Abuse liability assessment of hydrocodone under current draft regulatory guidelines

Journal of Pharmacological and Toxicological Methods 75 (2015) 118–129

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Original article

Abuse liability assessment of hydrocodone under current draft regulatory guidelines David V. Gauvin ⁎, Margaret McComb, Robert Code, Jill A. Dalton, Theodore J. Baird Department of Safety Pharmacology and Neurobehavioral Sciences, MPI Research Inc., Mattawan, MI, United States

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Article history: Received 11 February 2015 Received in revised form 10 April 2015 Accepted 4 May 2015 Available online 9 May 2015 Keywords: Hydrocodone Self administration Drug discrimination Drug dependence Discontinuation syndrome Withdrawal Morphine Oxycodone Abuse liability Methods

a b s t r a c t Introduction: The abuse liability of hydrocodone was assessed in male Sprague–Dawley rats under the European Medicines Agency, the International Commission on Harmonisation, and the U.S. Food & Drug Administration draft guidelines for the non-clinical investigation of the dependence potential of medicinal products. Methods: Self-administration, drug discrimination, and repeat-dose two week dependence liability studies were conducted to compare hydrocodone to the prototypical opiates, morphine and oxycodone. Results: Hydrocodone was self-administered, produced an opiate-like subjective discriminative generalization profile and produced a significant discontinuation syndrome following abrupt treatment cessation that was quantitatively and qualitatively similar to morphine and/or oxycodone. Conclusion: Hydrocodone has abuse liability more similar to Schedule II opiates than other Schedule III compounds currently controlled under the U.S. Controlled Substance Act.

© 2015 Elsevier Inc. All rights reserved.

1. Introduction Guidelines for regulatory review of all new psychoactive substances for both human and veterinary approval have been disseminated by the European Monitoring Centre for Drug and Drug Addiction (EMCDDA, 2009), the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMEA, 2006), the International Conference on Harmonisation (ICH, M3[R2], 2009), and the United States Food and Drug Administration (FDA, 2010). These guidelines are intended to 1) help define the scope of the term “psychoactive substances” and, 2) to put in place a sound methodological and procedural basis for carrying out risk assessments in regard to health and social risks of the use of, manufacture of, and traffic in these new psychoactive substances that involve member states of both the 1961 United Nations Single Convention on Narcotic Drugs and the 1971 United Nations Convention on Psychotropic Substances. A three-part, evidence-based preclinical risk assessment plan requires standardized behavioral assays of self-administration, drug discrimination, and dependence potential to be conducted in either rodents (contemporarily considered the primary model) or non-human ⁎ Corresponding author at: Dept NBS, MPI Research Inc., 54943 North Main Street, Mattawan, MI 49071, United States. Tel.: +1 269 668 3336x1613; fax: +1 269 668 4151. E-mail address: [email protected] (D.V. Gauvin).

http://dx.doi.org/10.1016/j.vascn.2015.05.003 1056-8719/© 2015 Elsevier Inc. All rights reserved.

primates. The results of these assays must be supplied prior to health agency approval of any new chemical entity that 1) crosses the blood brain barrier; 2) is pharmacologically similar to any known drug of abuse; 3) has a novel mechanism of action; 4) produces psychoactive effects such as sedation, euphoria, or mood changes; or 5) has any direct or indirect actions on other neurotransmitter systems associated with abuse potential, such as dopamine, norepinephrine, GABA, acetylcholine, opioid, NMDA, and cannabinoid. The chemical 4,5α-epoxy-3-methoxy-17-methyl-morphinan-6-one was given the drug name, dihydrocodeinone, when it was first marketed in Germany in the early 1920's it sold under the proprietary name of Dicodid®. It was never screened for abuse liability prior to approval as a medicinal product. As translated and cited by Eddy, Halbach, and Braenden (1957), hydrocodone addiction was reported as early as 1927: Müller de la Fuenta said that cases of addiction to dicodid were known in 1927; 17 of the 280 questionnaires analysed by Wolff, in 1928, reported dicodid addiction; and in 1930 Richtzenhain warned that “dicodidismus” was then so often observed that one should be as cautious with dicodid injection as one would be with morphine. In the United States the nonproprietary or generic name adopted for the drug was simply, hydrocodone. Hydrocodone combination products

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(i.e., Vicodin™, Hycotuss™), the only legitimate U.S. drug products on the market at the time, were placed into Schedule III of the Controlled Substances Act (21 USCA, Chapter 13 §801-971) in spite of differential control of its analgesic equivalents oxycodone (Roxicodone™, Percocet™) and morphine (Kadian™, MS-Contin™) into Schedule II. The differential scheduling action was approved under the premise that the likelihood of acetaminophen toxicity would limit or minimize the abuse of hydrocodone pharmaceutical products (cf Commission on Narcotic Drugs: The Single Convention on Narcotic Drugs; Schedules: E/CN.7/AC.3/9/Add.1; 18 November 1958; Eddy, Halbach, & Braenden, 1956). In the conclusions of these early reviews, hydrocodone was considered to be pharmacologically equivalent to morphine with respect to analgesia, CNS depression, and dependence potential. Reports in the published literature and by the U.S. National Institute on Drug Abuse indicate that hydrocodone-combination products are consumed in large quantities without concomitant and significant changes in liver function profiles that might have been predicted based on, for example, acetaminophen content. The therapeutic effects of hydrocodone, its abuse potential, and actual abuse history in the US were thoroughly reviewed during the requisite 8-factor analyses for schedule control actions. Full literature reviews have been conducted by both U.S. DEA (2014) and U.S. Department of Health and Human Services (2014). More recently, hydrocodone single entity and combination products have been administratively placed into Schedule II (Drug Enforcement Administration, 2014). The present studies were designed to systematically assess and compare the relative abuse liability of hydrocodone and the prototypical CII opiates, morphine and oxycodone, using an integrative approach consistent with the current standardized international regulatory guidelines.

