With the rise of combination therapy – the use drugs with different mechanisms of action to combat a specific disease state – comes the need to address medical costs and reimbursement issues. Joint negotiation of package deals with government and health insurers may prove useful, particularly for companion diagnostics and treatment of chronic conditions. Companies that share drug development risks and costs (preclinical, clinical trials, sales and marketing, etc.) with each other are not only better positioned to negotiate for reimbursement but are also better poised to defend against competition. Multiple collaborations, however, increase the risk of legal complexity for all concerned.
A previous entry detailed Dried Blood Spot Analysis: Preclinical Pros and Cons. Additional preclinical considerations include the ambiguity of acceptance by global regulatory agencies, none of which have issued definitive rulings on how they’ll handle New Drug Applications (NDA) that use the technique. Furthermore, although validation standards and regulatory guidance exist for liquid assays, many of the suggested parameters (e.g., reproducibility after freezing and thawing of samples) are not applicable to dried blood spot analyses, where samples are dried and stored at room temperature.
Physical parameters also affect dried matrix spotting. Blood spot size is partly dependent on hematocrit, the percentage of the blood volume composed of red blood cells. Hematocrit is not only variable between individuals but also varies daily within a given individual. Therefore given sample dilution based on variable hematocrit, analyte levels can vary widely between individual samples. As a further development, the heightened analytical sensitivity used in nonclinical drug development (relative to the more traditional clinical uses) has mandated more stringent standards for blotter paper.
Another preclinical use for this technique is analysis of other limited-volume body fluids (e.g., synovial fluid, tears, and cerebrospinal fluid), some of which have not been routinely sampled preclinically in the past due to inefficient methodology. For example, arthritis mostly affects biomarkers in synovial fluid. In rodent preclinical models, however, only a few microliters of synovial fluid exist in each joint. This has forced preclinical scientists to rely on surrogate markers in the animal’s plasma to monitor drug efficacy/toxicity. By utilizing dried matrix spotting, rodent joints can now be sampled directly. Furthermore, due to the generally colorless nature of alternate fluids, proprietary paper treatments have been identified to allow for color changes that facilitate spot identification. As an additional benefit, alternate fluid analyses lack the inherent variability due to hematocrit.
Dried matrix spotting is quickly overcoming perceived challenges. It remains to be seen whether the heralded FDA Strategic Priorities for 2011-2015, which include advancing the field of Regulatory Science, will promote advancement/acceptance of dried matrix spotting as part of it’s mandate to develop new tools, standards, and approaches to assess the safety, effectiveness, quality, and performance of FDA-regulated products. Stay tuned…!
Any new drug that penetrates the central nervous system must receive some preclinical analysis of abuse liability potential (Draft Guidance). Usually, it is determined through prior knowledge of chemistry and/or pharmacology of the candidate’s drug class that compounds have abuse liability. For example, if a test article stimulates release of dopamine in the nucleus accumbens of the brain, most likely it will be abused by humans (Koob and Volkow, 2010). Discussion of nonclinical abuse liability testing requirements with regulators prior to submission of any formal materials, however, is always advised.
Behavioral pharmacology is only one facet for determining abuse liability; however, preclinical drug discrimination and self-administration data speak loudly. As outlined in O’Connor et. al. (2011), several factors can influence nonclinical drug self-administration data. Animal strain, training regimen, food restriction, duration of access, rate of infusion, and training doses can all influence self-administration data (Baladi et. al., 2010; Banks and Negus, 2010; Caroll, 1985; Kosten et. al., 1997; Lynch et. al., 2010; Woolverton, 1992). Misleading self-administration data can lead to program-killing false-positives or underestimated abuse liability that will manifest during clinical trials. Something as “unimportant” as the dose of the training compound can impact drug discrimination. Too high or too low of a training dose may alter the interoceptive cue of test article and shift dose response curves accordingly when the test article is screened (e.g., Mumford and Holtzman, 1991). These results would drastically affect interpretation of safety margin. Unchecked variables can significantly impact analysis and delay submissions. Although regulators are savvy to these variables, to the classically trained chemist, for example, these variables can seem like smoke and mirrors without the proper experience.
