Opioid and Cannabinoid Interactions

Opioids and cannabinoids interactions

Xavier Nadal Roura, PhD R&D – Extraction manager

4th ISN Latin American School of Advanced Neurochemistry October 28th, 2017, Montevideo, Uruguay INTRODUCTION Endogenous opioid system

Opioid receptors Endogenous opioid peptides PROOPIOMELANOCORTIN  (MOPR) -endorphin (, )

 (DOPR) PROENKEPHALIN Leu-enkephalin () Met-enkephalin

k (KOPR) PRODYNORPHIN Dinorphin A (k) Dinorphin B -neoendorphin -neoendorphin Leu-encekephalin (, ) INTRODUCTION Endocannabinoid system

Figura 1. Mecanismo de acción Figura 1. Mecanismo de acción retrograda de los endocanabinoides. retrograda de los endocanabinoides. A: DSE en neuronas cerebelares. Una B: DSI en interneuronas gabérgicas. despolarización, mediante la entrada Una despolarización, mediante la de calcio, que estimula la biosíntesis de entrada de calcio, produce la biosíntesis anandamida que se produce en dos de 2-AG. La principal ruta tiene dos pasos: 1) acilación de la pasos: 1) catabolismo del fosfatidiletanolamina con el ácido fosfatidilinositol a 1,2-DAG por la PLC; araquidónico catalizada por la N- 2) catabolismo del 1,2-DAG a 2-AG por aciltransferasa; 2) catabolismo de la N- la DGL. Este 2-AG atravesará el araquidonoil-PE por la PLD para espacio sináptico para activar los liberar ANA. Esta ANA atravesara el receptores CB1 postsinápticos; 3) así se espacio sináptico para activar los provoca el cierre de los canales de Ca2+ receptores CB1 postsinapticos; 3) así mediante las subunidads g de la se provoca mediante la subunidad  de proteína G; 4) provocando la inhibición de la proteína G la apertura de los canales de la liberación de GABA. El 2-AG sobrante es K+; 4) dejándolo salir de la neurona y transportado al interior celular por el tANA; provocando la disminución de la 5) al igual que la ANA. 6) Allí es entrada de Ca2+; 5) y la consecuente degradado a AA y glicerol por la MGL. 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. (Nadal & Baños, 2005) Interactions opioid and cannabinoid systems Early evidences of the interaction between opioids and cannabinoids

• 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) Interactions opioid and cannabinoid systems Evidences of the interaction between opioids and cannabinoids

• Analgesia in central and peripheral level

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

• Emotion and cognition

• Immunomodulation

(Review: Parolaro et al., 2010) Pharmacological interaction with opioid and cannabinoid in reward Scheme of common neurobiological substrates between endocannabinoid system and endogenous opioid system

THC

CB1

THC

(Modified from: Yaksh & Wallace, Opioids, Analgesia, and Pain Management in Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 12th Edition.) Interactions between Endogenous opioid and cannabinoid systems Implication of endogenous opioid system in cannabinoid analgesia using “Ko” mice

• 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. Interactions between Endogenous opioid and cannabinoid systems Implication of endogenous cannabinoid system in opioid analgesia using “Ko” mice

• 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). Interactions between Endogenous opioid and cannabinoid systems Implication of endogenous opioid and cannabinoid systems in pharmacological cannabinoid or opioid analgesia

(Nadal et al., 2013) Interactions between Endogenous opioid and cannabinoid systems Modifications of endogenous opioid and cannabinoid systems in “Ko” mice for receptors of endogenous cannabinoid or opioid systems.

