Safety Assessment Of Pharmaceuticals Research Articles

Screening a novel class of anticoagulants for multi‐organ toxicity using Fischer 344 rats: involvement of clinical and anatomic pathologists in the early drug development process

Veterinary clinical and anatomic pathologists play a critical role in assessing the safety of new molecules. The process for evaluation of candidate molecules in drug discovery may vary markedly, depending on the unique characteristics of the compound class. The goal of this report is to describe the evaluation process for assessing the potential toxicity of 2 anticoagulant compounds that were representative of molecules tested in early screening studies in Fisher rats, and to use these studies as an example of the strategic approach used by veterinary pathologists in pharmaceutical safety assessment. Groups of 3 rats were given vehicle alone or one of several doses of compound A or B by oral gavage daily for 4 consecutive days. Survival; clinical signs; body and organ weight measurements; hematologic, coagulation, and clinical biochemical testing; and gross and histologic findings at necropsy were assessed. Transmission electron microscopy was used to characterize unique findings in the liver of rats treated with compound B. Both compounds caused dose-dependent prolongation of the prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin clotting time (TCT). Hepatobiliary and intestinal toxicity were identified by alterations in serum chemistry data, and by histopathologic findings. Electron microscopy and tissue inorganic phosphorus analysis revealed phospholipidosis in rats treated with compound B. Pharmacologically mediated or "on target" effects for these molecules were characterized by dose-progressive prolongation of the PT, APTT, and TCT. Nonpharmacologically mediated or "off-target" toxicity consisted of hepatoxicity and enterotoxicity. These liabilities required that scientists alter the original molecular scaffold to reach the desired therapeutic target and minimize toxicity.

Minimisation of dog use in the safety assessment of pharmaceuticals: Progress with the EFPIA/RSPCA/FRAME dog project

Minimisation of dog use in the safety assessment of pharmaceuticals: Progress with the EFPIA/RSPCA/FRAME dog project

Human Tissue for In Vitro Research as an Alternative to Animal Experiments: A Charitable “Honest Broker” Model to Fulfil Ethical and Legal Regulations and to Protect Research Participants

Research with human tissue offers the possibility not only of improving preclinical pharmaceutical research and safety assessment, but also of the substitution of some animal experiments. Surgically removed human tissue is discarded after pathological evaluation. This tissue would be of enormous value for research, especially in the pharmaceutical branch, if it were readily available in an ethically and legally approved manner. But there are public concerns about the use of human tissue, especially for "commercial" purposes, such as in the pharmaceutical industry. The question is whether the ethical boundaries are sufficiently respected in the course of striving for industrial profit. To overcome this problem, a clear procedure for tissue donation, collection, supply and allocation must be established, which is guaranteed to be independent of special interests. The persisting problem seems to be the lack of an authority which asks for informed consent, coordinates tissue as well as blinded data collection, and supplies research facilities with tissue samples in a transparent manner. Therefore, a charitable, state-controlled foundation acting as an "honest broker" was initiated, to cover the ethical and legal aspects, as well as to protect the research participants in their use of human tissue as an alternative to animal experiments.

CDNA cloning, expression, purification, distribution, and characterization of biologically active canine alanine aminotransferase-1

Alanine aminotransferase (ALT) is a pyridoxal enzyme found mainly in the liver and kidney, but also in small amounts in the heart, muscle, fat, and brain. Serum aminotransferase activities have been used broadly as surrogate markers for tissue injury and disease in human and veterinary clinical settings and in safety assessment of chemicals and pharmaceuticals. Because of its relative abundance in liver, increased serum ALT activity is generally considered indicative of liver damage. Two ALT isoenzymes, ALT1 and ALT2, are known and have been cloned and sequenced from human, rat, and mouse. In this study, we have cloned the complementary DNA encoding the canine orthologue of ALT1 (cALT1). The complete cDNA sequence comprised 1852 bases and contained a 1485-base open reading frame, which encodes a polypeptide of 494 amino acid residues. Canine ALT1 shares 87.7, 87.2, and 87.0% amino acid identity to its human, mouse, and rat orthologues, respectively. The cDNA was expressed in Escherichia coli, with a N-terminal His (6×) tag, and the recombinant enzyme was purified using immobilized metal-affinity chromatography. The final yield of the purified recombinant cALT1 was greater than 5 mg/L culture. The alanine transaminase activity of purified cALT1 was 229.81 U/mg protein, which is approximately 38-fold higher than that of total soluble recombinant E. coli cell lysate, confirming that the enzyme is a functional ALT. Evaluation of various canine tissues by RT-PCR revealed that the level of ALT1 expression is in the order of: heart > liver > fat ∼ brain ∼ gastrocnemius > kidney. The purified cALT1 will be helpful to develop isoenzyme-specific anti-bodies, which could further improve the diagnostic resolution of current ALT assays in drug safety studies.