2. Material and methods 2.1. Subjects Male Sprague–Dawley rats ordered from Charles River Laboratories (Portage, MI), 7–8 weeks of age and weighing approximately 230–260 g were used in these experiments. The preponderance of the published reports of drug abuse models in animal species has indicated a selective use of male animals. It is generally assumed that the chronic nature of dosing regimens used in these studies render equi-effective responses in animal subjects; no gender-differences were expected in the direction, duration, or magnitude of behavioral and physiological effects induced by the procedures set forth in the study plans, and accordingly only males were used. Animals in the self-administration and drug discrimination studies were singly housed in solid bottom poly-boxes with non-aromatic bedding. Animals in the drug dependence study were singly housed in standard stainless-steel wire-bottom cages. Solid-bottom cages bedding materials were not used in this latter study because 1) of the potential to induce pica (Batra & Schrott, 2011) and the incidence of copraphagia (Barnes & Fiala, 1958a,b, 1959; Barnes, Fiala, McGehee, & Brown, 1957; Iwomoto & Klaassen, 1977; Lugo & Kern, 2002; March & Elliott, 1954; Mullis, Perry, Finn, Stafford, & Sadée, 1979) in rats. Access to fecal boli containing behaviorally active concentrations of opiate and opiate-related metabolites, therefore, represents a significant experimental confound in such a study plan, and was avoided by the use of alternate wire bottom caging. Fluorescent lighting was provided via an automatic timer for approximately 12:12 hour light:dark cycle per day. Temperature and humidity were monitored and recorded daily and maintained according to standard operating procedures between 64 to 79 °F and 30 to 70%, respectively. The basal diet was block Lab Diet® Certified Rodent Diet #5002 (PMI Nutrition International, Inc.). The diet and tap water were available ad libitum unless designated otherwise (see below). The

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protocols governing these studies had prior approval of MPI Research Institutional Animal Care and Use Committee. 2.2. Equipment The self-administration and drug discrimination studies were conducted in standard rat two-lever operant chambers (ENV-008CT; Med Associates, Inc. NH, USA) with a modified top for self-administration (MED-008CT-B2) equipped with a syringe pump (PHM-100) located in a specially constructed and locked box located on top of the soundattenuating cubicle (ENV-018MD). Each chamber was equipped with two stimulus lamps (ENV-221M), two retractable levers (ENV112CM), house lamp (28 V DC, 100 mA, ENV 215M), a modular pellet dispenser (ENV 203N-45), and exhaust fan. The operant chamber was interfaced (SmartCtrl 8 Input, 16 Output Package) with an IBM-based personal computer system capable of controlling 16 chambers. An operant control and data collection software program for both drug discrimination and self-administration procedures (R. Code, MPI Research, Inc.) was written and validated using MED-PC language. A total of thirty-two identically-equipped chambers were used in these studies that were located in two security-controlled, video monitored, key-card accessed rooms of the test facility. 2.3. Surgery Sterile surgical implantation of jugular catheters to enable the self-administration study was conducted by Charles River Laboratories (Portage, MI) using specially designed and manufactured catheters (MPI Research, Inc.). Eighty-six percent of all implanted catheters remained patent for up to 6 months under current laboratory standards and procedures (Gauvin, Dalton, Baird, & Faqi, 2013). Catheters were flushed regularly with normal sterile saline for injection (USP) and locked with heparinized solutions (30–100 IU/mL) of either 50% dextrose or saline throughout the life of the catheter to prolong patency. Saline flushing occurred immediately prior to and after selfadministration sessions. Patency was verified daily with presession and postsession flushing of catheters. The resistance to flow was used as the first indicator of possible catheter occlusion. If catheter occlusion was suspected, a systems check on the viability of the implanted catheters was conducted. Technicians would administer a 5 mg/kg dose of methohexital (Brevital™), or any equivalent short-onset, short-lived barbiturate through the catheter. Animals were monitored for at least 5 min post injection for signs of lethargy, malaise, or unconsciousness. If the catheter was patent, the animal would appear anesthetized shortly following the infusion. Recovery from the system check would take approximately 15 min. 2.4. Procedures 2.4.1. Self-administration Details of the self-administration training and testing procedure are similar to those previously described by Briscoe et al. (1999). The rat self-administration procedure that has been adopted by the industry and FDA is described as a single lever operant lever press response under a fixed-ratio 10 (FR10) schedule of cocaine deliveries with session lengths of at least 1 h duration. Once animals demonstrated day-to-day stability in responding for cocaine deliveries (less than 20% day-to-day variability in the total number of training drug deliveries for three consecutive days). Once each animal demonstrated stable operant responding for cocaine infusion (0.56 mg/kg/injection) for three contiguous days of training or maintenance sessions a series of test sessions were planned (A-B-A study design). The first series of test sessions was conducted with the maintenance dose of cocaine (0.56 mg/kg/injection) — in this session the animal, for the first time, was allowed to respond for an unlimited number of injections over a one-hour access period on each of three consecutive days. Following

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the completion of the maintenance dose tests, an “extinction test” with saline (vehicle) was scheduled. These operant conditioning procedures make explicit use of the fact that drug-reinforced responding undergoes extinction and eventually decreases in probability when a reinforcing drug dose is replaced with vehicle (saline) or an ineffective drug reinforcer or drug dose. Complete dose–response functions were conducted for the training drug (cocaine) and morphine, hydrocodone, and oxycodone for comparison. Using 32 rats each dose of a 5 dose test article generalization function in a subset of 6 animals per dose can be tested without repeat. Each test was preceded by at least 3 contiguous days of cocaine maintenance self-administration with 0.56 mg/kg/injection of cocaine with less than 20% day-to-day variability. That is, all drug tests are conducted following 3 cocaine baseline sessions (i.e. A–B–A design; where “A” refers to the maintenance dose of cocaine, and “B” refers to a novel test condition). Following the completion of each three-day substitution test, the animals self-administered the “maintenance” dose of 0.56 mg/kg/injection until stable behavioral output was observed permitting the next scheduled substitution test. 2.4.2. Drug discrimination Details of the training and test sessions are similar to Gauvin, Harland, Michaelis, and Holloway (1989), Gauvin, Criado, Moore, and Holloway (1990), and Harland et al. (1989). As previously highlighted by Glennon and Young (2011), the administration of drugs can exert effects on behavior via sites of action that are apart or aside from the CNS (p. 62). In preclinical abuse liability studies it is imperative to “think outside the box” and be open to alternative approaches to drug administration outside the restricted scope of the IND enabling study design. Risk assessments must include actual abuse patterns in the real world with the knowledge that drug abusers will often not use the intended route of administration as used in preclinical screening study designs. Rectal administration of tablets (Coon, Miller, Kaylor, & Jones-Spangle, 2005; Stevenson & Hume, 1991), chewing of transdermal patches (Mrvos et al., 2012; Prosser, Jones, & Nelson, 2010), or the insufflation of crushed and pyrolyzed opiate tablets (chasing the dragon) were not part of any preclinical IND-enabling studies of opiates, but they are common routes-ofadministration in the opiate abusing population. The NCE might affect behavioral activity via an action at neuromuscular junction, the peripheral or spinal opiate receptor, the autonomic nervous system, or the site of (an accidentally inferior) injection. As described by Järbe (2011) the onset, offset, and potency of the discriminative properties of a drug may covary with the route-of-administration. In risk assessment reviews of preclinical animal-related data it is imperative to include variations in the route-of-administration in order to demonstrate or assess the relative risk of abuse in the real world when the drug is used nonmedically (Rush, Vansickel, & Stoops, 2011). Thus, an important part of the characterization of the mechanism of a druginduced change in behavior is the assessment of the relative contribution of rates-of-change, metabolic processes, and distributional actions that covary with routes of administration. One of these approaches is to train and test via differing routes of administration (Glennon & Young, 2011; Schechter, 1973). For example, Craft and Howard (1998) have previously shown equal potency between oral, subcutaneous, and intraperitoneal administration of nicotine with respect to the discriminative stimulus properties of 0.5 mg/kg nicotine in rats. Therefore, differential routes of administration were used for training and testing in this study (oral, sc, and ip). One group of 32 male rats were trained to discriminate between 20 mg/kg morphine or tap water administered orally (per os, po) 60 min prior to the training sessions in daily 30 minute two-lever operant sessions, under a fixed-ratio 10 schedule of food reinforcement (Group 1). Another group of 32 male rats was trained to discriminate between 1.0 mg/kg oxycodone or saline administered subcutaneously (sc) 30 min prior to the sessions (Group 2). Daily training sessions

