Safety differentiation: emerging competitive edge in drug development.

With increasing expectations to provide evidence of drug efficacy, safety, and cost-effectiveness, best-in-class drugs are a major value driver for the pharmaceutical industry. Superior safety is a key differentiation criterion that could be achieved through better risk:benefit profiles, safety margins, fewer contraindications, and improved patient compliance. To accomplish this, comparative safety assessments using innovative and adaptive nonclinical and clinical outcome-based approaches should be undertaken, and continuous strategic adjustments must be made as the risk:benefit profiles evolve. Key success criteria include scientific expertise and integration between all disciplines during the full extent of the drug development process.

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.

Integrated risk assessment of suicidal ideation and behavior in drug development.

Treatment-related suicidal ideation and behavior (SIB) adverse events are under increasing public, legal and regulatory scrutiny. Prospective assessment of SIB is emerging as a challenging safety requirement by health authorities for the development of drugs but the underlying risk factors remain ill defined. To help with the understanding of risk factors that trigger a prospective assessment of SIB in clinical trials, we present an industry consensus framework for risk assessment and decision making of SIB during drug development. Application of this strategy is based on chemical and pharmacological similarities of compounds with clinical evidence of suicidal intent, target or indication classes associated with high incidence of SIB, in vitro neuropharmacological activity profile, in vivo ADME properties, patient population of the underlying indication and regulatory precedents.

The determination and interpretation of the therapeutic index in drug development.

A key part of drug discovery and development is the characterization and optimization of the safety and efficacy of drug candidates to identify those that have an appropriately balanced safety-efficacy profile for a given indication. The therapeutic index (TI)--which is typically considered as the ratio of the highest exposure to the drug that results in no toxicity to the exposure that produces the desired efficacy--is an important parameter in efforts to achieve this balance. Various types of safety and efficacy data are generated in vitro and in vivo (in animals and in humans), and these data can be used to predict the clinical TI of a drug candidate at an early stage. However, approaches to systematically and quantitatively compare these types of data and to apply this knowledge more effectively are needed. This article critically discusses the various aspects of TI determination and interpretation in drug development for both small molecule drugs and biotherapeutics.

Comparative requirements for exploratory clinical trials -- eIND, eCTA and microdosing.

Exploratory clinical trials provide a strategy for rapid human entry of investigational drugs. Such clinical studies are typically conducted during early clinical development in phase I as first-in-human studies, have no therapeutic intent, are not intended to examine clinical tolerability and involve a small number of human subjects at limited dose/exposure. Early decision data derived from such clinical studies may include PK, PD and/or biomarker-based translational medicine endpoints as well as PK/PD modeling approaches. This review critically discusses the various exploratory clinical trial strategies, their advantages and disadvantages as well as the regulatory safety requirements. In this respect, strategies for exploratory Investigational New Drugs (eIND), exploratory Clinical Trial Applications (eCTA) and microdosing are highlighted and compared in view of the new ICH M3(R2) guideline including options for biotechnology-derived pharmaceuticals such as monoclonal antibodies.

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.

Effect of Vitamin E on Cytochrome P450 mRNA Levels in Cultured Hepatocytes (HepG2) and in Rat Liver.

Vitamin E has been described in the literature as a regulator of gene expression. The gene-regulatory activity of vitamin E with regard to genes encoding cytochrome P450 (CYP) enzymes, which play a pivotal role both in the metabolism of xenobiotics and vitamin E, has not been conclusively characterised. The objective of the current study was, therefore, to elucidate the short- and long-term effects of natural and synthetic vitamin E on CYP gene expression using Affymetrix GeneChip® technology. To this end, HepG2 cells were incubated with 0, 10, 30, 80 and 300 µM RRR-α-tocopheryl acetate (natural vitamin E) or all rac-α-tocopheryl acetate (synthetic vitamin E) for 7 days and the mRNA of CYP genes was quantified. The expression of only one (CYP20A1) of 14 CYP genes with detectable mRNA levels was dose-dependently up-regulated. No differences in gene-regulatory activity were observed between RRR- and all rac-α-tocopheryl acetate. To study the role of vitamin E in CYP gene expression in vivo, Fisher 344 rats were randomly assigned to either a vitamin E-enriched (60 mg/kg RRR-α-tocopheryl acetate) or - deficient (1.7 mg/kg RRR-α-tocopheryl acetate) diet for 290 days. Neither in the vitamin E-enriched, nor in the vitamin E-deficient rats, were significant changes in the liver CYP, mRNA levels observed. In conclusion, our data indicated that vitamin E does not appear to modulate cytochrome P450 mRNA expression in HepG2 cells or in rats.

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.

Large heterogeneity of mutations in the gene encoding the low-density lipoprotein receptor in subjects with familial hypercholesterolaemia.

Molecular genetic testing for presymptomatic identification of subjects affected by familial hypercholesterolaemia (FH) is difficult due to the heterogeneity of the mutations in the gene encoding the low-density lipoprotein receptor (LDLR) in most populations. This investigation presents a detailed analysis of comparable, country-specific prevalence data of LDLR mutations in subjects with clinically defined FH and assesses the heterogeneous mutation diversity observed in most geographic regions.

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.

Expression of COX-2 and Wnt pathway genes in adenomas of familial adenomatous polyposis patients treated with meloxicam.

