Synergy between Δ9-tetrahydrocannabinol and morphine in the arthritic rat

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    nares et al., 1999) through actions at cannabinoid and opioid

    the periaqueductal grey, amygdala, and thalamus (Mansour et al.,1988; Martin et al., 1999). These structures are part of a

    Endogenous opioids have been shown to mediate cannabi-

    1992; Smith et al., 1998a). A later study (Cichewicz et al., 1999)confirmed that a non-antinociceptive oral dose of 9-THCenhances the potency of an acute oral dose ofmorphine, as well asother opioid analgesics. Furthermore, a full isobolographic

    European Journal of Pharmacologydescending pain control circuit that mediates pain suppressivereceptors, members of the G-protein-coupled receptor family.Activation of G-protein-coupled receptors produces intracellularevents such as inhibition of adenylate cyclase activity (Sharmaet al., 1975; Howlett and Fleming, 1984), decreased calciuminflux, and increased potassium efflux (Morita and North, 1982;Hescheler et al., 1987; Felder et al., 1992). Cannabinoid CB1receptors and mu-opioid receptors have been reported to co-localize in brain areas involved in nociceptive responses such as

    noid-induced antinociception. 9-THC-induced antinociceptionwas found to be modulated by mu-opioid receptors supraspinally,while kappa-opioid receptors were involved in spinal antinoci-ception (Smith et al., 1994; Reche et al., 1996). Spinallyadministered 9-THC releases dynorphin A in the spinal cordof the rat (Mason et al., 1999). 9-THC and morphineadministration by any combination of routes significantlyenhances the potency of morphine in mice (Welch and Stevens,interaction between 9-THC and morphine in both the non-arthritic and the arthritic rats. Since Freund's adjuvant-induced alteration inendogenous opioid tone has been previously shown, our data indicate that such changes did not preclude the use of 9-THC and morphine incombination. As with acute preclinical pain models in which the 9-THC/morphine combination results in less tolerance development, theimplication of the study for chronic pain conditions is discussed.Published by Elsevier B.V.

    Keywords: Isobologram; Synergy; Arthritis; 9-THC; Morphine

    1. Introduction

    Cannabinoids and opioids share several pharmacologicaleffects, including hypothermia, sedation, analgesia, and theinhibition of motor activity (Bloom and Dewey 1978; Manza-

    actions of both opioids and cannabinoids (Basbaum and Fields,1984; Meng et al., 1998). In the spinal cord, opioid andcannabinoid receptors are co-localized in areas of the dorsalhorn where they are involved in nociceptive control (Salio et al.,2001).for the fixed ratios (total dose) in relation to the ED50 value of the drugsinflammatory pain models, such as Freund's complete adjuvant-induced arthritic model. One fixed-ratio combination was chosen for testing theinteraction between 9-THC and morphine in the Freund's adjuvant-induced arthritic model. This combination represented a 1:1 ratio of the drugsand thus consisted of equieffective doses ranging from 0.1 to 5 mg/kg 9-THC and from 0.1 to 5 mg/kg morphine. The combination ED50 value

    alone was determined. The isobolographic analysis indicated a synergisticSynergy between 9-tetrahydrocanna

    Melinda L. Cox, Victoria

    Department of Pharmacology and Toxicology, Virginia Commo

    Received 8 February 2007; received in revisAvailable onli


    We have shown in past isobolographic studies that a small aantinociception in mice. However, previous studies of the 9-THC/moracute thermal antinociception. Less is known about cannabinoid and Corresponding author. P.O. Box 980524, Richmond, VA 23298, UnitedStates. Tel.: +1 804 828 8446; fax: +1 804 828 2117.

    E-mail address: (V.L. Haller).

    0014-2999/$ - see front matter. Published by Elsevier B.V.doi:10.1016/j.ejphar.2007.04.010inol and morphine in the arthritic rat

    Haller , Sandra P. Welch

    alth University, Richmond, Virginia 23298-0524, United States

    orm 27 March 2007; accepted 1 April 20070 April 2007

    unt of 9-tetrahydrocannabinol (9-THC) can enhance morphinene interaction were performed using normal mice or rats and evaluatedioid interactions involved in mechanical nociception and in chronic

    567 (2007) of the interaction between oral 9-THC and morphineor codeine provided evidence of synergy between 9-THC andthese opioids (Cichewicz and McCarthy, 2003).

  • of PPrevious studies of the 9-THC/morphine interaction wereperformed using normal mice or rats and evaluated acutethermal antinociception. Less is known about cannabinoid andopioid interactions involved in mechanical nociception and inchronic inflammatory pain models, such as Freund's completeadjuvant-induced arthritic model. Freund's adjuvant treatmentproduces chronic inflammation, edema, and hyperalgesia in rats(Millan et al., 1986a). Sofia et al. (1973) demonstrated that 9-THC is effective in the paw-pressure test for mechanicalnociception in rats. In Freund's adjuvant-induced arthritic rats,9-THC-elicited antinociceptive efficacy was no different fromthat in normal rats (Smith et al., 1998b). However, 9-THCmodulation of dynorphin Awas found to differ in normal versusarthritic rats. While 9-THC was shown to trigger an increasein release of dynorphin A in normal rats, arthritic rats had highlevels of dynorphin A and 9-THC normalized dynorphin Alevels to that of normal animals (Cox and Welch, 2004). Sinceendogenous opioid release has been shown to play a major rolein the enhancement of morphine by 9-THC in acute painmodels (Pugh et al., 1996), our study was designed to determineif this enhancement was observed in arthritic and normal ratsusing mechanical stimuli.