Embryonic Stem Cells

The predictive toxicity of chemicals to humans is largely based on scientific investigation in animal models. However, interspecies variability impacts the ability of certain animal models to emulate human response, which ultimately influences attrition rates in pharmaceutical development and safety assessment of new drugs. The availability of scalable human in vitro models for predictive toxicology may increase our confidence in risk assessment of chemicals in parallel to animal studies.

The Cinderella story of metabolic profiling: does metabolomics get to go to the functional genomics ball?

To date most global approaches to functional genomics have centred on genomics, transcriptomics and proteomics. However, since a number of high-profile publications, interest in metabolomics, the global profiling of metabolites in a cell, tissue or organism, has been rapidly increasing. A range of analytical techniques, including 1H NMR spectroscopy, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), Fourier Transform mass spectrometry (FT-MS), high performance liquid chromatography (HPLC) and electrochemical array (EC-array), are required in order to maximize the number of metabolites that can be identified in a matrix. Applications have included phenotyping of yeast, mice and plants, understanding drug toxicity in pharmaceutical drug safety assessment, monitoring tumour treatment regimes and disease diagnosis in human populations. These successes are likely to be built on as other analytical and bioinformatic approaches are developed to fully exploit the information obtained in metabolic profiles. To assist in this process, databases of metabolomic data will be necessary to allow the passage of information between laboratories. In this prospective review, the capabilities of metabolomics in the field of medicine will be assessed in an attempt to predict the impact this 'Cinderella approach' will have at the 'functional genomic ball'.

Role of natural killer cells in immunotoxicity: an update

As natural killer cells play a significant role in innate immunity against viruses and neoplasia, the measurement of natural killer cell activity has long been recommended for nonclinical immunotoxicity evaluation. A number of agrochemical, industrial and environmental chemicals have been shown to impair natural killer cell activity. However, direct evidence for clinically significant pathologic consequences, such as infections or immunosuppression-related cancer in human beings exposed to these chemicals, is lacking. In addition, extremely few pharmaceuticals, including potent immunosuppressive drugs, have been shown to depress natural killer cell activity reproducibly. Due to this ambiguous situation, the value of measuring natural killer cell activity for the prediction of immunotoxicity is debatable, as reflected by recommendations included in recent guidelines for the nonclinical safety assessment of human pharmaceuticals. Limitations of current analyses of natural killer cell activity may explain this situation. Progress is expected from the utilization of most recent techniques to identify more relevant natural killer cell activation markers or from studying underlying mechanisms.

Gene expression profiling for pharmaceutical safety assessment

Toxicogenomics is the application of gene expression profiling technology to toxicology. This results in the generation of very large and complex gene expression data sets associated with the development of toxicities. It is widely assumed that this data can be deconvoluted to reveal novel insights into toxicological processes that are of value to the task of risk assessment. More specifically, it is hoped that toxicogenomics will aid in the prediction of the toxic potential and mechanisms of toxicity of novel chemical entities. On the basis of such promise, the pharmaceutical industry has invested heavily in this area, as the perceived rewards in terms of improved pipeline efficiency and safer drugs are immense. Consequently, a great deal of groundwork has been done over the past several years to establish working methods in toxicogenomics, both within industry and academia, demonstrating utility in proof-of-concept studies, generating the databases on which some approaches depend, and developing new data analysis tools. Despite such activity, there is little reported evidence to suggest that toxicogenomics is making a significant impact on the discovery and development of drugs. This may partly reflect the understandable reluctance of pharmaceutical industries to share information in a competitive environment. It may also partly reflect difficulties in bridging the gap between theory and practice, as is required to deliver real value to the industry. This review will assess the successes and shortcomings of toxicogenomics, and consider how it can be usefully applied to a drug discovery pipeline.

