Value in global standardization of outbred rats for regulatory pharmaceuticals studies.

As a result of a revolution in globalization over the last 10 years, few pharmaceutical companies conduct their non-clinical studies within a single region and virtually none conduct all non-clinical studies in house. The increased activity of product in-licensing at all stages of development results in further segmentation of toxicological source information. Juxtaposed to this segmental collection of toxicology information is the nature of toxicological testing for pharmaceuticals, an iterative process, carefully building subsequent testing designs and analyses upon the foundation of previously identified outcomes and associated safety issues. As a consequence of the segmentation of global product development, today's analyses of toxicological outcomes for single pharmaceutical projects often involve a conglomeration of results from studies conducted in several world regions using a variety of sources of animals. Such practices can lead to discordant study results and difficulty in understanding or rationalizing a compound's toxicological and pharmacological profile, and eventually how this relates to human risk. Despite the variety in source information, an integration of the total toxicology/pharmacology data must be made by industry, and/or by health authorities. To improve this integration, the development and use of better standardized genetics for rodent species used in pharmaceutical testing is more imperative today than in the past, as a result of a constellation of changes in industry policies.     

Practical aspects of mutagenicity testing strategy: an industrial perspective

Genetic toxicology studies play a central role in the development and marketing of new chemicals for pharmaceutical, agricultural, industrial, and consumer use. During the discovery phase of product development, rapid screening tests that require minimal amounts of test materials are used to assist in the design and prioritization of new molecules. At this stage, a modified Salmonella reverse mutation assay and an in vitro micronucleus test with mammalian cell culture are frequently used for screening. Regulatory genetic toxicology studies are conducted with a short list of compounds using protocols that conform to various international guidelines. A set of four assays usually constitutes the minimum test battery that satisfies global requirements. This set includes a bacterial reverse mutation assay, an in vitro cytogenetic test with mammalian cell culture, an in vitro gene mutation assay in mammalian cell cultures, and an in vivo rodent bone marrow micronucleus test. Supplementary studies are conducted in certain instances either as a follow-up to the findings from this initial testing battery and/or to satisfy a regulatory requirement. Currently available genetic toxicology assays have helped the scientific and industrial community over the past several decades in evaluating the mutagenic potential of chemical agents. The emerging field of toxicogenomics has the potential to redefine our ability to study the response of cells to genetic damage and hence our ability to study threshold phenomenon.     

The emergence of the genetically engineered animal models in carcinogenic risk assessment of pharmaceuticals: a case study of process innovation

In this article, we explain the emergence of new short-term tests for carcinogenicity involving genetically engineered animals for the purposes of pharmaceutical regulation. Drawing on some long-standing theories of technological innovation, we argue that the alteration of carcinogenic risk assessment of pharmaceuticals, which occurred from 1998, did not result solely, or perhaps even mainly, from internal logical and technical developments in the experimental sciences of toxicology or genetics. Rather, this process innovation in regulatory science resulted from a complex interaction between scientist activism around molecularization of toxicology in powerful US government institutions, on the one hand, and a powerful research-based trans-national pharmaceutical industry committed to deregulatory mobilization, on the other, seeking to reduce carcinogenicity testing of its products and capable of marshalling significant support from governments and regulators, especially in Europe and Japan, to achieve that goal. The new techno-scientific basis for regulatory decisions about whether pharmaceuticals are carcinogenic risks to the public was, in effect, an accommodation in “bio-political” trading between two power-blocks of expert scientists. Those from industry and their regulatory allies in Europe and Japan, who sought reductions in “the burden” of drug testing, on the one hand, and FDA scientists, who did not accept the simple “reduction” agenda, but were interested in shifting the paradigm of carcinogenicity testing toward geneticization, on the other.     

Integrating computer-based de novo drug design and multidimensional filtering for desirable drugs

In the pharmaceutical industry today, many of the potent compounds discovered using expensive technologies are eventually rejected because of poor physicochemical or absorption, distribution, metabolism, excretion and toxicology (ADME/Tox) properties. This problem can be addressed by placing fast and accurate computational technologies at the heart of drug discovery. Chemically diverse and potent compounds generated by de novo design algorithms are scored for ADME/Tox properties using rigorously validated statistical models. Every molecule passing through this in silico pipeline is thus associated with a wealth of predicted properties, thereby allowing for rapid assessment to determine which molecule should be further developed. Critical to this idea is a platform that allows for the efficient exchange of in silico and experimental data between all scientists regardless of specialization. By bridging the gap between the in silico and experimental cultures in this fashion, an information-driven, cost-effective drug discovery program can be realized.     

