Welch 99, Synergy With Opioids

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Brain Research 848 1999 . 183190www.elsevier.comrlocaterbres

Interactive report


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Synergistic interactions of endogenous opioids and cannabinoid systems1

Sandra P. Welch ), Micah EadsDepartment of Pharmacology and Toxicology, Box 980613, Virginia Commonwealth Uniersity, Richmond, VA 23298-0613,USA

Accepted 29 July 1999AbstractCannabinoids and opioids are distinct drug classes historically used in combination to treat pain. D9-THC, an active constituentinmarijuana, releases endogenous dynorphin A and leucine enkephalin in the production of analgesia. The endocannabinoid,anandamide AEA . , fails to release dynorphin A. The synthetic cannabinoid, CP55,940, releases dynorphin B. Neither AEA nor CP55,940enhancesmorphine analgesia. The CB1 antagonist, SR141716A, differentially blocks D9-THC versus AEA. Tolerance to D9-THC, but notAEA,involves a decrease in the release of dynorphin A. Our preclinical studies indicate that D9-THC and morphine can be useful inlow dosecombination as an analgesic. Such is not observed with AEA or CP55,940. We hypothesize the existence of a new CB receptor

differentially linked to endogenous opioid systems based upon data showing the stereoselectivity of endogenous opioid release.Such areceptor, due to the release of endogenous opioids, may have significant impact upon the clinical development ofcannabinoidropioidcombinations for the treatment of a variety of types of pain in humans. q1999 Elsevier Science B.V. All rights reserved.Keywords: Opioid; Cannabinoid; Dynorphin; Enkephalin; Analgesia; Endocannabinoid

1. IntroductionThe cannabinoidropioid interaction differs in thatcannabinoids generally fall into two categories thosethat enhance the antinociceptive effects of morphine in thespinal cord D 9 -THC THC 4 for example . and those that donot enhance spinally administered morphine CP55,940

CP55 4 for example . w 48 x . The mechanisms by which thecannabinoids produce antinociception are as yet unclear.We believe that our data indicate that the mechanism bywhich the cannabinoids produce antinociception involvesdynorphin release spinally and that the greater than addi-tive effects of the cannabinoids with morphine w 1,36 x andthe delta opioid, DPDPE, are due to the initial release ofdynorphin A peptides and the subsequent breakdown ofthe dynorphin A to leucine enkephalin w 28 x . We hypothe-size that the functional coupling of the murdelta andmurkappa receptors leads to enhanced antinociceptiveeffects of morphine and DPDPE by the cannabinoids. Weenvision cannabinoid-induced release of dynorphins as anindirect process due to the disinhibition of yet unknown)Corresponding author. Tel.: q1-804-828-8424; Fax: q1-804-828-2117; E-mail: [email protected] on the World Wide Web on 10 August 1999.

neuronal processes. The localization of the cannabinoid


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receptors involved in dynorphin release are not known. Wehypothesize that in the spinal cord, cannabinoids produceantinociceptive effects via the direct interaction of thecannabinoid receptor with Giro proteins resulting in adecreased c-AMP production w 47 x , as well as hyperpolar-ization via interaction with specific potassium channels w 3 x .

Thus, the cannabinoids may produce disinhibition by de-creasing the release of an inhibitory neurotransmitter indynorphinergic pathways. The net result of such an effectmay be an increase in dynorphin release. The events which

precede and follow the release of dynorphin remain un-clear. The dynorphin most likely is a modulator of otherdown-stream systems possible substance P release orinteraction with NMDA-mediated events . which culminatein antinociception upon administration of cannabinoids.What has proved intriguing is the observation that cannabi-noids differ in their interactions with dynorphins and

subsequently with mu and delta opioids . w 16,17 x .THC appears to interact with the dynorphin A systemw 28,44 x , while CP55 appears to interact with and releasedynorphin B w 27 x , although CP55 is clearly cross-tolerantto THC w 6 x . THC is not cross-tolerant to dynorphin B, butis cross-tolerant to the dynorphins of the A type w 44 x . Inaddition, as animals are rendered tolerant to THC, the0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.PII: S0006-8993 99 . 01908-3

ELSEVIER

BRAIN RESEARCH INTERACTIVE


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( 184

S.P. Welch, M. EadsrBrain Research 848 1999 ) 183190 levels of dynorphin A released are decreased. Thus, toler-


