A Commentary on Fasting of Nonclinical Research Animals.

This commentary discusses the implementation of fasting in nonclinical animal experimental subjects. The short-term removal of food from cages of experimental animals is in all respects innocuous. The term "stress" is ill-defined and the statutes and regulations governing animal research laboratories that exert their authority in the performance of their operations do so without substantive grounds to base compliance. The legislative and administrative history of the implementation of the Animal Welfare Act (AWA) has evolved into the development of laboratory management strategies that focus on the reduction of the biological cost of stress to the animals and the determination of when subclinical stress (eustress) becomes distress. Animal welfare is based on the tenet that in laboratories conducting animal research in compliance with Good Laboratory Practices (Title 21 USC, Chapter 13,§58), it is the study protocol and the study director that establish procedures and processes that are approved by each Institutional Animal Care and Use Committee to ensure the humane care and use of animals in research, teaching, and testing and to ensure compliance with guidelines and regulations. This approval process establishes the justification of eustress in the environment that do not rise to the threshold of distress under the AWA.

De-risking in Tier I CNS safety assessments is the primary function of study design and technical training of laboratory staff observers.

Derisking is not a pharmaceutical industry strategy to reduce time, effort, or costs in drug development. Derisking strategies originated within the National Institutes of Health as a predicate to good science. There is a growing sentiment within drug development programs to diminish the importance of behavioral measures in toxicological studies and in the Tiered Safety assessment plans of the U.S. Regulatory Agencies and the International Commission on Harmonization. The validity and reliability of the Functional Observational Batter (FOB) is critically dependent on consistency and technical quality in each risk assessment plan. US Federal and International drug approval organizations have universally adopted the concept of principles of test construction rather than delineating specific behavioral assay endpoints for inclusion of the FOB in nonclinical safety protocols. The validity and reliability of behavioral observations in standardized neurotoxicity screening is critically dependent on the FOB developed by the Study Director with the Sponsor throughout all stages of testing.. The individual risk factors selected for observation to be included in the early Tier 1 safety program should be determined by the mechanism and mode of action of the test article. The results of Tier I testing are the basis for Tier II testing designs. Critical to the compliance with Good Laboratory Practices is the documentation of training of the operational staff scheduled to conduct all aspects of the established protocol.

Distortion Product Otoacoustic Emission Test is Not the Test to Use in Nonclinical Safety Assessment.

Ototoxicity and ocular toxicity screening are but two examples of specialty product lines that are often employed as Tier II or III nonclinical safety/hazard screening assessments. Compared to the regulatory guidelines that govern over standard toxicology or neurotoxicology programs, there is a paucity of regulatory strategies to address these specialized product lines. With respect to ototoxicity testing, we argue for the inclusion of the "least burdensome principles" adopted by the US FDA in providing the most pragmatic, efficient, and directed identification of potential harm to auditory function in the nonclinical safety arena. We argue for the exclusive use of the auditory brainstem response and the exclusion of the distortion product otoacoustic emissions (DPOAEs) in these Tiered II safety assessment programs. The inclusion of both are a burden on operational staff and, due to the extended episodes of anesthesia required to conduct both assays, this strategy poses a health and welfare concern for the selected animal species to be used. The DPOAE does not provide any sufficiently valid or reliable data above and beyond the gold standard ABR data, followed by complete oto-histopathology and cytocochleogram combination designs.

Nonclinical evaluation of abuse liability of the dual orexin receptor antagonist lemborexant.

Lemborexant is a dual orexin receptor antagonist (DORA) approved in multiple countries including the United States, Japan, Canada and Australia for the treatment of adults with insomnia. As required for marketing approval of new compounds with central nervous system activity with sedating effects, the abuse potential of lemborexant was assessed in accordance with regulatory guidelines, which included three nonclinical studies. These assessments comprised physical dependence and drug discrimination studies in rats and a self-administration study in rhesus monkeys. There was no evidence of withdrawal signs following abrupt drug discontinuation, indicating that lemborexant does not induce physical dependence. In the drug discrimination study, lemborexant at doses up to 1000 mg/kg administered orally did not cross-generalize to the zolpidem training stimulus, although another DORA included in the same experiment, suvorexant, showed partial generalization with zolpidem. In rhesus monkeys, lemborexant treatment did not induce any gross behavioral changes, and there was no increase in self-administration rates compared with control, indicative of a lack of reinforcing effects of lemborexant. Collectively, these nonclinical studies support the position that lemborexant, which has been placed in Schedule IV by the United States Drug Enforcement Administration, has a low risk of abuse in humans.

