opioids and cannabinoids interactions€¦ · dor activity is enhanced in the presence of cbr...

Xavier Nadal Roura, PhDR&D – Extraction manager

Opioids and cannabinoids interactions

4th ISN Latin American School of Advanced NeurochemistryOctober 28th, 2017, Montevideo, Uruguay

https://www.neurochemistry.org/

http://phytoplant.es/

Opioid receptors

(MOPR)

(DOPR)

k (KOPR)

Endogenous opioid peptides

PROOPIOMELANOCORTIN-endorphin (, )

PROENKEPHALINLeu-enkephalinMet-enkephalin

PRODYNORPHIN

Dinorphin A

Dinorphin B

-neoendorphin

-neoendorphin

Leu-encekephalin (, )

()

(k)

Endogenous opioid system

INTRODUCTION

Endocannabinoid system

INTRODUCTION

(Nadal & Baños, 2005)

Figura 1. Mecanismo de acción

retrograda de los endocanabinoides.

A: DSE en neuronas cerebelares. Una

despolarización, mediante la entrada

de calcio, que estimula la biosíntesis de

anandamida que se produce en dos

pasos: 1) acilación de la

fosfatidiletanolamina con el ácido

araquidónico catalizada por la N-

aciltransferasa; 2) catabolismo de la N-

araquidonoil-PE por la PLD para

liberar ANA. Esta ANA atravesara el

espacio sináptico para activar los

receptores CB1 postsinapticos; 3) asíse provoca mediante la subunidad dela proteína G la apertura de los canales deK+; 4) dejándolo salir de la neurona y

provocando la disminución de la

entrada de Ca2+; 5) y la consecuente

inhibición de la liberación de glutamato.

6) La ANA sobrante es transportada al

interior celular por el tANA; 7) y allí es

degradada a AA y etanolamina por la

FAAH.

Figura 1. Mecanismo de acción

retrograda de los endocanabinoides.

B: DSI en interneuronas gabérgicas.

Una despolarización, mediante la

entrada de calcio, produce la biosíntesis

de 2-AG. La principal ruta tiene dos

pasos: 1) catabolismo del

fosfatidilinositol a 1,2-DAG por la PLC;

2) catabolismo del 1,2-DAG a 2-AG por

la DGL. Este 2-AG atravesará el

espacio sináptico para activar los

receptores CB1 postsinápticos; 3) así se

provoca el cierre de los canales de Ca2+

mediante las subunidads g de laproteína G; 4) provocando la inhibición dela liberación de GABA. El 2-AG sobrante estransportado al interior celular por el tANA;5) al igual que la ANA. 6) Allí es

degradado a AA y glicerol por la MGL.

• Allosteric modulation of the opioid receptor by delta 9-THC is the result of a direct interaction with the receptor protein or with a specific protein-lipid complex and not merely the result of a perturbation of the lipid bilayer of the membrane (Vaysse et al., 1987)

• Cannabinoids and opioids might interact at the level of their signal-transduction mechanisms (Manzanares et al. 1999a)

• Opioid and cannabinoid receptors are coupled to similar intracellular signaling systems, i.e. reduction in adenylyl cyclase activity and blockage of calcium currents, through activation of G proteins (Childers et al. 1992; Howlett 1995; Reisine et al. 1996)

• Co-expression of CB1 cannabinoid receptors and -opioid receptors in the same striatal cells has been reported (RodrBguez et al. 2001)

• Existence of a direct effect of cannabinoid compounds on the synthesis and release of endogenous opioid peptides (Corchero et al. 1997a, 1997b; Manzanares et al. 1998; Valverde et al. 2001)

Early evidences of the interaction between opioids and cannabinoids

Interactions opioid and cannabinoid systems

• Analgesia in central and peripheral level

• Drug addiction: implications in reward, dependence, and withdrawal

• Emotion and cognition

• Immunomodulation

Evidences of the interaction between opioids and cannabinoids

Interactions opioid and cannabinoid systems

(Review: Parolaro et al., 2010)

Scheme of common neurobiological substrates between endocannabinoid system and endogenous opioid system

(Modified from: Yaksh & Wallace, Opioids, Analgesia, and Pain Management in Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 12th Edition.)

Pharmacological interaction with opioid and cannabinoid in reward

THC

THC

CB1

CB1

• THC-induced antinociception in the tail immersion and hot plate test was not modified in MOPRKO, DOPRKO, KOPRKO (Ghozland et al., 2002), double mutant MOPRKO/DOPRKO (Castañe et al., 2003),

• The development of tolerance to this antinociceptive effects was not modified in these knockout lines (Ghozland et al., 2002).