continued until each rat emitted less than 20 responses prior to the delivery of the first food pellet and greater than 80% of total session responses were emitted on the stimulus appropriate lever. Training and test sessions ended after 50 reinforcer deliveries or 30 min, whichever occurred first. Test sessions were identical to training sessions except for the dose of drug administered before the session and during the test session 10 consecutive responses on either lever produced a food reward. Tests were conducted with various doses of orally administered morphine and hydrocodone (Group 1) or orally, intraperitoneal (ip), and subcutaneous injections of oxycodone or hydrocodone (Group 2). These time intervals were selected from published PK studies conducted with oxycodone and hydrocodone (Chan, Edwards, Wyse, & Smith, 2008; Cone, Darwin, Gorodetzkey, & Tan, 1978; Huang, Edwards, & Smith, 2005; Lelas, Wegert, Otton, Sellers, & France, 1999; Tomkins et al., 1997). 2.4.3. Dependence liability Methods for assessing the dependence potential of morphine in rats were developed by Akera and Brody (1968). While the procedure for other species varies, the basic method adopted by the College on Problems of Drug Dependence (Brady & Lukas, 1984) is as follows: Ideally, two evenly spaced injections per day are administered. The specific patterns of dosing (qd, bid, tid, etc.) must be derived from established pharmacokinetic data from the species used in these dependence liability studies. The Cmax, Tmax, and metabolic half-life of the compound will determine the actual number of dose administrations and the interdose intervals that should be used to adequately ensure that plasma concentrations are achieved and maintained throughout the full repeat dosing cycle (Cone et al., 1978; Lelas et al., 1999; Tomkins et al., 1997). Due to well characterized respiratory depression and self-mutilation that is induced in rat subjects by even moderate doses of opiates, the initial dose administered in these types of repeat-dose study designs is limited. Using an “equivalent dosing strategy” (see below), as tolerance to the sedative, and respiratory depressive effects develops to the initial dosing schedule, the maintenance dose was increased. Rats were dosed over 14 days. The initial dose of both drugs was 20 mg/kg administered twice per day. The morning dose was, in effect, a loading dose to achieve chronic equivalence from the start of treatment in order to provide a reference point to evaluate chronic tolerance development. Based on the acute time course of action of this dose previously reported in the scientific literature, the dosing times of (7:00 am and 5:00 pm) were selected allow an approximate 12 h average dose interval. These dose intervals (10 and 14 h) were used to ensure relative continuity of CNS drug exposure with adequate recovery between drug administrations. In order to ensure that the selected dosing schedule achieved both equivalency of peak responses as well as continuity of drug effect between doses, a behavioral scoring system of ataxia assessment, first developed to measure CNS depression (Boisse & Okamoto, 1978a,b; Okamoto & Boisse, 1981; Okamoto, Boisse, Rosenberg, & Rosen, 1978; Okamoto, Hinman, & Aaronson, 1981) was used, in part, to escalate the dose of morphine and hydrocodone from day to day up from an initial 20 mg/kg to a maximum approaching 300 mg/kg/day. Group mean scores on the ataxia scoring system were determined daily at 1:00 pm (6 h following the first daily dose) during the chronic dosing period. The lowest possible score of “0” implies no CNS impairment or depression. The highest score of “11” implies “severely depressed”. On observation of scores of 6 or less, the daily dose of morphine and hydrocodone was increased to the next incremental level. Scores of 7 or greater required additional review/consideration of the clinical presentation by the study director to determine potential need for increment. Accordingly for hydrocodone and morphine treatment groups, over the course of 15 days, the second of two daily doses of drug was increased from an initial dose of 20 mg/kg to the maximal tolerable dose (defined as the overt expression of clinical signs of toxicity; scores of

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11 or 12 on the ataxia scale). The morning daily loading dose may have been different from the afternoon dose. When, or if, a maximum tolerable dose was reached the dose was maintained for the remainder of the 15 day treatment regimen. Based on previously published reports of opiate dependence in rats, the maximum doses were expected to approximate levels as high as 300 mg/kg/day (expressed as the base). Doses were administered for 14 days and achieved a combined daily dose of 300 mg/kg by Day 13 (often 150 mg/kg/rat twice daily). On Days 15 through 18 animals received sham injections of saline to control for handling and injection conditioning. Complete functional observational batteries (Moser, McCormick, Creason, & MacPhail, 1988) were then conducted during the initial “withdrawal” phase of the study (Days 16, 17, and 18 of the study plan). Each rat was assessed as follows: 2.4.3.1. Functional observational battery. Each animal was observed for a minimum of 3 min in a black plexiglass, open-field observation box measuring 20 in. by 20 in. by 8 in. Parameters evaluated were based on those outlined in Moser et al. (1988) and Moser and Ross (1996). 2.4.3.2. Expected clinical signs. Based on the preponderance of scientific reports appearing in peer-reviewed scientific journals chronic opiate administration was expected to produce pica, gastroparesis, gastric distention, constipation, aggression, irritability, and self-mutilation. Treatments with a natural stimulant laxative (Senna) was initiated prior to and during the chronic dosing regimen. This was intended to mimic the standardized treatments of human patients receiving long-term high dose opiates and to minimize the deleterious effects of the opiates on gut motility during the course of drug administration on this study. Placement of aspen wood blocks, Nyla™ bones, and metal chain rings into the cages were used as added environmental enrichments for singly housed rats per MPI Research SOPs and also was intended to help ameliorate the incidences of self-mutilation. Peripheral histamine is released during opiate administrations in both humans and animal subjects. Histamine-induced itching in the limbs typically results in scratching, picking (crank bugs), and biting in human opiate addicts and self-mutilation of paws and limbs in “morphinized” animals. Cleansing with Novasan™ and the presence of the additional environmental enrichments were intended to reduce the potential need for medical intervention during this study. Part of a clear demonstration and validation for “dependence of the opiate type” were intended to be requisite clinical findings. During the “withdrawal phase” of the study it was expected that animals in both drug treatment groups would display marked or severe diarrhea and an expected body weight loss that could achieve 15 g/day — these are considered prototypical withdrawal signs from opiate dependence and an important target to be demonstrated in this study. This bodyweight loss is usually self-limiting, short-lived, and was expected to abate by the 3rd or 4th day of the withdrawal phase of the study. No need for treatment or medical intervention was expected. All of these expected clinical signs were presented to the full IAUC committee review of the protocol (Classification “D” of pain and distress) which approved and initiated the treatment plan and scheduled veterinary consultations throughout the conduct of the dependence liability study. 2.4.3.3. Food consumption. Each animal's food was weighed every other day in order to get a rudimentary impression of food intake. With rodent block diet, it is understood that such consumption would not be a perfectly accurate measure of actual nutritional intake for each rat. However, on the assumption that rats would lose or waste similar amounts of block diet each day, the food consumption measure was used to help discern incidences of pica, motoric effects, and gut motility properties potentially associated with the chronic dosing regimens.