    2. Materials and methods

    2.1. Animals

    Male SpragueDawley rats (Harlan Laboratories, Indiana-polis, IN), which weighed 350 to 375 g were housed in ananimal care facility maintained at 222 C on a 12-h light/darkcycle with free access to food and water. All experiments wereconducted according to guidelines established by the Institu-tional Animal Care and Use Committee of Virginia Common-wealth University and adhere to the guidelines of theCommittee for Research and Ethical Issues of IASP.

    2.2. Freund's adjuvant-induced arthritis treatment

    A volume of 0.1 ml of vehicle (mineral oil) or Freund'scomplete adjuvant (heat-killed Mycobacterium butyricum;5 mg/ml) was injected intradermally into the base of the tail.Animals remained in their cages for 12 days and wereacclimated daily to paw-pressure testing until day 19, onwhich they were tested. Acclimation consisted of a briefpressure stimulus, less than that necessary to elicit pawwithdrawal, to the hind paw of the rat using the paw-pressureapparatus. Inflammation proceeds into a generalized polyar-thritis within 19 days. Paw-pressure baseline measurements onday 19 indicated that arthritic rats are more sensitive tomechanical nociception than non-arthritic rats.

    2.3. Paw-pressure test

    The paw-pressure test consisted of gently holding the body

    126 M.L. Cox et al. / European Journalof the rat while the hind paw was exposed to increasingmechanical pressure. The Analgesy-Meter (Ugo-Basile, Varese,Italy) is designed to exert a force on the paw that increases at aconstant rate, similar to the Randall and Sellito (1957) test ofmechanical nociception. Force was applied to the hind paw thatwas placed under a small plinth under a cone-shaped plungerwith a rounded tip. The operator depressed a pedal-switch tostart the mechanism that exerted force. The force in grams atwhich the rat withdrew its paw was defined as the paw-pressurethreshold. The baseline paw-pressure was measured beforeinjecting vehicle or drug. Non-arthritic rats that had a baselinepaw-pressure greater than 100 g (average=1776.42 g) wereused in further testing. Arthritic rats that had a baseline paw-pressure less than 100 g (average=713.05 g) were used infurther experimentation. The upper limit of 500 g was imposedfor the experiments to allow the foot to not become immobilizeddue to undue pressure.

    2.4. Drug administration protocol

    For the generation of doseresponse curves using the paw-pressure test of antinociception in arthritic and non-arthritic rats,morphine and 9-THC were administered i.p. The doseresponse curves for morphine and 9-THC alone weredetermined. Morphine was prepared in distilled water and 9-THC was prepared in a solution of emulphor, ethanol, and salineat a 1:1:18 ratio. Morphine (0.5, 1, 2, and 4 mg/kg) or distilledwater vehicle was administered 30 min prior to antinociceptivetesting. 9-THC (0.5, 1, 2 and 4 mg/kg) or 1:1:18 vehicle wasadministered 30 min prior to antinociceptive testing. The peaktimes for antinociception produced by 9-THC and morphinehave been previously determined to be 30 min post adminis-tration (Cox and Welch, 2004).

    2.5. Percent maximum possible effect determination

    The average paw-pressure threshold in grams was deter-mined before drug administration (baseline) and the selectedtimes (test) following drug administration. The maximumpossible effect (%MPE) was determined for each rat accordingto the following formula using a 500 g maximum pressure: %MPE= [test (g)baseline (g) / 500 gbaseline (g)] 100.Doseresponse curves were generated using three or fourdoses of test drug. ED50 values and 95% confidence limits weredetermined using the methods of Tallarida and Murray (1987).Injection of distilled water (i.p., 0.1 cc/100 g body weight)resulted in a %MPE less than 51%. Injection of 1:1:18emuphor:ethanol:saline vehicle resulted in a %MPE of 82%.

    2.6. Isobolographic analysis

    The use of the isobologram has been reviewed extensively inthe context of drug combination studies (Wessinger, 1986;Tallarida et al, 1989; Tallarida, 2001). The method used in thepresent studies is similar to that reported by Kimmel et al.(1997). Doseresponse curves were generated for each drugalone, and ED50 values (dose which yields 50% effect) and

    harmacology 567 (2007) 125130standard error (S.E.M.) were computed using unweighted least-squares linear regression as modified from procedures 5 and8 described by Tallarida and Murray (1987). The ED50 values of

  • 2.7. Statistical analysis

    The average paw-pressure threshold in grams was deter-

    Fig. 1. Doseresponse curve of9-THC (i.p.) in non-arthritic (indicated by clearsquares) and arthritic rats (indicated by black diamonds). Animals were treatedwith 9-THC (i.p.) alone were tested 30 min later using the paw-pressure test.The data are presented as %MPES.E.M using an N=68 rats per dose.Separate rats were used for each dose.