• Attenuation of acute THC-induced antinociception in the tail immersion test was observed in PenkKO(Valverde et al., 2000b) and PdynKO (Zimmer et al., 2001; Gardell et al., 2002).

• The antinociceptive responses of THC in the hot plate test and the development of tolerance to THC antinociception were not modified in these knockout lines (Valverde et al., 2000b; Zimmer et al., 2001).

• The antinociceptive effects of the CB2 agonist AM1241 in the plantar test were suppressed in MOPRKO, indicating the possible participation of MOP receptor in the CB2 mediated analgesia (Ibrahim et al., 2003).

• These data demonstrate that the suppression of the opioid receptors has not major effects in cannabinoid-induced antinociception and on the development of cannabinoid antinociceptive tolerance.

Implication of endogenous opioid system in cannabinoid analgesia using “Ko” mice

Interactions between Endogenous opioid and cannabinoid systems

• The antinociceptive effects of different selective MOP, DOP and KOP receptors agonists and the development of tolerance to morphine antinociception were not modified in CB1RKO (Ledent et al., 1999; Miller et al., 2011; Valverde et al., 2000a).

• Stress-induced analgesia mediated by endogenous opioid mechanisms was absent in CB1RKO (Valverde et al., 2000a).

• Morphine antinociception was not modified in CB2RKO, suggesting that the lack of CB2 receptor would not produce a general disruption of opioid-mediated antinociception (Ibrahim et al., 2006).

• The administration of a KOP receptor antagonist (nor-binaltorphimine) in FAAHKO reduced the tail flick latencies, indicating an endocannabinoid–KOP receptor interaction in the tonic control of pain (Haller et al.,2008).

Implication of endogenous cannabinoid system in opioid analgesia using “Ko” mice


Implication of endogenous opioid and cannabinoid systems in pharmacological cannabinoid or opioid analgesia


(Nadal et al., 2013)

• The lack of CB1 receptor decreased basal levels of DOP and KOP receptors gene expression in the spinal cord.(La Porta et al., 2013)

• The lack of CB2 receptor decreased the basal level of MOP receptor gene expression in the spinal cord and increase the levels of KOP receptor gene expression under basal (La Porta et al., 2013).

• The lack of MOP receptor or DOP receptor modifies the density and activity of CB1 receptor in motor and reward brain áreas (Berrendero et al., 2003).

• The lack of KOP recetor do not produce alterations in CB1 receptors (Berrendero et al., 2003).


Modifications of endogenous opioid and cannabinoid systems in “Ko” mice for receptors of endogenous cannabinoid or opioid systems.

Fig. 4 Effects of (-)-Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and rimonabant (SR) on 3H-DAMGO (a) or 3H-NTI (b) equilibrium binding to rat cerebral cortical membranes. Unspecific binding was determined usingnaloxone 10 μM(a) and naltrindole 10 μM(b), respectively. Means±SEM from 4 experiments in triplicate. Error bars are not shown when they are smaller than the symbols

(Kathmann etal., 2006)

Cannabinoids can bind or modify the binding of opioid agonist to the MOR and DOR opioid receptors

Interaction between opioid and cannabinoid at same receptor

Figure 4. Determination of the affinity of test compounds for hMORs stably expressed in CHO cells by competition receptor binding performed with membrane homogenates Specific binding was determined as described in the Materials and Methods by incubating 1 nM of [3H]DAMGO with increasing concentrations of morphine (filled squares), AM-251(open triangles), rimonabant (open circles) or AM-281 (open squares) and 100 μg of membranes prepared from CHO-hMOR cells. The Cheng-Prusoff equation (Cheng and Prusoff, 1973) was used to convert the experimental ICvalues obtained from competition receptor binding experiments to Ki 50 values, a quantitative measure of receptor affinity (presented in Table 1). (Seely et al., 2013)

AM-251 and Rimonabant Act as Direct Antagonists at Mu-Opioid Receptors:


Figure 6. Modulation of morphine-stimulated G-protein activity by test compounds utilizing [35S]GTPγS binding in CHO-hMOR cell homogenates. Morphine produced a concentration-related activation of G-proteins in CHO-hMOR membranehomogenates that was significantly shifted-to-the-right by co-incubation with rimonabant (10 μM; Panel B) and AM-251 (1and 10 μM; Panel C), but not by AM-281 (10 μM; Panel A). AM-251 competitively antagonizes G-protein activation bymorphine with a Schild slope of 1.02 and a K value of 719 nM (Panel C; inset). Data are expressed as percent specific[35S]GTPγS binding normalized to the average maximal response produced by morphine and individual ED50 values arediscussed in the Results section.

