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.

Source: ScienceBlog

Original Article: Clinical Hemorheology and Microcirculation (free pdf)

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.”

Source: Chemical & Engineering News

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.

Source: New England Journal of Medicine

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.

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

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.

Source: ProteoMonitor and MedHealthWorld

The Federal Register and the ICH S9 Guidance for the nonclinical evaluation for anticancer pharmaceuticals issued today.  The guidance provides recommendations for preclinical studies for the development of pharmaceuticals, including both drugs and biotechnology derived products, intended to treat patients with advanced cancer.  The recommendations describe the type and timing of preclinical studies to support an investigational new drug application (IND) and the submission of a new drug application (NDA) or biologics license application (BLA).

To verify the availability of pharmacokinetic parameters in cynomolgus monkeys, hepatic availability (Fh) and the fraction absorbed (Fa) multiplied by intestinal availability (Fg) were evaluated to determine their contributions to absolute bioavailability (F) after intravenous and oral administrations. These preclinical results were compared with those for humans using 13 commercial drugs for which human pharmacokinetic parameters have been reported.  In addition, in vitro studies of these drugs, including membrane permeability, intrinsic clearance, and p-glycoprotein affinity, were performed to classify the drugs on the basis of their pharmacokinetic properties.

In the present preclinical study, monkeys had a markedly lower F than humans for 8 of 13 drugs.  Although there were no obvious differences in Fh between humans and monkeys, a remarkable species difference in FaFg was observed.  These results suggest that first-pass intestinal metabolism is greater in cynomolgus monkeys than in humans, and that bioavailability in cynomolgus monkeys after oral administration may be unsuitable for predicting pharmacokinetics in humans.  A rough correlation was also observed between in vitro metabolic stability and Fg in humans.

Key: F (bioavailability), Fa (fraction absorbed), Fg (intestinal availability), Fh (hepatic availability).

Drugs examined: amitriptyline, dexamethasone, digoxin,  hydrochlorothiazide, ibuprofen,  lithium carbonate, midazolam, nifedipine,  propranolol, quinidine,  tacrolimus, timolol, and verapamil.

Source: Drug Metabolism and Disposition