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Original article

Abuse liability assessment of hydrocodone under current draftregulatory guidelines

David V. Gauvin , Margaret McComb, Robert Code, Jill A. Dalton, Theodore J. BairdDepartment of Safety Pharmacology and Neurobehavioral Sciences, MPI Research Inc., Mattawan, MI, United States

a b s t r a c ta r t i c l e i n f o

Article history:Received 11 February 2015

Received in revised form 10 April 2015Accepted 4 May 2015Available online xxxx

Keywords:HydrocodoneSelf administrationDrug discriminationDrug dependenceDiscontinuation syndromeWithdrawalMorphineOxycodoneAbuse liabilityMethods

Introduction: The abuse liability of hydrocodone was assessed in male Sprague Dawley rats under the EuropeanMedicines Agency, the International Commission on Harmonisation, and the U.S. Food & Drug Administrationdraft 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 wereconducted to compare hydrocodone to the prototypical opiates, morphine and oxycodone.Results: Hydrocodone was self-administered, produced an opiate-like subjective discriminative generalizationpro le and produced a signi cant discontinuation syndrome following abrupt treatment cessation that wasquantitatively and qualitatively similar to morphine and/or oxycodone.Conclusion: Hydrocodone has abuse liability more similar to Schedule II opiates than other Schedule IIIcompounds 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 substancesforboth human andveterinary approvalhave been disseminated by theEuropean 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 InternationalConference on Harmonisation (ICH, M3[R2], 2009 ), and the UnitedStates Food and Drug Administration (FDA, 2010) . These guidelinesare intended to 1) help de ne the scope of the term psychoactive sub-stances and, 2) to put in place a sound methodological and proceduralbasis for carrying out risk assessments in regard to health and social

risks of the use of, manufacture of, and traf c in these new psychoactivesubstances that involve memberstates of both the 1961 United NationsSingle Convention on Narcotic Drugs and the 1971 United NationsConvention on Psychotropic Substances.

A three-part, evidence-based preclinical risk assessment planrequires standardized behavioral assays of self-administration, drugdiscrimination, and dependence potential to be conducted in either ro-dents (contemporarily considered the primary model) or non-human

primates. The results of these assays must be supplied prior to healthagency approval of any new chemical entity that 1) crosses the bloodbrain barrier; 2) is pharmacologically similar to any known drug of abuse;3) hasa novel mechanismof action;4) produces psychoactiveef-fects such as sedation, euphoria, or mood changes; or 5) has any director indirect actions on other neurotransmitter systems associated withabuse potential, such as dopamine, norepinephrine, GABA, acetylcho-line, opioid, NMDA, and cannabinoid.

The chemical 4,5 -epoxy-3-methoxy-17-methyl-morphinan-6-onewas given the drug name, dihydrocodeinone, when it was rstmarketed in Germany in the early 1920's it sold under the proprietaryname 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 reportedas early as 1927:

Mller de la Fuenta said that cases of addiction to dicodid were knownin 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 ascautious with dicodid injection as one would be with morphine.

In the United States thenonproprietaryor generic name adopted forthe drugwas simply, hydrocodone. Hydrocodonecombination products

Journal of Pharmacological and Toxicological Methods xxx (2015) xxx xxx

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).

JPM-06270; No of Pages 12

http://dx.doi.org/10.1016/j.vascn.2015.05.003

1056-8719/ 2015 Elsevier Inc. All rights reserved.

Contents lists available at ScienceDirect

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Please cite this article as:Gauvin, D.V., et al., Abuse liability assessment of hydrocodone under current draft regulatory guidelines, Journalof Phar-macological and Toxicological Methods (2015), http://dx.doi.org/10.1016/j.vascn.2015.05.003
http://dx.doi.org/10.1016/j.vascn.2015.05.003http://dx.doi.org/10.1016/j.vascn.2015.05.003http://dx.doi.org/10.1016/j.vascn.2015.05.003mailto:[email protected]://dx.doi.org/10.1016/j.vascn.2015.05.003http://www.sciencedirect.com/science/journal/10568719http://www.elsevier.com/locate/jpharmtoxhttp://dx.doi.org/10.1016/j.vascn.2015.05.003http://dx.doi.org/10.1016/j.vascn.2015.05.003http://www.elsevier.com/locate/jpharmtoxhttp://www.sciencedirect.com/science/journal/10568719http://dx.doi.org/10.1016/j.vascn.2015.05.003http://localhost/var/www/apps/conversion/tmp/scratch_3/Journal%20logomailto:[email protected]://dx.doi.org/10.1016/j.vascn.2015.05.003


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(i.e., Vicodin , Hycotuss ), the only legitimate U.S. drug products onthe market at the time, were placed into Schedule III of the ControlledSubstances Act (21 USCA, Chapter 13 801-971) in spite of differentialcontrol of its analgesic equivalents oxycodone (Roxicodone ,Percocet ) and morphine (Kadian , MS-Contin ) into Schedule II.

The differential scheduling action was approved under the premisethat the likelihood of acetaminophen toxicity would limit or minimizethe 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 con-sidered to be pharmacologically equivalent to morphine with respect toanalgesia, CNS depression, and dependence potential. Reports in thepublished literature and by the U.S. National Institute on Drug Abuseindicate that hydrocodone-combination products are consumed inlarge quantities without concomitant and signi cant changes in liverfunction pro les thatmight have been predicted based on, for example,acetaminophen content. The therapeutic effects of hydrocodone, itsabuse potential, and actual abuse history in the US were thoroughlyreviewed during the requisite 8-factor analyses for schedule controlactions. 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 productshave been administratively placed into Schedule II ( Drug EnforcementAdministration, 2014 ).

The present studies were designed to systematically assess andcompare therelativeabuseliability of hydrocodone andthe prototypicalCII opiates, morphine and oxycodone, using an integrative approachconsistent with the current standardized international regulatoryguidelines.

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 approximately230 260 g were used in these experiments. The preponderance of thepublished reports of drug abuse models in animal species has indicateda selective use of male animals. It is generally assumed that the chronicnature of dosing regimens used in these studies render equi-effectiveresponses in animal subjects; no gender-differences were expected inthe direction, duration, or magnitude of behavioral and physiologicaleffects induced by the procedures set forth in the study plans, andaccordingly only males were used.