(Seely et al., 2013)

AM-251 and Rimonabant Act as Direct Antagonists at Mu-Opioid Receptors


Figure 7. Modulation of forskolin-stimulated adenylyl cyclase activity by acute administration of test compounds in intact CHO-hMOR cells. C) Inhibition of adenylyl cyclase activity produced by 10 nM morphine was significantly attenuated by co-incubation with either the neutral MOR antagonist naloxone (1 μM), AM-251 (10 μM), rimonabant (10 μM) or AM-281 (10 μM). Values designated with different letters above the error bars are significantly different (P<0.05, one-way ANOVA followed by a Newman-Keuls post-hoc test, mean ± SEM).

(Seely et al., 2013)

AM-251 and Rimonabant Act as Direct Antagonists at Mu-Opioid Receptors “in vitro” and “in vivo”

Figure 9. Antagonism of in vivo morphine analgesia in two different strains of mice by test compounds utilizing the tail-flick procedure. B) B6SJL or C) C57BL/6J mice by intra-peritoneal (i.p.) injection of naloxone (4 mg/kg) or AM-251 (10 mg/kg), but not AM-281 (10 mg/kg) significantly reduced analgesia produced by 5 mg/kg morphine. The test doses of naloxone, AM-251, rimonabant or AM-281 had no effect on basal tail-flick latencies when administered alone (data not shown). All data are expressed as the percent of maximum possible effect (% MPE). Values designated with different letters above the error bars are significantly different (P<0.05, one-way ANOVA followed by a Newman-Keuls post-hoc test, mean ± SEM).


Figure 7. DOR activity is enhanced in the presence of CBR ligands in cortical membranes from lesioned animals. A, Membranes from cortices oflesioned animals were treated with 10 pM – 10 mM DPDPE in the absence of presence of 1 pM Hu-210, or with 1 pM Hu-210 alone for 1.5 hours.[35S]GTPcS binding to membranes was detected using a scintillation counter. Basal [35S]GTPcS binding in vehicle treated membranes istaken as 100%.Data represent Mean 6 SEM (n = 3 individual animals in triplicate). Statistically significant differences between 10 mM DPDPE alone and 10 mMDPDPE+1 pM Hu-210 are indicated ***, p,0.001, (t test). B, Membranes from cortices of sham and lesioned animals were treated with 10 mM DPDPE inthe absence of presence of 1 pM Hu-210, or with 1 pM Hu-210 alone for 1.5 hours. [35S]GTPcS binding to membranes was detected using a scintillationcounter. Basal [35S]GTPcS binding in vehicle treated membranes is taken as 100%. Data represent Mean 6 SEM (n = 4 individual animals in triplicate).Statistically significant differences between 10 mM DPDPE alone and 10 mM DPDPE+1 pM Hu-210 are indicated **, p,0.01, (ttest).

Cannabinoid-Opioid Heterodimer in Neuropathic Pain

(Bushlin et al., 2012)

Pharmacological interaction with opioid and cannabinoid in analgesia

Figure 7. DOR activity is enhanced in the presence of CBR ligands in cortical membranes from lesioned animals. C, Membranes from cortices oflesioned animals were treated with 10 mM DPDPE in the absence of presence of 1 mM PF-514273, or with 1 mM PF-514273 alone for 1.5 hours.[35S]GTPcS binding to membranes was detected using a scintillation counter. Basal [35S]GTPcS binding in vehicle treated membranes is taken as100%. Data represent Mean 6 SEM (n = 3 individual animals in triplicate). Statistically significant differences between 10 mM DPDPE alone and 10 mMDPDPE+1 mM PF-514273 are indicated *, p,0.05, (t test). D, Membranes from cortices of lesioned animals were treated with 10 mM DPDPE in theabsence of presence of 1 pM Hu-210, or 10 nM DAMGO or 10 nM U69593 for 1.5 hours. [35S]GTPcS binding to membranes was detected using ascintillation counter. Basal [35S]GTPcS binding in vehicle treated membranes is taken as 100%. Data represent Mean 6 SEM (n = 3 individual animals intriplicate). Statistically significant differences between 10 mM DPDPE and 10 mM DPDPE+ligand are indicated **, p,0.01, (t test).