Daily monitoring of behavioral data and animals (weights, response patterns, and general health) is necessary to determine whether preclinical studies are being carried out properly and are subsequently valid. One must be aware that self-administration and drug discrimination studies usually take several months to complete, with animals generating data daily. Failure to incorporate appropriate controls such as presenting “inactive” levers and recording inactive lever responses can render a study invalid; this practice serves as an index of accuracy (O’Connor et. al., 2011). Additionally, catheter patency in rats used for self-administration studies is not a trivial concern. An impaired catheter can seriously alter response patterns. The same animal may alter behavior over time due to time-dependent physiological changes (e.g., behavioral tolerance) or a faulty catheter. Behavioral criteria must be established well in advance in order to accurately track animal response patterns. Frequent catheter patency tests should regularly occur.
Several nonclinical laboratories (especially academic) combat less than aseptic conditions with daily administration of antibiotics to their experimental animals to maintain catheter patency and animal health for lengthy self-administration experiments. Body weights must be maintained at certain levels to ensure motivated animals. If an animal is food restricted for eight months and administered daily antibiotics, will this create problems with your compound? Concomitant effects can potentially lead to additional toxicology studies if you have unexpected clinical signs or abnormal clinical pathology findings.
Some contract research organizations (CRO) may suggest using their “trained” animals, usually non-human primates, for preclinical drug discrimination and self-administration studies. Will the drug history of these animals pose a problem? Should you instead consider use of rats over non-human primates? At this point in time, if the metabolism and kinetics of your compound are similar in rats and humans, use of non-human primates is not necessary and may not be justified from an animal welfare standpoint (O’Connor et. al., 2011). Moreover, the behavioral database for rats is just as strong as for non-human primates (O’Connor et. al., 2011). The benefits of using non-human primates, however, are multiple. A CRO can maintain a small colony of non-human primates that are trained to self-administer or discriminate drugs of abuse for years. For this reason, animals are essentially ready for screening at initiation of the study. One should consider, however, that non-naive animals may have impacted health due to long histories of handling, laboratory conditions, implanted devices (in self-administration animals), and a history of drugs that may impact physiology and/or behavior. Will this confluence of factors negatively interact with your compound?
In conclusion, behavioral pharmacology studies should not be taken lightly, and possession of the necessary expertise and skills to navigate these challenges is necessary. Lack of experience in what was once considered a “soft science” can be extremely detrimental in drug development, costing additional time and money. Just like any scientific assessment, there are “correct” and “incorrect” ways of conducting behavioral pharmacology experiments. For this reason, many large pharmaceutical companies and CROs now have expert working groups for abuse liability screening.
References Cited
Baladi MG, Newman AH, France CP. Dopamine D3 receptors mediate the discriminative stimulus effects of quinpirole in free-feeding rats. J Pharmacol Exp Ther. 2010 Jan; 332(1):308-15.
Draft Guidance for Industry Assessment of Abuse Potential of Drugs (January, 2010) prepared by the Controlled Substance Staff (CSS) in the Center for Drug Evaluation and Research (CDER) at the Food and Drug Administration.
Kosten TA, Miserendino MJ, Haile CN, et. al. Acquisition and maintenance of intravenous cocaine self-administration in Lewis and Fischer inbred rat strains. Brain Res. 1997; 778(2):418-29.
Lynch WJ, Nicholson KL, Dance ME, et. al. Animal models of substance abuse and addiction: implications for science, animal welfare, and society. Comp Med. 2010; 60(3):177-88.
Mumford GK, Holtzman SG. Qualitative differences in the discriminative stimulus effects of low and high doses of caffeine in the rat. J Pharmacol Exp Ther. 1991 Sep; 258(3):857-65.
O’Connor EC, Chapman K, Butler P, Mead AN. The predictive validity of the rat self-administration model for abuse liability. Neurosci Biobehav Rev. 2011; 35(3):912-38.
