4 T cells was more effective with decreasing histocompatibility barriers. In contrast, CD4 T cells were critical in second-set rejections, but CD8 T cells were not. Rejection of xenogeneic tumors was also T-cell dependent as demonstrated by anti-CD3 mAb, dependent on both CD4 and CD8 T cells as demonstrated using MHC-I- and MHC-II-deficient mice, but was more vigorous as demonstrated by the lack of effectiveness of immunosuppressive drugs in this model. CONCLUSIONS: This model can be used to define dominant and partial effects of immunologic pathways as well as synergistic interactions of agents to develop immunosuppressive strategies.

Kidney slices of human and rat to characterize cisplatin-induced injury on cellular pathways and morphology.

Kidney slices represent an in vitro model that has the cellular complexity of in vivo tissue to provide insights into mechanisms of organ injury, as shown in this study with the model nephrotoxicant cisplatin. Cell pathways altered by cisplatin exposure are assessed by gene expression analysis, cell function, and morphology in human and rat kidney slices in comparison to rat kidney from an in vivo study. The acute nephrosis of the tubular epithelium induced by cisplatin in vivo was reproduced in both human and rat kidney slices, while the glomerulus appeared resistant even at high concentrations. Kidney gene expression changes of in vivo and in vitro samples were indicative of transcription, DNA damage, cell cycle, proliferation, and apoptosis that are in agreement with the mechanism of cisplatin causing DNA damage, growth arrest, and apoptosis; while genes indicative of protein damage, the disruption of transport and calcium homeostasis, cellular metabolism, and oxidative stress are pathways linked with cisplatin binding to various cellular proteins and macromolecules. Both concentration and time-dependent gene expression changes evident in the in vitro model preceded a change in tissue morphology. Functional assays confirming cell dysfunction and increased apoptosis revealed the rat kidney to be more sensitive to the effects of cisplatin than human kidney as demonstrated by significant decreases in slice ATP and GSH levels, significant increases in caspase 9 and 3 activity, p53 protein levels, and increased DNA laddering. The regional markers of proximal and distal tubular injury, alpha- and pi-glutathione S-transferases, were shown for the human kidney slices to be significantly increased by cisplatin. In this study, cisplatin-induced nephrotoxicity was demonstrated morphologically in rat and human kidney slices, and the associated gene expression and functional changes characterized the cellular pathways involved.

Organ slice viability extended for pathway characterization: an in vitro model to investigate fibrosis.

Liver slice viability is extended to 96 h for rat, expanding the use of this in vitro model for studying mechanisms of injury and repair, including pathways of fibrosis. The contributing factors to increased organ slice survival consist of the use of a preservation solution for liver perfusion and slice preparation, obtaining rats that are within the weight range of 250-325 g, placing a cellulose filter atop the titanium mesh roller-insert to support the slice, and maintaining the slices in an optimized culture medium which is replaced daily. The liver slices remain metabolically active, synthesizing adenosine triphosphate (ATP), glutathione, and glycogen, and exhibit preserved organelle integrity and slice morphology. Slice preparation results in 2-cut surfaces which likely triggers a repair and regenerative response. The fibrogenic pathways are evident by the activation of stellate cells, the proliferation of myofibroblast-like cells, and an increased collagen deposition by 48 h. Markers indicative of activated stellate cells, alpha-smooth muscle actin, collagen 1a1, desmin, and HSP47 are substantiated by real time-PCR. Increased staining of alpha-smooth muscle actin initially around the vessels and by 72-96 h in the tissue is accompanied by increased collagen staining. Microarray gene expression revealed extracellular matrix changes with the up-regulation of cytoskeleton, filaments, collagens, and actin genes; and the down-regulation of genes linked with lipid metabolism. The improvements in extending liver slice survival, in conjunction with its three-dimensional multi-cellular complexity, increases the application of this in vitro model for investigating pathways of injury and repair, and fibrosis.

Timing of supplementation with the antioxidant N-acetyl-L-cysteine reduces tumor multiplicity in novel, cancer-prone p53 haploinsufficient Tg.AC (v-Ha-ras) transgenic mice but has no impact on malignant progression.

Epidemiological studies support the protective role of dietary antioxidants in preventing cancer. However, emerging evidence suggests that antioxidant supplements may actually exacerbate carcinogenesis. We explored this paradox in a model containing two common genotypic characteristics of human cancers. We selected p53 haploinsufficient Tg.AC (v-Ha-ras) mice as a model, because it contains an activated, carcinogen-inducible ras oncogene and an inactivated p53 tumor suppressor gene. These mice develop chemically induced benign and malignant skin tumors rapidly. Mice were fed basal diet with or without 3% N-acetyl-L-cysteine (NAC) before and after topical application of the carcinogen benzo[a]pyrene (64 micrograms twice per week for 7 wk) until 50% of mice within a group displayed at least one lesion. Half each of mice fed the basal and the NAC-supplemented diet were then switched to the alternate diet. Mice fed the NAC-supplemented diet or switched from the NAC-supplemented to the basal diet displayed 38% and 26% reductions, respectively, in tumor multiplicity and a 15% reduction if switched from the basal to the NAC-supplemented diet. Although latency was unaffected, NAC induced a lag in tumor incidence, which exceeded 90% at 10 wk for all groups. The timing of NAC supplementation did not affect malignant progression. Thus dietary NAC was chemoprotective by slowing tumorigenesis but did not affect malignant conversion.

Cytologic and histologic features of a poorly differentiated glioma in a dog.

A 5-year old female Boxer with a 1-week history of progressive paresis and paraplegia had a T10-13 subarachnoid filling defect on myelography. Exploratory hemilaminectomy revealed an intramedullary spinal cord tumor which was subsequently diagnosed as a poorly differentiated glioma, most likely an anaplastic ependymoma. The cytologic, histologic, and immunocytochemical staining characteristics of this neoplasm are described. Differential diagnoses, including primary and secondary tumors involving the central nervous system are discussed.