Figure 8. CB1R-DOR heteromer-specific antibody blocks enhancement of DOR activity. A, Membranesfrom cortices of lesioned animals were treated with 10 mM DPDPE without or with Hu-210 in theabsence or presence of 1 mg of indicated antibodies. [35S]GTPcS binding to membranes was detectedusing a scintillation counter. Basal [35S]GTPcS binding in vehicle treated membranes is taken as 100%.Data represent Mean 6 SEM (n = 3 individual animals in triplicate). Statistically significant differencesbetween 10 mM DPDPE+1 pM Hu-210 and 10 mM DPDPE+1 pM Hu210+1 mg antibody are indicated***, p,0.001, (t test). B, In the absence or presence of 1 mg CB1R-DOR heteromer-specific antibody,membranes from cortices of lesioned animals were treated with 10 mM DPDPE without or with 1 pMHu-210. [35S]GTPcS binding to membranes was detected using a scintillation counter. Basal [35S]GTPcSbinding in vehicle treated membranes is taken as 100%. Data represent Mean 6 SEM (n = 3 individualanimals in triplicate). Statistically significant differences between 10 mM DPDPE and 10 mM DPDPE+1pM Hu-210 are indicated ***, p,0.001, (t test).






Figure 10. DOR binding is enhanced by CB1R ligands in membranes of cells expressing CB1R and DOR. A, Membranes from N2A-DOR cells were incubatedwith 100 fM – 10 mM Hu-210 in the presence of 0.5 nM [3H]DPDPE 6 1 mg CB1R-DOR monoclonal antibody for 1 hour. Ligand binding assay was carried out asdescribed in ‘‘Methods’’ and [3H]DPDPE binding to membranes was detected using a scintillation counter. Data represent Mean 6 SEM (n = 3 experiments intriplicate). C, Membranes from N2A DOR cells were treated with 100 fM – 10 mM PF-514273 in the presence of 0.5 nM [3H]DPDPE along with the presence orabsence of 1 mg CB1R-DOR monoclonal antibody for 1 hour. Ligand binding assay was carried out as described in ‘‘Methods’’ and [3H]DPDPE binding tomembranes was detected using a scintillation counter. Data represent Mean 6 SEM (n = 3 experiments in triplicate).


Figure 1. (A) Antinociceptive effects of cumulative doses of CP55940 and spiradoline alone (open symbols) and in combination (filled symbols) with 50°C water in rats (n = 7) with an interinjection interval of 30 minutes. The ratio of CP55940 to spiradoline in the mixture, 1:3 (diamonds), 1:1 (squares), and 3:1 (inverted triangles), varied across tests. Abscissae: dose in milligrams per kilogram body weight. Ordinate: % maximum possible effect (MPE) ± 1 SEM (see “Data Analyses” for details). (B) Isobologram for mixtures of CP55940 and spiradoline (same data presented in A). Open symbols indicate the ED50 valued for CP55940 alone (triangle) and spiradoline alone (circle); the solid line connecting the open symbols indicates the line of additivity. Filled symbols indicate the ED50 values formixtures of CP55940 and spiradoline (same ratios and symbols presented in A). Abscissae: ED50 for spiradoline in milligrams per kilogram body weight (± 95% confidence interval). Ordinate: ED50 for CP55940 in milligrams per kilogram body weight (± 95% confidence interval).

(Maguire & France., 2016)

Pharmacological additive effects in tail flick analgesia between Cannabinoids and Opioids


Fig. 2. Isobologram of 9-THC/morphine drug combinations. The pointsdesignated z1 and z2 represent the ED50 values for each drug alone, and the lineconnecting these points contains all dose pairs that are simply additive. Points Aand B represent the theoretically additive value for the combinations z1/z2 and0.1z1/0.9z2, respectively. Point C represents the experimentally determined valuefor the combination z1/z2, and point D represents the experimentally determinedvalue for the combination 0.1z1/0.9z2. Since both points C and D fall to left andbelow the line of theoretical additivity, they indicate synergy between 9-THC andmorphine.

Fig. 3. Isobologram of 9-THC/codeine drug combinations. The pointsdesignated z1 and z3 represent the ED50 values for each drug alone, andthe line connecting these points contains all dose pairs that are simplyadditive. Points A and B represent the theoretically additive value for thecombinations z1/z3 and 0.2z1/0.8z3, respectively. Point C represents theexperimentally determined value for the combination z1/z3, and point Drepresents the experimentally determined value for the combination0.2z1/0.8z3. Since both points C and D fall to left and below the line oftheoretical additivity, they indicate synergy between 9-THC and codeine.

