Prospective identification and potential amelioration of cardiotoxicity is a critical component of contemporary drug development, particularly for targeted therapies (e.g., tyrosine kinases) in oncology that are designed to inhibit critical signaling pathways shared by both the tumor cell and the cardiac myocyte (e.g., HER2 and C-Abl). Current preclinical approaches to cardiac safety, which often focus primarily on ion channel testing (e.g., hERG), need to broaden the in vitro test menu to assess other cellular functions that are critical to cardiac cell health. Accordingly, effective nonclinical cardiotoxicity screening programs need to be implemented earlier in the development process.
Stem-cell technologies offer induced pluripotent stem-cell-derived (iPSC) cardiac myocytes that are pure, functionally relevant (exhibit electrical profiles in culture and are amenable to patch-clamp-like studies that monitor electrical potentials and voltage-gated ion channel function), and are human in origin. The following would comprise an effective preclinical cardiac safety testing program utilizing iPSC-derived cardiac myocytes:
Determining influences on key cardiac metabolic pathways focusing on AMPK;
Evaluating changes in fatty acid beta-oxidation;
Measuring changes in mitochondrial health , reactive oxygen species production, and ATP levels;
Assessing drug-induced apoptosis;
Survey potential off-target effects using a comprehensive kinase profiling platform.
In addition to the above, the preclinical program should identify compounds that demonstrate cardio-protective effects with regard to mitochondrial health and energy homeostasis.
Glossary
ABL1 = a proto-oncogene which encodes a cytoplasmic (C-ABl) and nuclear protein tyrosine kinase. Implicated in processes of cell differentiation, cell division, cell adhesion, and stress response.
AMPK = a metabolic sensor of cellular ATP. Controls fatty acid oxidation and glucose uptake in skeletal muscle, heart, and liver.
ATP = adenosine-5′-triphosphate, a multifunctional nucleoside triphosphate used in cells as a coenzyme. Responsible for intracellular energy transfer.
HER2 = “Human Epidermal growth factor Receptor 2,” a receptor required for healthy heart function.
hERG = the human Ether-à-go-go Related Gene. Codes for a potassium ion channel protein.
Zebrafish offer a nonclinical model for the high-throughput screening of drug compounds, including toxicity assessment, with resolution at the cellular level in living vertebrate organisms. These small, freshwater, tropical fish share genetic and biochemical similarity to humans, in addition to similar organ system development. Vertebrate disease models (e.g., Parkinson’s, epilepsy, wound repair) are available , as are 3-D image resolution and data analysis capabilities. Live-imaging options, unparalleled in other vertebrate organisms, are possible using the transparent larvae. Furthermore, live-cell microscopy can provide views of the inner complexity and workings at the cellular level. For purposes of disease modeling, researchers can create and screen genetic mutants in the zebrafish that are linked to human immune diseases. Neurological assessments using the live, transparent, zebrafish larvae allow visualization of themechanisms of myelination. In conclusion, the zebrafish preclinical model owes much of its popularity to the transparent nature and relevant ease of imaging of vertebrate larvae. Optimization of data analyses for these varied indications is ongoing.
Posted by cdavenport on Thursday Aug 4, 2011 Under Drug Safety, TigerU
Drug development is a complicated, often convoluted process. The ability to predict drug toxicity in humans from nonclinical data remains a major challenge. Since you can’t “erase” an adverse event, optimization of preclinical dose selection is essential. This presentation outlines the process for dealing with adverse preclinical / nonclinical events in order to 1) optimize the chances of successful drug development, or 2) to create a scientific basis for early termination of drug development. Conclusion: Experience counts! There is no single answer for all problems. Use of sound scientific and business judgement generally yields the best outcome.
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…!
Biomarker use in translational medicine is predicated upon preclinical qualification and validation – 2 distinct steps in the biomarker development process. Prior to issue in 2009 (EMA) and 2010 (FDA, PMDA) of the renal-specific DRAFT qualification guidelines, there was no clear direction by the U.S. Food and Drug Administration (FDA) or European Medicines Agency (EMA) of how companies should qualify new biomarkers for disease progression or clinical trial endpoints. The trend in biomarker use is multivariant analysis, the tracking of subtle changes in multiple biomarkers simultaneously, often utilizing various tissue types. While the new guidance addresses biomarker qualification, analytical validation of new biomarkers remains undefined. This review updates the reader of the status of both qualification and validation of translational biomarkers.
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.
By utilizing the basic principles of hemodynamics and hydraulics, research suggests that fluid retention is detrimental for the cardiovascular system because it increases the likelihood of turbulent blood flow, regardless of whether or not blood pressure is raised. Increased turbulence promotes endothelial dysfunction, thereby contributing to the development of atherosclerotic cardiovascular disease. Fluid retention induces hypertension in some individuals, increases stroke volume (the amount of blood that is ejected by the heart with each contraction) in others, and causes edema. Some blood pressure lowering medications also increase stroke volume and cause edema but prevent heart attacks and strokes when used to treat hypertension. For drugs that increase the risk of adverse cardiovascular events, it may be possible to reduce or neutralize the increased risk by simultaneous diuretic administration.
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.”
All medical products pose risks and postmarketing surveillance is critical to expanding the limited evidence base that exists when new drug products are approved. Through initiation of the Sentinel Initiative (May 2008), the Food and Drug Administration (FDA) is developing the capacity for actively monitoring the safety/toxicity of approved medical products using the electronic health information in claims systems, inpatient and outpatient medical records, and patient registries. The pilot program, Mini-Sentinel, uses a distributed data network (rather than a centralized database) of health plans and other organizations to create data files in a standard format while maintaining physical and operational control over their own patient-level data, thus ensuring patient privacy. Laying the groundwork for that system has required input from both public and private organizations. These data partners can obtain full-text medical records, when necessary, to confirm diagnoses or exposures and to determine the existence or severity of risk factors.
The initial focus of Mini-Sentinel has been on developing the ability to use medical claims data. Over the next year, laboratory-test results and vital signs will be added. The FDA will soon begin to actively monitor the data, seeking answers to specific questions (e.g., frequency of myocardial infarction among users of oral hypoglycemic agents). Using the Mini-Sentinel system, the FDA will also be able to obtain rapid responses to new questions about medical products and, eventually, to evaluate the health effects of its regulatory actions.
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