Table Of ContentANALGESICANDIMMUNOMODULATORYEFFECTSOF
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
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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).
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