Pharmacological Synergy in tail flick analgesia between Cannabinoids and Opioids

Cichewicz & McCarthy., 2003

Pharmacological interaction with opioid and cannabinoid in analgesisa

Fig. 3. Isobologram of Δ9-THC-THC/morphine drug combination in arthriticrats. The points designated z1 and z2 represent the ED50 values for eachdrug alone, and the line connecting these points contains all dose pairs that are simply additive. Point A represents the theoretically additive value for the combinations z1:z2. Point B represents the experimentally determined value for the combination z1:z2. Since point B falls to left and below the line of theoretical additivity, synergy between Δ9-THC and morphine has occurred.

Fig. 4. Isobologram of Δ9-THC-THC/morphine drug combination in arthriticrats. The points designated z1 and z2 represent the ED50 values for eachdrug alone, and the line connecting these points contains all dose pairs that are simply additive. Point A represents the theoretically additive value for the combinations z1:z2. Point B represents the experimentally determined value for the combination z1:z2. Since point B falls to left and below the line of theoretical additivity, synergy between Δ9-THC and morphine has occurred.

(Cox et al., 2007)


Pharmacological Synergy in paw latency analgesia between Cannabinoids and Opioids in arthritic rats

Fig. 1 Effect of an acute dose of morphine (2.5 mg/kg s.c.) with CP-55,940 (0.2 mg/kg i.p.) on analgesia expressed as área under the analgesic curve (AUC). Data are mean±SEM of at least five animals. *p<0.001 vs control, °p<0.001 vs morphine alone, #p<0.001 vs CP-55,940 alone (Tukey’s test)

Pharmacological Synergy in analgesia between Cannabinoids and Opioids

(Viganò et al., 2005)


(Wilson-Poe et al., 2013)

Figure 4.Hot plate latency following an acutemicroinjection of a CB1 receptor agonist and/ormorphine into the RVM. Microinjection into theRVM of CB1 receptor agonists (HU-210 or WIN55,212-2) combined with morphine caused anantinociceptive effect that was greater thanadministration of either drug alone (*p = 0.05).Rats that became sick with repeated injectionswere not included in this analysis so as not toconfound neurotoxic and receptor mediatedeffects.

Pharmacological Synergy in analgesia between Cannabinoids and Opioids


Pharmacological Synergy in acute termal analgesia between Cannabinoids and Opioids in humans

(Roberts et al., 2006)

Fig. 1. Single agent effects and dual agent interactions of morphine and Δ9-THC upon sensory (A) and affective (B) pain responses to a painful stimulus. Effects calculated using the average response per subject minus the average baseline response. Interactions calculated from a mixed-effects model as described in Methods.Verticalaxis represents displacement of responses on a scale of 100 mm.


Pharmacological Synergy in acute termal analgesia between Cannabinoids and Opioids in humans

(Roberts et al., 2006)

Fig. 2. Mean sensory and affective responses at three thermal stimulus temperatures. Conditions are: placebo/placebo (♦), placebo/morphine (▪), Δ-THC/placebo(▴), Δ9-THC/morphine (×).



Alteration in Cannabinoid receptor binding and functionality in Opioid tolerant animals.


Fig. 4 Effect of CP-55,940 chronic treatment (0.4mg/kg i.p., twice a day for 6.5 days) on opioidreceptor binding (a) and on net DAMGO-stimulated[35S]GTPγS binding determined by subtracting basal[35S]GTPγS binding (b) in different brain regions.Gray levels obtained with densitometric analysiswere transformed into fmol/mg tissue using[3H]standards.Bars indicate the mean±SEM of atleast five animals. *p<0.05 vs vehicle (Student’s ttest). Cpu Caudate putamen, NAc nucleusaccumbens, ctx cortex, TC central thalamus, TLlateral thalamus, TV ventral thalamus, hippohippocampus, amyg anterior amygdala, PAGperiaqueductal gray, coll colliculus, nigra substantianigra


Alteration in Opioid receptor binding and functionality in cannabinoid tolerant animals.


(Cichewicz & Welch., 2003)

Increase of Antinociceptive effects of Cannabinoids in Opioid tolerant mice.




Figure 2.Enhanced cannabinoid antinociception in thePAG of morphine tolerant rats. Microinjection ofthe CB1 receptor agonist HU-210 into the PAGproduced minimal antinociception in vehicle-pretreated rats, but significantly greaterantinociception in rats pretreated with morphineinto the PAG twice a day for 2 days (*p < 0.05).Cumulative doses of HU-210 were administeredone day after the last morphine injection.