In further defense of nonclinical abuse liability testing of biologics.

Risk assessment is not a choice. Drug Abuse Liability (DAL) is mandated under international and national drug control statutes for all drugs targeting the CNS. Once administered to humans many biologics may have long-lived or permanent physiological effects that make DAL testing arduous. We respond to premises of a recently published position on DAL testing of biologics by de Zafra et al. (2018). We propose that, at a minimum, Sponsors submitting a Biologics Licensure Application (BLA) must think "outside the box" and include differential study designs for the same three core small NME assays detailed in the current DAL guidelines (self-administration, drug discrimination, and dependence liability). Abuse liability testing for drug scheduling decisions for marketing approval are not excluded or limited from risk assessment analysis simply because the entity is a biologic. In fact, more robust study designs may be necessary to address alterations in the reinforcing and discriminative stimulus effects of common drugs of abuse, as well as the dependence liability of the biologic, itself.

Predicting the Need for a Tier II Ototoxicity Study From Early Renal Function Data.

History has established that many drugs, such as the antibiotics, chemotherapies, and loop diuretics, are capable of inducing both nephrotoxicity and ototoxicity. The exact mechanisms by which cellular damage occurs remain to be fully elucidated. Monitoring the indices of renal function conducted in the Food and Drug Administration's prescribed set of early investigational new drug (IND)-enabling studies may be the first signs of ototoxicity properties of the new drug candidate. In developing improved and efficacious new molecular entities, it is critically necessary to understand the cellular and molecular mechanisms underlying the potential ototoxic effects as early in the drug development program as possible. Elucidation of these mechanisms will facilitate the development of safe and effective clinical approaches for the prevention and amelioration of drug-induced ototoxicity prior to the first dose in man. Biomarkers for nephrotoxicity in early tier I or tier II nonclinical IND-enabling studies should raise an inquiry as to the need to conduct a full auditory function assay early in the game to clear the pipeline with a safer candidate that has a higher probability of continued therapeutic compliance once approved for distribution.

CNS Safety Screening Under ICH S7A Guidelines Requires Observations of Multiple Behavioral Units to Assess Motor Function.

In the adoption of behavior as a critical end point in safety pharmacology and neurotoxicity screening, federal regulatory agencies have shifted the predominating scientific perspective from pharmacology back to the experimental analysis of behavior (psychology). Nowhere is this more evident than in tier I safety assessment of the central nervous system (CNS). The CNS and peripheral nervous system have multiple behavioral units of general activity. A complete picture of the motor control neural pathways cannot be measured by any one single approach. The CNS safety protocols under International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use S7A are required to be conducted in accordance with Good Laboratory Practices by trained technical staff. The CNS safety assessments necessitate the inclusion of a thorough and detailed behavioral analysis of home cage activity, the response to handling, and transportation to and observations within an open-field apparatus with ancillary measures of basal muscle tone, muscle strength, and tremor in a functional observation battery, as well as quantitative measurements of 3-dimensional activity in an automated photobeam arena. Cost-cutting initiatives or a radical application of the "reduce use" principle of the 3 Rs only jeopardize the spirit, intent, and predictive validity of tier I safety testing assays dictated by current drug safety guidelines.

"Not Everything That Shakes Is a Seizure": Making the Determination of Test Article-Induced Convulsions in Toxicology Studies.

