Threats to Validity in the Design and Conduct of Preclinical Efficacy Studies: A Systematic Review of Guidelines for In Vivo Animal Experiments

Background: The vast majority of medical interventions introduced into clinical development prove unsafe or ineffective. One prominent explanation for the dismal success rate is flawed preclinical research. We conducted a systematic review of preclinical research guidelines and organized recommendations according to the type of validity threat (internal, construct, or external) or programmatic research activity they primarily address. Methods and Findings: We searched MEDLINE, Google Scholar, Google, and the EQUATOR Network website for all preclinical guideline documents published up to April 9, 2013 that addressed the design and conduct of in vivo animal experiments aimed at supporting clinical translation. To be eligible, documents had to provide guidance on the design or execution of preclinical animal experiments and represent the aggregated consensus of four or more investigators. Data from included guidelines were independently extracted by two individuals for discrete recommendations on the design and implementation of preclinical efficacy studies. These recommendations were then organized according to the type of validity threat they addressed. A total of 2,029 citations were identified through our search strategy. From these, we identified 26 guidelines that met our eligibility criteria—most of which were directed at neurological or cerebrovascular drug development. Together, these guidelines offered 55 different recommendations. Some of the most common recommendations included performance of a power calculation to determine sample size, randomized treatment allocation, and characterization of disease phenotype in the animal model prior to experimentation. Conclusions: By identifying the most recurrent recommendations among preclinical guidelines, we provide a starting point for developing preclinical guidelines in other disease domains. We also provide a basis for the study and evaluation of preclinical research practice. Background: The development process for new drugs is lengthy and complex. It begins in the laboratory, where scientists investigate the causes of diseases and identify potential new treatments. Next, promising interventions undergo preclinical research in cells and in animals (in vivo animal experiments) to test whether the intervention has the expected effect and to support the generalization (extension) of this treatment–effect relationship to patients. Drugs that pass these tests then enter clinical trials, where their safety and efficacy is tested in selected groups of patients under strictly controlled conditions. Finally, the government bodies responsible for drug approval review the results of the clinical trials, and successful drugs receive a marketing license, usually a decade or more after the initial laboratory work. Notably, only 11% of agents that enter clinical testing (investigational drugs) are ultimately licensed. Why Was This Study Done?: The frequent failure of investigational drugs during clinical translation is potentially harmful to trial participants. Moreover, the costs of these failures are passed onto healthcare systems in the form of higher drug prices. It would be good, therefore, to reduce the attrition rate of investigational drugs. One possible explanation for the dismal success rate of clinical translation is that preclinical research, the key resource for justifying clinical development, is flawed. To address this possibility, several groups of preclinical researchers have issued guidelines intended to improve the design and execution of in vivo animal studies. In this systematic review (a study that uses predefined criteria to identify all the research on a given topic), the authors identify the experimental practices that are commonly recommended in these guidelines and organize these recommendations according to the type of threat to validity (internal, construct, or external) that they address. Internal threats to validity are factors that confound reliable inferences about treatment–effect relationships in preclinical research. For example, experimenter expectation may bias outcome assessment. Construct threats to validity arise when researchers mischaracterize the relationship between an experimental system and the clinical disease it is intended to represent. For example, researchers may use an animal model for a complex multifaceted clinical disease that only includes one characteristic of the disease. External threats to validity are unseen factors that frustrate the transfer of treatment–effect relationships from animal models to patients. What Did the Researchers Do and Find?: The researchers identified 26 preclinical guidelines that met their predefined eligibility criteria. Twelve guidelines addressed preclinical research for neurological and cerebrovascular drug development; other disorders covered by guidelines included cardiac and circulatory disorders, sepsis, pain, and arthritis. Together, the guidelines offered 55 different recommendations for the design and execution of preclinical in vivo animal studies. Nineteen recommendations addressed threats to internal validity. The most commonly included recommendations of this type called for the use of power calculations to ensure that sample sizes are large enough to yield statistically meaningful results, random allocation of animals to treatment groups, and “blinding” of researchers who assess outcomes to treatment allocation. Among the 25 recommendations that addressed threats to construct validity, the most commonly included recommendations called for characterization of the properties of the animal model before experimentation and matching of the animal model to the human manifestation of the disease. Finally, six recommendations addressed threats to external validity. The most commonly included of these recommendations suggested that preclinical research should be replicated in different models of the same disease and in different species, and should also be replicated independently. What Do These Findings Mean?: This systematic review identifies a range of investigational recommendations that preclinical researchers believe address threats to the validity of preclinical efficacy studies. Many of these recommendations are not widely implemented in preclinical research at present. Whether the failure to implement them explains the frequent discordance between the results on drug safety and efficacy obtained in preclinical research and in clinical trials is currently unclear. These findings provide a starting point, however, for the improvement of existing preclinical research guidelines for specific diseases, and for the development of similar guidelines for other diseases. They also provide an evidence-based platform for the analysis of preclinical evidence and for the study and evaluation of preclinical research practice. These findings should, therefore, be considered by investigators, institutional review bodies, journals, and funding agents when designing, evaluating, and sponsoring translational research. Additional Information: Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001489.