Fig. 2 Analgesic response induced by different doses of CP -55,940 (0.2or 0.4 mg/kg i.p.) in rats made tolerant to morphine (5 mg/kg s.c., twicea day for 4.5 days). Data are expressed as area under the analgesic curve(AUC) and are the mean±SEM of at least five animals. *p<0.001,**p<0.0001 vs control, °p<0.001 vs acute morphine, $p<0.05, $$p<0.001vs chronic morphine, #p<0.001 vs acute CP-55,940 (Tukey’s test)





Increase of Antinociceptive effects of Opioids in Cannabinoid tolerant mice.

Figure 3.Enhanced morphine antinociception followingrepeated systemic administration of THC.Administration of cumulative doses of morphinecaused a dose-dependent increase in hot platelatency in both pretreatment groups. However,morphine potency was enhanced in THCpretreated rats even though morphine wasinjected 16 hours after the last of four THCinjections.


Fig. 3 Analgesic response induced by morphine (5 mg/kg s.c.) in ratsmade tolerant to CP-55,940 (0.4 mg/kg i.p., twice a day for 6.5 days).Data are expressed as area under the analgesic curve (AUC) and are themean±SEM of at least five animals. *p<0.001 vs control, °p<0.01 vs acutemorphine, #p<0.01 vs acute CP-55,940 (Tukey’s test)


Lack of increase of Antinociceptive effects of Opioids in Cannabinoid tolerant mice.


Fig. 2. A dose of 20 mg/kg 9-THC p.o. attenuates the development of oral morphine tolerance. Micereceived distilled water vehicle, morphine (200mg/kg p.o., days 1–2; 300 mg/kg p.o., days 3 9-THC p.o.twice daily for 7 days. At 12 h after the last drug administration, various doses of morphine wereadministered p.o. and the mice were tested 30 min later in the tail-flick test. Each data point represents anaverage % MPE S.E.M. of six mice.

Reduction of Antinociceptive tolerance to Opioids by Cannabinoids.

Cichewicz & Welch., 2003




Bidirectional Antinociceptive effects of Opioids and Cannabinoid in tolerance.

Fig. 2 Antinociceptive effects expressed by area under the curve (AUC) values of endomorphin-1 (EM1) and 2-arachidonoyl-glycerol (2-AG) in different doses by themselves and in combinations (10 : 1 fixed dose-ratio). Each point denotes the mean ± standard mean error of the results. Symbol * indicates significant (P < 0.05) difference compared to the saline-treated group (Tukey–Kramer post-hoc analysis). # denotes a significant difference from EM1 treated groups. · denotes a significant difference from 2-AG treated groups. Number of animals in the different groups are indicated in parentheses. MPE, maximum possible effect.

Peripheral antinociceptive effect of 2-arachidonoyl-glycerol and its interaction with endomorphin-1 in arthritic rat ankle joints

(Mecs et al., 2010)


Figure 1 Plasma concentration–time curves for sustained-release (a) morphine and (b) oxycodone before and after exposure to inhaled cannabis.

(Abrams et al., 2011)

Cannabinoid–Opioid Interaction in chronic Pain in humans


Figure 2 Subjective highs experienced when cannabis was combined with (a) morphine and (b) oxycodone on day 5.

Hour Hour

(Abrams et al., 2011)

Cannabinoid–Opioid Interaction in chronic Pain in humans


(Paquette & Olmstead, 2005)


(modified from Fields, 2004)


Figure 5: Shifts in nociceptive modulatorystate during morphine analgesia and acutenaloxone-induced abstinence.a | Morphineactivates off cells to inhibit pain (lower left),whereas when naloxone is used to precipitateacute abstinence, on cells are activated andproduce a hyperalgesic state (lower right). b |Synaptic distribution of opioid receptors withinthe rostral ventromedial medulla (RVM). -Opioid receptor (MOR) is located on GABA (-aminobutyric acid)-releasing terminals at offcells and the somadendritic region of on cells.Both cell classes have somadendritic opioidreceptor-like (ORL1) receptors and both areexcited by -opioid receptor (KOR)-bearingglutamatergic terminals (glut) that arise fromdifferent input neurons. Whereas MORagonists produce anti-nociceptive effects byinhibiting on cells and disinhibiting off cells,ORL1 and KOR agonists acting in the RVM canblock analgesia by inhibiting off cells or blockhyperalgesia by inhibiting on cells. PAG,periaqueductal grey.

Diagram of interaction of endogenous opioid and cannabinoid systems in analgesis

THCCB1 antagonist

CB1R

Fig. 2. AM1241 (i.p.) produced dose-dependent antinociception in wildtype (+/+) mice but not in -opioid receptor knockout (-/-) mice. Data are expressed as mean SEM. n = 6 per group. #, P 0.05 compared with WT mice.