Spontaneous unexpected events occasionally develop during the course of rodent preclinical toxicology studies. The presentation of serious adverse events on animal studies may require notification of these events to the Food and Drug Administration if the events are most likely the direct result of test article administration. Classical conditioning of emotional responses may occur over the course of a repeat-dose study and clinical observation calls of "convulsions" are reported to the study director and/or staff veterinarians. In the current heightened environment of most research laboratories related to general animal welfare issues, it is imperative to have an action plan that will help to elucidate the potential origins of these motor events. We provide 10 factors that should be considered to help the study director determine the most likely cause of these motor attacks as being organic or psychogenic in origin.

Ototoxicity: The Radical Drum Beat and Rhythm of Cochlear Hair Cell Life and Death.

The function and structure of the auditory information processing system establishes a unique sensory environment for the "perfect storm." The battle between life and death pits the cascade of an apoptotic storm, programmed cell death cascades, against simple cell death (necrosis) pathways. Live or die, the free radical biology of oxygen and hydroxylation, and the destruction of transition metal migration through the mechanical gate sensory processes of the hair cell lead to direct access to the cytoplasm, cytoplasmic reticulum, and mitochondria of the inner workings of the hair cells. These lead to subsequent interactions with nuclear DNA resulting in permanent hearing loss. The yin and yang of pharmaceutical product development is to document what kills, why it kills, and how do we mitigate it. This review highlights the processes of cell death within the cochlea.

NKTR-181: A Novel Mu-Opioid Analgesic with Inherently Low Abuse Potential.

The increasing availability of prescription opioid analgesics for the treatment of pain has been paralleled by an epidemic of opioid misuse, diversion, and overdose. The development of abuse-deterrent formulations (ADFs) of conventional opioids such as oxycodone and morphine represents an advance in the field and has had a positive but insufficient impact, as most opioids are still prescribed in highly abusable, non-ADF forms, and abusers can tamper with ADF medications to liberate the abusable opioid within. The abuse liability of mu-opioid agonists appears to be dependent on their rapid rate of entry into the central nervous system (CNS), whereas analgesic activity appears to be a function of CNS exposure alone, suggesting that a new opioid agonist with an inherently low rate of influx across the blood-brain barrier could mediate analgesia with low abuse liability, regardless of formulation or route of administration. NKTR-181 is a novel, long-acting, selective mu-opioid agonist with structural properties that reduce its rate of entry across the blood-brain barrier compared with traditional mu-opioid agonists. NKTR-181 demonstrated maximum analgesic activity comparable to that of oxycodone in hot-plate latency and acetic-acid writhing models. NKTR-181 was distinguishable from oxycodone by its reduced abuse potential in self-administration and progressive-ratio break point models, with behavioral effects similar to those of saline, as well as reduced CNS side effects as measured by the modified Irwin test. The in vitro and in vivo studies presented here demonstrate that NKTR-181 is the first selective mu-opioid agonist to combine analgesic efficacy and reduced abuse liability through the alteration of brain-entry kinetics.

Preclinical assessment of the abuse potential of the orexin receptor antagonist, suvorexant.

Suvorexant (Belsomra®) is a dual orexin receptor antagonist approved for the treatment of insomnia. Because of its pharmacology within the central nervous system, intended therapeutic indication, and first-in-class status, an assessment of suvorexant abuse liability potential was required prior to marketing approval. The nonclinical abuse liability potential studies for suvorexant included: 1) rat drug-dependence model to assess physical dependence following abrupt cessation; 2) rat drug-discrimination model to examine the potential similarity of the interoceptive or subjective effects of suvorexant to those elicited by zolpidem and morphine; 3) self-administration model to assess the relative reinforcing efficacy of suvorexant in rhesus monkeys conditioned to self-administer methohexital. No significant signs of spontaneous drug withdrawal or 'discontinuation syndrome' were observed in rats following abrupt discontinuation of suvorexant. Suvorexant did not elicit complete cross-generalization to either a zolpidem or morphine training/reference stimuli in rats, and suvorexant was devoid of behavioral evidence of positive reinforcing efficacy in monkeys. These nonclinical findings suggested that suvorexant will have low abuse potential in humans. In the final regulatory risk assessment, suvorexant was placed into Schedule IV, likely due to its first-in-class status, its sedative properties, and the outcome of the clinical abuse potential assessment.