High-throughput screening: update on practices and success

High-throughput screening (HTS) has become an important part of drug discovery at most pharmaceutical and many biotechnology companies worldwide, and use of HTS technologies is expanding into new areas. Target validation, assay development, secondary screening, ADME/Tox, and lead optimization are among the areas in which there is an increasing use of HTS technologies. It is becoming fully integrated within drug discovery, both upstream and downstream, which includes increasing use of cell-based assays and high-content screening (HCS) technologies to achieve more physiologically relevant results and to find higher quality leads. In addition, HTS laboratories are continually evaluating new technologies as they struggle to increase their success rate for finding drug candidates. The material in this article is based on a 900-page HTS industry report involving 54 HTS directors representing 58 HTS laboratories and 34 suppliers.     

The selection of marmoset monkeys (Callithrix jacchus) in pharmaceutical toxicology

Prior to controlled clinical trials in human volunteers or patients it is required that novel pharmaceuticals are evaluated for pre-clinical safety in a rodent and a non-rodent ('second') species. In most cases the rodent species used has been the rat and the second species has been the dog or macaque (usually cynomolgus or rhesus) monkey. However, there is an increasing trend within the United Kingdom (UK) pharmaceutical industry to use the common marmoset (Callithrix jacchus) for pre-clinical toxicology programmes. This paper examines the practicality of using the common marmoset (henceforth referred to as 'the marmoset') in toxicological testing and reviews metabolic and pharmacodynamic similarities between this species and humans. It then discusses some of the advantages and disadvantages of the use of this species when compared with two other alternatives to the dog and macaque, namely the ferret and minipig. In particular, the marmoset has clear advantages over the macaque in terms of animal welfare and practicality. There is regulatory acceptance of this species for Investigational New Drug (IND), Clinical Trial Exemption (CTX), New Drug Application (NDA) and Marketing Authorization Application (MAA) registrations. Whilst the dog is likely to be maintained as the primary non-rodent species in toxicology, the marmoset has been, and will likely continue to be, adopted as an additional non-rodent species in pre-clinical toxicology programmes where appropriate.     

Government Regulations and the Use of Drugs

I have tried to trace the new drug development pattern from 1766, when Withering obtained his medical degree, to the present.The role of governmental authority as defined by the 1962 Kefauver-Harris amendments to the 1906 law and the subsequently issued regulations has been summarized. Four phases of testing in man have been detailed.Something of the scientific or research capability of the pharmaceutical industry has been presented.It is concluded that in the period of over two hundred years of medical education in the United States, the university hospital has become more and more the focus of medical research, teaching and practice in the community. The safety and effectiveness in the use of drugs in the future will depend upon the liaison and rapport of the industry physicians, government officials and the university hospital teacher-clinical investigators (phase 1 and 2) in designing the most critical studies of the safety and effectiveness of new drugs.Whether the medical profession as we know it will participate more in the future than has been possible since 1962 in mass clinical trial (phase 3) before new drug approval by governmental authority remains to be seen.The final approbation or disapproval of a drug after NDA approval (phase 4) will continue to be in the hands of the participating physician as long as he can establish scientifically that the drug is the best possible agent for him to use in healing the sick and comforting the dying.     

Toxicogenomics in drug discovery: from preclinical studies to clinical trials

Gene expression analysis applied to toxicology studies, also referred to as toxicogenomics, is rapidly being embraced by the pharmaceutical industry as a useful tool to identify safer drugs in a quicker, more cost-effective manner. Studies have already demonstrated the benefits of applying gene expression profiling towards drug safety evaluation, both for identifying mechanisms underlying toxicity, as well as for providing a means to identify safety liabilities early in the drug discovery process. Furthermore, toxicogenomics has the potential to better identify and assess adverse drug reactions of new drug candidates or marketed products in humans. While much still remains to be learned about the relevance and the application of gene expression changes in human toxicology, the next few years should see gene expression technologies applied to more stages and more programs of the drug discovery and development process. This review will focus on how toxicogenomics can or has been applied in drug discovery and development, and will discuss some of the challenges that still remain.     