Paul Kruzich is an experienced abuse liability and safety pharmacology consultant. He has extensive industrial/CRO experience as a study director and academic experience as a tenure-track faculty member at the Medical College of Georgia. His professional affiliations include the College on Problems of Drug Dependence, Safety Pharmacology Society, Society of Toxicology, and Society for Neuroscience. Dr. Kruzich has authored over 23 peer-reviewed articles and 2 book chapters and has served as a reviewer for over 5 scientific journals.
In order to keep our competitive edge, the Federal Drug Administration (FDA) is placing increased emphasis on strengthening both the field and application of regulatory science relative to pharmaceutical research, development, review, and post-market surveillance. The FDA also has a mandate to recognize areas of unmet public health need and try to galvanize action to move appropriate new products through the pipeline and into the market. The FDA has the responsibility, therefore, not just to review and approve products if the data support that decision, but also to follow these products once marketed to answer critical questions about efficacy and safety. Examination of products across their life cycle enables not only the identification and analysis of emerging safety signals, but also facilitates the continual balancing of risks and benefits.
Research studies, both preclinical and clinical, that form the basis for approval of medical products are increasingly being performed in other countries and often in networks of other countries. For this reason, international recognition of both the scientific appropriateness and ethical conduct of those studies becomes increasingly important to global regulatory bodies. A key understanding is that if a safety concern develops for an approved drug, it does not necessarily reflect that a mistake was made. It may instead reflect new emerging knowledge about that drug in practical use. Regulatory safety has to be a dynamic process. The desire is to proactively ensure that the right studies are done so that the best possible decisions result. However, there isn’t always an absolute, clear decision to be made; resolution, therefore, requires a dynamic balancing of risks and benefits. Questions need to be asked about whether certain subpopulations of patients may benefit from targeted use of a drug, or whether the safety concerns are sufficient to mean a more active withdrawal of a product from the market. Advances in science and technology need to be better incorporated into the regulatory process, with a key area being safety science. To continue to strengthen the science of regulatory safety, the need is to broaden not only the kinds of preclinical and clinical studies that can be done to deepen our understanding of safety, but also to broaden our understanding of how to apply and weight that data to further the science of risk management.
Source: Interview between Dr. Eli Adashi, Professor of Medical Science at Brown University and host of Medscape One-on-One, and Dr. Margaret Hamburg, Commissioner of the US Food and Drug Administration. MedScape Today.
Both advantages and challenges exist for use of dried blood spots during preclinical drug development. Advantages include small sample volumes coupled with easy shipment and storage. The amount of blood per spot varies (10 to 100 μL), but use of 15 to 20 μL seems to be most common. With larger blood spots, although multiple analyses are possible from each spot, the spots are less homogeneous. For this reason, it is suggested to have 3-4 smaller spots (of 20 μL or less) which are more homogeneous, thus increasing inherent sample quality.
The small sample volumes required for dried blood spot analysis mean that fewer animals – and therefore less drug – are needed during preclinical studies relative to conventional blood analysis (milliliters of blood often required). Blood samples spotted and dried on cards don’t need to be frozen, thereby simplifying the procedures for both sampling and shipping, with subsequent cost savings. Provided a compound is stable in blood, which must be demonstrated for each compound, dried blood spot samples can be shipped in an envelope at room temperature.
In addition to the ethical and financial benefits, use of dried blood spot analysis can also improve preclinical data quality. Typically, use of multiple small animals is necessary to generate drug concentration-time curves in typical pharmacokinetic and toxicology studies, due to insufficient blood volume per animal, thus introducing a potential source of undesirable variation in the data. That source of variability can be eliminated with dried blood spot analysis. The smaller volumes associated with the technique mean that serial sampling can be performed with each animal, thereby enhancing preclinical data quality. In addition, some researchers have found that the relatively high stability of compounds in dried blood spots, especially prodrugs and their metabolites, is a key advantage of the technology.