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ance to THC involves a decrease in the release of dynor-phin A w 16 x .The kappa antagonist, nor-binaltorphimine nor-BNI . ,and dynorphin antisera block THC-induced antinocicep-tion, but do not block THC-induced catalepsy, hypother-mia, or hypoactivity w 28,38,43 x . In addition, the discovery

of the bi-directional cross tolerance of THC and CP55 tokappa agonists using the tail-flick test w 38 x and to dynor-

phin A w 44 x , indicates that cannabinoids interact in ayet-to-be-determined manner with kappa opioids. The at-tenuation of the antinociceptive effects of THC by anti-sense oligonucleotides to the cloned kappa-1 opioid recep-tor further implicates the release of endogenous kappaopioids in the mechanism of action of the cannabinoidsw 26 x . Dynorphin antibodies block THC-induced antinoci-ception, and prevention of the metabolism of dynorphin A 117 . to dynorphin 18 . or to leucine enkephalin pre-

vents the enhancement of morphine-induced antinocicep-tion by the THC w 28 x .The endogenous cannabinoid, AEA, appears to differfrom THC and CP55 in its lack of interactions withdynorphinergic systems w 38,44 x . Despite similarities in the

profile of action to classical cannabinoids, distinct differ-ences between AEA and other cannabinoids in terms of

behavioral effects have been reported w 28,38,44,45 x . AEAappears to differ from the traditional cannabinoids in that itis not active following icv. administration in several behav-iors which are characteristic of cannabinoids. Other differ-ences between anandamide and THC have been observed

in tasks involving learning and memory w 13 x , drug discrim-ination w 49 x and modulation by agonists and antagonists ofclassical neurotransmitter systems w 45 x . AEA which isneither blocked by the kappa antagonist, nor-BNI, norcross-tolerant to any dynorphins w 37,44,45 x is cross-tolerantto THC and CP55 and displaces binding of the traditionalcannabinoids w 4,33,37,45 x . Anandamide fails to enhancethe activity of any opioid and does not release dynorphinA w 28,44,45 x .Hence three cannabinoids, representing three differentclasses, induce antinociceptive activity via the cannabinoid

receptor, yet differentially modulate dynorphinergic sys-tems. These differences may reflect differences in theinteractions of receptor or activities cannabinoids with the of functional subtypes cannabinoid of the cannabi-CB1

noid envision CB 1such receptor diverse in the dynorphin spinal cord. release It is difficult to

profiles for thedrugs through actions at one receptor subtype. The mecha-


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nisms underlying the differential release of dynorphins byTHC versus CP55 and AEA thus remains unknown.Two distinct cannabinoid receptors have been cloned,the CB1 receptor which is predominantly located in thecentral nervous system w 18 x , and the CB2 receptor which isfound on immune cells and on peripheral tissues w 21 x . In

addition, a splice variant of the CB1 receptor termed theCB1A receptor has been identified w 34 x . The discovery ofthe cannabinoid CB1 receptor antagonist, SR141716A w 30x and the discovery of the first endogenous cannabinoid-likeligand, anandamide AEA 4 , w 4 x greatly facilitated workwith the cannabinoids and complements the discovery andcloning of the cannabinoid receptors. The newly describedcannabinoid be of great help CB 2in receptor elucidating antagonist, cannabinoid SR144528 receptor w 31 x , sub-willtypes. Receptor-ligand binding studies have produced evi-

dence tor suggesting the existence of cannabinoid subtypes w 41 x . We evaluated THC, CP55, CB and 1recep-AEAalone and in combination with SR141716A SR. , a CB1antagonist, in order to better characterize potential diver-sity in interactions of the cannabinoids with the cannabi-noid CB1 . receptor. The effects of SR on AEA-inducedantinociception were mixed. The maximum attenuation ofAEA-induced antinociception intrathecally administered,i.t. .by SR i.t. . was only 38%. SR administered intraperi-toneally, i.p. .blockade of ANA was complete, but theAD THC 50

or was CP55. nearly In 15-fold addition, higher SR than i.p. that required to blockor i.t. . failed to blockthe hypothermic effects of AEA i.t. . , while completelyreversing the hypothermic effects of THC i.t. . . Such dataare suggestive of either a differential interaction of thecannabinoids at the CB1 receptor or the existence ofsubtypes of the CB1 receptor w 46 x .In addition to the use of the CB1 antagonist,SR141716A, in this paper we report the stereoselectivity ofopioid peptide release by the administration of levo-nantradol, a synthetic cannabinoid which we have shown

to produce antinociception and enhance the activity ofmorphine w 48 x , and dextronantradol, its inactive stereoiso-mer. We chose this pair of stereoisomers since the drugshad been previously evaluated in several test systems inmice and rats. Clearly, many other stereoisomeric pairs ofcannabinoids have been tested in other systems see Ref.w 15 x for review .but most have not been tested via the i.t.route of administration and have not been evaluated for the