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2.4.3.4. Body weights. Body weights were measured and recorded within 3 days of arrival, prior to randomization to treatment conditions, and every day during the chronic dosing regimen. Body weights measured following arrival are not included in this report. Animals were weighed daily during the chronic dosing and withdrawal phases of the stud, and as part of the individually scheduled FOBs. 2.5. Drugs Normal sterile saline for injection (USP) was used as the vehicle for all study designs and for sham injections in the dependence liability study. Hydrocodone bitartrate, oxycodone hydrochloride, and morphine sulfate pentahydrate were purchased from Mallinkrodt, Inc. (Covidian Pharma, Inc., St. Louis, MO). Doses were weighed and expressed as the base. For the oral morphine drug discrimination task, normal tap water was used as the control “vehicle” condition. As stated above, chronic high dose opiate treatments are known to induce constipation, weight gain and, and in some cases, gastroparesis in both human and animal research subjects. For the health and safety of the animals on this study there was no need to further demonstrate the full spectrum and magnitude of this major pharmacological effects of the opiates used as positive control articles (i.e., morphine and hydrocodone) by administration of an expanded range of (including higher) doses. As a preemptive treatment, two days prior to the initiation of dosing and daily (approximately 1:00 pm) during the 15 day chronic dosing regimen, each rat in Groups 2 & 3 received doses of a natural plant derived stimulant laxative, senna, to ensure gut motility was maintained during dose regimen. An initial dose of 210 mg/kg (5.97 mL/kg volume) was selected for administration. This initial dose was escalated upward in 50 mg/kg increments, up to a maximum dose of 600 mg/kg to maintain fecal output over the 2 week dosing period. Mengs, Mitchell, McPherson, Gregson, and Tigner (2004) have previously demonstrated the relative safety of up to 1500 mg/kg/day of senna treatments in rats exposed to daily doses for 13 weeks with no signs of toxicity or rebound phenomena during abrupt cessation of treatments. 3. Results 3.1. Self-administration All 32 rats were conditioned to initiate and maintain lever press responding to receive a single iv dose of 0.56 mg/kg/infusion of cocaine. Less than 20% day-to-day variability was maintained by such cocaine doses throughout the study plan. Fig. 1 compares the results of three consecutive daily substitution tests conducted with various doses of cocaine (upper left panel), morphine (upper right panel), hydrocodone (lower left panel), and oxycodone (lower right panel) in rats conditioned to initiate and maintain stable patterns of self-administration of 0.56 mg/kg/infusion of cocaine in the regulatory standard self-administration behavioral assay. Under the recommendations of the current FDA guidance document, substitution tests are conducted from a stable behavioral cocaine-selfadministration baseline. That is, all substitution tests are initiated following the demonstration of a day-to-day stability of cocaine self-administration. Substitution tests with saline, a non-reinforcer, engendered a typical “extinction” burst on Day 1 of substitution with a downward staircase pattern of responding during Day 2 and Day 3 with respect to the total number of injections administered (all four panels). The upper left panel shows the result of tests conducted with various doses of cocaine tested in ascending order Cocaine tests demonstrated a typical “inverted U-shaped” pattern of dose-effect function across the 1.25 log unit dose response function. Moderate doses of 0.32 and 0.1 mg/kg/injection maintained a higher number of selfinjections when compared to the higher doses of 0.32, 0.56, and

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Fig. 1. The group mean total number of infusions self-administered during one-hour daily sessions is plotted as a function of available cocaine dose expressed in mg/kg/injection in 32 rats conditioned to self-administer 0.56 mg/kg/infusion of cocaine in daily sessions (upper left panel). For comparisons, the group mean total number of infusions during substitution tests conducted with three opiate derivatives: morphine (upper right panel), hydrocodone (lower left panel) and oxycodone (lower right panel) in animals conditioned to self-administer 0.56 mg/kg/infusion of cocaine. Each unlimited access cross-generalization test was conducted following at least 3 consecutive days of cocaine self-administration baselines. Bars represent the daily mean intake of 32 rats (saline and 0.56 mg/kg/infusion cocaine) or six randomly selected rats tested for three consecutive days in one-hour unlimited access sessions at each selected drug and drug dose. The open circles represent the grand mean of all three days of substitution. The averaged total number of infusions self-administered during one-hour daily sessions is plotted as a function of available drug dose expressed in mg/kg/infusion. All drugs engendered a typical inverted U-shaped dose–response function. All three opiates were self-administered in rats conditioned to self-administer cocaine. Cocaine and oxycodone data adapted from Gauvin, Dalton and Baird (2013, p 455).

1.0 mg/kg/injection of cocaine. All five tested doses of cocaine sustained a similar pattern and number of injections over the three days of substitution when compared to vehicle control tests. The upper right panel of Fig. 2 shows the results of substitution tests conducted with the classic mu opioid agonist, morphine, in those rats trained to self-administer cocaine (Fig. 1, upper left panel). Note the abbreviated ordinal axis. Morphine produced a blunted or flattened “inverted U shaped” dose response function compared to the training drug, cocaine. While consistent day-to-day patterns of self-injections were demonstrated for each dose of morphine tested in this study, the patterns and numbers of injections engendered in these tests were very low across the tested dose range and were attributed to the pharmacodynamic properties of morphine in this limited one hour access period study design. Similar low patterns of responding for morphine in similar 1 hour access test sessions (e.g., less than 10 infusions) have been demonstrated by Mierzejewski, Koroś, Goldberg, Kostowski, and Stefański (2003) in Sprague–Dawley rats and by Sánchez-Cardoso et al. (2007), Sánchez-Cardoso et al. (2009) and Garcia-Lecumberri et al. (2011) in Lewis and Fischer 344 rats. With the catheter tip of the indwelling catheters placed at or very near the entrance of the right atrium, these low patterns of morphine intake may be related to the direct dynamic cardiovascular effects of intravenously administered morphine in the rat (Fennessy & Rattray, 1971; Randich, Robertson, & Willingham, 1993; Thurston, Starnes, & Randich, 1993). The lower left panel of Fig. 1 shows the results of substitution test sessions conducted with ascending doses of hydrocodone in rats conditioned to maintain stable day-to-day intakes of 0.56 mg/kg/infusion of cocaine interspersed between the specific dose substitution tests. Similar to cocaine, the maintenance drug and morphine, the prototypic

