Secondary pharmacology: screening and interpretation of off-target activities - focus on translation.

Secondary pharmacology is an essential component of drug discovery and is used extensively in the pharmaceutical industry for achieving optimal specificity of new drugs via early hazard identification and off-target mitigation. The importance of this discipline has been achieved by increasing its translational value, based on the recognition of biological target-drug molecule-adverse drug reaction (ADR) associations and integration of secondary pharmacology data with pharmacokinetic parameters. Information obtained from clinical ADRs, from recognition of specific phenotypes of animal models and from hereditary diseases provides increasing regulatory confidence in the target-based approach to ADR prediction and mitigation. Here, we review the progress of secondary pharmacology during the past decade and highlight and demonstrate its applications and impact in drug discovery.

Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies.

Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease and as such, are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human starting dose is discussed.

The minimum anticipated biological effect level (MABEL) for selection of first human dose in clinical trials with monoclonal antibodies.

Dose selection for first-in-human (FIH) clinical trials with monoclonal antibodies (mAbs) is based on specifically designed preclinical pharmacology and toxicology studies, mechanistic ex vivo/in vitro investigations with human and animal cells and pharmacokinetic/pharmacodynamic (PK/PD) modeling approaches and requires a thorough understanding of the biology of the target and the relative binding and pharmacological activity of the mAb in animals and humans. These investigations provide the essential information required for the selection of a safe starting dose and escalation for FIH trials based on toxicology and pharmacology data and the minimal anticipated biological effect level (MABEL) by integrating all available in vivo and in vitro data. In this review, strategies for estimation of the MABEL for mAbs specific for both membrane and soluble targets are presented and the scientific and regulatory challenges highlighted.

Tissue-specific, non-invasive toxicity biomarkers: translation from preclinical safety assessment to clinical safety monitoring.

Limited sensitivity and limited target organ specificity are the major drawbacks for most peripheral clinical pathology parameters traditionally used for monitoring organ integrity both during preclinical toxicological assessment and clinical safety testing of investigational drugs. Several novel toxicity biomarkers have emerged as sensitive tools for detection, monitoring, quantification and prediction of solid organ toxicity. These tissue-specific, non-invasive biomarkers may provide valuable information for decision making during toxicological assessment and may be used for sensitive and specific target organ monitoring during clinical trials. This review critically discusses the opportunities and limitations of these biomarkers with respect to their translation into (first-in-human) clinical trials. A comprehensive overview is provided on serum- and urine-based biomarkers for hepatotoxicity, nephrotoxicity, cardiotoxicity, gonadotoxicity, pancreatic toxicity, vascular toxicity and phospholipidosis including species-specific assay availabilities and sampling regimens. In addition, the current regulatory status is presented and discussed in view of recent changes in regulatory acceptance by health authorities.

Comparative quantification of pharmacodynamic parameters of chiral compounds (RRR- vs. all-rac-alpha tocopherol) by global gene expression profiling.

Pharmacologically active compounds (e.g. from the groups of pharmaceutical drugs, cofactors or vitamins) often consist of two or more stereoisomers (enantiomers or diastereoisomers) which may differ in their pharmacodynamic/kinetic, toxicological and biological properties. A well-known example is vitamin E which is predominantly administered as two different forms, one derived from natural sources (mainly soybeans), and one from production by chemical total-synthesis. While vitamin E from natural sources occurs as a single stereoisomer (RRR-alpha-tocopherol), synthetic vitamin E (all-rac-alpha-tocopherol) is an equimolar mixture of eight stereoisomers. Based on a number of animal studies it has been suggested that the biological potency of natural-source vitamin E is 1.36 greater compared to its counterpart produced by chemical synthesis. In this study, we have used the Affymetrix GeneChip technology to evaluate the feasibility of a new bio-assay where the gene regulatory activities of RRR-alpha-tocopherol and all-rac-alpha-tocopherol were quantified and compared on the genome-wide level. For this purpose, HepG2 cells were supplemented with increasing amounts of RRR- or all-rac-alpha-tocopherol for 7 days. Genes showing a dose-related induction/repression were identified by global gene expression profiling. Our findings show that RRR- and all-rac-alpha-tocopherol share an identical transcriptional activity, i.e. induce/repress the expression of the same set of genes. Based on the transcriptional dose-response data, EC50 and IC50 values were determined for each of these genes. The feasibility of calculating a "transcriptional potency factor" of RRR- vs. all-rac-e-tocopherol was evaluated by dividing the EC50/IC50 of RRR-alpha-tocopherol by the corresponding EC50/IC50 of all-rac-alpha-tocopherol for every of the vitamin E responsive genes. Using this approach we have calculated 215 single biopotency ratios. Subsequently, the mean of all potency ratios was found to be 1.05. In the present work we propose a new assay for the analysis and comparison of the biological activity and potency of chiral compounds in vivo.

Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats.

The recent discovery that vitamin E (VE) regulates gene activity at the transcriptional level indicates that VE may exert part of its biological effects by mechanisms which may be independent of its well-recognised antioxidant function. The objective of this study was the identification of hepatic vitamin E-sensitive genes and examination of the effects of VE on their corresponding biological endpoints. Two groups of male rats were randomly assigned to either a VE-sufficient diet or to a control diet deficient in VE for 290 days. High-density oligonucleotide microarrays comprising over 7000 genes were used to assess the transcriptional response of the liver. Differential gene expression was monitored over a period of 9 months, at four different time-points, and rats were individually profiled. This experimental strategy identified several VE-sensitive genes, which were chronically altered by dietary VE. VE supplementation down-regulated scavenger receptor CD36, coagulation factor IX and 5-alpha-steroid reductase type 1 mRNA levels while hepatic gamma glutamyl-cysteinyl synthetase was significantly up-regulated. Measurement of the corresponding biological endpoints such as activated partial thromboplastin time, plasma dihydrotestosterone and hepatic glutathione substantiated the gene chip data which indicated that dietary VE plays an important role in a range of metabolic processes within the liver.