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ability to enhance the antinociceptive effects of morphine.We hypothesized that levonantradol, which was the most

potent cannabinoid that we tested w 48 x would release moredynorphin and produce antinociceptive effects at lowerdoses in the rat than THC, while dextronantradol wouldnot release dynorphin. Such work was designed to show

the stereoselectivity and thus, receptor mediation, of thedynorphin release.2. Materials and methods2.1. Animal husbandryThese studies were conducted using male SpragueDawley rats, weighing between 450 and 500 g, obtainedfrom Harlan Laboratories. Subjects were housed individu-ally and maintained on a fixed 12 h light cycle at a


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( S.P. Welch, M. EadsrBrain Research 848 1999 ) 183190 185

temperature of 22"28C. Water and food Harlan Rat


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Chow . were provided ad libitum.2.2. Intrathecal administration of drugs and opioid collec-tionIntrathecal drug administration and opioid collectionwere performed using a modified version of techniquesdescribed by Mason et al. w 16,17 x , Tseng et al. w 42 x and

Yaksh w 50 x . Subjects were selected for partitioning intoexperimental groups at random and anesthetized via i.p.injection of sodium barbital 375 mgrkg . and a separatei.p. injection of 2 mgrkg atropine methyl nitrate. Theanesthetized rats were placed in stereotaxis and an incisionmade on the atlanto-occipito membrane to expose thecisterna magna. A catheter of PE-10 polyethylene tubingwas inserted through the exposed cisternal cavity, cau-dally, into the subarachinoid space of the spinal cord. Thecatheter contained an artificial cerebrospinal fluid, com-

posed of 125 mM Naq; 2.6 mM Kq; 0.9 mM Mg2q; 1.3

mM Ca2q; 122.7 mM Cly; 21.0 mM HOCy; 2.4 mMHOP2y ; 0.5 mgrml bovine serum albumen, bacitracin 30 mgrml . , 0.01% Triton X and effervesced with 95%O caudally 2 and 5% 8.5 CO cm 2. passing Positioned through as such, the the catheter extendedthoracolumbar regionto an area just above the sacral enlargement. Followingcatheter implantation, animals were allowed to acclimateapproximately 30 min on a heating pad. Following accli-mation, base-line tail-flick latency was assessed. Onlyanimals exhibiting normal tail-flick response to noxiousstimuli, greater than 1.5 s, but less than 4 s latency, were

used. Test compounds were administered in a 20 ml bolusof vehicle, via spinal catheter, at a rate of 30 mlrmin.Subjects were then segregated into groups for cere-

brospinal fluid sampling and tail-flick latency assessment10 min post administration of the test compound. Cere-

brospinal fluid collection entailed rapid perfusion of thespinal cavity with artificial cerebrospinal fluid culminatingin the collection of 1.5 ml of the eluting artificial cere-

brospinal fluid from the open cisternal space. This is anopen system and the sampling technique is similar to the

push pull cannula technique commonly employed in the

mouse. Collected fractions were boiled for 12 min andcentrifuged at a rate of 10000 rpm for 10 min. Thesupernatant was collected, frozen at y708C andlyophilized. Samples were reconstituted in 250 ml radioim-munoassay buffer before dynorphin A- 117 . , dynorphinB, methionine met 4 enkephalin, or leucine leu 4 enkephalin

peptide measurement.2.3. Measurement of opioid peptides


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Measurement of opioid peptide release pgrml . wasaccomplished using a specific radioimmunoassay kits ob-tained from Peninsula Laboratories, Inc. The reconstitutedsamples were analyzed in duplicate. The manufacturerreported cross-reactivity of dynorphin A- 117 . antibodyas 100% versus dynorphin- 124 . , a parent compound,and less than 2% versus smaller peptide fragments. Wefound no cross-reactivity of the antibody to dynorphinA- 18 . , dynorphin A- 113 . , dimethyl sulfoxide or D 9 -te-trahydrocannabinol. Similarly, the dynorphin B antibodyhad less than 3% cross-reactivity to dynorphin A or to themet or leu enkephalins. The met enkephalin antibody hadless than 1% cross-reactivity to the leu-enkephalin anti-

