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ANALGESICANDIMMUNOMODULATORYEFFECTSOF CODEINEANDCODEINE6-GLUCURONIDE By VINAYAKJAYASRINIVASAN ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 1996 i.~'.... >\ ?^ ; i -- / ACKNOWLEDGMENTS IwouldliketotakethisopportunitytooffermyheartfeltappreciationtoDr. IanTebbett.Hiscontinousguidanceandencouragementwasthedrivingforce forthisresearchproject.Iwouldliketoexpressmysincerethankstotheother membersofmycommittee-Dr.DonnaWielbo,Dr.KennethSloan,Dr.Hartmut DerendorfandDr.RogerBertholf-foralltheiradviceandsuggestionsthroughout theproject. IwanttothankDr.JamesSimpkins,Dr.JanetKarlixandDr.Guenther Hochhausforgivingmeanopportunitytoworkintheirlabsinordertogenerate someofmydata.IalsowouldliketothankJimKetchamforhisinvaluablehelp inputtingallmyposters,papersandthisdissertationtogether. IwouldalsoliketoconveymythankstoShawnToffoloandBecky Frieburgerfortheirhelpinmywork.Ialsodeeplyappreciatethesupportofall thegraduatestudentsandtheotherfacultymembersandsecretariesinthe department. Iwanttoexpressmydeepestgratitutetomybrother,myrelativesandall myfriendswhohavestayedbymethroughthethickandthin.Finally,Iwantto dedicatethisworktomyparentsbecauseIdiditforthem. TABLEOFCONTENTS pagg ACKNOWLEDGMENTS ii ABSTRACT vi CHAPTERS 1.BACKGROUNDANDSIGNIFICANCE 1 1.1 OpioidsandPain 2 1.1.1 PainTransnnission 3 1.1.2.PainPerception 4 1.2 Codeine 4 1.2.1AdministrationandDosage 5 1.2.2PharmacologicalActions 6 1.2.3Toxicity 7 1.2.4TherapeuticUses 7 1.2.5DrugDependenceandTolerance 8 1.2.6AnalyticalTechniques 9 1.2.7PharmacokineticsinMan 10 1.2.7.1Absorption 11 1.2.7.2Distribution 11 1.2.7.3Metabolism 12 1.2.7.4Elimination 14 1.2.8PharmacokineticsinRats 16 1.2.8.1Absorption 16 1.2.8.2Distribution 17 1.2.8.3Metabolism 18 1.2.8.4Elimination 19 1.3DrugGlucuronidation 20 1.3.1Overview 20 1.3.2DirectPharmacologicalActivity 21 1.4EvaluationofAnalgesiainSmallAnimals 26 1.4.1 Introduction 26 III 1.4.2TimeCourseofAnalgesicEffect 27 1.5GeneticPolymorphism 28 1.6Immunomodulation 29 1.6.1OpioidReceptors 30 1.6.2EffectsonLymphocytes 31 1.6.3EffectsonMyleoidCells 32 1.6.4EffectsonNaturalKillerCells 34 1.6.5MechanismofAction 34 1.7ReceptorBinding 37 1.8Hypotheses 39 1.9SpecificObjectives 39 2.METHODS 41 2.1 SpecificObjective#1:AnalyticalMethod 41 2.1.1 Materials 41 2.1.2ExtractionProcedure 41 2.1.2.1 Humanurine 41 2.1.2.2Ratplasma 42 2.1.2.3Ratbrain 42 2.1.3ChromatographicConditions 43 2.1.3.1 HPLCsystem1 43 2.1.3.2HPLCsystem2 44 2.2SpecificObjective#2:SynthesisofCodeine6-glucuronide 45 2.2.1 ReactionStepI 45 2.2.1.1 Drybenzene 47 2.2.1.2Freshsilvercarbonate 47 2.2.2ReactionStepII 48 2.2.3ReactionStepIII 48 2.3SpecificObjective#3:AnalgesicActivitiesofCodeineand Codeine6-glucuronide 48 2.3.1 IntracerebroventhcularRouteStudies 49 2.3.1.1 Surgery 50 2.3.1.2Tailflickmethod 50 2.3.2SubcutaneousRouteStudies 51 2.3.3IntravenousRouteStudies 51 2.3.4Statistics 52 2.4SpecificObjective#4:ImmuneStudieswithHuman TLymphocytes 52 2.4.1 Method 53 2.4.2Statistics 55 2.5SpecificObjective#5:ReceptorBindingStudies 55 IV 2.5.1 Materials 55 2.5.2Method 56 2.6SpecificObjective#6:PlasmaandBrainConcentrations 57 3.RESULTS 59 3.1HPLCDevelopment 59 3.1.1 ExtractionRecoveries 61 3.1.2Range/LinearityofStandardCurve 61 3.1.3Specificity 62 3.1.4Sensitivity/LimitofDetectionandQuantitation 63 3.1.5PrecisionandAccuracy 63 3.1.6Stability 65 3.2Synthesis 65 3.3AnalgesiaStudies 66 3.3.1 IntracerebroventricularRoute 66 3.3.2SubcutaneousRoute 70 3.3.3IntravenousRoute 83 3.4ImmuneStudies 105 3.5ReceptorBindingStudies 111 3.6PlasmaandBrainConcentrations 116 4.DISCUSSION 121 REFERENCES .....:...... 