Regulatory considerations on new adjuvants and delivery systems

New and improved vaccines and delivery systems are increasingly being developed for prevention, treatment and diagnosis of human diseases. Prior to their use in humans, all new biological products must undergo pre-clinical evaluation. These pre-clinical studies are important not only to establish the biological properties of the material and to evaluate its possible risk to the public, but also to plan protocols for subsequent clinical trials from which safety and efficacy can be evaluated. For vaccines, evaluation in pre-clinical studies is particularly important as information gained may also contribute to identifying the optimum composition and formulation process and provide an opportunity to develop suitable indicator tests for quality control. Data from pre-clinical and laboratory evaluation studies, which continue during clinical studies, is used to support an application for marketing authorisation. Addition of a new adjuvant and exploration of new delivery systems for vaccines presents challenges to both manufacturers and regulatory authorities. Because no adjuvant is licensed as a medicinal product in its own right, but only as a component of a particular vaccine, pre-clinical and appropriate toxicology studies need to be designed on a case-by-case basis to evaluate the safety profile of the adjuvant and adjuvant/vaccine combination. Current regulatory requirements for the pharmaceutical and pre-clinical safety assessment of vaccines are insufficient and initiatives are in place to develop more specific guidelines for evaluation of adjuvants in vaccines.

Safety pharmacology: an essential interface of pharmacology and toxicology in the non-clinical assessment of new pharmaceuticals

Safety pharmacology studies are defined as the studies that investigate the potential undesirable pharmacodynamic effects of a substance on physiological functions in relation to exposure. In consequence, these studies are an integral part of the non-clinical safety assessment of new pharmaceuticals, in association with toxicological studies. A retrospective shows the evolution of the discipline in these last years. Safety pharmacology studies are of special interest, and some drawbacks and pitfalls must be considered (i.e. invasive methods, difficulties related to GLP (good laboratory practices) requirements, choice of a strategy). In the future, some priority should be given to education, promotion of scientific activities, reinforcement of the links between pharmacologists and toxicologists and implementation of relevant guidelines.

Safety pharmacology: an essential interface of pharmacology and toxicology in the non-clinical assessment of new pharmaceuticals

Safety pharmacology studies are defined as the studies that investigate the potential undesirable pharmacodynamic effects of a substance on physiological functions in relation to exposure. In consequence, these studies are an integral part of the non-clinical safety assessment of new pharmaceuticals, in association with toxicological studies. A retrospective shows the evolution of the discipline in these last years. Safety pharmacology studies are of special interest, and some drawbacks and pitfalls must be considered (i.e. invasive methods, difficulties related to GLP (good laboratory practices) requirements, choice of a strategy). In the future, some priority should be given to education, promotion of scientific activities, reinforcement of the links between pharmacologists and toxicologists and implementation of relevant guidelines.

I POINTS TO CONSIDERi REGARDING SAFETY ASSESSMENT OF BIOTECHNOLOGY-DERIVED PHARMACEUTICALS IN NON-CLINICAL STUDIES (ENGLISH TRANSLATION

Regulatory and industrial scientists collaborated to publish a "points to consider" document regarding the safety assessment of biotechnology-derived pharmaceuticals in non-clinical studies in 2002 (Pharmaceutical Non-clinical Investigation Group, 2002). The collaboration team intended to clarify the interpretation of ICH-S6 guideline and furthermore share recent Japanese practices on this matter. However, the document was written in Japanese. Thus, we share here an English translation of the document so that non-native Japanese correctly understand the contents.

167 Approaches to the minimisation of dog use in the safety assessment of pharmaceuticals: An industry/animal welfare initiative

The cytotoxicity of the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) reference chemicals was investigated by measuring the increased reactive oxygen species (ROS) formation in rat hepatoma-derived Fa32 cells. ROS formation was measured with 2′,7′-dichlorodihydrofluorescein diacetate as a fluorescent probe. The results were quantified by determining the ROS50. This is the concentration of test compound required to increase the ROS formation with 50% compared with control cells. An extremely high ROS formation was observed with ferrous sulfate. Of a total of 44 chemicals, an increased ROS formation was observed for 24. This was not the case for the 20 other chemicals. When the ROS formation in Fa32 cells was compared with human toxicity, the correlation coefficient was clearly higher than for human hepatoma-derived Hep G2 cells, at least when the extremely sensitive ferrous sulfate was withdrawn from the comparison. The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Consequently, the ROS formation assay in Fa32 cells has a high predictive value for human toxicity, with the drawback that only ROS increasing chemicals can be evaluated.

Safety assessment of biotechnology-derived pharmaceuticals: ICH and beyond.

