Development of Predictive in vivo Screening Systems for Phenotypic Drug Discovery for Smoking Cessation

Summary Phenotypic drug discovery is a complement to target-based drug discovery and provides an alternative approach that begins by querying more complex cellular of physiological systems instead of specific targets. The possible advantage resides in the fact that a relevant biological context is interrogated without predisposed bias toward mechanism(s). Thus, an opportunity is created to identify compounds that may interact with one or more targets or pathways not anticipated by a single mechanism-driven hypothesis. In essence, phenotypic approaches screen multiple mechanisms and targets simultaneously. This solicitation for Small Business Innovation Research (SBIR) contract proposals invites contract proposals from small business concerns (SBCs) for a multi-pronged research program to develop and evaluate a weighted battery of animal behavioral tests that can be used for the phenotypic drug discovery and development of smoking cessation medications. The battery should be, preferably, high- or medium- throughput and designed to improve the predictive validity of in vivo screening of drug candidates. Since people continue to smoke for many different reasons - withdrawal relief, pleasure, taste, improvement in concentration, weight control, stress control, arousal, etc. – the battery should be capable of assessing the multiple components (reinforcement, affective, cognitive, etc.) that underlie smoking. The offeror is expected to identify and propose the behavioral domains of particular importance to human tobacco dependence, and to develop the necessary animal behavioral protocols to test multiple processes believed to be contributing to smoking in humans. It is not necessary that the behavioral measures model the aspects of smoking in precise fashion. That is, this contract solicitation focuses on the predictive (and construct) validity of the behavioral battery, not on its face validity. The SBC must screen in uniform fashion and, then, build a large database of pharmacological signatures of currently available medications (with known anti-smoking efficacy in humans) comprised of measures reflecting the reinforcement, affective, cognitive, etc. functions. The critical part of this solicitation is the focus on biostatistical/computational solutions and modeling. Bioinformatics algorithms are to be developed which will complement and enhance the statistical power to recognize phenotypic profiles of drugs. Proposals are expected to include animal behavior tests only. Background Information Despite major developments in computational, in vitro and ex vivo model systems, in vivo animal testing remains a necessary part of drug development. This solicitation invites offers from SBCs interested in developing a weighted battery of animal behavioral tests/assays (rat or mouse) to function as a screening system for preclinical development of smoking cessation medications. In this solicitation, behavioral assay is defined as a means of qualifying a dependent biological variable. In this solicitation, animal model is defined as a theoretical description of the way a system (or disease) works. The animal model induces under- or over-expression of a biological variable which assay quantifies. This solicitation seeks to establish a battery of carefully selected behavioral tests/assays that will provide a comprehensive behavioral assessment of domains of particular importance in tobacco smoking and quitting. A battery must be comprised of currently used and available tests whose appropriateness and validation could be assessed through literature search. Applications proposing new animal models or assays are not allowed. This assay battery will be used to obtain standardized data on the responses to existing first-line pharmacotherapies that have been approved by FDA as aids for smoking cessation (i.e. varenicline, bupropion ), and second-line pharmacotherapies (i.e. clonidine, nortriptyline). Measuring several domains and several medications will result in obtaining a rich dataset, which is to be systematically analyzed for identification of critical parameters or axes, selecting the most useful features and identifying underlying variables. The critical parameters in each domain are to be selected to be combined into final, “lean”, standardized and validated battery of assays, with the goal to provide superior reliability, greater statistical power and higher throughput than standard methods. This validated battery of assays will be used for in vivo screening of anti-smoking drug candidates to advance future research and discovery in this area. Because of the importance of tobacco-dependence treatment to national tobacco control efforts, the National Cancer Institute and the National Institute on Drug Abuse (NIDA) convened a meeting, entitled “Translational Medication Development for Nicotine Dependence Workshop.” Participants from industry, academia and government agencies provided their views on the greatest opportunities for accelerating the translational efforts in the area of nicotine dependence. One of the challenges in medications development for addictive disorders is to determine the predictive validity of preclinical and clinical pharmacology models of various aspects of drug dependence. Several pharmacotherapies are, in fact, available to support treatment of nicotine (tobacco) dependence, including ones with an overt nicotinic mechanism of action, such as nicotine replacement therapies and varenicline (Chantix®, Champix®), others, in which nicotinic mechanisms may be a component, such as bupropion (Zyban®), and still others, for which there is no obvious nicotinic component to their action (e.g., the second-line medication clonidine, an α2 adrenergic agonist). Each of these drugs significantly improves the quit-rate compared to placebo. With these currently available medications, however, only about 20% of smokers are able to maintain long-term abstinence, and more efficacious pharmacotherapies need to be developed promptly. Scientific knowledge to be achieved through research supported by this contract Basic research into the mechanisms of nicotine addiction has the benefit of the availability of a wide array of preclinical animal behavioral models and assays. A number of these tests have seen extensive use as research tools, and, as a result, they are known to have utility in studying the mechanisms and processes related to addiction and drug abuse relapse. In addition, some of these models, such as drug-self administration and drug discrimination, have demonstrated translational utility in providing preclinical data in support of the development of varenicline. However, given that varenicline is the only smoking cessation pharmacotherapy to have been developed through the typical pharmaceutical industry drug discovery approach, there is little evidence for the predictive validity of any of these animal behavioral models for medication development for drug addiction. Indeed, since inadequate efficacy remains a significant cause of failure in the drug development process, improvements in the ability to predict efficacy at various stages across the drug development process are warranted. The intent of this contract solicitation is to fund small business concerns (SBCs) capable to undertake screening of anti-smoking medications (with various mechanisms of action and known to demonstrate some efficacy in humans) in multiple animal behavior paradigms. Although addiction is frequently and preferentially conceptualized in terms of reward and reinforcement, this contract solicitation seeks to collect data simultaneously in other domains that may be relevant to drug addiction and drug abuse relapse, namely cognition and affect. For each of these domains, there exist well-established assays and models that have validity within the domain (e.g., for reward and reinforcement – drug self-administration, intra-cranial self-stimulation; for cognition - pre-pulse inhibition, 5 choice serial reaction time; affect - open-field test, elevated plus-maze). It is important to note that these examples are not intended to be exclusive or prescriptive; other models may be worthy of validation in this effort. The purpose of this initiative is to support research to estimate if individually and/or collectively these tests are predictive of medication efficacy in clinical trials. A critical part of this solicitation focuses on improving the reproducibility of preclinical data which will be obtained. Given the high cost of clinical drug development, factors such as low reproducibility and translatability, or heterogeneity in study design that hinder the comparison of preclinical data are major disincentives for investment in development of novel treatments. Diseases of addiction are not among the priorities for pharmaceutical companies. In addition, a major concern highlighted in other CNS- and non-neurologic disease areas is the poor reproducibility of preclinical data for compounds progressing from academic laboratories to industrial development programs and, ultimately, to clinical trials. The reasons for these obstacles are multiple and varied, but methodological issues related to the design, execution, and reporting of preclinical studies are important components. Thus, offerors are expected to meticulously address methodological issues in their applications and, if successfully selected, in execution of this contract. Fig 1 illustrates the basics of the concept. Please note that the examples of behavioral paradigms are not intended to be exclusive or prescriptive. The offeror is expected to identify and propose the behavioral domains of particular importance, and to develop the necessary animal behavioral protocols to test multiple processes believed to be contributing to smoking in humans. In general, the tests chosen should be ones that have been used pre-clinically, so that reliability of the data generated has been established and that there exists some conceptual understanding of what the test measures. In addition, while considering models for the reward and positive reinforcement domain, investigators must be aware that certain animal behavioral tests appear not to generate reliable data with nicotine, and these should not be proposed (e.g., conditioned place preference, nicotine-primed reinstatement). Equally, it is important to highlight the fact that this contract solicitation is not intended as a mechanism to support the development of new behavioral models, assays or tests, or to uncover novel behavioral processes or neurobiological mechanisms. Nor is it intended to be directed only to investigators with addiction research expertise. NIDA welcomes the interest of small businesses who have expertise with animal models that have not seen wide use in addiction research (e.g., potentiated startle, elevated plus maze), but that might be useful as part of a screening battery. Offerors must provide the rationale for: • Selecting the model system. A detailed description of the animal model characteristics such as a definition of study population using a common strain and individual commercial stock designations of each animal provider, diet, housing conditions, microbial status and handling should be described. Before using compounds in the rat or mouse, detailed information about the cross-reactivity of the compounds with the target in the rat or mouse is necessary. For example, lower binding affinity in mice would require higher dosing which, in turn, might compromise target specificity and affect the results of testing. In addition, other interspecies differences should be taken into consideration. Bupropion is metabolized differently in the rat compared to human and mouse. Therefore, offerors need to specify how they will address this and similar species differences (e.g., comparison of dosing with bupropion compared to its metabolite over a sufficient dose/time range). • Selecting the assays/models for the behavioral domains to be studied. Provide the rationale for the selected domains, assays, models and end points. The proposed tests must be validated for their specificity and selectivity to measure clinically relevant symptom(s) or physiological parameters. It is expected that, regardless of the model or assay chosen for testing, investigators will propose an approach that is sensitive to the need for relatively high throughput assessment. Approaches that require lengthy training or assessment periods will be deemed not feasible. For behavioral domains different from Reward, testing in nondependent animals is expected. Testing in animal models of nicotine dependence is not required, although may be proposed if justified and deemed consistent with high- and medium-throughput. There are a variety of behavioral tests in the behavioral neuroscience field that could be adapted to this purpose of phenotypic drug screening. For the selection and validation of the tests, it is important to recognize that some drugs that provide similar phenotypic effects have differential effectiveness in aiding smoking cessation. For example, nicotine improves attentional function and does help with smoking cessation, whereas amphetamine also improves attentional function but there is no evidence for it aiding smoking cessation. Buproprion is an antidepressant and aids smoking cessation but sertraline which also is an antidepressant has not been found to help smoking cessation. It is thus important to integrate the phenotypic information with the neuropharmacological information to help develop a more integrated neurobehavioral approach. Importantly, the selected behavioral tests/models must provide internal behavioral validation such as learning rate or habituation which offers assurance that the conditions of the tests are appropriate. • Adequacy of controls. Verification that interventional drugs reached and engaged the target. A potential concern about the specificity of drug effects in this screen – establishing a balance between sensitivity and specificity- should be addressed through selection of appropriate drug controls. The evaluation of both of these components, sensitivity and specificity, of predictive validity is vital in work of this type to avoid costly clinical studies of false-positive drugs that affect behaviors in preclinical screens but are ineffective in humans that are addicted to tobacco. • Selecting the dose, timing and route of administration for the medications being tested. A drug administration and dose regimen that is adapted to the pharmacokinetic properties of the drug, and to the duration of the study requires careful consideration. The rationale for dosing the animal must be evident from appropriate pharmacodynamic and pharmacokinetic assays, published in the literature or obtained in applicants’ laboratories. The route of administration is important not only because of animal welfare, but also out of consideration that the oral route is the desired route of administration in humans. Hence, oral dosing is preferable whenever possible. Frequent subcutaneous, intraperitoneal or intravenous injections lead to considerable stress in the animals and could influence test outcome. Assistant application devices, such as minipumps, should be avoided wherever possible, as they require surgery, additional control groups and can cause technical complications. For testing, a minimum of 3 doses of first and second line medications should be proposed. All of the medications are to be screened in each model proposed for evaluation. • Selecting the experimental design. o Must describe methods of blinding, strategies for randomization and /or stratification. To improve the reliability of the proposed studies, the offeror must use randomization to eliminate systematic differences between treatment groups; induce the condition under investigation without knowledge of whether or not the animal will get the drug of interest; and assess the outcome in a blinded fashion. o Demonstrate the reconciliation between statistical needs for the detection of biological effects and constraints of animal welfare, cost and time. To guard against 'underpowered' studies, researchers must calculate the number of animals required to have a reasonable chance of detecting the anticipated effect given the expected variance of the data. A detailed discussion about the use of appropriate statistics must be provided, including the evidence that applicants consulted a statistician to obtain information about the sufficient group size, and the appropriate statistical method to be applied for data analysis for each behavioral test and adapting the statistical approach for combining data across behavioral tests in developing an optimized predictive ‘screening system.’ o Detailed description of statistical methods used in analysis and interpretation of results • Guidelines on nicotine dose selection for in vivo research. If the models, in which in vivo nicotine dosage is warranted, are selected for this project, an evidence for species-specific nicotine dosage ranges must be presented. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. Guidelines on nicotine dose selection for in vivo research must be consulted (Psychopharmacology (2007) 190:269–319). Phase I Activities and Expected Deliverables • Select the behavioral assays and models representing the behavioral domains of particular importance believed to be contributing to smoking and quitting in humans. • Develop the necessary animal behavioral protocols to test multiple processes in those identified domains. Must finalize the selection of model systems, controls, experimental design, the dose, timing and route of administration, and provide verification that interventional drugs reached and engaged the target. • Demonstrate that the selected behavioral tests are capable of high or medium throughput. • Must agree and comply with good reporting practices, such as ARRIVE Guidelines. The ARRIVE guidelines consist of a checklist of 20 items describing the minimum information that all scientific publications reporting research using animals should include, such as the number and specific characteristics of animals used (including species, strain, sex, and genetic background); details of housing and husbandry; and the experimental, statistical, and analytical methods (including details of methods used to reduce bias such as randomization and blinding). All the items in the checklist have been included to promote high-quality, comprehensive reporting to allow an accurate critical review of what was done and what was found (see: Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research, PloS Biology, 2010, Volume 8, issue 6, 1-5.) • To eliminate bias, all results, negative and positive, must be reported. • Must provide data interpretation, including alternative interpretations. Must include the review of relevant literature in support or in disagreement with results. Phase II Activities and Expected Deliverables • Establish the battery of carefully selected behavioral tests. • Demonstrate that the battery of carefully selected behavioral tests is capable of high or medium throughput. Demonstrate that this battery can make data collection and analysis more efficient without compromising the quality of the phenotypic assessment. • Demonstrate that the battery of carefully selected behavioral tests is sufficiently sensitive to produce a comprehensive behavioral assessment in functional domains of cardinal importance for tobacco dependence. • In selected model systems, using appropriate controls, experimental design, the dose, timing and route of administration, obtain standardized data on the responses to existing first-line pharmacotherapies that have been approved by FDA as aids for smoking cessation (i.e. varenicline, bupropion ), and second-line pharmacotherapies (i.e. clonidine, nortriptyline). • Establish, test and validate the bioinformatics algorithms/processes which are able to quickly and reliably recognize phenotypic profiles produced by varenicline, bupropion, clonidine and nortriptyline. The proposed bioinformatic approach must aid the predictions of the therapeutic effect of candidate compounds for smoking cessation to be developed in the future. • Confirm that multivariate and/or bioinformatics algorithms are able to discriminate between varenicline-, bupropion-, clonidine- and nortriptyline-treated animals and at least two positive and two negative controls. • Demonstrate the compliance with good reporting practices, such as ARRIVE Guidelines. The ARRIVE guidelines consist of a checklist of 20 items describing the minimum information that all scientific publications reporting research using animals should include, such as the number and specific characteristics of animals used (including species, strain, sex, and genetic background); details of housing and husbandry; and the experimental, statistical, and analytical methods (including details of methods used to reduce bias such as randomization and blinding). All the items in the checklist have been included to promote high-quality, comprehensive reporting to allow an accurate critical review of what was done and what was found (see: Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research, PloS Biology, 2010, Volume 8, issue 6, 1-5.) • Collect and report all results, negative and positive. • Must provide data interpretation, including alternative interpretations. Must include the review of relevant literature in support or in disagreement with results. • Creating the all-inclusive platform which is comprised of hardware that evaluates the behaviors of domains of importance, and software that recognizes and analyzes behavior is encouraged.