132 BIOGRAPHICALSKETCH 149 -,f ^t AbstractofDissertationPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy ANALGESICANDIMMUNOMODULATORYEFFECTSOF CODEINEANDCODEINE6-GLUCURONIDE By VINAYAKJAYASRINIVASAN May1996 Chairman:Dr.IanRonaldTebbett MajorDepartment:Pharmaceutics The interactions between opioid analgesics and the human immune system can have important clinical consequences. A better understanding of theseinteractionsisneededduetothewidespreaduseandabuseofopiates.In recentyears,anincreasedknowledgeandawarenessinthisareahasgenerated aconsiderablesurgeinresearch. Narcoticsarepredominantlyusedtoalleviate pain and discomfort in patients with trauma or undergoing major surgery. However, they are also known to cause impairment of the immune system. Subsequently, this could lead to patients becoming predisposed to infectious diseasesasaresultoftheimmunosuppressiveeffectsofnarcotics. AnHPLCsystemwassuccessfullydevelopedfortheanalysisofcodeine anditsmetabolitesinvariousbiologicalsamples,thatis,plasma,urineandbrain tissue.Codeine6-glucuronideandanintermediatecompoundweresynthesized usingamodificationoftheKoenigs-Knorrreaction.Thesyntheticprocedurewas VI efficientand reproducible. Analgesia studieswith thetail flick method showed that codeine 6-glucuronide and the intermediate exhibited a higher analgesic activity compared to codeine when administered intracerebroventricularly. However,bothcompoundswerenotasactiveascodeinewhenadministeredby subcutaneousand intravenousroutes. Immunomodulatorystudies showedthat the glucuronide metabolites of codeine and morphine were less immunosuppressive compared to their parent compounds, especially at physiologicallyrelevantconcentrations. Receptorbinding profilesofcodeine6- glucuronide and the intermediatewere similarto codeine, indicating thatthey possessedactivitytowardsthe|i-opioidreceptors. The overall goal of the project was to correlate the analgesic and immunomodulatory effects of codeine and codeine 6-glucuronide. This would resultinabetterunderstanding ofthesignificanceofhigh levelsofcodeine6- glucuronidepresentintheplasmaandurineinmanaftercodeineadministration. Further, this may lead to the development of glucuronide analogs for the managementandtreatmentofpaininimmunocompromisedpatients. VII CHAPTER 1 BACKGROUNDANDSIGNIFICANCE Pain is an unpleasant sensation that can disturb the comfort, thought, sleep, and normaldailyactivityofaperson. Painsignalsareconsideredtobe partofaprotectivemechanismdesignedtoindicatethepresenceofapotentially dangerouscondition.Thus, itisconsideredtobesymptomaticofanunderlying conditionthatrequiresattentionandtreatment.Painistheneteffectofcomplex interactions of ascending and descending neurosystems which include biochemical, physiological, psychological, and neurocortical processes. Also, since pain is a very