ECVAM-ICCVAM: prospects for future collaboration

The level and complexity of testing for hazard and risk assessment of marketed products and environmental agents has increased substantially over time, resulting in the use of greater numbers of both animals and humans for testing. Today, industry and regulatory bodies worldwide face increasing pressures to demonstrate responsible utilisation of laboratory animals, to limit their use, and to employ alternative non-animal tests. Institutions have also been established to identify, encourage development of, conduct research on, and validate new, improved, and surrogate test methods that will reduce and replace animal use. Two such organisations are ECVAM and the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM). As the evolutionary changes occurring in the field of toxicology result in an unprecedented increase in the introduction of alternative methodologies, these will strain the capacities of such alternative methods institutions. That realisation is causing a shift in thinking and creating an impetus to seek approaches by which to collaborate and develop more-efficient operational procedures for the validation and regulatory acceptance of alternative methods. Similarities in objectives, functions, scientific standards, and commitment to the principles of validation and animal welfare support the value of a cooperative arrangement between ECVAM and ICCVAM, to minimise duplication of effort, maximise productivity, and influence the international adoption of alternative tests. Opportunities for ECVAM-ICCVAM collaboration are discussed, which illustrate the feasibility and potential benefits of such a partnership.     

Mysids in toxicity testing — a review

The use of mysid shrimp, particularly the genusMysidopsis, along with specific testing procedures, has become accepted in aquatic toxicology. Investigators have developed methodologies for both culture and testing of these organisms. Acute and chronic (life cycle) toxicity tests in addition to dredge spoil and effluent tests with mysids are now becoming common. Attempts have been made to use mysids as test organisms in behavioral, physiological, nutritional, and food-chain studies. In general,Mysidopsis spp. have been shown to be as sensitive or more sensitive to toxic substances than other marine species tested. The ease of handling and culture, relative sensitivity to toxicants, short life cycle, small size and direct larval development make these organisms desirable for research purposes. Continued research using mysid species will probably demonstrate even greater usefulness of these organisms in assessment of pollutant impacts on estuarine or marine communities.     

Short-term testing--are we looking at wrong endpoints?

Short-term testing has been performed and interpreted on the basis of correlation between these tests and animal carcinogenicity. This empirical approach has been the only feasible one, due to a lack of knowledge of the actual genetic endpoints of relevance in carcinogenicity. However, the rapidly growing information on genetic alterations actually involved in carcinogenicity and in particular activation of oncogenes, provides facts of basic importance for the strategy of short-term testing. The presently used sets of short-term tests focus on standard genetic endpoints, mainly point mutations and chromosomal aberrations. Little attention has been paid in that connection to other endpoints, which have been shown or suspected to play an important role in carcinogenicity. These endpoints include gene amplification, transpositions, hypomethylation, polygene mutations and recombinogenic effects. Furthermore, indirect effects, for instance via radical generation and an imbalance of the nucleotide pool, may be of great significance for the carcinogenic and cocarcinogenic effects of many chemicals. Modern genetic and molecular technology has opened entirely new prospects for identifying genetic alterations in tumours and in its turn these prospects should be taken advantage of in order to build up more sophisticated batteries of assays, adapted to the genetic endpoints actually demonstrated to be involved in cancer induction. Development of new assay systems in accordance with the elucidation of genetic alterations in carcinogenicity will probably constitute one of the most important areas in genetic toxicology in the future. From a regulatory point of view the prerequisite for a development in this direction will be a flexibility of the handling of questions concerning short-term testing also at a bureaucratic level.     