(Ibrahim et al., 2005)

CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral releaseof endogenous opioids2

Fig. 3. The CB2receptor-selective agonist AM1241 stimulated –endorphin release from glabrous paw skin. (B) Mouse paw skin. AM1241 (10M) stimulated -endorphin release from the skin of wild-type (CB2-/-) but not from CB2 receptor-knockout (CB2+/+) mice. Data are expressedas mean SEM. n 12 per group., * P<0.05 compared with vehicle; #, P<0.05 compared with 10M AM1241 alone.

Interaction with opioid and cannabinoid in peripheral analgesia

Fig. 4. AM1241 stimulated -endorphin release from cultured human keratinocytes (HaCaT) cells. AM630 (1M) inhibited the effects of AM1241 (1M). AM630 had no effect in the absence of AM1241. Data are expressed as percent of release in medium alone and presented as mean SEM. n 12 per group. *, P 0.05 compared with medium alone. #, P 0.05 compared with 1M AM1241.


Fig. 3. The CB2receptor-selective agonist AM1241 stimulated –endorphin release from glabrous paw skin. release from glabrous paw skin. (A) Rat paw skin. The CB2 receptor antagonist AM630 (10M) prevented the effects of AM1241 (10 M). AM630 had no effect on-endorphin release in the absence of AM1241. Data are expressed as mean SEM. n 12 per group., * P<0.05 compared with vehicle; #, P<0.05 compared with 10M AM1241 alone.

CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral releaseof endogenous opioids


Fig. 6. Double-labeling immunofluorescence with antibodies against CB2, -endorphin, and ETRB in the epidermis of glabrous skin from the hindpaw of a rat.The images are from sections that alternate with those in Fig. 5.


CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral releaseof endogenous opioids


Figure 1 Time course of the effects of b-FNA on the antinociceptive actions of AM1241 on thermal hyperalgesia and mechanical allodynia in rats. A, Effects ofb-FNA on the AM1241 action on thermal withdrawal latency in response to heat stimulus applied to the inflamed paw. C, Effects of b-FNA on analgesic action ofAM1241 on mechanical withdrawal threshold in response to von Frey filaments applied to the inflamed paw. Time 0 represents baseline values before CFAinjection. AM1241 or its vehicle (50 μL) was injected subcutaneously into the dorsal surface of the left hindpaw of rats, as indicated by white arrows. b-FNA orits vehicle was injected 24 hours before AM1241. Data are presented as means ± SEM (n = 8 rats in each group). *P < 0.05, compared with the vehicle controlgroup; #P < 0.05, compared with the CFA+vehicle of AM1241 group; &P < 0.05, compared with the CFA+AM1241 group (Two-way ANOVA followed byBonferroni’s test).

(Su et al., 2011)

Cannabinoid CB2 Receptors Contribute to Upregulation of b-endorphin in Inflamed Skin Tissues


Figure 2 Effects of AM1241, EA, AM1241 plus AM630 on the mRNA level of POMC (A) and the protein level of b-endorphin (C, D) in inflamed skin tissues. A,summary data show the relative mRNA level of POMC in the skin tissues obtained from the vehicle control (CONT), CFA+vehicle of AM1241 (CFA), CFA+AM1241(AM1241), and CFA+AM1241+AM630 (AM1241+AM630) groups. C, a representative gel image showing the protein level of b-endorphin in the skin tissuesobtained from 7 groups of rats. b-actin was used as a loading control. The protein band at 3.5 kDa corresponds to the b-endorphin protein. D, summary datashow the % increase in the b-endorphin protein level by AM1241 with and without AM630. Data are expressed as means ± SEM (n = 6 rats in each group). * P <0.05, compared with the vehicle control group; # P < 0.05, compared with the CFA+vehicle of AM1241 group; & P < 0.05, compared with the CFA+AM1241group (One-way ANOVA followed by Tukey’s test).

(Su et al., 2011)



Figure 3 Effects of AM1241, AM1241 plus AM630 on the mRNA (A, B) and the protein (C, D, E) levels of µ-opioid receptor-1 in inflamed skin tissues. A, summary data show the relative mRNA level of µ-opioid receptor-1 in the skin tissues obtained from the vehicle control (CONT), CFA+vehicle of AM1241 (CFA), CFA+AM1241 (AM1241) and CFA+AM1241+AM630 (AM1241+AM630) groups. C, a representative gel image showing the protein level of µ-opioid receptor-1 inthe skin tissues obtained from those 7 groups. b-actin was used as a loading control. Both two bands correspond to the µ-opioid receptor-1 protein. D, summary data show the % increase in the µ-opioid receptor-1 protein level by AM1241 with and without AM630. Data are expressed as means ± SEM (n = 6 rats in each group). * P < 0.05, compared with the vehicle control group; # P < 0.05, compared with the CFA+vehicle of AM1241 group; & P < 0.05, compared with the CFA+AM1241 group (One-way ANOVA followed by Tukey’s test).