subjective experience, only the patient can describe its intensity.Thissubjectivitymakesitdifficulttoassesstheactivityofanalgesicsin humans. Analgesicsaredefinedasdrugsthatcanrelievepainwithoutcausingloss ofconsciousness.Themostpotentanalgesicsarereferredtoasnarcoticsand actdirectlyonthecentralnervoussystem.Narcoticsasagroupincludethe opioids,whichareconsideredtobethemosteffectiveanalgesicsavailable.The opioidfamily,whosenamederivesfromopium,includesagentssuchas morphine,codeine,meperidineandmethadone.Whileopioidisageneralterm foranydrug,naturalorsynthetic,thathasactionssimilartomorphine,theterm opiateismorespecificandappliesonlytocompoundspresentinopiumsuchas morphineandcodeine.Apartfromactingasanalgesics,opioidsproducea varietyofpharmacologicalactionsonvarioustissuesinthebody. 1.1OpioidsandPain Opioidsrepresentthemainclassofdrugsintheclinical managementof mild to moderate pain in various cases of medical illness, and relieve pain primarily through direct actions on receptors in the central nervous system. Opioid analgesics include natural alkaloids from opium (morphine, codeine), synthetic surrogates (methadone, meperidine) and endogenous peptides (enkephalins,p-endorphins). , . Opioidsactatreceptorsitesbothwithinandoutsidethecentralnervous system. Binding studieswith variousdrugs and ligands in the brain and other tissues suggest the presence of a multitude of opioid receptors. The three importantreceptortypesaredesignatedasmu{\x),kappa(k)anddelta(5).The effects mediated bythe |i receptors include supraspinal analgesia, respiratory depressionandeuphoria.Thekreceptorsmediateanalgesiaatthelevelofthe spinalcord,alongwithsedationandmiosis.The5receptorsarealsothoughtto beinvolvedinanalgesia,bothatthespinalandsupraspinalsites.However,their roleinthisregardremainscontroversial(JaffeandMartin,1985). The body produces three families of peptides that are capable of interacting with opioid receptors-enkephalins, (3-endorphins and dynorphins. These endogenous opioids have a high affinity forthe ^, k and 5 receptors, respectively.Ttieyarepresentthroughoutthebodyandserveashormonesand neurotransmitters. Itisthoughtthatmorphineandotheropioidanalgesicsmimic the actions ofthese endogenous ligands by binding to the opioid receptors. These interactionsarepresumedtogive risetotheobserved pharmacological effects. 1.1.1 PainTransmission Pain generally begins with a noxious stimulus that injures or destroys tissues. Endogenous chemical substances such as histamine, bradykinins, prostaglandins, and others are then released from the damaged tissues and nerveterminals.Thereleasedchemicalsbindto"painreceptors"ornociceptors presentalongtheafferentnervefibers, depolarizingthenervemembranesand initiatinganactionpotential.Thiscausesthegenerationofapainimpulsewhich isthentransmittedviatheafferentfiberstothespinalcordasshowninthefigure below(Figure 1-1).Whenthepainsignalsarriveatthespinalcord,theyarein turnrelayedtothehighercentersofthebrain-thalamusandcortex. DMeosdcuelnatdiionng Pen-AQu«^uctalOray MIDBRAIN LFaotremraalliRoenticular RapNtiueclMeaugsnus MFeodrimaaltiRoenticular MEDULLA Sp4r«oT-rTahctalamic From PPariinmaFriybrAefsferent Figure1-1 :Thepainmodulatingsystem(adaptedfromPuntillo,1988). »> *h