• 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). Interaction between opioid and cannabinoid at same receptor Cannabinoids can bind or modify the binding of opioid agonist to the MOR and DOR opioid receptors

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 using naloxone 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) Interaction between opioid and cannabinoid at same receptor AM-251 and Rimonabant Act as Direct Antagonists at Mu-Opioid Receptors:

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 IC values obtained from competition receptor binding experiments to Ki 50 values, a quantitative measure of receptor affinity (presented in Table 1). (Seely et al., 2013) Interaction between opioid and cannabinoid at same receptor 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 membrane homogenates that was significantly shifted-to-the-right by co-incubation with rimonabant (10 μM; Panel B) and AM-251 (1 and 10 μM; Panel C), but not by AM-281 (10 μM; Panel A). AM-251 competitively antagonizes G-protein activation by morphine 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 are discussed in the Results section. (Seely et al., 2013) Interaction between opioid and cannabinoid at same receptor AM-251 and Rimonabant Act as Direct Antagonists at Mu-Opioid Receptors “in vitro” and “in vivo”

Figure 7. Modulation of forskolin-stimulated adenylyl cyclase Figure 9. Antagonism of in vivo morphine analgesia in two different strains of mice activity by acute administration of test compounds in intact CHO- by test compounds utilizing the tail-flick procedure. B) B6SJL or C) C57BL/6J mice by hMOR cells. C) Inhibition of adenylyl cyclase activity produced by 10 intra-peritoneal (i.p.) injection of naloxone (4 mg/kg) or AM-251 (10 mg/kg), but not nM morphine was significantly attenuated by co-incubation with AM-281 (10 mg/kg) significantly reduced analgesia produced by 5 mg/kg morphine. either the neutral MOR antagonist naloxone (1 μM), AM-251 (10 The test doses of naloxone, AM-251, rimonabant or AM-281 had no effect on basal μM), rimonabant (10 μM) or AM-281 (10 μM). Values designated tail-flick latencies when administered alone (data not shown). All data are expressed with different letters above the error bars are significantly different as the percent of maximum possible effect (% MPE). Values designated with different (P<0.05, one-way ANOVA followed by a Newman-Keuls post-hoc letters above the error bars are significantly different (P<0.05, one-way ANOVA test, mean ± SEM). followed by a Newman-Keuls post-hoc test, mean ± SEM). (Seely et al., 2013) Pharmacological interaction with opioid and cannabinoid in analgesia Cannabinoid-Opioid Heterodimer in Neuropathic Pain

Figure 7. DOR activity is enhanced in the presence of CBR ligands in cortical membranes from lesioned animals. A, Membranes from cortices of lesioned 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 mM DPDPE+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 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 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). (Bushlin et al., 2012) Pharmacological interaction with opioid and cannabinoid in analgesia Cannabinoid-Opioid Heterodimer in Neuropathic Pain

Figure 7. DOR activity is enhanced in the presence of CBR ligands in cortical membranes from lesioned animals. C, Membranes from cortices of lesioned 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 as 100%. Data represent Mean 6 SEM (n = 3 individual animals in triplicate). Statistically significant differences between 10 mM DPDPE alone and 10 mM DPDPE+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 the absence 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 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 differences between 10 mM DPDPE and 10 mM DPDPE+ligand are indicated **, p,0.01, (t test). (Bushlin et al., 2012) Pharmacological interaction with opioid and cannabinoid in analgesia

Cannabinoid-Opioid Heterodimer in Neuropathic Pain

Figure 8. CB1R-DOR heteromer-specific antibody blocks enhancement of DOR activity. A, Membranes from cortices of lesioned animals were treated with 10 mM DPDPE without or with Hu-210 in the absence or presence of 1 mg of indicated antibodies. [35S]GTPcS binding to membranes was detected using 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 differences between 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 pM Hu-210. [35S]GTPcS binding to membranes was detected using 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 differences between 10 mM DPDPE and 10 mM DPDPE+1 pM Hu-210 are indicated ***, p,0.001, (t test).