body and vice versa. We found no cross-reactivity of themet- or leu- enkephalin antibody to dynorphin A- 18 . ,dynorphin A- 113 . , dimethyl sulfoxide or D 9 -tetrahydro-cannabinol. Only the linear portion of the radioimmunoas-

say standard curve, between 1 pgrml and 64 pgrml of thestandard peptide, was used to calculate dynorphin concen-tration.2.4. Assessment of tail-flick latencyAntinociceptive behavior was assessed using a modifiedversion of that described by DAmour and Smith w 2 x . Eachanimal was acclimated in the laboratory 24 h prior toexperimentation. Tail-flick latency was not found to besignificantly increased by sodium barbital or catheteriza-tion in comparison to unanesthetized or non-catheterizedanimals. Base latencies were measured as 1.54 s withmaximal post-drug latency set at 10 s after which the

noxious heat stimulus was terminated. Antinociception wasmeasured in terms of percent maximal possible effect %MPE . defined by Harris and Pierson w 9 x as:%MPEs [ test latencycontrol latency ] = 100%w 10 s-control latencyx Each parameter i.e. test or control tail-flick latencyvalue . represents the mean of three recordings at 10 sintervals.2.5. Statistical analysisUsing a randomized design, analysis of data concerningtail-flick latency or peptide concentration was done using

ANOVA analysis of variance . followed by two-tailedDunnetts t-test w 5 x .2.6. Drugs and ehicleFor i.t. challenges, levonantradol 5 mgrrat . and dex-tronantradol 30 mgrrat . were administered using a 100%dimethyl sulfoxide vehicle DMSO . at 15 min prior tocollection of the CSF. At 15 min post administration, the

peak antinociceptive effects of levonantradol were ob-


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served. The concentration of DMSO has been used innumerous studies and has no effect on the animals behav-ior in awake, non-anesthetized animals . or on dynorphinrelease in our anesthetized rats w 16,17 x . Drugs were pro-vided by Dr. Lawrence Melvin, Pfizer PharmaceuticalResearch. The dose chosen for levonantradol was that


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( ) S.P. Welch, M. EadsrBrain Research 848 1999 183190 186

which produced 100% antinociceptive effect in the tail-flick


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. test. The dose chosen for dextronantradol 30 mgrrat wasthe highest dose we could run with the available drugsupply.3. ResultsFig. 1 indicates the results of antinociceptive testing onthe rats subsequently utilized for the collection of CSF as

described in the Materials and Methods section. The clearbars represent the average %MPE for Ns9 rats subse- . quently used to quantitate dynorphin A- 117 release.The striped bars represent the average %MPE for Ns10rats subsequently used to quantitate the release of dynor-

phin B. Drugs were administered i.t. at 15 min prior to testing using the tail-flick test. Levonantradol Ns9 rats;. dose of 5 mgrrat in a 20 ml volume produced 87.6"12.3% MPE versus 2.5"2.2%MPE in dextronantradol- treated rats Ns10; dose of 30 mgrrat in a 20 ml. volume and 4.8"2.9%MPE in DMSO-treated rats Ns

. 9; dose in a 20 ml volume p-0.0001 from both DEX-

. TRO and DMSO . Immediately following testing in thetail-flick test CSF was removed for testing of the endoge-nous opioid release from the same rats. Separate groups of

Fig. 1. Antinociceptive effects of i.t. levonantradol and dextronantradol inrats. Antinociceptive behavior was assessed using a modified version ofthat described by DAmour and Smith w 2 x as described in the Materialsand Methods using the tail-flick test. %MPE was quantified for each rat.The average % MPE"standard error of the mean is reported for separategroups of rats Nsat least 8 rats per group . . Clear bars represent theaverage %MPE for rats subsequently evaluated for the release of dynor-

phin A- 117 . ; striped bars represent the average %MPE for rats subse-quently evaluated for release of dynorphin B data shown in Fig. 3 . .DMSO indicates the vehicle-treated rats. LEVO indicates rats treated with5 mgrrat levonantradol. DEXTRO indicates rats treated with dextro-nantradol 30 mgrrat . . Treatments were i.t. at 15 min prior to testing.Significance was determined using ANOVA followed by the post hocDunnetts t-test unpaired, two-tailed .. U

p-0.05 from DMSO-treatedrats. Similar antinociceptive effects were observed in separate groups ofrats in which met- and leu-enkephalin release was quantified Fig. 2 . .