Small Compartment Toxicity: CN VIII and Quality of Life: Hearing Loss, Tinnitus, and Balance Disorders.

Life experiences, industrial/environmental exposures, and administration of Food and Drug Administration (FDA)-approved drugs may have unintended but detrimental effects on peripheral and central auditory pathways. Most relevant to the readership of this journal is the role that drug treatments approved by the FDA as safe and effective appear to interact with 3 independent modes of toxicity within the small compartment of the ear. What may seem to be trivial drug-induced toxicity has the potential to change important measures of quality of life and functional capacity of mid- to late-life patients. Drugs meant to treat can become the source of interference in the activities of daily living, and as a result, treatment compliance may be jeopardized. Ototoxicity has been defined as the tendency of certain therapeutic agents and other chemical substances to cause functional impairments and cellular degeneration of the tissues of the inner ear resulting in hearing loss. However, one of the largest contributors to hospitalizations is fall-related injuries in the elderly patients associated with disorders of vestibular function linked to progressive and drug-induced toxicities. Tinnitus affects 35 to 50 million adults representing approximately 25% of the US population, with 12 million seeking medical care and 2 to 3 million reporting symptoms that were severely debilitating. This review is intended to highlight these targets of neurotoxicity that threaten the usefulness of drug treatments deemed safe and effective prior to access by the general public.

Preclinical assessment of abuse liability of biologics: In defense of current regulatory control policies.

Current regulatory policies of both the US Food and Drug Administration and Drug Enforcement Administration do not delineate automatic exceptions for biologics with respect to preclinical assessments for abuse liability of all new entities. As defined in current guidance documents and drug control policies, an exception may be given upon thorough review of available data, therapeutic target and in consultation with the Controlled Substances Staff within the Center for Drug Evaluation and Research of the FDA, but a blanket exception for all biological entities is not currently available. We review the abuse liability testing of four known biologics with definitive positive abuse liability signals in the three core abuse liability assays, self-administration, drug discrimination, and dependence potential described in the FDA draft guidance document. Interestingly, while all four examplars have positive abuse liability signals in all three assays, two of these biologics are controlled under the Comprehensive Drug Abuse and Control Act (CSA, 1970) and the other two are not currently controlled. Admittedly, these four biologics are small molecule entities. However, there is no reference to "molecular size" in the legally-binding statutory definition of biologics under the FD&C act or in the Controlled Substances Act. Neither of these drug control policy mandates have a bifurcated control status in which to make exceptions based solely on molecular size. With the current pharmaceutical focus on new technologies, such as "Trojan Horses", targeting the active transport of large molecule entities directly into the CNS, an argument to automatically exempt new molecular entities solely on molecular size is untenable. We argue that for the safety and health of general public the current regulatory control status be maintained until definitive criteria for exceptions can be identified and amended to both the FD&CA and CSA, if warranted.

REL-1017 (esmethadone; D-methadone) does not cause reinforcing effect, physical dependence and withdrawal signs in Sprague Dawley rats.

REL-1017 (esmethadone, D-methadone) is the opioid-inactive d-isomer of racemic D,L-methadone. REL-1017 may exert antidepressant effects via uncompetitive N-methyl-D-aspartate receptor (NMDAR) channel block. As REL-1017 is expected to exert central nervous system activity, full characterization of its abuse potential is warranted. We evaluated lack of reinforcing effect, physical dependence, and withdrawal of REL-1017 in Sprague Dawley rats. (1) Self-administration Study Rats were trained to self-administer oxycodone intravenously (IV) and then were subjected to 3-day substitution tests where saline, oxycodone, and REL-1017 were self-delivered IV by a fixed number of lever presses; (2) Drug Discontinuation Study Rats were treated for 30 days by oral gavage with vehicle, REL-1017, ketamine or morphine and evaluated for withdrawal with functional observational batteries (FOBs). In the self-administration study, rats treated with saline, vehicle, and all REL-1017 doses showed the typical "extinction burst" pattern of response, characterized by an initial rapid increase of lever-pressing followed by a rapid decrease over 3 days. Rats treated with oxycodone maintained stable self-injection, as expected for reinforcing stimuli. In the withdrawal study, REL-1017 did not engender either morphine or ketamine withdrawal signs over 9 days following abrupt discontinuation of drug exposure. REL-1017 showed no evidence of abuse potential and did not engender withdrawal symptomatology.