Animals in the self-administration and drug discrimination studieswere singly housed in solid bottom poly-boxes with non-aromaticbedding. Animals in the drug dependence study were singly housed instandard stainless-steelwire-bottomcages. Solid-bottom cagesbeddingmaterialswere notused in this latterstudybecause 1)of thepotentialto

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, & Sade, 1979 ) in rats. Accessto fecal boli containing behaviorally active concentrations of opiateand opiate-related metabolites, therefore, represents a signi cantexperimental confound in such a study plan, and was avoided by theuse of alternate wire bottom caging.

Fluorescent lighting was provided via an automatic timer forapproximately 12:12 hour light:dark cycle per day. Temperature andhumidity were monitoredand recorded daily andmaintainedaccordingto standard operating procedures between 64 to 79 F and 30 to 70%,respectively. The basal diet was block Lab Diet Certi ed Rodent Diet#5002 (PMI Nutrition International, Inc.). The diet and tap water were

available ad libitum unless designated otherwise (see below). The

protocols governing these studies had prior approval of MPI ResearchInstitutional Animal Care and Use Committee.

2.2. Equipment

The self-administration and drug discrimination studies were con-ducted in standard rat two-lever operant chambers (ENV-008CT; MedAssociates, Inc. NH, USA) with a modi ed top for self-administration

(MED-008CT-B2) equipped with a syringe pump (PHM-100) locatedin a specially constructed and locked box located on top of the sound-attenuating cubicle (ENV-018MD). Each chamber was equipped withtwo stimulus lamps (ENV-221M), two retractable levers (ENV-112CM), house lamp (28 V DC, 100 mA, ENV 215M), a modular pelletdispenser (ENV 203N-45), and exhaust fan. The operant chamber wasinterfaced (SmartCtrl 8 Input, 16 Output Package) with an IBM-basedpersonal computer system capable of controlling 16 chambers. Anoperant control and data collection software program for both drugdis-crimination 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 studiesthat 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 theself-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 cathetersremained patent for up to 6 monthsunder current laboratory standardsand procedures ( Gauvin, Dalton, Baird, & Faqi, 2013 ). Catheters were

ushed regularly with normal sterile saline for injection (USP) andlocked with heparinized solutions (30 100 IU/mL) of either 50%dextrose or salinethroughout thelife of thecatheter to prolongpatency.Saline ushing occurred immediately prior to and after self-administration sessions. Patency was veri ed daily with presessionand postsession ushing of catheters. The resistance to ow was usedas the rst indicator of possible catheter occlusion. If catheter occlusionwas suspected, a systems check on the viability of the implanted cathe-ters was conducted. Technicians would administer a 5 mg/kg dose of methohexital (Brevital ), or any equivalent short-onset, short-livedbarbiturate through the catheter. Animals were monitored for at least5 min post injection for signs of lethargy, malaise, or unconsciousness.If the catheter was patent, the animal would appear anesthetizedshortly following the infusion. Recovery from the system check wouldtake approximately 15 min.

2.4. Procedures

2.4.1. Self-administrationDetails of the self-administration training and testing procedure are

similar to those previously described by Briscoe et al. (1999) . The ratself-administration procedure that has been adopted by the industryand FDA is described as a single lever operant lever press responseunder a xed-ratio 10 (FR10) schedule of cocaine deliveries withsession lengths of at least 1 h duration. Once animals demonstratedday-to-day stability in responding for cocaine deliveries (less than20% day-to-day variability in the total number of training drug deliver-ies for three consecutive days). Once each animal demonstrated stableoperant responding for cocaine infusion (0.56 mg/kg/injection)for three contiguous days of training or maintenance sessions a seriesof test sessions were planned (A-B-A study design). The rst series of test sessions was conducted with the maintenance dose of cocaine(0.56 mg/kg/injection) in this session the animal, for the rst time,was allowed to respond for an unlimited number of injections over a

one-hour access period on each of three consecutive days. Following

2 D.V. Gauvin et al. / Journal of Pharmacological and Toxicological Methods xxx (2015) xxx xxx

Please cite this articleas: Gauvin,D.V., et al., Abuse liability assessment of hydrocodone under current draft regulatoryguidelines, Journal of Phar-macological and Toxicological Methods (2015), http://dx.doi.org/10.1016/j.vascn.2015.05.003
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the completion of the maintenance dose tests, an extinction test withsaline (vehicle) was scheduled. These operant conditioning proceduresmake explicit useof thefact that drug-reinforcedresponding undergoesextinction and eventually decreases in probability when a reinforcingdrug dose is replaced with vehicle (saline) or an ineffective drug rein-forceror drug dose. Complete dose response functions wereconductedfor the training drug (cocaine) and morphine, hydrocodone, andoxycodone for comparison. Using 32 rats each dose of a 5 dose test

article generalization function in a subset of 6 animals per dose can betested without repeat.Each test was preceded by at least 3 contiguous days of cocaine

maintenance self-administration with 0.56 mg/kg/injection of cocainewith less than 20% day-to-day variability. That is, all drug tests areconducted following 3 cocaine baseline sessions (i.e. A B A design;where A refers to the maintenance dose of cocaine, and B refers toa novel test condition). Following the completion of each three-daysubstitution test, the animals self-administered the maintenance

dose of 0.56 mg/kg/injection until stable behavioral output wasobserved permitting the next scheduled substitution test.

2.4.2. Drug discriminationDetails of the training and test sessions are similar to Gauvin,

Harland, Michaelis, and Holloway (1989) , Gauvin, Criado, Moore, andHolloway (1990) , and Harland et al. (1989) .

As previouslyhighlighted by Glennon andYoung(2011) , the admin-istrationof drugs canexert effects on behavior viasites of actionthat areapart or aside from the CNS (p. 62). In preclinical abuse liability studiesit is imperative to think outside the box and be open to alternativeapproaches to drug administration outside the restricted scope of theIND enabling study design. Risk assessments must include actualabuse patterns in the real world with the knowledge that drug abuserswill often not use the intended route of administration as used inpreclinical 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 insuf ation of crushed and pyrolyzedopiate tablets (chasing the dragon) were not part of any preclinicalIND-enabling studies of opiates, but they are common routes-of-administration in the opiate abusing population.