Dried blood sample analysis has some drawbacks in that analysis is more time-consuming than that required for liquid samples, but still includes liquid chromatography and tandem mass spectrometry. The limit of resolution is not yet adequate for low-exposure drugs (e.g., pg/mL), and components of the cards on which spots are collected can interfere with some analyses. Some researchers have determined that the additional time necessary for analysis is a detriment to the speed required in discovery-phase research. In some organizations, the decision to use dried blood spots is currently being made on a program-by-program basis as drug candidates move from discovery into early-stage development. One holdup has been the impracticality of switching late-stage compounds with a long history of analyses in plasma over to dried blood spot analysis. The pharmacokinetic values obtained from liquid plasma and from dried blood are not directly comparable, and “bridging” studies are required to switch between matrices. “Even though you can generate an in vitro number for converting between blood and plasma, it doesn’t always work,” Neil Spooner, director of bioanalytical science and development at GlaxoSmithKline in Ware, England said.
Perhaps the most pressing detriment to use of dried blood spots is the need for improved automation, although some automation is available. Fully automated techniques are generally available for fluid samples, thus enabling high throughput analysis of thousands of samples. Direct analysis methods for dried blood spots, which bypass the need to create a paper punch, are under development.
To date, it is undetermined how global regulatory bodies will respond to data obtained from dried blood spot analysis. Some feel that the European Union may be more accepting than the Federal Drug Administration (FDA). The FDA declined to comment citing “insufficient experience with the technology.” Although international guidelines state that kinetics can be measured in blood, plasma, or serum, specific US guidelines for use of dried blood spot analyses are absent. Richard M. LeLacheur, vice president at PharmaNet USA, a contract research organization in Princeton, N.J., says “As the comfort level, regulatory experience, and infrastructure grow, people will realize it’s not a big leap to go into dried blood spots, and the benefits are worth it.”
For pharmaceutical companies, is personalized medicine more of a threat than an opportunity? In addition to the development of new drugs, genetic information can also help target the use of current medications (e.g., Plavix). The use of genetic (or other) information to target patient population subsets is expected to increase drug safety and render cost savings to both insurer and patient, but can it also be expected to limit the potential market and lower pharmaceutical sales? By potentially enhancing drug safety, personalized medicine is expected to elicit fewer adverse drug reactions, thereby leading to fewer liability claims against drug companies. Drug development costs rise, however, if preclinical scientists also must isolate a genetic trigger and develop a companion test for a treatment, even if the size of clinical trials can potentially be reduced and additional income can be expected through purchase of both medication and companion diagnostic. Even when a drug is utilized in target populations, how much risk will be deemed acceptable? Whether personalized medicine stimulates or inhibits pharmaceutical drug development remains to be determined.
Posted by cdavenport on Tuesday Nov 9, 2010 Under Drug Safety, ICH
The Australian TGA has adopted the EU Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (ICH M3(R2)bb). The effective date was November 5, 2010. The purpose of this document is to recommend international standards for, and promote harmonization of, the nonclinical/preclinical safety studies recommended to support human clinical trials of a given scope and duration as well as marketing authorization for pharmaceuticals. This guidance should facilitate the timely conduct of clinical trials, reduce the use of animals in accordance with the 3R (reduce/refine/replace) principles, and reduce the use of other drug development resources.
During the past 30 years, genetic toxicology testing has evolved technologically to play an important safety assessment role in the progression of chemical candidates through the drug discovery and development process. Prior to application of the battery of regulatory tests, high-throughput screening assay methods are now used to reduce costs by terminating compounds with undesirable characteristics (mutagenic hazard or potential carcinogen). With few exceptions, compounds found to be mutagenic in these assays are dropped from development, and clastogenic compounds result in unfavorable labeling, require disclosure in clinical trial consent forms, and can greatly impact the marketability of a new drug. Furthermore, in vitro clastogenicity responses can delay drug development by requiring additional testing to determine the in vivo relevance, although these assays can at times be integrated into other in vivo toxicity studies to expedite the progression of drugs to clinical trials. Thus, genetic toxicology testing at the drug discovery and optimization stages serves to quickly identify mutagenic compound so that they can be quickly dropped from development.