(Bushlin et al., 2012) Pharmacological interaction with opioid and cannabinoid in analgesia Cannabinoid-Opioid Heterodimer in Neuropathic Pain

Figure 10. DOR binding is enhanced by CB1R ligands in membranes of cells expressing CB1R and DOR. A, Membranes from N2A-DOR cells were incubated with 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 as described in ‘‘Methods’’ and [3H]DPDPE binding to membranes was detected using a scintillation counter. Data represent Mean 6 SEM (n = 3 experiments in triplicate). 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 or absence of 1 mg CB1R-DOR monoclonal antibody for 1 hour. Ligand binding assay was carried out as described in ‘‘Methods’’ and [3H]DPDPE binding to membranes was detected using a scintillation counter. Data represent Mean 6 SEM (n = 3 experiments in triplicate). (Bushlin et al., 2012) Pharmacological interaction with opioid and cannabinoid in analgesia Pharmacological additive effects in tail flick analgesia between Cannabinoids and Opioids

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 for mixtures 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 interaction with opioid and cannabinoid in analgesisa Pharmacological Synergy in tail flick analgesia between Cannabinoids and Opioids

Fig. 2. Isobologram of 9-THC/morphine drug combinations. The points Fig. 3. Isobologram of 9-THC/codeine drug combinations. The points designated z1 and z2 represent the ED50 values for each drug alone, and the line designated z1 and z3 represent the ED50 values for each drug alone, and connecting these points contains all dose pairs that are simply additive. Points A the line connecting these points contains all dose pairs that are simply and B represent the theoretically additive value for the combinations z1/z2 and additive. Points A and B represent the theoretically additive value for the 0.1z1/0.9z2, respectively. Point C represents the experimentally determined value combinations z1/z3 and 0.2z1/0.8z3, respectively. Point C represents the for the combination z1/z2, and point D represents the experimentally determined experimentally determined value for the combination z1/z3, and point D value for the combination 0.1z1/0.9z2. Since both points C and D fall to left and represents the experimentally determined value for the combination below the line of theoretical additivity, they indicate synergy between 9-THC and 0.2z1/0.8z3. Since both points C and D fall to left and below the line of morphine. theoretical additivity, they indicate synergy between 9-THC and codeine. Cichewicz & McCarthy., 2003 Pharmacological interaction with opioid and cannabinoid in analgesia Pharmacological Synergy in paw latency analgesia between Cannabinoids and Opioids in arthritic rats

Fig. 3. Isobologram of Δ9-THC-THC/morphine drug combination in arthritic Fig. 4. Isobologram of Δ9-THC-THC/morphine drug combination in arthritic rats. The points designated z1 and z2 represent the ED50 values for each rats. The points designated z1 and z2 represent the ED50 values for each drug alone, and the line connecting these points contains all dose pairs that drug alone, and the line connecting these points contains all dose pairs that are simply additive. Point A represents the theoretically additive value for are simply additive. Point A represents the theoretically additive value for the combinations z1:z2. Point B represents the experimentally determined 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 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 of theoretical additivity, synergy between Δ9-THC and morphine has occurred. occurred. (Cox et al., 2007) Pharmacological interaction with opioid and cannabinoid in analgesia Pharmacological Synergy in analgesia between Cannabinoids and Opioids

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)

(Viganò et al., 2005) Pharmacological interaction with opioid and cannabinoid in analgesia Pharmacological Synergy in analgesia between Cannabinoids and Opioids

Figure 4. Hot plate latency following an acute microinjection of a CB1 receptor agonist and/or morphine into the RVM. Microinjection into the RVM of CB1 receptor agonists (HU-210 or WIN 55,212-2) combined with morphine caused an antinociceptive effect that was greater than administration of either drug alone (*p = 0.05). Rats that became sick with repeated injections were not included in this analysis so as not to confound neurotoxic and receptor mediated effects.

(Wilson-Poe et al., 2013) Pharmacological interaction with opioid and cannabinoid in analgesia Pharmacological Synergy in acute termal analgesia between Cannabinoids and Opioids in humans

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.Vertical axis represents displacement of responses on a scale of 100 mm.