BACKGROUND: Familial adenomatous polyposis (FAP) is an autosomal, dominantly inherited predisposition to colorectal cancer caused by germline mutations within the adenomatous polyposis coli (APC) gene, a key member of the Wnt signalling pathway. A new class of non-steroidal anti-inflammatory drugs (NSAIDs), the specific cyclooxygenase 2 (COX-2) inhibitors, have recently been applied for the treatment of FAP patients. PATIENTS AND METHODS: The expressions of the Wnt members and targets APC, c-myc, cyclin D1 and COX-2, as measured by real-time quantitative RT-PCR, have been evaluated in fresh samples of normal colorectal mucosa and matched adenoma tissue of six unrelated FAP patients before and after treatment with meloxicam. RESULTS: A significant up-regulation of COX-2 in adenomas after treatment with meloxicam was found. Furthermore, in adenomas, a down-regulation of APC after treatment and a tight correlation of the expressions of the two Wnt targets, c-myc and cyclin D1, in both stages of treatment were observed. CONCLUSION: A feedback loop mediated by the peroxisome proliferator-activated receptor (PPAR) gamma is discussed as being responsible for the up-regulation of COX-2

Indinavir inhibits sterol-regulatory element-binding protein-1c-dependent lipoprotein lipase and fatty acid synthase gene activations.

BACKGROUND: A syndrome characterized by hypertriglyceridaemia, hypercholesterolaemia, hyperinsulinaemia, and lipodystrophy has been found to be associated with highly active antiretroviral treatment (HAART) including protease inhibitors. A marker predicting this syndrome has been previously identified in the gene encoding the sterol-regulatory element-binding protein (SREBP)-1c, a regulator of triglycerides, cholesterol, insulin, and adipocytes. OBJECTIVE: A possible inhibition of SREBP-1c-dependent genes by the protease inhibitor indinavir and its possible reversal by the lipid-lowering drug simvastatin were studied. METHODS: The effects of indinavir and simvastatin on the inhibition/activation of SREBP-1c-dependent genes were compared with the effects of indinavir and simvastatin on the inhibition/activation of SREBP-1c-independent genes. RESULTS: Indinavir inhibited the SREBP-1c-dependent genes encoding the lipoprotein lipase (103 nmol/l resulted in an inhibition of 12.4%; P = 0.0051) and the fatty acid synthase (103 nmol/l resulted in an inhibition of 30.3%; P = 0.036) in a dose-dependent fashion but not the SREBP-1c-independent gene encoding the low-density lipoprotein receptor. Simvastatin antagonized the indinavir-induced SREBP-1c-inhibition. CONCLUSIONS: Indinavir inhibits important effector genes of the SREBP-1c pathway, explaining major HAART-related adverse effects.

Sterol-regulatory element-binding protein (SREBP)-2 contributes to polygenic hypercholesterolaemia.

Sterol-regulatory element-binding protein (SREBP)-2 is a key regulator of cholesterol. When cells are deprived of cholesterol, proteolytic cleavage releases the NH(2)-terminal domain of SREBP-2 that binds and activates the promoters of SREBP-2-regulated genes including the genes encoding the low-density lipoprotein (LDL) receptor, 3-hydroxymethyl-3-glutaryl-(HMG-)CoA-synthase, and HMG-CoA-reductase. Thus, SREPB-2 gene activation leads to enhanced cholesterol uptake and biosynthesis. A novel protein polymorphism (SREBP-2-595A/G) discovered in the regulatory domain of human SREBP-2 was investigated regarding its impact on cholesterol homeostasis. In human embryonic kidney (HEK)-293-cells, the cleavage-rate of the SREBP-2-595A-isoform was slightly decreased compared to that of the SREBP-2-595G-isoform. Since cleavage of SREBP-2 activates the LDL receptor-mediated uptake of plasma cholesterol, we hypothesized the LDL receptor-mediated uptake to be decreased in homozygous SREBP-2-595A-carriers and thus, plasma total cholesterol (TC) to be higher than in SREBP-2-595G-carriers. Multiple linear regression analysis of population samples from Switzerland (N=1334) and Israel (N=923) demonstrated a significant positive, gene dose-dependent association of the SREBP-2-595A-isoform with higher plasma TC (P=0.001). This cholesterol-modulating effect was present in hypercholesterolaemic (DeltaTC=1.05 mmol/l, 14.4%; P=0.002; N=477), but absent in normocholesterolaemic subjects (DeltaTC=0.06 mmol/l, 1.4%; P=0.334; N=1780). In summary, a slightly but constantly decreased cleavage-rate of the SREBP-2-595A-isoform compared to that of the SREBP-2-595G-isoform may lead to a reduced transcriptional activation of the LDL receptor-gene weakening the SREBP-mediated compensation mechanisms, and may, therefore, be a critical factor in the development of polygenic hypercholesterolaemia.

Processing of gene expression data generated by quantitative real-time RT-PCR.

Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.

Overexpression of Wnt target genes in adenomas of familial adenomatous polyposis patients.

Germline mutations within the adenomatous polyposis coli (APC) gene, a key member of the Wnt signalling pathway, have been shown to cause adenoma development in familial adenomatous polyposis (FAP), a dominantly inherited predisposition to colorectal cancer. Although it has been suggested for several years that alterations within the Wnt pathway are the underlying events for the development of colorectal adenomas in FAP patients, no detailed analysis of the gene expressions of Wnt pathway members has been available in fresh colorectal tissue of FAP patients, so far. Thus, we investigated potential differences in the expressions of APC and its Wnt partners conductin, beta-catenin, cyclin D1, and c-myc in normal colorectal mucosa and matched adenoma tissue of 14 FAP patients using real-time quantitative PCR. The expressions of both Wnt target genes, cyclin D1 and c-myc, were significantly increased in adenoma compared to matched normal mucosa. Furthermore, the overexpressions of these two genes showed a highly significant positive correlation. Our data suggest that the concomitant overexpression of the Wnt targets and cell cycle regulators cyclin D1 and c-myc plays an important role in the neoplastic proliferation of adenomas in FAP patients.