The NCE might affect behavioral activity via an action at neuromus-cular junction, the peripheral or spinal opiate receptor, the autonomicnervous system, or the site of (an accidentally inferior) injection. Asdescribed by Jrbe (2011) the onset, offset, and potency of the discrimi-native properties of a drugmay covary with the route-of-administration.In risk assessment reviews of preclinical animal-related data it is imper-ative to include variations in the route-of-administration in order todemonstrate or assess the relative risk of abuse in the real world whenthe drug is used nonmedically ( Rush, Vansickel, & Stoops, 2011 ). Thus,an important part of the characterization of the mechanism of a drug-induced change in behavior is theassessmentof the relativecontributionof rates-of-change, metabolic processes, and distributional actions that

covary with routes of administration. One of these approaches is totrain and test via differing routes of administration ( Glennon & Young,2011; Schechter, 1973 ). For example, Craft and Howard (1998) havepreviously shown equal potency between oral, subcutaneous, and intra-peritoneal administration of nicotine with respect to the discriminativestimulus properties of 0.5 mg/kg nicotine in rats. Therefore, differentialroutes of administration were used for training and testing in thisstudy (oral, sc, and ip).

One group of 32 male rats were trained to discriminate between20 mg/kg morphine or tap water administered orally (per os, po)60 min prior to the training sessions in daily 30 minute two-lever oper-ant sessions, under a xed-ratio 10 schedule of food reinforcement(Group 1). Another group of 32 male rats was trained to discriminatebetween 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 thedelivery of the rst food pellet and greater than 80% of total session re-sponses were emitted on the stimulus appropriate lever. Training andtest sessions ended after 50 reinforcer deliveries or 30 min, whicheveroccurred rst. Test sessions were identical to training sessions exceptfor the dose of drug administered before the session and during thetest session 10 consecutive responseson either lever produced a foodre-ward. 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 conduct-ed with oxycodone and hydrocodone ( Chan, Edwards, Wyse, & Smith,2008; Cone, Darwin, Gorodetzkey, & Tan, 1978; Huang, Edwards, &Smith, 2005; Lelas, Wegert, Otton, Sellers, & France, 1999; Tomkinset al., 1997 ).

2.4.3. Dependence liabilityMethods for assessing the dependence potential of morphine in rats

were developed by Akera and Brody (1968) . While the procedure forother species varies, the basic method adopted by the College on Prob-lems of Drug Dependence ( Brady & Lukas, 1984 ) is as follows: Ideally,two evenly spaced injections per day are administered. The speci cpatterns of dosing (qd, bid, tid, etc.) must be derived from establishedpharmacokinetic data from the species used in these dependence liabil-ity studies. TheC max , Tmax , and metabolic half-life of the compound willdetermine the actual number of dose administrations and the inter-dose intervals that should be used to adequately ensure that plasmaconcentrations are achieved and maintained throughout the full repeatdosing cycle ( Cone et al., 1978; Lelas et al., 1999; Tomkins et al., 1997 ).

Due to wellcharacterized respiratory depression and self-mutilationthat is induced in ratsubjectsby even moderatedoses of opiates, theini-tial dose administered in these types of repeat-dose study designs islimited. Using an equivalent dosing strategy (see below), as toleranceto the sedative, and respiratory depressive effects develops to the initialdosing schedule, the maintenance dose was increased.

Rats were dosed over 14 days. The initial dose of both drugs was20 mg/kg administered twice per day. The morning dose was, in effect,a loading dose to achieve chronic equivalence from the start of treat-ment in order to providea referencepoint to evaluate chronic tolerancedevelopment. Based on the acute time course of action of this dose pre-viously reported in thescienti c literature, thedosing times of (7:00 amand 5:00 pm) were selected allow an approximate 12 h average doseinterval.These doseintervals (10 and14 h) were used to ensurerelativecontinuity of CNS drug exposure with adequate recovery between drugadministrations.

In order to ensure that the selected dosing schedule achieved bothequivalency of peak responses as well as continuity of drug effectbetween doses, a behavioral scoring system of ataxia assessment, rstdeveloped 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 aninitial 20 mg/kg to a maximum approaching 300 mg/kg/day.

Group mean scores on the ataxia scoring system were determineddaily at 1:00 pm (6 h following the rst daily dose) during the chronicdosing period. The lowest possible score of 0 implies no CNS impair-ment or depression. The highest score of 11 implies severelydepressed . On observation of scores of 6 or less, the daily dose of mor-phine and hydrocodone was increased to the next incremental level.Scores of 7 or greater required additional review/consideration of theclinical presentation by the study director to determine potential needfor increment.

Accordingly for hydrocodone and morphine treatment groups, overthe course of 15 days, the second of two daily doses of drug was in-creased from an initial dose of 20 mg/kg to the maximal tolerable dose

(de ned as the overt expression of clinical signs of toxicity; scores of

3D.V. Gauvin et al. / Journal of Pharmacological and Toxicological Methods xxx (2015) xxx xxx



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11 or 12 on the ataxia scale). The morning daily loading dose may havebeen different from the afternoon dose. When, or if, a maximum tolera-ble dose was reached the dose wasmaintained for the remainder of the15 day treatment regimen. Based on previously published reports of opiate dependence in rats, the maximum doses were expected toapproximate levels as high as 300 mg/kg/day (expressed as the base).

Doses were administered for14 daysand achieved a combined dailydose of 300 mg/kg by Day 13(often 150 mg/kg/rattwicedaily). On Days

15 through 18 animals received sham injections of saline to control forhandling 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 studyplan). Each rat was assessed as follows:

2.4.3.1. Functional observational battery. Each animal was observed for aminimum of 3 min in a black plexiglass, open- eld observation boxmeasuring 20 in. by 20 in. by 8 in. Parameters evaluated were basedon those outlined in Moser et al. (1988) and Moser and Ross (1996) .

2.4.3.2. Expected clinical signs. Based on the preponderance of scienti creports appearing in peer-reviewed scienti c journals chronic opiate

administration was expected to produce pica, gastroparesis, gastricdistention, constipation, aggression, irritability, and self-mutilation.

Treatments with a natural stimulant laxative (Senna) was initiatedprior to and during the chronic dosing regimen. This was intended tomimic the standardized treatments of human patients receivinglong-term high dose opiates and to minimize the deleterious effects of the opiates on gut motility during the course of drug administrationon this study.

Placement of aspen wood blocks, Nyla bones, and metal chainrings into the cages were used as added environmental enrichmentsfor singly housed rats per MPI Research SOPs and also was intended tohelp ameliorate the incidences of self-mutilation.