Drug delivery and nanoparticles:applications and hazards

The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly. Currently many substances are under investigation for drug delivery and more specifically for cancer therapy. Interestingly pharmaceutical sciences are using nanoparticles to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient. The kind of hazards that are introduced by using nanoparticles for drug delivery are beyond that posed by conventional hazards imposed by chemicals in classical delivery matrices. For nanoparticles the knowledge on particle toxicity as obtained in inhalation toxicity shows the way how to investigate the potential hazards of nanoparticles. The toxicology of particulate matter differs from toxicology of substances as the composing chemical(s) may or may not be soluble in biological matrices, thus influencing greatly the potential exposure of various internal organs. This may vary from a rather high local exposure in the lungs and a low or neglectable exposure for other organ systems after inhalation. However, absorbed species may also influence the potential toxicity of the inhaled particles. For nanoparticles the situation is different as their size opens the potential for crossing the various biological barriers within the body. From a positive viewpoint, especially the potential to cross the blood brain barrier may open new ways for drug delivery into the brain. In addition, the nanosize also allows for access into the cell and various cellular compartments including the nucleus. A multitude of substances are currently under investigation for the preparation of nanoparticles for drug delivery, varying from biological substances like albumin, gelatine and phospholipids for liposomes, and more substances of a chemical nature like various polymers and solid metal containing nanoparticles. It is obvious that the potential interaction with tissues and cells, and the potential toxicity, greatly depends on the actual composition of the nanoparticle formulation. This paper provides an overview on some of the currently used systems for drug delivery. Besides the potential beneficial use also attention is drawn to the questions how we should proceed with the safety evaluation of the nanoparticle formulations for drug delivery. For such testing the lessons learned from particle toxicity as applied in inhalation toxicology may be of use. Although for pharmaceutical use the current requirements seem to be adequate to detect most of the adverse effects of nanoparticle formulations, it can not be expected that all aspects of nanoparticle toxicology will be detected. So, probably additional more specific testing would be needed.     

The use of functional human tissues in drug development

Fresh, functional human tissues have long been considered the closest possible model of human in vivo function and can be used to measure a wide range of pharmacological responses. Despite this, relatively little drug development is conducted using fresh human tissue because of the logistical and ethical difficulties surrounding the availability of tissue and practicalities of experimental work. Most tests of drug activity require a living test system comprising cells, tissues or whole organisms. In some instances, “living” (fresh) human tissues have the potential to reduce or replace animal tests through superior prediction of drug safety and efficacy. Before functional human tissue tests become a routine part of drug development, two factors must co-exist. Firstly, organisations such as Biopta must continue to create compelling evidence that human tissues are more predictive than alternative models; such evidence will drive demand from the pharmaceutical industry for human tissue-based tests. Secondly, the vast number of tissues and organs residual to surgery or unsuitable for transplant must be routinely consented for medical research and made available to all researchers in an equitable and timely manner. This requires a concerted effort throughout the NHS and consistent demand as well as financial support from researchers, particularly within industry. It is our view that the next 5–10 years will generate compelling evidence of the value of functional human tissue-based tests and recognition that more efficient use of residual or non-transplantable tissues and organs is an urgent priority for the development of new medicines.     

High-throughput human metabolism and toxicity analysis

Poor drug candidate safety profiles are often identified late in the drug development process, manifesting themselves in the preclinical and clinical phases and significantly contributing to the high cost and low yield of drug discovery. As a result, new tools are needed to accelerate the assessment of drug candidate toxicity and human metabolism earlier in the drug development process, from primary drug candidate screening to lead optimization. Although high-throughput screens exist for much of the discovery phase of drug development, translating such screening techniques into platforms that can accurately mimic the human in vivo response and predict the impact of drug candidates on human toxicology has proven difficult. Nevertheless, some success has been achieved in recent years, which may ultimately yield widespread acceptance in the pharmaceutical industry.     

Framework for validation and implementation of in vitro toxicity tests

Summary The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community—academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.     

Genetic toxicology: can we design predictive in vivo assays?

The present analysis examines the assumptions in, the perceptions and predictivity of and the need for short-term tests (STTs) for genotoxicity in light of recent findings that most noncarcinogens from the National Toxicology Program are genotoxic (i.e., positive in one or more in vitro STTs). Reasonable assumptions about the prevalence for carcinogens (1-10% of all chemicals), the sensitivity of these STTs (ca. 90% of all carcinogens are genotoxic) and their estimated "false positive" incidence (60-75%) imply that the majority of chemicals elicit genotoxic responses and, consequently, that most in vitro genotoxins are likely to be noncarcinogenic. Thus, either the usual treatment conditions used in these in vitro STTS are producing a large proportion of artifactual and meaningless positive results or else in vitro mutagenicity is too common a property of chemicals to serve as a useful predictor of carcinogenicity or other human risk. In contrast, the limited data base on in vivo STTs suggests that the current versions of these assays may have low sensitivity which appears unlikely to improve without dropping either their 'short-term' aspect or the rodent carcinogenicity benchmark. It is suggested that in vivo genotoxicity protocols be modified to take into consideration both the fundamentals of toxicology as well as the lessons learned from in vitro genetic toxicology. In the meantime, while in vivo assays are undergoing rigorous validation, genetic toxicology, as currently practiced, should not be a formal aspect of chemical or drug development on the grounds that it is incapable of providing realistic and reliable information on human risk. It is urged that data generated in new, unvalidated in vivo genotoxicity assays be exempted from the normal regulatory reporting requirements in order to encourage industry to participate in the laborious and expensive development of this next phase of genetic toxicology.     