Many scientific discussions, especially in the past 8 yr, have focused on definition of criteria for the optimal assessment of the preclinical toxicity of pharmaceuticals. With the current overlap of responsibility among centers within the Food and Drug Administration (FDA), uniformity of testing standards, when appropriate, would be desirable. These discussions have extended beyond the boundaries of the FDA and have culminated in the acceptance of formalized, internationally recognized guidances. The work of the International Committee on Harmonisation (ICH) and the initiatives developed by the FDA are important because they (a) represent a consensus scientific opinion, (b) promote consistency, (c) improve the quality of the studies performed, (d) assist the public sector in determining what may be generally acceptable to prepare product development plans, and (e) provide guidance for the sponsors in the design of preclinical toxicity studies. Disadvantages associated with such initiatives include (a) the establishment of a historical database that is difficult to relinquish, (b) the promotion of a check-the-box approach, i.e., a tendancy to perform only the minimum evaluation required by the guidelines, (c) the creation of a disincentive for industry to develop and validate new models, and (d) the creation of state-of-the-art guidances that may not allow for appropriate evaluation of novel therapies. The introduction of biotechnology-derived pharmaceuticals for clinical use has often required the application of unique approaches to assessing their safety in preclinical studies. There is much diversity among these products, which include the gene and cellular therapies, monoclonal antibodies, human-derived recombinant regulatory proteins, blood products, and vaccines. For many of the biological therapies, there will be unique product issues that may require specific modifications to protocol design and may raise additional safety concerns (e.g., immunogenicity). Guidances concerning the design of preclinical studies for such therapies are generally based on the clinical indication. Risk versus benefit decisions are made with an understanding of the nature of the patient population, the severity of disease, and the availability of alternative therapies. Key components of protocol design for preclinical studies addressing the risks of these agents include (a) a safe starting dose in humans, (b) identification of potential target organs, (c) identification of clinical parameters that should be monitored in humans, and (d) identification of at-risk populations. One of the distinct aspects of the safety evaluation of biotechnology-derived pharmaceuticals is the use of relevant and often nontraditional species and the use of animal models of disease in preclinical safety evaluation. Extensive contributions were made by the Center for Biologics Evaluation and Research to the ICH document on the safety of biotherapeutics, which is intended to provide worldwide guidance for a framework approach to the design and review of preclinical programs. Rational, scientifically sound study design and early identification of the potential safety concerns that may be anticipated in the clinical trial can result in preclinical data that facilitate use of these novel therapies for use in humans without duplication of effort or the unnecessary use of animals.

Effects of Diet and Overfeeding On Body Weight and Survival in the Rodent Bioassay: the Impact On Pharmaceutical Safety Assessment

Uncontrolled body weight due to ad libitum (AL) overfeeding is the most significant variable affecting the rodent bioassay. The correlation between AL food consumption, the resultant excessive adult body weight, and the low 2-yr survival in Sprague-Dawley (SD) rats was highly significant. However, initial body weight does not correlate with adult body weight or survival. AL feeding of diets that varied in protein, fiber, and energy content did not improve low 2-yr survival. Only moderate dietary restriction (DR) of all diets tested significantly improved survival and delayed the onset or severity of spontaneous degenerative diseases and tumors. Moderate DR-fed rats had a similar incidence of tumors as AL-fed rats by 2 yr; however, the tumors were more likely to be incidental and not fatal. There was a decreased age-adjusted incidence of pituitary and mammary-gland tumors, but tumor volume and growth time were similar between AL and moderate DR groups. These data indicate a similar tumor progression, but only a delay in tumor onset. Moderate DR did not significantly alter drug-metabolizing enzyme activities nor the toxicologic response to five pharmaceuticals tested at maximum tolerated doses (MTDs). However, moderate DR did require higher doses of compounds to be given before classical MTDs were produced with four drug candidates. Toxicokinetic studies of two of these compounds demonstrated steady-state systemic exposures that were equal or higher in moderate DR-fed rats. Biochemical markers of oxidative stress (lipid peroxidation, protein oxidation) were decreased and cytoprotective markers were increased by moderate DR. These and other data indicate that moderate DR is the most appropriate method of body weight control when used to assess pharmaceuticals for human safety.