Peripheral histamine is released during opiate administrations inboth humans and animal subjects. Histamine-induced itching in the

limbs typically results in scratching, picking (crank bugs), and bitingin 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 reducethe potential need for medical intervention during this study. Part of aclear demonstration and validation for dependence of the opiatetype were intended to be requisite clinical ndings.

During the withdrawal phase of the study it was expected that ani-mals in bothdrug treatment groupswould display markedor severediar-rhea and an expected body weight loss that could achieve 15 g/day

these are considered prototypical withdrawal signs from opiate depen-dence and an important target to be demonstrated in this study. Thisbodyweight loss is usually self-limiting, short-lived, and was expectedto 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 IAUCcommittee review of the protocol (Classi cation D of pain and dis-tress) which approved and initiated the treatment plan and scheduledveterinary consultations throughout the conduct of the dependenceliability study.

2.4.3.3. Food consumption. Each animal's food was weighed every otherday in order to get a rudimentary impression of food intake. Withrodent block diet, it is understood that such consumption would notbe a perfectly accurate measure of actual nutritional intake for eachrat. However, on the assumption that rats would lose or waste similaramounts of block diet each day, the food consumption measure wasused to help discern incidences of pica, motoric effects, and gut motility

properties potentially associated with the chronic dosing regimens.

2.4.3.4. Body weights. Body weights were measured andrecorded within3 days of arrival, prior to randomization to treatment conditions, andevery day during the chronic dosing regimen. Body weights measuredfollowing arrival are not included in this report. Animals were weigheddaily during the chronic dosing and withdrawal phases of the stud, andas part of the individually scheduled FOBs.

2.5. Drugs

Normal sterile saline for injection (USP) was used as the vehicle forall study designs and for sham injections in the dependence liabilitystudy. Hydrocodone bitartrate, oxycodone hydrochloride, andmorphine sulfate pentahydrate were purchased from Mallinkrodt, Inc.(Covidian Pharma, Inc., St. Louis, MO). Doses were weighed andexpressed 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 toinduce constipation, weight gain and, and in some cases, gastroparesisin both human and animal research subjects. For the health and safetyof the animals on this study there was no need to further demonstratethe full spectrum and magnitude of this major pharmacological effectsof the opiates used as positive control articles (i.e., morphine andhydrocodone) by administration of an expanded range of (includinghigher) doses.

As a preemptive treatment, two days prior to the initiation of dosingand daily (approximately 1:00 pm) during the 15 day chronic dosingregimen, each rat in Groups 2 & 3 received doses of a natural plantderived stimulant laxative, senna, to ensure gut motility was maintainedduring dose regimen.

An initial dose of 210 mg/kg (5.97 mL/kg volume) was selected foradministration. This initial dose was escalated upward in 50 mg/kgincrements, up to a maximum dose of 600 mg/kg to maintain fecaloutput over the 2 week dosing period. Mengs, Mitchell, McPherson,Gregson, and Tigner (2004) have previously demonstrated the relativesafety of up to 1500 mg/kg/day of senna treatments in rats exposed todaily doses for 13 weeks with no signs of toxicity or rebound phenom-ena during abrupt cessation of treatments.

3. Results

3.1. Self-administration

All 32 rats were conditioned to initiate and maintain lever pressresponding 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 cocainedoses throughout the study plan.

Fig. 1 compares the results of three consecutive daily substitutiontests conducted with various doses of cocaine (upper left panel),morphine (upperrightpanel), hydrocodone (lowerleft panel), andoxy-codone (lower right panel) in rats conditioned to initiate and maintainstable patterns of self-administration of 0.56 mg/kg/infusion of cocaine

in the regulatory standard self-administration behavioral assay. Underthe recommendations of the current FDAguidance document, substitu-tion tests are conducted from a stable behavioral cocaine-self-administration baseline. That is, all substitution tests are initiatedfollowing the demonstration of a day-to-day stability of cocaineself-administration. Substitution tests with saline, a non-reinforcer,engendered a typical extinction burst on Day 1 of substitution witha downward staircase pattern of responding during Day 2 and Day 3with respect to the total number of injections administered (all fourpanels). The upper left panel shows the result of tests conducted withvariousdosesof cocaine tested in ascendingorder Cocaine tests demon-strated a typical inverted U-shaped pattern of dose-effect functionacross the 1.25 log unit dose response function. Moderate doses of 0.32 and 0.1 mg/kg/injection maintained a higher number of self-

injections when compared to the higher doses of 0.32, 0.56, and




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1.0mg/kg/injection of cocaine. All ve testeddoses of cocaine sustaineda similar pattern and number of injections over the three days of substitution when compared to vehicle control tests.

The upper rightpanel of Fig. 2 shows the results of substitution testsconducted with the classic mu opioid agonist, morphine, in those ratstrained to self-administer cocaine ( Fig. 1, upper left panel). Note theabbreviated ordinal axis. Morphine produced a blunted or attened inverted U shaped dose response function compared to the trainingdrug, cocaine. While consistent day-to-day patterns of self-injectionswere demonstrated for each dose of morphine tested in this study, thepatterns and numbers of injections engendered in these tests werevery low across the tested dose range and were attributed to the phar-macodynamic properties of morphine in this limited one hour accessperiod study design. Similar low patterns of responding for morphine

in similar 1 hour access test sessions (e.g., less than 10 infusions) havebeen demonstrated by Mierzejewski, Koro , Goldberg, Kostowski, andStefa ski (2003) in Sprague Dawley rats and by Snchez-Cardosoet al. (2007) , Snchez-Cardoso et al. (2009) and Garcia-Lecumberriet al. (2011) in Lewis and Fischer 344 rats. With the catheter tip of theindwelling cathetersplaced at or very near theentranceof theright atri-um, these low patterns of morphine intake may be related to the directdynamic cardiovascular effects of intravenously administered morphinein 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 testsessions conducted withascending doses of hydrocodone in ratscondi-tioned to maintain stable day-to-day intakes of 0.56 mg/kg/infusion of cocaine interspersed between the speci c dose substitution tests. Simi-

lar to cocaine, the maintenance drug and morphine, the prototypic

mu-opioid, hydrocodone engendered an inverted U-shaped functionwhen the total number of infusions self-administered was plotted as afunction of the selected test doses. The total number of injections foreach dose test was greater than those engendered during morphinesubstitution tests.

Finally, the lower right panel of Fig. 1 shows the results of substitu-tion tests conducted with oxycodone. Similar to hydrocodone,oxycodone engendered more robust responding for self-injectionscompared to the parent mu opioid, morphine. An inverted U-shapedfunction was engendered across the 1.5 common log unit dose rangetested in this study.