Study design criteria for regulatory-based drug control action: Drug discrimination.

This "methods paper" focusses on one specific and limited aspect of drug safety evaluations required for all new drug entities that affect the central nervous system - the drug discrimination (DD) assay. We focus on three critical factors involved in experimental design and protocol development for the conduct of DD studies for abuse liability risk assessment that comply with the Good Laboratory Practice Guidelines (GLPs). The selection of 1) the reference drug(s) choice, 2) training dose selection, and 3) the selected route-of-administration will determine the applicability of the data to meet the regulatory expectations of the 8-factors determinative of schedule control recommendations. The study conduct and resulting data submission to the FDA are intended for drug scheduling review by the Controlled Substances Staff in the Center for Drug Evaluation and Research (CDER) at the US Food & Drug Administration (FDA). These animal studies are required to meet the statutory requirements of the Controlled Substances Act of 1970. The abuse liability study is conducted during Phase II and III of human clinical trials. Procedural or method-based errors this late in drug development can result in a significant economic and business threat to the program.

FOB vs modified Irwin: What are we doing?

There is a general sentiment in the nonclinical safety assessment literature and the proponents of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), that the "Modified Irwin" and the Functional Observation Battery are distinct and unique assays for the nonclinical assessment of the central nervous system (CNS). We identify and defend the position that the Irwin screen was developed as an FOB and both terms refer to a single, unitary functional assay. In giving credit to one prominent contributor to any one significant discipline of science for a specific assay, orientation, or theory may have an exclusionary influence on the merits of other prominent contributors within the same research arena. Scientific organizations as well as journal and textbook editors have attempted to unify the nomenclature used within a scientific discipline to make the disciplines conform to non-attributional surname nomenclatures. For example, the Salk-Sabin immunization is simply referred to as the polio vaccine. The "Skinner box" is now the "operant chamber" and "Pavlovian conditioning" is now "respondent conditioning". In 1968, Samuel Irwin established an operational method of analysis used for measuring drug effects in purpose bred laboratory animals. We present and defend the view that the behavioral screening assay developed by Irwin is, for all intents and purposes, a functional observational battery (FOB). We take the position that in standardizing nomenclature without "surnames" the FOB is simply the contemporary name for the data collection system in use under the harmonized safety pharmacology guidelines.

Establishing performance characteristics for positive control article selection in drug self-administration studies.

The selection of a controlled substance (CS) for use as the positive control article in a nonclinical drug abuse liability (DAL) assessment study should be contemplated carefully and with full understanding of the stated intent of the study design. Any CS that can maintain day-to-day stable baseline responding of voluntary intravenous intakes in animals may be selected under the current guidelines. Schedule I - IV CNS stimulants, depressants, and sedative/hypnotics can serve as maintenance drugs in these protocols, but not all of these compounds will provide comparatively efficient, robust, and stable daily intakes. Each Sponsor is directed to select a positive control article and training dose that will provide the most balanced, predictive, and scientifically-sound comparison consistent with the mechanism of action or therapeutic target of the test article. The SA study design is not a "one-size-fits-all" assay. This is a discussion of the critical design factors to be considered in selecting the most appropriate positive control article to use for a SA study.

NOEL and NOAEL: A retrospective analysis of mention in a sample of recently conducted safety pharmacology studies.