mu-opioid, hydrocodone engendered an inverted U-shaped function when the total number of infusions self-administered was plotted as a function of the selected test doses. The total number of injections for each dose test was greater than those engendered during morphine substitution tests. Finally, the lower right panel of Fig. 1 shows the results of substitution tests conducted with oxycodone. Similar to hydrocodone, oxycodone engendered more robust responding for self-injections compared to the parent mu opioid, morphine. An inverted U-shaped function was engendered across the 1.5 common log unit dose range tested in this study. For comparison purposes, Fig. 2 shows the results of the data in Fig. 1, but expressed as the grand mean of the total drug intake over all three days of substitution for each dose and drug tested to assess the relative reinforcing properties on this study. The total drug intake induced by all four drugs demonstrated a linear monotonic function when plotted as a function of the tested doses. The dashed line in the cocaine dose-effect function shows the typical training dose used in standard cocaine 2-choice drug discrimination studies, which is based on the interoceptive properties of the drug (Gauvin et al., 1989, 1990; Harland et al., 1989). Rats self-administering iv cocaine titrated their drug intake to a cumulative dose that was clearly above an equivalent dose of ip administered cocaine required to establish discriminative stimulus control in rats. The dashed lines in the morphine, hydrocodone, and oxycodone dose-effect functions represent the 95% confidence limits for the ED50 values for full generalization to a 0.04 mg/kg fentanyl discriminative stimulus cue in rats (Meert & Vermeirsch, 2005). All three opiates were self-administered via the iv route to dose levels that exceeded the equi-effective subjective dose

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Fig. 2. The averaged 3-day total amount of drug self-administered during the three consecutive one-hour daily sessions shown in Fig. 4. Data are plotted as a function of available cocaine (upper left panel), morphine (upper right panel), hydrocodone (lower left panel) and oxycodone (lower right panel) in rats conditioned to self-administer a maintenance dose of 0.56 mg/kg/infusion of cocaine. Each point on the graphs represents the three-day averaged intake of 32 rats (saline and 0.56 mg/kg/injection) or six randomly selected rats from the pool of trained rats. As described in Fig. 4, each drug and dose was tested for three consecutive days in one hour unlimited access sessions at each selected dose shown. The closed circles represent the mean of all three days of substitution. The dashed lines show 1) the standard training dose of cocaine (10 mg/kg) in drug discrimination assays (upper left panel) or the 95% confidence limits for the ED50 (threshold) fentanyl-like cross-generalizations between morphine (upper right panel), hydrocodone (lower left panel) or oxycodone (lower right panel) generated in a separate study in which rats were trained to discriminate the presence versus absence of 0.04 mg/kg of fentanyl by Meert and Vermeirsch (2005). The data show that each drug tested in the self-administration paradigm induced self-injected doses that were equivalent to those that produce a subjective state that would establish and maintain stimulus control of behavior.

associated with the s.c. administration of fentanyl in a standard FR10 two-lever fentanyl vs saline discriminative stimulus assay in rats. With respect to the reinforcing properties of two commonly abused Schedule II opiates, morphine and oxycodone, these data have demonstrated that hydrocodone has qualitatively and quantitatively similar positive hedonic/rewarding effects in the standard regulatory required behavioral self-administration assay. 3.2. Drug discrimination The drug discrimination assay has been used to assess the relative similarity of interoceptive stimuli associated with drug administrations. Fig. 3 shows the subjective similarity of hydrocodone and morphine in rats trained to discriminate between 20 mg/kg orally administered morphine and water administered 60 min preceding sessions. Hydrocodone engendered complete generalization to the morphine training stimulus (top panel) with similar behaviorally disruptive effects on rates-ofresponding during the test sessions (lower panel). Morphine and hydrocodone were equivalent in both subjective and rate-disruptive effects on operant performance under a fixed ratio 10 schedule of food delivery. Fig. 4 shows the cross generalization profiles for various doses of oxycodone administered by three routes of administration (sc, ip and po) in rats trained to discriminate the presence versus absence of 1.0 mg/kg oxycodone administered subcutaneously 30 min before the operant test sessions. All three routes of administration produced subjectively similar but differential potency dose–response generalization functions with corresponding changes in the rates-of-responding for food deliveries during the sessions. The route-of-administration demonstrated an ordered sensitivity relationship to oxycodone's subjective and rate-altering effects of sc b ip b po and is consistent with the known absorption-distribution-metabolism profile of oxycodone in rats.

Similarly, Fig. 5 shows the cross generalization profile for hydrocodone administered by sc, ip, and po administrations in those rats trained to discriminate the presence versus absence of 1.0 mg/kg sc administered oxycodone shown in Fig. 4, above. Hydrocodone engendered equivalent and complete cross generalization with the 1.0 mg/kg oxycodone training stimulus with a similar route-ofadministration potency relationship of sc b ip b oral with respect to both the subjective interoceptive (top panel) and rate-altering effects (bottom panel) under the fixed ratio 10 schedule of food deliveries used in this study. With respect to the subjective and motoric effects of two commonly abused Schedule II opiates, morphine and oxycodone, the data have demonstrated that hydrocodone has qualitatively and quantitatively similar subjective and rate-altering effects in the standard regulatory required behavioral drug discrimination assay. 3.3. Dependence liability 3.3.1. Clinical observations Clinical signs related to stimulation of the autonomic nervous system were salivation, lacrimation, chromodacryorrhoea (secretion of red fluid around eyes), and piloerection. The clinical finding of “material around the eyes” was a routine, non-significant finding related to grooming. The lacrimal secretions are due to the Harderian gland secretions. The glands are located behind the rat's eyes and the secretions are the result of porphyrin secretion that usually goes unseen except during periods of poor grooming (illness, stress, lethargy, sleep, etc.; cf., Sharp & LaRegina, 1998). Calls of “material around nose-red” were documented during predose, dosing, and withdrawal phases of the study plan. There were no distinctive or physiologically significant clinical findings reported over the two week dosing period in the two opiate treated groups. The “equivalent” dosing strategy maintained the normal appearance and clinically observable health status of the rats on study.