For comparison purposes, Fig. 2 shows the results of the data inFig. 1, but expressed as the grand mean of the total drug intake overall three days of substitution for each dose and drug tested to assess

the relative reinforcing properties on this study. The total drug intakeinduced by all four drugs demonstrated a linear monotonic functionwhenplotted asa function ofthe tested doses. The dashed linein the co-caine dose-effect function shows the typical training dose used in stan-dard cocaine 2-choice drug discrimination studies, which is based onthe interoceptive properties of the drug ( Gauvin et al., 1989, 1990;Harland et al., 1989 ). Rats self-administering iv cocaine titrated theirdrug intake to a cumulative dose that was clearly above an equivalentdose of ip administered cocaine required to establish discriminativestimulus control in rats. Thedashed lines in themorphine,hydrocodone,and oxycodone dose-effect functions represent the 95% con dencelimits for the ED50 values for full generalization to a 0.04 mg/kgfentanyl 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

Fig. 1. Thegroup meantotalnumber of infusions self-administeredduringone-hour daily sessionsis plottedas a functionof available cocaine doseexpressedin mg/kg/injection in 32 ratsconditioned 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 testsconducted with three opiate derivatives: morphine (upper right panel), hydrocodone (lower left panel) and oxycodone (lower right panel) in animals conditioned to self-administer0.56mg/kg/infusionof cocaine.Each unlimited accesscross-generalizationtest wasconducted following at least3 consecutive daysof cocaineself-administration baselines. Bars representthe 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 eachselected 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-hourdaily 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 opiateswere self-administered in rats conditioned to self-administer cocaine.Cocaine and oxycodone data adapted from Gauvin, Dalton and Baird (2013, p 455) .




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associated with the s.c. administration of fentanyl in a standard FR10two-lever fentanyl vs saline discriminative stimulus assay in rats.

With respect to the reinforcing properties of two commonly abusedSchedule II opiates, morphine and oxycodone, these data havedemonstrated that hydrocodone has qualitatively and quantitativelysimilar positive hedonic/rewarding effects in the standard regulatoryrequired behavioral self-administration assay.

3.2. Drug discrimination

The drug discrimination assay has been used to assess the relativesimilarity of interoceptive stimuli associated with drug administrations.Fig. 3 shows the subjective similarity of hydrocodone and morphine inrats trained to discriminate between 20 mg/kg orallyadministeredmor-phine andwater administered 60 minpreceding sessions. Hydrocodoneengendered complete generalization to the morphine training stimulus(top panel) with similar behaviorally disruptive effects on rates-of-responding during the test sessions (lower panel). Morphine and

hydrocodone were equivalent in both subjective and rate-disruptiveeffects on operant performance under a xed ratio 10 schedule of fooddelivery.

Fig. 4 shows the cross generalization pro les for various doses of oxycodone administered by three routes of administration (sc, ip andpo) in rats trained to discriminate the presence versus absence of 1.0 mg/kg oxycodone administered subcutaneously 30 min before theoperant test sessions. All three routes of administration produced sub- jectively similar but differential potency dose response generalizationfunctions with corresponding changes in the rates-of-responding forfood deliveries during the sessions. The route-of-administrationdemonstrated an ordered sensitivity relationship to oxycodone'ssubjective and rate-altering effects of sc b ip b po and is consistentwith the known absorption-distribution-metabolism pro le of

oxycodone in rats.

Similarly, Fig. 5 shows the cross generalization pro le forhydrocodone administered by sc, ip, and po administrations in thoserats trained to discriminate the presence versus absence of 1.0 mg/kgsc administered oxycodone shown in Fig. 4, above. Hydrocodoneengendered equivalent and complete cross generalization with the1.0 mg/kg oxycodone training stimulus with a similar route-of-administration potency relationship of sc b ip b oral with respect toboth the subjective interoceptive (top panel) and rate-altering effects(bottom panel) under the xed ratio 10 schedule of food deliveriesused in this study.

With respect to the subjective and motoric effects of two commonlyabused Schedule II opiates, morphine and oxycodone, the data havedemonstrated that hydrocodone has qualitatively and quantitativelysimilar subjective and rate-altering effects in the standard regulatoryrequired behavioral drug discrimination assay.

3.3. Dependence liability

3.3.1. Clinical observationsClinical signs related to stimulation of the autonomic nervous

system were salivation, lacrimation, chromodacryorrhoea (secretion of red uidaroundeyes),andpiloerection. Theclinical nding of materialaround the eyes was a routine, non-signi cant nding related togrooming. The lacrimal secretions are due to the Harderian gland secre-tions.Theglands are located behindthe rat's eyes and thesecretions arethe resultof porphyrinsecretion that usuallygoes unseenexcept duringperiods of poor grooming (illness, stress, lethargy, sleep, etc.; cf., Sharp& LaRegina, 1998 ). Calls of material aroundnose-red were document-ed during predose, dosing, and withdrawal phases of the study plan.

There were no distinctive or physiologically signi cant clinical nd-ings reported over the twoweek dosingperiod in the twoopiate treatedgroups. The equivalent dosing strategy maintained the normal ap-

pearance and clinically observable health status of the rats on study.

Fig. 2. The averaged3-day total amount of drug self-administeredduringthe three consecutive one-hourdaily sessionsshown in Fig. 4. Data are plotted as a function ofavailablecocaine(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 thepool of trainedrats. As described in Fig. 4, eachdrug and dosewas tested for threeconsecutive days in one hourunlimitedaccess sessions at each selected dose shown. The closed circlesrepresent the mean of allthree daysof substitution. The dashedlines show1) the standard training doseof cocaine (10mg/kg)in drugdiscrimination assays (upper left panel) or the 95%con dence 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 trainedto discriminate the presenceversusabsence of 0.04 mg/kg of fentanylby Meert and Vermeirsch (2005) . Thedata show thateachdrug 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 stimuluscontrol of behavior.




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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 16morphine treated rats were considered hypersensitive to touch .Vocalization scores increased each day of withdrawal in both thehydrocodone treated (3/16 to 7/16 calls) and morphine treated (3/16to 7/16 calls) rats on Day 16 to Day 18, respectively. Both of these clin-ical signs are considered to represent classic signs of opiate withdrawalin the rat.