Genetic toxicology was the first branch of toxicology to fully embrace in vitro test methods, notably through the visionary work of Bruce Ames and coworkers with the development of the Salmonella typhimurium tester strains. These prokaryotic assays demonstrated good correlation with rodent carcinogenicity results. The Ames test is generally used as the first screening method to assess chemical genotoxicity. Although it provides extensive information on DNA reactivity, the Ames assay is not suitable for detecting nongenotoxic carcinogens. In time, in vitro assays were developed for the detection of gene mutations, chromosomal aberrations, and micronuclei formation. The mouse lymphoma assay in particular has been developed to the point that both gene mutations and chromosomal aberrations can be detected and quantified following exposure to test chemicals, when compared with known direct-acting mutagens and promutagens.
Current Perspectives
Assay Predictivity
The performance of a combination of the 3 most commonly used in vitro genotoxicty tests – the Ames, the mouse lymphoma, and the in vitro micronucleus or chromosomal aberration tests – have been evaluated for their ability to discriminate rodent carcinogens from non-carcinogens using a database of over 700 chemicals (Kirkland et al., 2005). Based on the relative predictivity measure (RP; the ratio of real:false positive results), that study demonstrated that positive results in all 3 tests indicated that a chemical is greater than 3 times more likely to be a rodent carcinogen than a non-carcinogen. Similarly, negative results in all three tests indicated that a chemical is more than two times more likely to be a rodent non-carcinogen than a carcinogen. But further evaluation of combinations of positive and negative results in this genotoxicity battery using the RP calculations indicated that it is not possible to predict outcome of a rodent carcinogenicity study when only 2/3 of the genotoxicity results are in agreement (Kirkland et al., 2006).
Assay Shortcomings
A basic if not critical shortcoming in all these mammalian in vitro assays is the lack of mammalian absorption, distribution, metabolism, and excretion (ADME) features. As summarized in a recent European Centre for the Validation of Alternative Methods (ECVAM) workshop (Kirkland et al., 2007), cell lines used for genotoxicity testing have a number of deficiencies that may contribute to a high false-positive rate. These include a lack of normal metabolism leading to reliance on exogenous metabolic activation systems (e.g., Aroclor-induced S9), impaired tumor protein 53 (p53) transcription factor function, and altered deoxyribonucleic acid (DNA) repair capacity. Also the use of excessive test chemical concentrations to achieve an empirical correlation between genotoxicity and carcinogenicity can result in “promiscuous activation.” Because these in vitro assays rely on such artificial activation systems, other enzymes that are relatively unimportant in vivo may take over the activation role, leading to the same or a different metabolite – hence, “promiscuous activation.” Recently, a risk assessment method has been proposed that is dependent upon the availability of quantitative human and rodent ADME data such that exposures to a metabolite of genotoxic concern can be estimated at the intended human efficacious dose and the maximum dose used in the 2-year rodent bioassay (Dobo et al., 2009).
Other notable genotoxicity testing methods are available for use in the drug discovery and lead-optimization process. The comet assay is a microgel electrophoresis technique for detecting DNA damage – in vitro and in vivo- at the level of a single cell. When used in vivo, DNA lesions can be measured in any organ, regardless of the extent of mitotic activity and under normal ADME conditions. The conventional mouse micronucleus test in the hematopoietic system is a simple method to assess the in vivo clastogenicity of chemicals if the chemical reaches the hematopoietic system. When multiple organs in the mouse were analyzed following exposure to 208 chemicals, the comparison of comet assay results and carcinogenicity suggested that the comet assay was more capable than the mouse micronucleus assay of detecting rodent carcinogens (Sasaki et al., 2000).