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Fig. 3. Dose–effect generalization functions for morphine (closed circles, solid lines) and hydrocodone (open circles and dashed lines) that were orally administered 60 min prior to test sessions. Thirty-two rats were trained in a two-choice lever-press response drug discrimination assay. Each rat was trained to discriminate the presence versus absence of 20 mg/kg orally administered morphine (MOR TD) under a fixed-ratio 10 (FR10) schedule of food reward. Top panel: Percentage of total session responses emitted on the morphine-appropriate lever following morphine or hydrocodone dose administrations. Bottom panel: Group mean rates-of-responding on either lever during the 30 minute test sessions expressed as a function of dose. The points about the “S” on the abscissa represent data from saline test sessions and the closed circle under the “MOR TD” represent the data from tests conducted with the 20 mg/kg morphine training dose (n = 32). All other points on the graphs represent the mean of 6 rats. Hydrocodone engendered qualitative and quantitative changes strikingly similar to morphine in this assay based on the subjective effects (top panel) and motor-impairing effects (bottom panel) produced by drug administrations.

Some calls of “swelling” were documented in 1 out of 16 rats in each of the hydrocodone and morphine treatments groups. During withdrawal 6 out of 16 hydrocodone rats and 7 out of 16 morphine treated rats were considered “hypersensitive to touch”. Vocalization scores increased each day of withdrawal in both the hydrocodone treated (3/16 to 7/16 calls) and morphine treated (3/16 to 7/16 calls) rats on Day 16 to Day 18, respectively. Both of these clinical signs are considered to represent classic signs of opiate withdrawal in the rat. During the FOBs conducted during the withdrawal phase of the study clinical signs were documented for the hydrocodone and morphine groups which included the following: a) pinpoint pupils, b) changes in gait — hindfeet walking on tip toes, or hunched body

Fig. 4. Dose–effect generalization functions for oxycodone tests conducted following subcutaneous (closed circles), intraperitoneal (open circles) and oral (half-filled circles) doses administered 30 min prior to test sessions. Thirty-two rats were trained in a two-choice lever-press response drug discrimination assay. Each of 32 rats was trained to discriminate the presence versus absence of 1.8 mg/kg subcutaneously administered oxycodone (TD) under a fixed-ratio 10 (FR10) schedule of food reward. Top panel: Percentage of total session responses emitted on the oxycodone-appropriate lever following various doses of oxycodone administered via three different routes. Bottom panel: Group mean ratesof-responding on either lever during the 30 minute test sessions expressed as a function of dose. The points about the “S” on the abscissa represent data from saline test sessions and the closed circle under the “TD” represent the data from tests conducted with the 1.8 mg/kg oxycodone training dose (n = 32). All other points on the graphs represent the mean of 6 rats. Oxycodone engendered dose-related monotonic dose–response functions for both the subjective effects (top panel) and motor-impairing effects (bottom panel) produced by each route of administration. The order of oxycodone thresholds was sc b ip b oral for both subjective and rate-altering effects. Adapted from Gauvin, Dalton and Baird (2013).

position, and c) rapid respirations. None of these calls were made in saline treated cohorts. 3.3.2. Body weights Fig. 6 shows the changes in body weights in the dependence liability study. Body weights are shown for each day of the 15-day escalating dosing period which was initiated at twice daily oral administrations of either tap water (control group) or 20 mg/kg of morphine or hydrocodone. The individual doses were incremented upward across

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Fig. 6. Changes in body weights from dependence liability study. Group mean body weights for three groups of rats treated for 15 days with oral gavaged tap water (closed black squares) or escalating doses of up to 300 mg/kg/day of hydrocodone (red filled circles) or morphine (green filled triangles), and for three days following abrupt cessation of opiate exposure (Days 16, 17, and 18). Each point represents the group mean and standard error of the mean of 16 rats treated with water, hydrocodone, or morphine.

in the morphine and hydrocodone treatment groups experiencing the initial onset of a classic opiate withdrawal syndrome. The degree of withdrawal based on the magnitude of decrements in bodyweights was limited by the intentional and preemptive treatment of a stimulant laxative (senna) over the course of opiate treatments for the sake of the health and safety of the rats, meant to diminish the intensity of constipation and/or gastroparesis known to be induced by opiates. Vehicle control cohorts showed a continued normal weight gain during the Day 16–18 study interval.

Fig. 5. Dose–effect cross-generalization functions for hydrocodone tests conducted following subcutaneous (closed circles), intraperitoneal (open circles) and oral (half-filled circles) doses administered 30 min prior to test sessions. Thirty-two rats were trained in a two-choice lever-press response drug discrimination assay. Each of 32 rats was trained to discriminate the presence versus absence of 1.8 mg/kg subcutaneously administered oxycodone under a fixed-ratio 10 (FR10) schedule of food reward. Top panel: Percentage of total responses emitted on the oxycodone-appropriate lever following various doses of hydrocodone administered via three different routes: subcutaneous, intraperitoneal, and oral gavage. Bottom panel: Group mean rates-of-responding on either lever during the 30 minute test sessions expressed as a function of hydrocodone test dose. The points about the “S” on the abscissa represent data from saline test sessions. All points on the graphs represent the mean of 6 rats tested with various doses of hydrocodone administered following one of three different routes. Hydrocodone engendered dose-related monotonic dose–response cross generalization functions for both the subjective effects (top panel) and motor-impairing effects (bottom panel) produced by each route of administration. The order of hydrocodone thresholds for oxycodone-like response choice and response rates was sc b ip b oral. Doses greater than 32 mg/kg PO hydrocodone were not tested for the sake of animal health and well-being.

successive days using an equi-effective dosing strategy to a final dose on Day 15 of twice daily administrations of 150 mg/kg of either morphine or hydrocodone. Vehicle control animals received equal volumes as their drug treated cohorts. The final dose administration occurred on the evening of Day 15. Body weights on Days 16, 17, and 18 reflect the first three days of the discontinuation syndrome. There was a bodyweight loss over the course of dosing in both drug treated groups when compared to tap water control animals (Day 1 to Day 15). A more dramatic weight loss was demonstrated on Days 16 through 18

3.3.3. Food consumption Fig. 7 shows the relative changes in food consumption over the study duration. Over the 15-day repeat dose incrementing phase of the study both morphine and hydrocodone treated rats consumed much less food than their vehicle control cohorts. While the control group showed stable intake over the study, both opiate treatments appeared to engender a similar drop in food consumption across the 15 day repeat dose phase of the study. The change in food consumption could have been attributed to the pica associated with opiate treatments in rats or a reduced motivation to eat. 3.3.4. Functional observational batteries FOBs can be organized into measures of functional domains. For organization and ease of reporting toxicology study designs are generally using these functional domains. is most often reported using stratified neurobehavioral functions. 3.3.4.1. Activity/arousal measurements. There were no functional differences between treatment groups on any measure of activity/arousal during the predose FOB evaluation. The “ease of removal” scores were lower in the hydrocodone and morphine treatment groups on Day 7 of dosing when compared to their vehicle control cohorts. On Day 15 the “ease of removal” scores were low for both opiate treatment groups, but only the morphine treated animals were statistically lower than vehicle controls. “General arousal” scores for both hydrocodone and morphine treated groups were statistically higher than their vehicle control cohorts on Day 15 following the highest dose of 150 mg/kg/dose administered on this study.