During the FOBs conducted during the withdrawal phase of thestudy clinical signs were documented for the hydrocodone andmorphine groups which included the following: a) pinpoint pupils,

b) changes in gait

hindfeet walking on tip toes, or hunched body

position, and c) rapid respirations. None of these calls were made insaline treated cohorts.

3.3.2. Body weightsFig. 6 shows thechanges in body weights in the dependence liability

study. Body weights are shown for each day of the 15-day escalatingdosing period which was initiated at twice daily oral administrationsof either tap water (control group) or 20 mg/kg of morphine or

hydrocodone. The individual doses were incremented upward across

Fig. 3. Dose effect generalization functions for morphine (closed circles, solid lines) andhydrocodone (open circles and dashed lines) that were orally administered 60 min priorto test sessions. Thirty-two rats were trained in a two-choice lever-press response drugdiscrimination assay. Each rat was trained to discriminate the presence versus absenceof20 mg/kg orallyadministered morphine (MORTD) under a xed-ratio 10 (FR10)sched-ule of food reward. Top panel: Percentage of total session responses emitted on the mor-phine-appropriate lever following morphine or hydrocodone dose administrations.Bottom panel: Group mean rates-of-responding on either lever during the 30 minutetest sessions expressed as a function of dose. The points about the S on the abscissa rep-resent data from saline test sessions and the closed circle under the MOR TD representthe data from tests conducted with the 20 mg/kg morphine training dose (n = 32). Allother points on the graphs represent the mean of 6 rats. Hydrocodone engendered quali-tative and quantitative changes strikingly similar to morphine in this assay based on thesubjective effects (top panel) and motor-impairing effects (bottom panel) produced bydrug administrations.

Fig. 4. Dose effect generalization functions for oxycodone tests conducted following sub-cutaneous (closedcircles), intraperitoneal (opencircles) and oral (half- lled circles) dosesadministered 30 min prior to test sessions. Thirty-two rats were trained in a two-choicelever-press responsedrugdiscriminationassay.Eachof 32rats wastrained to discriminatethe presence versus absence of 1.8 mg/kg subcutaneously administered oxycodone (TD)under a xed-ratio 10 (FR10) schedule of food reward. Top panel: Percentage of totalsession responses emitted on the oxycodone-appropriate lever following various dosesof oxycodone administered via three different routes. Bottom panel: Group mean rates-of-responding on either lever during the 30 minute test sessions expressed as a functionof dose. The points about the S on the abscissa represent data from saline test sessionsand the closed circle under the TD represent the data from tests conducted with the1.8 mg/kg oxycodone training dose (n = 32). All other points on the graphs representthe mean of 6 rats. Oxycodone engendered dose-related monotonic dose responsefunctions for both the subjective effects (top panel) and motor-impairing effects (bottompanel) produced by each route of administration. The order of oxycodone thresholds wassc b ip b oral for both subjective and rate-altering effects.Adapted from Gauvin, Dalton and Baird (2013) .




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successive days using an equi-effectivedosingstrategy to a nal doseonDay 15 of twice daily administrations of 150 mg/kg of either morphineor hydrocodone. Vehicle control animals received equal volumes astheir drug treated cohorts. The nal dose administration occurred onthe evening of Day 15. Body weights on Days 16, 17, and 18 re ect the

rst three days of the discontinuation syndrome. There was abodyweight loss over the course of dosing in both drug treated groupswhen compared to tap water control animals (Day 1 to Day 15). A

more dramatic weight loss was demonstrated on Days 16 through 18

in the morphine and hydrocodone treatment groups experiencing theinitial onset of a classic opiate withdrawal syndrome. The degree of withdrawal based on the magnitude of decrements in bodyweightswas limited by the intentional and preemptive treatment of a stimulantlaxative (senna) over thecourse of opiate treatments for thesake of thehealth and safety of the rats, meant to diminish the intensity of consti-pation and/or gastroparesis known to be induced by opiates. Vehiclecontrol cohorts showed a continued normal weight gain during theDay 16 18 study interval.

3.3.3. Food consumptionFig. 7 shows therelative changes in food consumption over thestudy

duration. Over the 15-day repeat dose incrementing phase of the studyboth morphine and hydrocodone treated rats consumed much less foodthan their vehicle control cohorts. While the control group showed sta-ble intake over the study, both opiate treatments appeared to engendera similar drop in food consumption across the 15 dayrepeat dose phaseof the study. Thechange in food consumption could have been attribut-ed to the pica associated with opiate treatments in rats or a reducedmotivation to eat.

3.3.4. Functional observational batteriesFOBs can be organized into measures of functional domains. For or-

ganization and ease of reporting toxicology study designs are generallyusing these functional domains. is most often reported using strati edneurobehavioral functions.

3.3.4.1. Activity/arousal measurements. There were no functional differ-ences between treatment groups on any measure of activity/arousalduring the predose FOB evaluation.

The ease of removal scores were lower in the hydrocodone andmorphine treatment groups on Day 7 of dosing when compared totheir vehicle control cohorts. On Day 15 the ease of removal scoreswere low for both opiate treatment groups, but only the morphinetreated animals were statistically lower than vehicle controls. Generalarousal scores for both hydrocodone and morphine treated groupswere statisticallyhigherthan their vehicle control cohorts on Day15 fol-

lowing the highest dose of 150 mg/kg/dose administered on this study.

Fig. 5. Dose effectcross-generalizationfunctions for hydrocodone testsconducted follow-ing subcutaneous (closed circles), intraperitoneal (open circles) and oral (half- lled cir-cles) doses administered 30 min prior to test sessions. Thirty-two rats were trained in atwo-choice lever-press response drug discrimination assay. Each of 32 rats was trainedto discriminate the presence versus absence of 1.8 mg/kg subcutaneously administeredoxycodone under a xed-ratio 10 (FR10) schedule of food reward. Top panel: Percentageof total responses emittedon the oxycodone-appropriate lever following various doses of hydrocodone administered via three different routes: subcutaneous, intraperitoneal, andoral gavage. Bottom panel: Group mean rates-of-responding on either lever during the30 minute test sessions expressed as a function of hydrocodone test dose. The pointsabout the S on the abscissa represent data from saline test sessions. All points on thegraphs represent the mean of 6 rats tested with various doses of hydrocodone adminis-

tered following one of three different routes. Hydrocodone engendered dose-relatedmonotonic dose response cross generalization functions for both the subjective effects(top panel) andmotor-impairingeffects (bottompanel)producedby eachroute ofadmin-istration. The order of hydrocodone thresholds for oxycodone-like response choice andresponse rates was sc b ip b oral. Doses greater than 32 mg/kg PO hydrocodone werenot tested for the sake of animal health and well-being.