Fig. 2. Lack of the release of met- and leu-enkephalin by the i.t.administration of levonantradol and dextronantradol in rats. Antinocicep-tive behavior was assessed using a modified version of that described byDAmour and Smith w 2 x as described in the Materials and Methodsusing the tail-flick test. %MPE was quantified for each rat Nsat least 8rats per group . . The rats were then spinally perfused and the CSFremoved as described in the Material and Methods. Methionine andleucine enkephalin were quantified using radioimmunoassay. Clear barsrepresent the average met-enkephalin release in pgrml of CSF . ; filled


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bars represent the average leu-enkephalin release in pgrml of CSF . .DMSO indicates the vehicle-treated rats. LEVO indicates rats treated with5 mgrrat levonantradol. DEXTRO indicates rats treated with dextro-nantradol 30 mgrrat . . Collection of CSF occurred at 15 min followingdrug or vehicle administration and concurrently with the tail-flick testing.Significance was determined using ANOVA followed by the post hoc

Dunnetts t-test unpaired, two-tailed .. Up-0.05 from DMSO-treatedrats.

rats were administered the drugs and CSF was removed forthe quantitation of met- or leu-enkephalin Ns1012 rats

per group . . The average %MPE for those groups of LEVO-or DEXTRO-treated rats did not differ from the results

presented in Fig. 1. data not shown . . LEVO producedsignificant antinociceptive effects in all groups, whileDEXTRO and DMSO produced no significant effects.Fig. 2 indicates the results of the radioimmunoassay

for met-enkephalin and leu-enkephalin. DMSO-inducedrelease of met-enkephalin was 16.4"2 pgrml. Levo-nantradol LEVO . and dextronantradol DEXTRO . - in-duced release was 19.2"1.1 and 22.7"2.7 pgrml, re-spectively, and did not differ significantly from each otheror DMSO control. LEVO and DEXTRO similarly did not

produced a significant increase in leu-enkephalin com-pared to DMSO 6.3"3; 5.8"2.7 and 3.7"2.3 pgrml,respectively . .Fig. 3 indicates the results of LEVO and DEXTRO on

both dynorphin A- 117 . and dynorphin B release. Therelease of dynorphin A- 117 .by DMSO was 3.6"0.4

pgrml versus 1.1"0.01 pgrml for DEXTRO and 1.8"0.2 for LEVO. LEVO significantly increased dynorphin Brelease from 4.0"0.8 pgrml for DMSO to 9.8"2.7

pgrml p-0.046 . . DEXTRO did not increase dynorphinB levels 3.7"0.6 pgrml . over those observed for DMSO.

DMSO LEVO DEXTRO Treatments

Ill T

DMSO LEVO DEXTRO Treatments


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Fig. 3. Release of dynorphin B, but not dynorphin A- 117 .by the i.t.administration of levonantradol, but not dextronantradol, in rats.Antinociceptive behavior was assessed using a modified version of that


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described by DAmour and Smith w 2 x as described in the Materials andMethods using the tail-flick test. %MPE was quantified for each ratNsat least 8 rats per group . . The rats were then spinally perfused andthe CSF removed as described in the Material and Methods. DynorphinA- 117 . clear bars . and dynorphin B striped bars . were quantifiedusing radioimmunoassay. DMSO indicates the vehicle-treated rats. LEVO

indicates rats treated with 5 mgrrat levonantradol. DEXTRO indicatesrats treated with dextronantradol 30 mgrrat . . Collection of CSF occurredat 15 min following drug or vehicle administration and concurrently withthe tail-flick testing. Significance was determined using ANOVA fol-lowed by the post hoc Dunnetts t-test unpaired, two-tailed .. U

p-0.05from DMSO-treated rats.