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Fig. 7. Changes in food consumption from dependence liability study. Group mean of the available food portions weighed daily over the 18 days of the dependence liability study. Each point represents the averaged food in the cages for three groups of rats treated for 15 days with tap water (black closed squares) or escalating doses of up to 300 mg/kg/day of hydrocodone (red filled circles) or morphine (green filled triangles), and for three days following abrupt cessation of opiate exposure (Days 16, 17, and 18). The lower food portions reported for hydrocodone and morphine treated rats reflect 1) the induction of pica by opiate administrations, 2) smaller meal sizes, or 3) opiate induced constipation (illness).

During the withdrawal phase hydrocodone and morphine treated rats demonstrated statistically significant increases in “ease of removal” and “handling reactivity” scores on all three days of withdrawal (Days 16, 17, and 18). “General arousal” scores were also elevated on the first and second day of withdrawal (Days 16 and 17) in the opiate treated groups when compared to vehicle control cohorts, but returned to “normal” by the third day of withdrawal (Day 18). The total number of “rearings” counted during the 3-minute open field arena test was also significantly higher in the hydrocodone and morphine treatment groups on the first two days of withdrawal (Days 16 and 17) when compared to saline treated control cohorts. Increases in measures of ease of removal, handling reactivity, general arousal, and rearings are considered to be classic signs of opiate withdrawal in rodents. 3.3.4.2. Autonomic measurements. There were no functional differences between treatment groups on any autonomic nervous system measurement during the predose FOB evaluations. There were no significant group differences in any measure of autonomic function on Day 7 of repeat dosing. On the last day of dosing, the morphine treated animals exhibited a statistically significant increase in the total number of fecal boli deposited within the open field following 3 min of monitoring. The difference of 1 fecal boli (between 4.8 and 3.8) in the opiate treatment groups was not considered to be physiologically significant or indicative of differential pharmacological activity across these treatment groups. During the withdrawal phase, group differences in the total number of fecal boli excreted and counted during the 3-minute open field assessments were not physiologically meaningful due to the use of the senna treatments during the chronic dosing phase to control or regulate fecal output in the hydrocodone and morphine treatment groups. Also relevant to the lack of differentiation in the open field assessment is the relative timing and brevity of this manipulation in relation to the temporal onset and full expression of the withdrawal syndrome. No other significant changes in autonomic measures were noted during the withdrawal phase of this study. 3.3.4.3. Neuromuscular measurements. There was a single neuromuscular function endpoint that demonstrated statistically significant differences

between the treatment groups. Stereotypy calls were distributed between scores of “1” and “2” in the vehicle controls (4/16 — “1” and 12/16 — “2”) compared to hydrocodone group (12/16 — “1” and 4/16 — “2”) and morphine groups (10/16 — “1” and 6/16 — “2”) cohorts. A score of “1” represents a call of “alert and periodic sniffing spell in the air” and a score of “2” represents a call of “constant sniffing on the wall or floor”. While statistically significant, the distributional differences between groups were not considered to be physiologically relevant or a reason to redistribute group cohorts prior to the initiation of dosing. During the Repeat Dose Phase of the study, there was no statistically significant group differences on any measure of neuromuscular function during FOBs conducted on Day 7 or 15. Following abrupt cessation of opiate treatments on the evening of Day 15, the gait assessment of rats in the hydrocodone treated group was reported to be abnormal on Days 2 and 3 (Days 16 and 17) of withdrawal, with corresponding gait alteration observed in the morphine group on Day 1 and Day 3 of withdrawal when compared to untreated cohorts (Days 16 and 18). Additionally, significantly higher “stereotypy” scores were recorded on the first two days of withdrawal in the opiate treatment groups when compared to their vehicle control cohorts. Grip strength and hindlimb splay scores remained unaffected during opiate withdrawal on this study. 3.3.4.4. Physiological measurements. There were no functional differences between treatment groups on any physiological measure during the predose (Day −1) or repeat dose phase of this study (Days 7 and 15). During the withdrawal phase of the study pinpoint pupils and rapid respirations were reported in the two opiate treated groups. Distributional score differences between the three treatment groups on measures of respiration were recorded on Day 1 of withdrawal (Study Day 16). Scores of “0” (normal respirations) and “1” (rapid or slowed breathing, to be identified by comment) were given on Day 16. All rats in the vehicle control group (16/16) received scores of “0”. The hydrocodone group received scores of “0” (10/16) and “1” (6/16), and the morphine treatment group received scores of “0” (13/16) and “1” (3/16). Other allowable scores representing labored, irregular or difficult breathing (score of 2), rales (score of 3) or dyspnea, unusually deep breathing (score of 4) were not documented in any group in this study. These distributed scores of “0” and “1” on Day 16 resulted in a statistically significant difference between the vehicle and hydrocodone treatment groups (Cochran–Mantel–Haenszel test, p b 0.05). Body weights also decreased during withdrawal as shown in Fig. 6. 3.3.4.5. Sensorimotor measurements. There were no functional differences between treatment groups on any sensorimotor measure during the predose FOB. Additionally, there were no statistically- or physiologically-significant differences between opiate treated and saline treated groups on Day 7 of dosing on any measure of sensorimotor function. On Day 15 distribution differences in scores of “0, 1, and 2” were noted between groups: the hydrocodone treated group had scores distributed between “1” (8/8) and “2” (8/8) compared to vehicle cohorts (“1”: 1/16, “2”: 14/16; “3”:1/16). A score of “1” refers to a “slight reaction” to an auditory click stimulus and a score of “2” refers to a “freeze or flinch” response. In isolation, this solitary statistically significant finding for this qualitatively subjective group difference in sensorimotor functioning was not considered to be physiologically significant or indicative of a meaningful difference in the pharmacology of morphine versus hydrocodone. During the withdrawal phase all four categorical measurements representing the sensorimotor domain were significantly higher in morphine and hydrocodone treatment groups when compared to tap water treated controls. Elevations in “approach response” were recorded for hydrocodone (Days 16 and 18) and morphine treated rats (Day 18). Responses to “tail pinch” and “touch” were equally affected in hydrocodone and morphine rats on all three days of withdrawal (Days 16, 17, and 18) and elevated responses to an auditory (mechanical clicker) stimulus were also reported on Days 1 and 3 of withdrawal in