Strategy for genotoxicity testing--metabolic considerations

The report from the 2002 International Workshop on Genotoxicity Tests (IWGT) Strategy Expert Group emphasized metabolic considerations as an important area to address in developing a common strategy for genotoxicity testing. A working group convened at the 2005 4th IWGT to discuss this area further and propose practical strategy recommendations. To propose a strategy, the working group reviewed: (1) the current status and deficiencies, including examples of carcinogens "missed" in genotoxicity testing, established shortcomings of the standard in vitro induced S9 activation system and drug metabolite case examples; (2) the current status of possible remedies, including alternative S9 sources, other external metabolism systems or genetically engineered test systems; (3) any existing positions or guidance. The working group established consensus principles to guide strategy development. Thus, a human metabolite of interest should be represented in genotoxicity and carcinogenicity testing, including evaluation of alternative genotoxicity in vitro metabolic activation or test systems, and the selection of a carcinogenicity test species showing appropriate biotransformation. Appropriate action triggers need to be defined based on the extent of human exposure, considering any structural knowledge of the metabolite, and when genotoxicity is observed upon in vitro testing in the presence of metabolic activation. These triggers also need to be considered in defining the timing of human pharmaceutical ADME assessments. The working group proposed two strategies to consider; a more proactive approach, which emphasizes early metabolism predictions to drive appropriate hazard assessment; and a retroactive approach to manage safety risks of a unique or "major" metabolite once identified and quantitated from human clinical ADME studies. In both strategies, the assessment of the genotoxic potential of a metabolite could include the use of an alternative or optimized in vitro metabolic activation system, or direct testing of an isolated or synthesized metabolite. The working group also identified specific areas where more data or experiences need to be gained to reach consensus. These included defining a discrete exposure action trigger for safety assessment and when direct testing of a metabolite of interest is warranted versus the use of an alternative in vitro activation system, a universal recommendation for the timing of human ADME studies for drug candidates and the positioning of metabolite structural knowledge (through in silico systems, literature, expert analysis) in supporting metabolite safety qualification. Lastly, the working group outlined future considerations for refining the initially proposed strategies. These included the need for further evaluation of the current in vitro genotoxicity testing protocols that can potentially perturb or reduce the level of metabolic activity (potential alterations in metabolism associated with both the use of some solvents to solubilize test chemicals and testing to the guidance limit dose), and proposing broader evaluations of alternative metabolic activation sources or engineered test systems to further challenge the suitability of (or replace) the current induced liver S9 activation source.     

Proof of Safety in Toxicology Based on the Ratio of Two Means for Normally Distributed Data

The aim of toxicology testing is the safety assessment of a substance such as a new pharmaceutical compound or a pesticide relative to a vehicle or negative control. This paper is concerned with the direct safety approach when safety is expressed in terms of the ratio of population means and when the toxicological endpoint is normally distributed. The methodology for power and sample size calculation is derived for the parallel group design and results are presented for various situations.     

Uses and future applications of flow cytometry in immunotoxicity testing.

Flow cytometry is an emerging technology that has numerous applications to immunotoxicity testing. The use and development of high-speed single-cell laser-based assays capable of quantitation of fluorescence, light scatter, and electrical impedance measurements can provide important information on xenobiotic-induced toxicity in defined target cell populations. The purpose of this article is to briefly review established and emerging immunotoxicology assays that use flow cytometry. In the coming years it is likely that many new flow cytometry assays will be developed and validated that will improve the sensitivity and perhaps specificity of immunotoxicity testing. Since flow cytometry is readily adaptable to high-throughput screening, it is also likely that this technology will increasingly find its place in the preclinical testing of drugs and chemicals in the pharmaceutical and chemical industries.