Fig. 6. Changes in body weights from dependence liability study. Group mean bodyweights for three groups of rats treated for 15 days with oral gavaged tap water (closedblack squares) or escalating doses of up to 300 mg/kg/day of hydrocodone (red lled

circles)or morphine(green lledtriangles), andfor three daysfollowing abrupt cessationof opiate exposure (Days 16, 17, and 18). Each point represents the group mean andstandard error of the mean of 16 rats treated with water, hydrocodone, or morphine.




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During the withdrawal phase hydrocodone and morphine treatedrats demonstrated statistically signi cant increases in ease of removal

and handling reactivity scores on all three days of withdrawal (Days16, 17, and 18). General arousal scores were also elevated on the

rst and second dayof withdrawal (Days 16 and 17) in theopiate treat-ed groups when compared to vehicle control cohorts, but returned to normal by the third day of withdrawal (Day 18). The total numberof rearings counted during the 3-minute open eld arena test wasalso signi cantly higher in the hydrocodone and morphine treatmentgroups on the rst two days of withdrawal (Days 16 and 17) whencompared to saline treated control cohorts. Increases in measures of ease of removal, handling reactivity, general arousal, and rearings areconsidered to be classic signs of opiate withdrawal in rodents.

3.3.4.2. Autonomic measurements. There were no functional differencesbetween treatmentgroups on any autonomicnervous systemmeasure-ment during the predose FOB evaluations. There were no signi cantgroup differences in any measure of autonomic function on Day 7 of repeat dosing. On the last day of dosing, the morphine treated animalsexhibited a statistically signi cant increase in the total number of fecalboli deposited within the open eld following 3 min of monitoring.The difference of 1 fecal boli (between 4.8 and 3.8) in the opiatetreatment groups was not considered to be physiologically signi cant

or indicative of differential pharmacological activity across thesetreatment groups.

During the withdrawal phase, group differences in the total numberof fecal boli excreted and counted during the 3-minute open eldassessments were not physiologically meaningful due to the use of thesenna treatments during thechronic dosingphase to control or regulatefecal output in the hydrocodone and morphine treatment groups. Alsorelevant to the lack of differentiation in the open eld assessment isthe relative timing and brevity of this manipulation in relation to thetemporal onset and full expression of the withdrawal syndrome. Noother signi cant changes in autonomic measures were noted duringthe withdrawal phase of this study.

3.3.4.3. Neuromuscularmeasurements. There wasa singleneuromuscular

function endpoint that demonstrated statistically signi cant differences

between the treatment groups. Stereotypy calls were distributedbetween scores of 1 and 2 in the vehicle controls (4/16 1 and12/16 2 ) compared to hydrocodone group (12/16 1 and4/16 2 ) andmorphine groups(10/16 1 and 6/16 2 ) cohorts.A score of 1 represents a call of alert and periodic snif ng spell in theair and a score of 2 represents a call of constant snif ng on the wallor oor . While statistically signi cant, the distributional differences be-tween groups were not considered to be physiologically relevant or a

reason to redistribute group cohorts prior to the initiation of dosing.During the Repeat DosePhase of the study, there wasno statisticallysigni cant group differences on anymeasure of neuromuscularfunctionduring 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 abnormalon Days 2 and 3 (Days 16 and 17) of withdrawal, with correspondinggait alteration observed in the morphine group on Day 1 and Day 3 of withdrawal when compared to untreated cohorts (Days 16 and 18).Additionally, signi cantly higher stereotypy scores were recorded onthe rst two days of withdrawal in the opiate treatment groups whencompared to their vehicle control cohorts. Grip strength and hindlimbsplay scores remained unaffectedduring opiate withdrawalon thisstudy.

3.3.4.4. Physiologicalmeasurements. There wereno functionaldifferencesbetween treatment groups on any physiological measure during thepredose (Day 1) or repeat dose phase of this study (Days 7 and 15).

During the withdrawal phase of the study pinpoint pupils and rapidrespirations were reported in the two opiate treated groups.

Distributional scoredifferences between the three treatment groupson measures of respiration were recorded on Day 1 of withdrawal(Study Day 16). Scores of 0 (normal respirations) and 1 (rapid orslowed breathing, to be identi ed by comment) were given on Day16. 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 ordif cult breathing (score of 2), rales (score of 3) or dyspnea, unusuallydeep breathing (score of 4) were not documented in any group in thisstudy.These distributedscores of 0 and 1 on Day 16resultedin a sta-tistically signi cant difference between the vehicle and hydrocodonetreatment 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 functionaldifferences between treatment groups on any sensorimotor measureduring the predose FOB. Additionally, there were no statistically- orphysiologically-signi cant differences between opiate treated and sa-line treated groups on Day 7 of dosing on any measure of sensorimotorfunction. On Day 15 distribution differences in scores of 0, 1, and 2

were noted between groups: thehydrocodone treated group hadscoresdistributed between 1 (8/8) and 2 (8/8) compared to vehicle co-horts ( 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 inch response. In isolation, this solitary statistically signi -cant nding for this qualitatively subjective group difference in sensori-motor functioning was not considered to be physiologically signi cantor indicative of a meaningful difference in the pharmacology of morphine versus hydrocodone.

During the withdrawal phase all four categorical measurementsrepresenting the sensorimotor domain were signi cantly higher inmorphine and hydrocodone treatment groups when compared to tapwater treated controls. Elevations in approach response were record-ed for hydrocodone (Days 16 and 18) and morphine treated rats (Day18). Responses to tail pinch and touch were equally affected inhydrocodone and morphine rats on all three days of withdrawal (Days16, 17, and 18) and elevated responses to an auditory (mechanical

clicker) stimulus were also reported on Days 1 and 3 of withdrawal in

Fig. 7. Changes in food consumption from dependence liability study. Group mean of theavailable food portions weighed daily over the 18 days of the dependence liabilitystudy.Each point represents theaveraged foodin thecages forthreegroupsof rats treatedfor 15 days with tap water (black closed squares) or escalating doses of up to300 mg/kg/day of hydrocodone (red lled circles) or morphine (green lled 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 re ect1) the induction of pica by opiate administrations, 2) smaller meal sizes, or 3) opiateinduced constipation (illness).




