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CANNABINOIDS AS POTENTIAL NEW ANALGESICS

Jose A. Fuentes, Mariano Ruiz-Gayo, Jorge Manzanares, Gema Vela, Ignacio Reche and Javier Corchero

Departamento de Farmacologia, Facultad de Farmacia, and Unidad de CartografIa Cerebral, Institute Phnidisciplinar, Universidad Complutense, 28040 Madrid, Spain.

Summary

Among other pharmacological properties analgesia is one of the important features of cannalrhoids with therapeutical prospects. Cannabinoids have been shown to produce antinociception in experimental animals and humans. Recently a new system of neuromodulation based upon the existence of camrabinoid receptors and their endog,enous agonists has emerged. This has been proposed as another of the endo8,enous pain control systems. Current evidence indicate an interaction between catmabinoid and opioid systems, the latter being of known relevance in nociception. The fact that either exogenous or endogenous opioids enhanced cannabinoid-induced antinociception suggests simultaneous activation of both opioid and cannabinoid receptors by drugs as a new analgesic strategy.

Key I%&: catmabinoids, opioids, nociception, morphine, enkephalindegrading enzyme inhibitors

1. Pharmacology of cannabinoids: antinociceptive effect.

Cannabinoids produce a mixture of psychotomimetic and depressant effects mainly by working upon the central nervous system, together with various centrally mediated, peripheral autonomic effects. These include a feeling of relaxation and well-being that can run along with a sharpened sensory awareness with increased visual and auditory perception that may become fantastic in nature. Other central effects also described are analgesia, depression of motor activity, catalepsy with occurs in rats and mice, and an antiemetic effect that is currently of therapeutic application. Stimulation of appetite, reduction of intraocular pressure, bronchodilation, vasodilation (particularly marked on the scleral and conjunctival vessels, producing a typical bloodshot appearance), tachycardia and inhibition of intestinal motility are the main signs of autonomic stimulation induced by cannabis.

Besides their current use as supressants of nausea and vomiting (nabilone) provoked by antitumor agents and also as appetite stimulant of AIDS patients (A’-tetrahydrocannabinol: A’-THC), the pharmacology of camtabinoids promise other potential applications in therapeutics. In fact, other putative uses as medicines are nowadays under study (1). In this paper we refer to the potential

Corresponding author: Jorge Manzanares, Ph.D., Unidad de Cartografia Cerebral, Instituto Pluridisciplinar, Universidad Comphrtense de Madrid, Paseo de Juan XXI& 1,28030 Madrid, SPAIN; (Phone)34-91-394-3254; (Fax)34-91-394-3264; (E-mail): jorgemr@eucmax.sim.ucm.es

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application of the cannabinoids as analgesics.

An antinociceptive action for camrabinoids has been described in rodents, primates and humans. A’-THC, WlN 55,212, CP-55,940 and anandamide were effective in increasing tail-flick latencies and reducing strecht reflexes induced by p-phenylquinone in rats and mice (2,3,4,5). Moreover, A’-THC and anandamide have been found to be effective as antinociceptive agents also in a model of tonic pain, the arthritic rat (6). Recently, Vivian et al. (7) characterized the antinociceptive action of A’-THC and WlN 55,212 in rhesus monkeys. Although a controversial issue as yet, clinical trials have revealed that cannabinoids behave as analgesics in cases of both cancer pain and acute post- operative pain (8,9,10). An interesting review has been recently published on the clinical experience with the cannabinoid nabilone for chronic pain (11).

High atity binding sites for cannabinoids have been characterized in the CNS (CB, receptor) and in the periphery (CB, receptor) (12,13). Further research lead to the identification of arachidonylethanolamide (called anandamide) as the main natural figand of the CB, receptor (14). Binding studies have shown that the CB, receptors are especially common in the basal ganglia, hippocampus and cerebellum. Anandamide synthase activity is highest in the hippocampus, followed by the thalamus, cortex and striatum (15). As it is the case of the periaqueductal gray matter (PAG) (16) other central regions, though less abundant in receptors, were found to be relevant for the antinociceptive action of camrabinoids. An important breakthrough in the field of camrabinoids was the discovery of a selective antagonist for the CB, receptor (17). The compound, named SR-14 1,716A has resulted in the delineation of a central cannabinoid system independently involved in nociception

(18).