(Roberts et al., 2006) Pharmacological interaction with opioid and cannabinoid in analgesia Pharmacological Synergy in acute termal analgesia between Cannabinoids and Opioids in humans

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

(Roberts et al., 2006) Pharmacological interaction with opioid and cannabinoid in analgesia Alteration in Cannabinoid receptor binding and functionality in Opioid tolerant animals.

(Viganò et al., 2005) Pharmacological interaction with opioid and cannabinoid in analgesia Alteration in Opioid receptor binding and functionality in cannabinoid tolerant animals.

Fig. 4 Effect of CP-55,940 chronic treatment (0.4 mg/kg i.p., twice a day for 6.5 days) on opioid receptor 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 analysis were transformed into fmol/mg tissue using [3H]standards.Bars indicate the mean±SEM of at least five animals. *p<0.05 vs vehicle (Student’s t test). Cpu Caudate putamen, NAc nucleus accumbens, ctx cortex, TC central thalamus, TL lateral thalamus, TV ventral thalamus, hippo hippocampus, amyg anterior amygdala, PAG periaqueductal gray, coll colliculus, nigra substantia nigra

(Viganò et al., 2005) Pharmacological interaction with opioid and cannabinoid in analgesia Increase of Antinociceptive effects of Cannabinoids in Opioid tolerant mice.

(Cichewicz & Welch., 2003) Pharmacological interaction with opioid and cannabinoid in analgesia Increase of Antinociceptive effects of Cannabinoids in Opioid tolerant mice.

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

(Wilson-Poe et al., 2013) Pharmacological interaction with opioid and cannabinoid in analgesia Increase of Antinociceptive effects of Cannabinoids in Opioid tolerant mice.

Fig. 2 Analgesic response induced by different doses of CP -55,940 (0.2 or 0.4 mg/kg i.p.) in rats made tolerant to morphine (5 mg/kg s.c., twice a 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<0.001 vs chronic morphine, #p<0.001 vs acute CP-55,940 (Tukey’s test) (Viganò et al., 2005) Pharmacological interaction with opioid and cannabinoid in analgesia Increase of Antinociceptive effects of Opioids in Cannabinoid tolerant mice.

Figure 3. Enhanced morphine antinociception following repeated systemic administration of THC. Administration of cumulative doses of morphine caused a dose-dependent increase in hot plate latency in both pretreatment groups. However, morphine potency was enhanced in THC pretreated rats even though morphine was injected 16 hours after the last of four THC injections.

(Wilson-Poe et al., 2013) Pharmacological interaction with opioid and cannabinoid in analgesia Lack of increase of Antinociceptive effects of Opioids in Cannabinoid tolerant mice.

Fig. 3 Analgesic response induced by morphine (5 mg/kg s.c.) in rats made 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 the mean±SEM of at least five animals. *p<0.001 vs control, °p<0.01 vs acute morphine, #p<0.01 vs acute CP-55,940 (Tukey’s test)

(Viganò et al., 2005) Pharmacological interaction with opioid and cannabinoid in analgesia Reduction of Antinociceptive tolerance to Opioids by Cannabinoids.

Fig. 2. A dose of 20 mg/kg 9-THC p.o. attenuates the development of oral morphine tolerance. Mice received 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 were administered p.o. and the mice were tested 30 min later in the tail-flick test. Each data point represents an average % MPE S.E.M. of six mice. Cichewicz & Welch., 2003 Pharmacological interaction with opioid and cannabinoid in analgesia Bidirectional Antinociceptive effects of Opioids and Cannabinoid in tolerance.

(Wilson-Poe et al., 2013) Pharmacological interaction with opioid and cannabinoid in analgesia Peripheral antinociceptive effect of 2-arachidonoyl-glycerol and its interaction with endomorphin-1 in arthritic rat ankle joints

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.