    Table 1ED50 valuesS.E.M. for

    9-THC and morphine (both i.p.) in the pawwithdrawal test antinociception in non-arthritic rats and in arthritic rats

    9-THC (micrograms/kg) Morphine (micrograms/kg)

    Non-arthritic rats Non-arthritic rats

    Z1=2100300 Z2=2400400Log z1=3.222(2.48) Log z2=3.38(2.60)

    9-THC (micrograms/kg) Morphine (micrograms/kg)

    Arthritic rats Arthritic rats

    Z1=2500500 Z2=2200400Log z1=3.40(2.70) Log z2=3.34(2.60)

    Amounts are total (in micrograms/kg for ease of log calculations).

    127M.L. Cox et al. / European Journal of Pharmacology 567 (2007) 125130the drugs alone are then plotted and a theoretical additive line isconstructed on an isobologram. Experimental values from thefixed-ratio design studies were also analyzed using linearregression and an ED50 value for each combination wasdetermined and plotted on the isobologram for comparison tothe theoretical additive value. This theoretical value, termedZadd, is calculated using the formula Zadd= fz1+gz2 (Tallaridaet al., 1997, Eq. (3)), where f+g=1 (the proportions of eachdrug) and z1 and z2 represent the ED50 values for each drugalone. The standard error for Zadd is determined from theformula: S.E.M.(Zadd)= [f2{S.E.M.(z1)}2+g2{S.E.M.(z2)}2]1/2(Tallarida et al., 1997, Eq. (4)). The Student's t-test was used todetermine statistical significance of the difference between thelogarithmic equivalents of the ED50 values (since a requirementof the t-test is the use of values that are normally distributed). Amore detailed explanation of the calculations used for the t-test

    can be found in the literature (Tallarida, 2001). A P value lessthan 0.05 indicated that the drugs produced a synergistic effect.

    Fig. 2. Doseresponse curve of morphine (i.p.) in non-arthritic (indicated byclear squares) and arthritic rats (indicated by black diamonds). Animals weretreated with morphine (i.p.) alone were tested 30 min later using the paw-pressure test. The data are presented as %MPES.E.M using an N=68 rats perdose. Separate rats were used for each dose.mined before drug administration (baseline) and the selectedtimes (test) following drug administration. The maximumpossible effect (%MPE) was determined for each rat accordingto the following formula using a 500 g maximum pressure: %MPE= [test (g)baseline (g) / 500 gbaseline (g)] 100.Doseresponse curves were generated using at least threedoses of test drug. ED50 value and 95% confidence limitswere determined using the methods of Tallarida and Murray(1987).

    2.8. Drugs

    Freund's complete adjuvant was prepared in mineral oilsupplied by Sigma Chemical Co. (St. Louis, MO). Freund'scomplete adjuvant contains heat-killed M. butyricum and issupplied by Difco Laboratories (Detroit, MI). Morphine and9-THC were obtained from the National Institute on DrugAbuse (Rockville, MD).Fig. 3. Isobologram of 9-THC-THC/morphine drug combination in non-arthritic rats. The points designated z1and z2 represent the ED50 values for eachdrug alone, and the line connecting these points contains all dose pairs that aresimply additive. Point A represents the theoretically additive value for thecombinations z1:z2. Point B represents the experimentally determined value forthe combination z1:z2. Since point B falls to left and below the line of theoreticaladditivity, synergy between 9-THC and morphine has occurred.

  • 3. Results

    3.1. Doseresponse analysis of drugs alone

    Figs. 1 and 2 show the doseresponse curves for the antino-ciceptive effects of morphine and 9-THC respectively alone inrats. ED50 values (z1, z2) and S.E.M. for each drug, as well aslogarithmic equivalent doses, are presented in Table 1. Each of theED50 values is in accordance with earlier studies (Cox andWelch,2004). These values represent the equieffective doses of the drugsin these studies. The ED50 value for morphine with 95%confidence limits was 2.4 mg/kg (2.22.8) in normal rats. Inarthritic rats, the ED50 value for morphine with 95% confidencelimits was 2.2mg/kg (1.92.4). The ED50 value for

    9-THCwith95% confidence limits was 2.1 mg/kg (1.82.5) in normal rats. Inarthritic rats, the ED50 value for

    9-THC with 95% confidencelimits was 2.5 mg/kg (2.23.0). Thus, both drugs were equipotentand equiefficacious in the non-arthritic and the arthritic rats.

    induced antinociception, a combination of a fixed low dose of -THC (0.5 mg/kg) with low doses of morphine was tested in thepaw-pressure test. Our results indicated that in both non-arthriticand arthritic rats, the morphine-doseresponse curve was shiftedto the left (dat...


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