Issues with introducing new immunotoxicology methods into the safety assessment of pharmaceuticals

Unfortunately, the principal routine toxicology methods employed for the safety assessment of new products are over 40 years old and rely primarily on histopathological evaluation. It is difficult to introduce newer immunotoxicology or molecular toxicology methods into toxicity assessment without extensive and time consuming validation requiring several years due to concern for standardization of methods, inter-laboratory replication of these methods and resistance to acceptance of new methods by both regulatory agencies and industry. During the past 15 years, significant progress has occurred in the fields of molecular biology and basic/clinical immunology which promoted the establishment of newer more sensitive methods to assess cell injury or immune system effects in humans and laboratory animals. This brings us to the challenges associated with trying to introduce new immunotoxicology or molecular toxicology methods into the safety assessment of new products. Our experience at Sanofi Research with immunotoxicity methods development or validation and approaches for molecular toxicology and their application to the preclinical development of new chemical drug entities (NCE) will be discussed. Laboratories to investigate immunotoxicity and molecular toxicology were established during the past 10 years among several industrial research groups for the evaluation of new chemicals and drug candidates. Immunotoxicology methods have been selected and optimized for rodent testing leading to four inter-laboratory collaborative studies to demonstrate the reproducibility and value of these methods for predicting toxicity. Our experience at Sanofi has led us to believe that these newer methods can represent an important part of drug development, should be applied on an as needed basis, and should be driven by data suggestive of an immune or molecular toxicology effect or by the class of the chemical being evaluated. Ex vivo and in vitro assays are selected from a menu of validated methods for application on a case-by-case basis. Results in this area with inter-laboratory validation of these methods will be discussed. Newer molecular toxicology and immunotoxicity methods are beginning to play a more important role in the safety evaluation and risk assessment process.

Evaluation of testicular toxicity in safety evaluation studies: the appropriate use of spermatogenic staging.

Toxicology of the male reproductive system has received increased interest in recent years partly fuelled by the growing reports of falling sperm counts and rising reproductive disorders in the human population. Recently revised regulatory guidelines for the safety assessment of pharmaceuticals and chemicals on reproduction and fertility have emphasized the importance of detailed histopathological examination of the testes as a sensitive method for detecting disturbances in spermatogenesis. Unfortunately this has been accompanied by a general confusion regarding a practical approach to undertaking such a detailed examination, particularly in respect to the use of spermatogenic or tubular staging to identify subtle disturbances in spermatogenesis. The ability to identify tubular stages of the spermatogenic cycle in sections of testis plus a good understanding of the spermatogenic process and its dynamics are essential in order to carry out a sensitive of testicular histopathology and to interpret the changes seen. A rational approach is required initially to detect and subsequently to characterize toxic effects to the male reproductive system. It is important that a distinction is made between these two objectives since different study designs are required and different methodology may be employed to produce the type of information or data required.

Safety assessment of pharmaceuticals: examples of inadequate assessments and a mechanistic approach to assuring adequate assessment.

For a conventional organic new chemical entity (NCE) being developed as a pharmaceutical, standard regulatory safety assessment studies are required. Early in development, an NCE should undergo a safety/benefit analysis to justify further development. This analysis is made easier and more effective when comprehensive nonclinical data are available. One of the most important aspects of nonclinical toxicologic studies is to provide information on absence of potential carcinogenicity in humans. To avoid human exposures to potentially carcinogenic agents, even in early development of an NCE, the Decision Point Approach to carcinogen testing provides a useful guide to acquisition of mechanistically relevant data for risk assessment.

Immunotoxicity of Pharmaceuticals: An Introduction

The immune-mediated side effects of pharmaceuticals include lymphomas and infectious complications related to immunosuppression and immunodepression; flu-like reactions, exacerbation of allergy to unrelated allergens, and de novo induction of autoimmunity, associated with immunoenhancement; drug-induced hypersensitivity (allergic) reactions; and finally, organ-specific or systemic autoimmune diseases. A number of pharmaceuticals have been reported to induce one or several of these immune-mediated side-effects. Even though few guidelines have been published, every effort should be paid to predicting these side effects as part of the preclinical safety assessment of new pharmaceuticals. Although they are relatively infrequent they are sometimes severe, or even life-threatening. Unexpected immunosuppression, and possibly immunoenhancement, can be best addressed by tiered protocols in rodents. Contact sensitization assays or systemic anaphylaxis models can be instrumental in several instances. Autoimmunity is...

Third International Course on the Safety Assessment of Pharmaceuticals

Third International Course on the Safety Assessment of Pharmaceuticals