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the hydrocodone treated groups of rats (Days 16 and 18). When com-pared to their water control cohorts, these changes in re exiveresponseto sensorimotor stimulation (auditory, haptic, and pain sensorysystems) in the morphine and hydrocodone groups are considered tobe consistent with classic signs of opiate withdrawal in the rat.

In summary of the dependence liability assessment, there were nophysiologically signi cant differences between water treated controlsubjects and their selected opiate treatment group cohorts prior to dos-

ing. The escalating dose strategy has been previously used in numerouspublished reports of drug dependence induction, appearing in scienti cpeer reviewed journals. This strategy was intended to allow for atemporal dose escalation procedure based on the animal's ongoingphysiological status and the rates of ongoing normal behaviors exhib-ited a few hours following the rst of two daily doses. The results of thecurrent study con rm that such dose escalation procedures can be con-ducted without serious or detrimental physiological consequences inthat no biologically signi cant differences were noted in morphine orhydrocodone treated rats, compared to water treated cohorts, duringthe repeat dosing procedure in which rats were receiving up to150 mg/kg twice daily (300 mg/kg/day, Day 15 FOBs) of the two opiatederivatives. The termination of relatively high doses of morphine(150 mg/kg, b.i.d.) produced signs of classic opiate withdrawalsummarized in Table 1 .

4. Discussion

Morphine has been generally regarded as the reference compoundagainst which other analgesics are assessed ( World HealthOrganization, 1972 ). From the available data, it may be concludedthat hydrocodone's abuse liability is most likely attributable to its ownintrinsic ef cacy at -type opioid receptors. During the period of November 1949 through March 1950, the Committee on DrugAddiction and Narcotics of the National Research Council was calledupon for evaluation and recommendations concerning the ef cacyand dependence producing potential of hydrocodone and othernarcotics. Hydrocodone was reviewed to be:

in all respects morphine-likeand, in spite of the chemical relationship tocodeine, closer to morphine than to codeine in its dependence liability(Fraser & Isbell, 1950; Isbell, 1949 ).

The data generated in this study are consistent with theseconclusions.

According to Shannon and Holtzman (1976) , the property of morphine which enables it to function as a discriminative stimulus intherat is analogous to thecomponentof action of morphine responsiblefor producing the subjective effects in man. For opiates, there is a high

correspondence between drugs that exert morphine-like discriminativeeffects in rats and those that evoke morphine-like subjective reports inhumans ( Colpaert, 1978; Shannon & Holtzman, 1977 ). Inasmuch as thesubjective effects of opioids play an important role in their relativelyhigh abuse potential (i.e. Fraser, 1968a,b; Jasinski,1977 ), discriminationprocedures provide a laboratory model in which to assess the opiate-like abuse potential of other NCEs (cf. Sannerud & Young, 1987 ). Oneof the best predictors of abuse liability of opiates is the demonstration

of similar drug discriminative generalization functions to thosegenerat-ed by a known and commonly abused opiate like morphine or oxyco-done ( Shannon & Holtzman, 1977 ). With reference to the drugdiscrimination assay in all respects the subjective and pharmacokineticeffects produced by hydrocodone are similar to both oxycodone andmorphine (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 cocaineself-injection baselines in rats, as suggested by the FDA draft guidancedocument. All three opiates initiated and maintained self-administrationover three consecutive days of substitution in rats conditioned to self-administer 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 drugself-delivered by theratsin this study waswithin therange thatproducedsigni cant interoceptive or subjective effects equivalent to a standard0.04 mg/kg subcutaneously administered fentanyl training dose in rats(Meert & Vermeirsch, 2005 ).

The results from this study con rm the abuse liability status of hydrocodone. Using a set of diverse behavioral assays promulgatedby the recent FDA draft guidance document, the abuse liability of hydrocodone is demonstrated to be equivalent in all respects to theprototypic 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 ).

Behavioralobservations in the FOB have been a component of safetyassessment studies in developmental, reproductive, and standardtoxicology since 1975 ( Irwin, 1968; Of ce of Technology Assessment,US Congress, 1990 ). The European Union (2009) , Controlled SubstancesStaff, Centerfor Drug Evaluation andResearch CDER at the UnitedStatesFood and Drug Administration (2010) , the U.S. Department of Healthand Human Services (2010) , and the International Commission onHarmonisation (M3[R2]: 2009; S7A: 2000 ) have adopted the FOB asthe preferred assay for the preclinical assessment of drug dependence/withdrawal. This study further validatesthe utilization of this procedureas a rst tier descriptive technique that is uniquely applicable to thecharacterization of drug withdrawal (including opiate withdrawal)states.

The use of a stimulant-based laxative over the course of chronicopiate dosing was similar to standardized treatments of chronic pain

patients prescribed long term opiate treatments and was used in thisstudy forthe safetyandhealthof theanimals.Thelaxatives demonstrat-ed that gut motility could be effectively maintained over the course of chronic opiate dosing regimen without affecting the quality or quantityof withdrawal signs produced upon abrupt withdrawal. The use of theescalating dose strategies allowed for high dose opiate exposure(300 mg/kg/day) without concomitant decrements in behavioral func-tion as assessed in the functional observational batteries conducted onDay 7 and Day 15 of exposures. And nally, escalating doses from20 mg/kg b.i.d. to 150 mg/kg b.i.d. dosing produced a series of classicsigns of opiate withdrawal in the rat consistent with those describedin the published literature appearing in peer-reviewed scienti c journals.

The data from this study clearly demonstrated an equivalent abuse

potential shared by morphine, oxycodone, and hydrocodone using

Table 1Summary of clinical signs of opiate withdrawal induced by hydrocodone and morphinefollowing 15 days of escalating doses (40 mg/kg/day up to 300 mg/kg/day).

Functionaldomain

Observation Hydrocodone Morphine

Activity/arousal Ease of removal General arousal Handling reactivity Rearing

Neuromuscular Forelimb grip strength Gait Stereotypy

(Snif ng &grooming)

(Snif ng &grooming)

Sensorimotor Approach response Click response Tail pinch response Touch response

Physiological Bodyweight Autonomic Defecation




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predictive behavioral assays described in the draft regulatory guidancedocument issued by the US FDA.

Acknowledgments

This work was conducted and funded by MPI Research, Inc., anindependently owned Contract Research Organization that employs allof the authors at the time of study conduct and manuscript preparation.

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