(Mecs et al., 2010) Pharmacological interaction with opioid and cannabinoid in analgesia Cannabinoid–Opioid Interaction in chronic Pain in humans

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) Pharmacological interaction with opioid and cannabinoid in analgesia Cannabinoid–Opioid Interaction in chronic Pain in humans

Hour Hour

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

(Abrams et al., 2011) Pharmacological interaction with opioid and cannabinoid in analgesia

(Paquette & Olmstead, 2005) Pharmacological interaction with opioid and cannabinoid in analgesia Diagram of interaction of endogenous opioid and cannabinoid systems in analgesis

Figure 5: Shifts in nociceptive modulatory state during morphine analgesia and acute naloxone-induced abstinence.a | Morphine activates off cells to inhibit pain (lower left), THC whereas when naloxone is used to precipitate CB1 antagonist acute abstinence, on cells are activated and produce a hyperalgesic state (lower right). b | Synaptic distribution of opioid receptors within the rostral ventromedial medulla (RVM). - Opioid receptor (MOR) is located on GABA (- aminobutyric acid)-releasing terminals at off cells and the somadendritic region of on cells. Both cell classes have somadendritic opioid receptor-like (ORL1) receptors and both are excited by -opioid receptor (KOR)-bearing glutamatergic terminals (glut) that arise from different input neurons. Whereas MOR agonists produce anti-nociceptive effects by inhibiting on cells and disinhibiting off cells, ORL1 and KOR agonists acting in the RVM can block analgesia by inhibiting off cells or block CB1R hyperalgesia by inhibiting on cells. PAG, periaqueductal grey.

(modified from Fields, 2004) Interaction with opioid and cannabinoid in peripheral analgesia CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral release of endogenous opioids 2

Fig. 2. AM1241 (i.p.) produced dose-dependent antinociception in Fig. 3. The CB2receptor-selective agonist AM1241 stimulated – wildtype (+/+) mice but not in -opioid receptor knockout (-/-) mice. endorphin release from glabrous paw skin. (B) Mouse paw skin. AM1241 Data are expressed as mean SEM. n = 6 per group. #, P 0.05 compared (10M) stimulated -endorphin release from the skin of wild-type (CB2-/-) with WT mice. but not from CB2 receptor-knockout (CB2+/+) mice. Data are expressed as mean SEM. n 12 per group., * P<0.05 compared with vehicle; #, P<0.05 compared with 10M AM1241 alone.

(Ibrahim et al., 2005) Interaction with opioid and cannabinoid in peripheral analgesia CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral release of endogenous opioids

Fig. 4. AM1241 stimulated -endorphin release from cultured human Fig. 3. The CB2receptor-selective agonist AM1241 stimulated – keratinocytes (HaCaT) cells. AM630 (1M) inhibited the effects of AM1241 endorphin release from glabrous paw skin. release from glabrous paw (1M). AM630 had no effect in the absence of AM1241. Data are skin. (A) Rat paw skin. The CB2 receptor antagonist AM630 (10M) expressed as percent of release in medium alone and presented as mean prevented the effects of AM1241 (10 M). AM630 had no effect on SEM. n 12 per group. *, P 0.05 compared with medium alone. #, P 0.05 -endorphin release in the absence of AM1241. Data are expressed as compared with 1M AM1241. mean SEM. n 12 per group., * P<0.05 compared with vehicle; #, P<0.05 compared with 10M AM1241 alone. (Ibrahim et al., 2005) Interaction with opioid and cannabinoid in peripheral analgesia CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral release of 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.

