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.

Source – Drug Discovery and Development

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 the mechanisms 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.

Source:  Genetic Engineering and Biotechnology News

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…!

Source: Drug Discovery and Development.

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.”

Source: Chemical & Engineering News

Historical Overview

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.

References:

Dobo KL, Obach RS, Luffer-Atlas D, et al.  A strategy for the risk assessment of human genotoxic metabolites.  Chem Res Toxicol. 2009;22(2):348-56.

International Conference for Harmonization Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use.  S2(R1), 6 March 2008.

Kirkland D, Aardema M, Henderson L, et al.  Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: I. Sensitivity, specificity and relative predictivity [published erratum appears in Mutation Research 2005;588(1):70].

Kirkland D; Aardema M; Müller L; et al.  Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles.  Mutation Research 2006;608(1):29-42.

Kirkland D; Pfuhler S; Tweats D; et al.  How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop.  Mutation Research 2007;628(1):31-55.

Matthews EJ, Kruhlak NL, Cimino MC, et al.  An analysis of genetic toxicity, reproductive and developmental toxicity, and carcinogenicity data: I.  Identification of carcinogens using surrogate endpoints.  Regul. Toxicol. Pharmacol. 2006;44(2):83-96.

Sasaki YF, Sekihashi K, Izumiyama F., et al.  The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC Monographs and U.S. NTP Carcinogenicity Database.  CRC Crit. Rev. Toxicol. 2000;30(6):629-799.

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.

Source:  Business Insights

Seventh Wave Laboratories now offer a battery of  gastrointestinal (GI) preclinical models (TIM) to help clients to assess the behavior of oral medications.  The in vitro models are licensed from TNO, a Dutch contract research organization.  The TIM system has proven useful in solving specific needs in formulation development and pharmacokinetics.

By accurately simulating the conditions in the human GI tract, the TIM system gives insight into the release, solubility, and availability for absorption of pharmaceuticals.  This state-of-the-art, validated system has a much higher predictive value than regular dissolution tests.  The computer-regulated model can simulate various physiologic states and helps scientists to determine the bioaccessibility of active compounds and predict resultant blood concentration after single or repeated intake of various dosage forms.

Sources:  Outsourcing-Pharma.com and ScientistLIVE

Although drugs are usually targeted to be selective for a single protein, it is recognized that other proteins may also be affected, a phenomenon called polypharmacology.  While off-target interactions can cause potentially harmful side-effects, in some cases, an effect on an unintended target might suggest new disease indications for established drugs. 

Chemoinformatics, statistical programs, and experimental techniques are being utilized by Brian Shoichet of the University of California San Francisco to predict off-target interactions.  Similarities between 3665 (existing and experimental) drugs and a set of over 65,000 known ligands to protein receptors in the body were examined.  In addition, the ligands were arranged into around 250 classes depending on the type of receptor to which they bind.  This exhaustive survey yielded hundreds of previously unrecognised potential interactions between drugs and protein receptors in the body.   A number of these potential interactions were confirmed by laboratory experiments, including identification of the key receptor that binds the hallucinatory drug dimethyltryptamine.

These new computational techniques should not only prove valuable to pharmaceutical companies to both expand existing pipelines and to reap additional benefits from current compounds, they should also aid researchers to anticipate and avoid unanticipated adverse events.

Source: Chemistry World