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the hydrocodone treated groups of rats (Days 16 and 18). When compared to their water control cohorts, these changes in reflexive response to sensorimotor stimulation (auditory, haptic, and pain sensory systems) in the morphine and hydrocodone groups are considered to be consistent with classic signs of opiate withdrawal in the rat. In summary of the dependence liability assessment, there were no physiologically significant differences between water treated control subjects and their selected opiate treatment group cohorts prior to dosing. The escalating dose strategy has been previously used in numerous published reports of drug dependence induction, appearing in scientific peer reviewed journals. This strategy was intended to allow for a temporal dose escalation procedure based on the animal's ongoing physiological status and the rates of ongoing “normal” behaviors exhibited a few hours following the first of two daily doses. The results of the current study confirm that such dose escalation procedures can be conducted without serious or detrimental physiological consequences in that no biologically significant differences were noted in morphine or hydrocodone treated rats, compared to water treated cohorts, during the repeat dosing procedure in which rats were receiving up to 150 mg/kg twice daily (300 mg/kg/day, Day 15 FOBs) of the two opiate derivatives. The termination of relatively high doses of morphine (150 mg/kg, b.i.d.) produced signs of classic opiate withdrawal summarized in Table 1. 4. Discussion Morphine has been generally regarded as the reference compound against which other analgesics are assessed (World Health Organization, 1972). From the available data, it may be concluded that hydrocodone's abuse liability is most likely attributable to its own intrinsic efficacy at μ-type opioid receptors. During the period of November 1949 through March 1950, the Committee on Drug Addiction and Narcotics of the National Research Council was called upon for evaluation and recommendations concerning the efficacy and dependence producing potential of hydrocodone and other narcotics. Hydrocodone was reviewed to be: in all respects morphine-like and, in spite of the chemical relationship to codeine, closer to morphine than to codeine in its dependence liability (Fraser & Isbell, 1950; Isbell, 1949). The data generated in this study are consistent with these conclusions. According to Shannon and Holtzman (1976), the property of morphine which enables it to function as a discriminative stimulus in the rat is analogous to the component of action of morphine responsible for producing the subjective effects in man. For opiates, there is a high Table 1 Summary of clinical signs of opiate withdrawal induced by hydrocodone and morphine following 15 days of escalating doses (40 mg/kg/day up to 300 mg/kg/day). Functional domain

Observation

Hydrocodone

Morphine

Activity/arousal

Ease of removal General arousal Handling reactivity Rearing Forelimb grip strength Gait Stereotypy

↑ ↑ ↑ ↑ ↑ ↑ ↑ (Sniffing & grooming) ↑ ↑ ↑ ↑ ↓ ↑

↑ ↑ ↑ ↑ ↑ ↑ ↑ (Sniffing & grooming) ↑ ↑ ↑ ↑ ↓ ↑

Neuromuscular

Sensorimotor

Physiological Autonomic

Approach response Click response Tail pinch response Touch response Bodyweight Defecation

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correspondence between drugs that exert morphine-like discriminative effects in rats and those that evoke morphine-like subjective reports in humans (Colpaert, 1978; Shannon & Holtzman, 1977). Inasmuch as the subjective effects of opioids play an important role in their relatively high abuse potential (i.e. Fraser, 1968a,b; Jasinski, 1977), discrimination procedures provide a laboratory model in which to assess the opiatelike abuse potential of other NCEs (cf. Sannerud & Young, 1987). One of the best predictors of abuse liability of opiates is the demonstration of similar drug discriminative generalization functions to those generated by a known and commonly abused opiate like morphine or oxycodone (Shannon & Holtzman, 1977). With reference to the drug discrimination assay in all respects the subjective and pharmacokinetic effects produced by hydrocodone are similar to both oxycodone and morphine (cf Beardsley et al., 2004; Cone et al., 1978; Lelas et al., 1999; Picker, Doty, Negus, Mattox, & Dykstra, 1990; Tomkins et al., 1997; Yan-Hua & Ji-Wang, 2000). The self-administration assay demonstrated reinforcing properties of morphine, oxycodone and hydrocodone using stable operant cocaine self-injection baselines in rats, as suggested by the FDA draft guidance document. All three opiates initiated and maintained self-administration over three consecutive days of substitution in rats conditioned to selfadminister 0.56 mg/kg/infusion of cocaine (Garcia-Lecumberri et al., 2011; van Ree, Slangen, & de Wied, 1978; Weeks, 1962; Weeks & Collins, 1964, 1979; Werner, Smith, & Davis, 1976). The total amount of drug self-delivered by the rats in this study was within the range that produced significant interoceptive or subjective effects equivalent to a standard 0.04 mg/kg subcutaneously administered fentanyl training dose in rats (Meert & Vermeirsch, 2005). The results from this study confirm the abuse liability status of hydrocodone. Using a set of diverse behavioral assays promulgated by the recent FDA draft guidance document, the abuse liability of hydrocodone is demonstrated to be equivalent in all respects to the prototypic mu opioid agonist, morphine (Beach, 1957; Blasig, Herz, Reinhold, & Zieglgansberger, 1973; Fuentes, Hunt, & Crossland, 1978; Mucha, Kalant, & Linseman, 1979; Schulteis, Markou, Gold, Stinus, & Koob, 1994). Behavioral observations in the FOB have been a component of safety assessment studies in developmental, reproductive, and standard toxicology since 1975 (Irwin, 1968; Office of Technology Assessment, US Congress, 1990). The European Union (2009), Controlled Substances Staff, Center for Drug Evaluation and Research CDER at the United States Food and Drug Administration (2010), the U.S. Department of Health and Human Services (2010), and the International Commission on Harmonisation (M3[R2]: 2009; S7A: 2000) have adopted the FOB as the preferred assay for the preclinical assessment of drug dependence/ withdrawal. This study further validates the utilization of this procedure as a “first tier” descriptive technique that is uniquely applicable to the characterization of drug withdrawal (including opiate withdrawal) states. The use of a stimulant-based laxative over the course of chronic opiate dosing was similar to standardized treatments of chronic pain patients prescribed long term opiate treatments and was used in this study for the safety and health of the animals. The laxatives demonstrated that gut motility could be effectively maintained over the course of chronic opiate dosing regimen without affecting the quality or quantity of withdrawal signs produced upon abrupt withdrawal. The use of the escalating dose strategies allowed for high dose opiate exposure (300 mg/kg/day) without concomitant decrements in behavioral function as assessed in the functional observational batteries conducted on Day 7 and Day 15 of exposures. And finally, escalating doses from 20 mg/kg b.i.d. to 150 mg/kg b.i.d. dosing produced a series of classic signs of opiate withdrawal in the rat consistent with those described in the published literature appearing in peer-reviewed scientific journals. The data from this study clearly demonstrated an equivalent abuse potential shared by morphine, oxycodone, and hydrocodone using

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