(Ibrahim et al., 2005) Interaction with opioid and cannabinoid in peripheral analgesia Cannabinoid CB2 Receptors Contribute to Upregulation of b-endorphin in Inflamed Skin Tissues

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 of b-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 of AM1241 on mechanical withdrawal threshold in response to von Frey filaments applied to the inflamed paw. Time 0 represents baseline values before CFA injection. 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 or its 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 control group; #P < 0.05, compared with the CFA+vehicle of AM1241 group; &P < 0.05, compared with the CFA+AM1241 group (Two-way ANOVA followed by Bonferroni’s test). (Su et al., 2011) Interaction with opioid and cannabinoid in peripheral analgesia 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 tissues obtained 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 data show 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+AM1241 group (One-way ANOVA followed by Tukey’s test). (Su et al., 2011) Interaction with opioid and cannabinoid in peripheral analgesia Cannabinoid CB2 Receptors Contribute to Upregulation of b-endorphin in Inflamed Skin Tissues

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 in the 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) Interaction with opioid and cannabinoid in peripheral analgesia Cannabinoid CB2 Receptors Contribute to Upregulation of b-endorphin in Inflamed Skin Tissues.

Keratinocytes immunorecative to b-endorphin Macrophages labeled with b-endorphin T-lymphocytes with b-endorphin in the skin tissue in the skin tissues Immunoreactivity in the skin tissue (Su et al., 2011) Interaction with opioid and cannabinoid in peripheral analgesia Involvement of peripheral cannabinoid and opioid receptors in b-caryophyllene-induced antinociception

Figure 2 Antagonistic action of AM630 or AM251 on antinociception Figure 3 Antagonistic action of naloxone hydrochloride or naloxone produced by intraplantar (i.pl.) injection of b-caryophyllene (BCP) in the methiodide on antinociception produced by intraplantar (i.pl.) capsaicin test. AM630 or AM251 i.pl. (B) 30 min prior to i.pl. injection BCP. injection of b-caryophyllene (BCP) in the capsaicin test. Naloxone Capsaicin (1.6 mg/paw) was injected s.c. into the plantar surface of hindpaw hydrochloride (A,B) or naloxone methiodide (C) was injected s.c. and i.pl. 10 min after BCP injection. Values represent the mean SEM for 10 mice per 15 min prior to i.pl. injection of BCP. Capsaicin (1.6 mg/paw) was group. **p < 0.01 when compared with jojoba wax-treated control. p < 0.01 injected subcutaneous (s.c.) into the plantar surface of hindpaw 10 min when compared with saline plus BCP (18.0 mg/paw). 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). (Katsuyama et al., 2013) Interaction with opioid and cannabinoid in peripheral analgesia Involvement of peripheral cannabinoid and opioid receptors in b-caryophyllene-induced antinociception

Figure 4 Antagonistic action of b-funaltrexamine (b-FNA), naltrindole Figure 5 Antagonistic action of local injection of antisera against b-endorphin on hydrochloride (NTI) or nor-binaltorphimine (nor-BNI) on antinociception antinociception produced by intraplantar (i.pl.) injection of b-caryophyllene produced by intraplantar (i.pl.) injection of b-caryophyllene (BCP) in the (BCP) in the capsaicin test. Antisera against b-endorphin were injected i.pl. 5 min capsaicin test. b-FNA or nor-BNI was injected subcutaneous (s.c.) (A) and prior to BCP injection. Capsaicin (1.6 mg/paw) was injected subcutaneous into i.pl. (B) 24 h prior to i.pl. injection of BCP. NTI was injected s.c. (A) and i.pl. the plantar surface of hindpaw 10 min after BCP injection. Values represent the (B) 30 min prior to BCP injection. Capsaicin (1.6 mg/paw) was injected s.c. mean SEM for 10 mice per group. **p < 0.01 when compared with jojoba wax- into the plantar surface of hindpaw 10 min after BCP injection. Values treated control. # p < 0.01, ## p < 0.05 when compared with saline plus BCP (18.0 represent the mean SEM for 10 mice per group. **p < 0.01 when compared mg/paw). with jojoba wax-treated control. # p < 0.01, ## p < 0.05 when compared with saline plus BCP (18.0 mg/paw). (Katsuyama et al., 2013) Interactions Between Endogenous Opioid and Cannabinoid Systems Peripheral effects of the activation of the endogenous opioid and cannabinoid systems in inflammation

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

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