In all experiments the use of experimental animals wasapproved by the Institutional Care and Use Committee inaccordance with all federal directives. Care was taken inall cases to minimize the numbers of animals used and the

pain or discomfort of the animals.4. DiscussionIt has been documented that the cannabinoids produceeffects which have much in common with the opiates, suchas antinociception, hypothermia, cross tolerance to mor-

phine, and attenuation of naloxone-precipitated withdrawalfrom morphine. Early experiments to evaluate the anal-gesic effects of the cannabinoids dealt mainly with anexamination of the effects of D9-THC, the principle activeingredient in cannabis. Studies in human subjects indicatedthat at oral doses of 10 and 20 mgrkg D9-THC was nomore effective than codeine as an analgesic, while produc-

ing a significant degree of dysphoric side effects w 23 x .When tested following intravenous administration to hu-man dental patients, D9-THC produced analgesia that wasaccompanied by dysphoria and anxiety w 29 x . Thus, in thesestudies it was evident that D9-THC analgesia could only beelicited at doses producing other behavioral side effects. Inaddition, D9-THC was no more potent than the morecommonly used opioid analgesics. Levonantradol has pre-

( ) S.P. Welch, M. EadsrBrain Research 848 1999 183190 187

viously been shown to produce antinociceptive effectsw x upon i.t. administration to rats 50 and spinal administra-w x tion to the dog 8 at doses devoid of other behavioral sideeffects. These investigators concluded that a spinal site ofaction might be involved in the antinociceptive effectsobserved.Recently the interaction of cannabinoids with opioidsw x has been extensively revisited and reviewed 14 in terms


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of the interactions of the drug classes in the prevention ofpain with fewer side effects of either drug class and thelack of induction of addiction. Our findings of the block-ade of cannabinoids by nor-BNI, and dynorphin antibodiesin spinal cord, but not the brain, implicates dynorphins inthe spinal mechanism of action of the cannabinoids. This

finding is exciting in that it provides a direct link betweenthe cannabinoids and opioid systems and is the first timethat the antinociceptive effects of the cannabinoids havew x been separated from other behavioral effects 38 .The potent, synthetic cannabinoid, CP55, was instru-mental in demonstrating that cannabinoid binding sites are

present in the substantia gelatinosa, an area involved withw x the transmission of pain signals 10 . In addition, CP55

produces many of the behavioral and physiologic effectscharacteristic of THC. Despite these similarities, we havefound that THC, levonantradol, and CP55 differ in their

w x interaction with morphine in the spinal cord 48 . Pretreat- . ment of mice with CP55 i.t. does not enhance the . antinociceptive effects of morphine i.t. , while pretreat-ment with THC produces a 10-fold decrease in the mor-

phine ED and levonantradol enhances the potency of50

. w x morphine i.t. by greater than 10-fold 48 . Our dataindicate that THC enhances the antinociception of mor-

phine through the release of endogenous dynorphin A andw x its breakdown to leu-enkephalin 28 . Dynorphin B, an-other product of the prodynorphin precursor, also containsa copy of leucine enkephalin. However, the breakdown of

dynorphin B to leucine enkephalin is less well documentedw x 12 and dynorphin B fails to enhance the activity ofw x morphine in antinociceptive tests 27 . It has been shownthat dynorphin B produces antinociception when adminis-w x tered i.t. as measured by the tail-flick test 22,40 . Since ithas been shown that kappa, delta, and mu ligands interactw x in the production of antinociceptive effects 11,20,32,39 ,the release of a delta or kappa opioid by the cannabinoidscould also alter the antinociceptive effects of the predomi-w x nantly mu opioid, morphine. Pugh et al. 28 and Welchw x . 44 demonstrated that dynorphin A- 113 and dynorphin

. . A- 18 increased tail-flick latency i.t. in the mouse andw x enhance the activity of morphine. Mason et al. 16,17 . have shown that THC releases dynorphin A- 18 in therat which would presumable enhance morphines effects.Dextronantradol, which produced no antinociceptive ef-fects and did not enhance the effects of morphine, washypothesized to not release opioids. Our hypothesis provedcorrect. However, we were not expecting levonantradol to

be inactive in release of dynorphin A. Since levonantradol


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I DMSO LEVO DEXTRO

Treatments


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S.P. Welch, M. EadsrBrain Research 848 1999 ) 183190potently enhanced the effects of morphine, we expected it