Regulatory Guidance
At present, the ICH/FDA Guidance Document S2(R1) outlines two GLP genotoxicity testing assay options. Option 1 requires completion of: (1) a test for gene mutation in bacteria., (2) a cytogenetic test for chromosomal damage (choice of three), and (3) an in vivo test for chromosome damage using rodent hematopoietic cells (either micronuclei or chromosomal aberrations in metaphase cells). Option 2 combines (1) the highly predictive gene mutation assay in bacteria with (2) an in vivo assessment in 2 tissues (e.g., micronuclei using rodent hematopoietic cells plus a second in vivo assay, such as the liver unscheduled DNA synthesis (UDS) assay, transgenic mouse assay, comet assay, etc. Thus, the ICH guidance allows for the registration of pharmaceuticals without the submission of data from in vitro mammalian genotoxicity tests (e.g., the in vitro micronucleus test, chromosomal aberrations, mouse lymphoma assay). This is important because some authors (Matthews et al., 2006) have indicated that 2 of the tests in the FDA battery show good correlation for carcinogenicity prediction (Ames and in vivo micronucleus) and 2 tests show poor correlation (mouse lymphoma and in vitro chromosomal aberrations).
High-Throughput Screens
With the trend towards the application of early pre-screening, high-throughput methods to eliminate potential mutagens/clastogens prior to application of the more resource-intensive and time-consuming regulatory testing methods, many pharmaceutical companies are using these screening methods early in the discovery/lead optimization process. Examples of modified or high-throughput methods for early screening include: (1) computer-assisted (in silico) structural activity relationship (SAR) methods for predictive toxicity screening, (2) modified assays such as the in vitro assessment of micronucleus induction in Chinese hamster ovary (CHO) cells, the Ames II assay (TA98 and TA Mix), the in vitro comet assay, or well-based (e.g., 96- or 384-well format) modifications of the yeast deletion (DEL) assay, or (3) proprietary assays such as Vitotox™ (mutagenicity), RadarScreen® (clastogenicity), and GreenScreen® HC (genotoxicity).
About the Author:
David Amacher is a senior investigative and biochemical toxicologist with extensive experience in the safety evaluation of human and animal health products. Dr. Amacher is a Diplomate of the American Board of Toxicology, a Fellow of the National Academy of Clinical Biochemistry, and serves as an Assistant Research Professor of Toxicology and Adjunct Professor in the Graduate School of the University of Connecticut. His professional affiliations include memberships in the American Society for Pharmacology and Experimental Therapeutics, Society of Toxicology, American Society of Biochemistry and Molecular Biology, International Society for the Study of Xenobiotics, American Association of Clinical Chemistry, and the American College of Toxicology.
Preclinical models are developed to test lead compounds for toxicity and efficacy. This report 1) explores novel preclinical models (in vivo, in vitro, in silico, and systems biology) that show promise to expedite and improve the target validation, lead optimization, and toxicity screening timelines, and 2) discusses the various advantages and disadvantages of Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) screening techniques. In addition, the report provides an outlook for preclinical testing over the next decade. It focuses on more than 60 companies that are involved in using or developing ADMET technologies to advance preclinical research and provides an update on how new models and systems have been employed to accelerate the discovery and development process.
Scope of this report
• Understand the basis of ADMET testing and why it is a necessary and important component of preclinical research
• Up-to-date information on the preclinical models and systems currently used in drug discovery and development.
• Evaluation of the key recent developments and activities of companies who are developing and licensing new ADMET technologies.
• Identification of existing models and how new ones are being developed to improve productivity and knowledge.
The recent approval by the Japanese Pharmaceuticals and Medical Devices Agency (PMDA) of the 7 protein biomarker panel for use in detecting drug-induced kidney damage means that the biomarkers are now qualified at the same level by all of the ICH regulatory agencies. In a Critical Path Initiative statement, this is the first biomarker qualification decision by the PMDA and means that the panel is qualified for voluntary use in nonclinical safety studies. Furthermore, data generated using the panel can be submitted to the PMDA on a case-by-case basis for use in monitoring drug-induced renal toxicity in humans. The 7 biomarker panel, composed of kidney injury molecule-1, albumin, total protein, β2-microglobulin, cystatin C, clusterin, and trefoil factor 3, can be utilized in conjuntion with the current standard renal biomarkers, serum creatine and blood-urea nitrogen. With the exception of trefoil factor 3, the PMDA stated that the new renal biomarkers outperformed the current standard biomarkers. The renal biomarker panel received approval in 2008 from the US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) for limited use in nonclinical and clinical drug development. Additional guidelines regarding biomarker qualification are expected in July 2010.
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