INTRODUCTION: The concept of characterizing adversity in relation to administered test article dose and/or exposure within toxicology studies has long been considered a normal aspect of the drug safety evaluation enterprise. The typical way this is done in drug safety investigations is by examining study data, often with focus on clinical signs, clinical pathology and histopathology, to determine a No-Observable-Effect-Level (NOEL) and/or a No-Observable-Adverse-Effect-Level (NOAEL). Once established, these, with other information, may be used to identify a safe starting dose in human clinical trials. Although safety pharmacology (SP) is concerned to identify and characterize potentially "adverse" functional effects, NOEL, and particularly NOAEL, traditionally do not have application in SP study interpretation and reporting. METHODS: An anonymized survey of a contract research laboratory master schedule was undertaken to appreciate recent usage of these concepts in GLP (Good Laboratory Practice) cardiovascular, respiratory, and neurobehavioral safety studies. RESULTS: Results across the sample of studies (N = 635) generally confirmed application of appropriate dose selection strategies, as there was a very low proportion (<1%) of observed severe adverse events (antecedent observations ultimately associated with morbidity/mortality). Data further indicated either no mention of NOEL/NOAEL (50%), or alternately, explicit identification of NOEL (28%), or NOAEL (21%). The majority of times a NOAEL was identified, it was also the case that this coincided with the highest dose administered (e.g., there may have been drug-related findings, but these were considered non-adverse across the dose range). DISCUSSION: While the concept of adversity is certainly relevant to nonclinical SP studies, actual current practices appear to reflect a history which generally avoids toxicologically-oriented classifications such as NOAEL. Questions remain regarding the applicability of NOAELs to safety pharmacology studies, including, importantly, the specific circumstances under which such designations of adversity may be considered to add value to understandings of relative risk and risk mitigation in early human clinical trials.

Extended recording of LMA in rats: Effects of IV catheters, "comfort jackets" and chamber lids on arterial blood gas parameters.

The standard infrared photobeam locomotor activity system has been used extensively in neurobiology and neuropharmacology to study the functional impact of direct manipulations of the nervous system. There is interest in using the activity monitors to assess the early stages of drug withdrawal in rodents. In a standard twice-daily dosing strategy animals would be dosed at 6:00 am and 5:00 pm for 15 to 30 days. There is interest in using the chambers to assess the early stages of the discontinuation syndrome. Placement of the rodents into the chambers following the scheduled sham or vehicle last dose of a 15- to 30-day subchronic dosing regimen (b.i.d., t.i.d., etc.) and monitoring overnight allows for a quantitative measure of the initial physiological homeostatic acclimation period during the lights-out period. By using the chambers there is no circadian dysrhythmia induced as an experimental confound and objectively verifiable data is generated during the period expected to correspond with the plasma drug levels approaching zero and the onset of discontinuation syndrome. We demonstrated that untreated "normal" rats showed a normal decelerating time-effect curve over the 12-hour monitoring period that was not compromised by restricted access to food and water. Arterial blood gas monitoring before and after 12 h of night-time activity chamber monitoring clearly demonstrated normal respiratory function with no clinical signs of any blood gas-based diagnosis of metabolic dysfunction.

Relative equivalence of CNS safety (FOB) assessment outcomes in male and female Wistar-Han and Sprague-Dawley rats.

In 2006 the National Toxicology Program (NTP) of the FDA shifted to the preferred use of Wistar-Han rats from the more commonly used Sprague-Dawley (SD) strain - and industry followed. While European laboratories preferred the Wistar-Han line, there was a paucity of relevant historical control data in many US research institutions for the new "industry standard" rat strain. In 2010 the NTP reversed its decision and shifted back to SD rats because of reproductive issues with the Wistar strain. For post hoc comparative analyses, we report minimal practical differences in Functional Observational Battery (FOB) data from a large sample of male and female Wistar-Han and SD rats. In summarizing data from the preclinical safety evaluations of the CNS effects of new drugs using the FOB, it is crucial to understand the value of not only how the functional expression of drug effects in the rat are predictive of the human response, but also how and why they differ. What we can predict from the behavioral and physiological response of the designated test system to drug administration is the foundation of "generalizability" to the human's response. Here, we conclude that the use of either SD or WH rat strains in standard CNS safety studies provide equivalent supportive data for CNS safety assessment required for IND approval under the harmonized guidelines.