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to potently release dynorphin A. Such was not the case.Levonantradol released only dynorphin B which was tem-

porally correlated to its antinociceptive effect. Since levo-nantradol-induced antinociception is blocked totally bynor-BNI w 43 x , the antinociceptive effects of levonantradolappear to be related in part to dynorphin B release and

subsequent interaction at the kappa-opioid receptor. CP55releases only dynorphin B w 27 x . Like levonantradol, it doesnot release met- or leu-enkephalin or dynorphin A- 18. data not shown . . Since dynorphin B does not enhancemorphine-induced antinociception, the enhancement ofmorphine by levonantradol appears to not be related todynorphin B release. Since levonantradol does not releasedynorphin A, which does enhance morphines effects, themechanism by which levonantradol acts to enhance mor-

phine is obviously unique from that of THC. However, theeffects of levonantradol on dynorphin B release are stere-

oselective and blocked by SR141716A and are thus, recep-tor- mediated.AEA-induced antinociception is mediated by a mecha-nism which appears to differ from THC, CP55 and levo-nantradol in terms of kappa-opioid receptor involvementw 16,17,38,44,45 x . Unlike THC, AEA does not significantlyincrease immunoreactive dynorphin A- 117 . concentra-tion, nor is its ability to increase tail-flick latency nor-BNI-sensitive. Given the lack of a nor-BNI block ofAEA-induced antinociception, it is unlikely to be a criticalcomponent in its antinociceptive effects w 17 x . Similar con-clusions were noted in the development of tolerance to

AEA w 44 x . AEA does not release any opioid peptide tested data not shown . . However, the attenuation of AEA-in-duced tail-flick latency by SR141716A indicates thatAEA-induced antinociception, like that of THC and CP55,is sity mediated of effects, via between the cannabinoid AEA and CB THC, 1receptor. Such diver-have been reportedin other models w 35,37 x . Such data are suggestive of eithera differential interaction of AEA versus the classicalcannabinoids at the CB1 receptor or the existence ofsubtypes of the CB1 receptor.

Thus, the mechanism of action of anandamide, its activ-ity in modulating pain, and its lack of interaction withopioids remains unclear and appears to differ from that ofthe THC, CP55, and levonantradol. Anandamide is but oneof a family of arachadonic acid derivatives which havecannabinoid-like effects w 7,19,24,25 x . The elucidation ofdistinct mechanisms by which the body produces andutilizes endogenous cannabinoid substances in the modula-


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tion of nociception could have a potentially importantimpact on the design of new analgesics for clinical use.The immediate benefits of elucidating the full scope of theinteractions of cannabinoids exogenous or endogenous. with opioids will likely come in the management of pain,

particularly chronic pain w 35 x . THC, in comparison to the

morphine derivatives, has a greater therapeutic range. Opi-oid use can lead to development of tolerance, undesirableside effects, and tolerance. Studies such as ours and thoseof others w 1,14,16,17,36 x may lead to new techniques bywhich manipulation of the endogenous opioid system bycannabinoids can be used to supplement exogenous opioidagents resulting in decreased dosage of opioids and aresulting decreased risk of toxicity, as well as manageabletolerance development. Alternatively, the elucidation ofthe mechanisms underlying the differences in the interac-tion of distinct cannabinoids with distinct endogenous

opioid systems may lead to the discovery of novel CBreceptors as potential targets for development of eitheropioid adjuncts or non-opioid analgesics.In summary, we have shown that THC and levo-nantradol enhance the antinociceptive effects of morphine.THC releases dynorphin A, but levonantradol releasesdynorphin B. CP55 and AEA do not enhance the effects ofmorphine spinally. CP55 releases dynorphin B. AEA re-leases no opioid peptides. Antinociceptive effects of THC,levonantradol, CP55, and AEA are all blocked by the CB1antagonist, SR141716A. Thus, four distinct cannabinoidsexert four distinct patterns of interaction or lack of interac-

tion with endogenous opioid systems. We hypothesize thatsuch a diversity of interactions may be indicative of CB1receptor subtypes in the spinal cord. Such subtypes may beclinically important in the development or use of cannabi-noids in the treatment of pain.AcknowledgementsThis work was supported by the National Institute ofDrug Abuse Grants a K02 DA00186, and DA05274.Referencesw x1 D.L. Cichewicz, Z. Martin, F.L. Smith, S.P. Welch, Enhancement ofopioid potency with inactive oral administration of D9-THC: Dose-response analysis and receptor characterization, J. Pharmacol. Exp.Ther. 289 1999 . 859867.w x2 F.E. DAmour, D.L. Smith, A method for determining loss of painsensation, J. Pharmacol. Exp. Ther. 72 1941 . 7479.w x3 S.A. Deadwyler, R.E. Hampson, B.A. Bennet, T.A. Edwards, J. Mu,M.A. Pacheco, S.J. Ward, S.R. Childers, Cannabinoids modulate

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