(Su et al., 2011)



(Su et al., 2011)

Keratinocytes immunorecative to b-endorphinin the skin tissue

Macrophages labeled with b-endorphin in the skin tissues

T-lymphocytes with b-endorphin Immunoreactivity in the skin tissue

Cannabinoid CB2 Receptors Contribute to Upregulation of b-endorphin in Inflamed Skin Tissues.


Figure 2 Antagonistic action of AM630 or AM251 on antinociceptionproduced by intraplantar (i.pl.) injection of b-caryophyllene (BCP) in thecapsaicin test. AM630 or AM251 i.pl. (B) 30 min prior to i.pl. injection BCP. Capsaicin (1.6 mg/paw) was injected s.c. into the plantar surface of hindpaw10 min after BCP injection. Values represent the mean SEM for 10 mice per group. **p < 0.01 when compared with jojoba wax-treated control. p < 0.01 when compared with saline plus BCP (18.0 mg/paw).

(Katsuyama et al., 2013)

Involvement of peripheral cannabinoid and opioid receptors in b-caryophyllene-induced antinociception


Figure 3 Antagonistic action of naloxone hydrochloride or naloxone methiodide on antinociception produced by intraplantar (i.pl.) injection of b-caryophyllene (BCP) in the capsaicin test. Naloxonehydrochloride (A,B) or naloxone methiodide (C) was injected s.c. and i.pl. 15 min prior to i.pl. injection of BCP. Capsaicin (1.6 mg/paw) was injected subcutaneous (s.c.) into the plantar surface of hindpaw 10 min after BCP injection. Values represent the mean SEM for 10 mice per group. **p < 0.01 when compared with jojoba wax-treated control. # p < 0.01, ##p < 0.05 when compared with saline plus BCP (18.0 mg/paw).

Figure 5 Antagonistic action of local injection of antisera against b-endorphin onantinociception produced by intraplantar (i.pl.) injection of b-caryophyllene(BCP) in the capsaicin test. Antisera against b-endorphin were injected i.pl. 5 minprior to BCP injection. Capsaicin (1.6 mg/paw) was injected subcutaneous intothe plantar surface of hindpaw 10 min after BCP injection. Values represent themean SEM for 10 mice per group. **p < 0.01 when compared with jojoba wax-treated control. # p < 0.01, ## p < 0.05 when compared with saline plus BCP (18.0mg/paw).

(Katsuyama et al., 2013)

Involvement of peripheral cannabinoid and opioid receptors in b-caryophyllene-induced antinociception


Figure 4 Antagonistic action of b-funaltrexamine (b-FNA), naltrindolehydrochloride (NTI) or nor-binaltorphimine (nor-BNI) on antinociceptionproduced by intraplantar (i.pl.) injection of b-caryophyllene (BCP) in thecapsaicin test. b-FNA or nor-BNI was injected subcutaneous (s.c.) (A) andi.pl. (B) 24 h prior to i.pl. injection of BCP. NTI was injected s.c. (A) and i.pl.(B) 30 min prior to BCP injection. Capsaicin (1.6 mg/paw) was injected s.c.into the plantar surface of hindpaw 10 min after BCP injection. Valuesrepresent the mean SEM for 10 mice per group. **p < 0.01 when comparedwith jojoba wax-treated control. # p < 0.01, ## p < 0.05 when comparedwith saline plus BCP (18.0 mg/paw).

2 Release of opioids from lymphocytes acting 2 Interaction with CB2 R in the mast cell membrane on overexpressed nociceptors 3 Avoiding liberation of proinflamatory citoquines

3 Preventing painful signal from being 4 Interaction with CB2 R in keratinocytes transmitted to higher centers of the CNS 5 Release of beta-endorphins

6 Interaction with opioid receptors preventing painful signal to reach CNS

Peripheral effects of the activation of the endogenous opioid and cannabinoid systems in inflammation

Interactions Between Endogenous Opioid and Cannabinoid Systems

1 Cytokine release by macrophages 1 On-demand synthesis of endocannabinoids in response to inflammation

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