2. Interaction opioids-cannabinoids

Endogenous opioids play a relevant role in nociception and the potent exogenous receptor ligand morphine is still considered as a reference compound for analgesics used to treat moderate to severe pain. Several lines of evidence indicate an interaction between opioid and cannabinoid systems in rodents. First, an abstinence syndrome may be provoked by administration with the opioid receptor antagonist naloxone to adult rata that were either chronically-treated with A9-THC (19) or perinatally exposed to this drug (20). Moreover, these latter rats when adults showed a decrease in sensitivity to the antinociceptive action of morphine (20). Second, the antiemetic action of nabilone is prevented by opioid receptor antagonists (21). Third, the abstinence syndrome induced by naloxone in mice tolerant to morphine may be reduced either by A9-THC (22) or treatment with the CB, receptor agonist anandamide (23). Fourth, early exposure to A9-THC enhanced met-enkephalin and beta-endorphin immunorreactivity of neonatal rats (24). Fith, different types of treatment with cannabinoids have been found to produce an induction of opioid precursor gene transcription in different brain areas. Studies in rats and mice have demonstrated that Ag-THC-induced antinociception is mediated at both spinal and supraspinal levels (25,26). Pugh et al. (3), have shown that acute administration of A9-THC in the subarachnoid space increases dynorphin A and leucine-enkephalin levels in the spinal cord, suggesting that A9-THC-induced antinociception mediated at this level involves the release of endogenous opioid ligands. In agreement with this hypothesis, our laboratory has shown that subchronic A9-THC administration increased PENK and prodynorphin gene expression in the spinal cord (Figure 1) (27). Recently, it has been shown an involvement of PAG in cannabinoid-mediated aritinociception (16). As suggested in the spinal cord, this effect may be mediated by activation of endogenous opioids in the PAG. Consistent with this idea, subchronic A9-THC administration markedly increases PENK gene expression in the PAG (Figure 2), suggesting that this enhancement may be part of the supraspinal molecular mechanisms involved in camrabinoid antinociception (28).

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PDYNmRNA

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Fig. 1 Panel A: Northern Blot analysis of hybridization to prodynorphin and preproenkephalin mRNAs in 15 ug spinal cord mRNA Poly A prepared from pools of 6 rats per group. Vehicle: (saline:ethanol:cremophor; 18: 1: 1, 1 ml/kg i.p. daily for five days). THC (THC dissolved in vehicle to 5 mgikg i.p. daily for five days). Blots were normalized with human S26 ribosomal cDNA probe. Panel B: Relative concentrations of prodynorphin and preproenkephalin mRNA levels in the spinal cord. The autoradiograms were analyzed as described in materials and methods. Values are presented as a percent of control values (defined as 100%).

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Effects of chronic administration of THC on PENK mRNA levels in the periaqueductal grey (PAG) . Rats were injected with THC (10 m&/day; 5 days;i.p.) or with its vehicle (Tween-saline solution) and were decapitated 20 min after the last administration. Columns represent means and vertical lines +l S.E.M. of PE mRNA levels in 5 rats. *, Values tiom THC-treated animals that are significantly different (P<O.OS) from vehicle-treated rats (Student t-test). Autoradiograms of coronal brain sections at the level of the PAG. Slides were apposed to f3hn (Kodak, Biomax, MR- 1) 1 month. Bar represents 1 mm.

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Fig.3 Dose-response curves for the antinociceptive activity of A9 -tetrahydrocamrabinol (A9- ‘?‘I-$C) alone (0) and in animals that received an ineffective dose of morphine Q. Anlagesic responses were evaluated in tha tail-flick (A) and hot palte (B: jumping; C: paw-lick) test. A’-tetrahidrocannabinol was given i.v. 20 min prior to the test. Morphine was given i.p. 30 min before the test. Percentage of analgesia corresponds to ((test latency-control latency)/(cutt-off time-control latency)) x 100. Values on the grajph are expressed as means + SEM and correspond to values for groups of 8-10 mice. *, PC 0.05; **, PcO.01. Newman-Keuls’test.

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The antagonism of camtabinoid-induced antinociception by opioid receptor antagonists deserves a special consideration. In the field of opioid pharmacology, blockade by a non-selective receptor antagonist is considered as an indication of opioid system involvement. Results obtained by using naloxone to explore the role of opioids involvement in the antinociceptive action of cannabinoids appear to depend on the experimental conditions. Some authors were not able to block the effects of A’-THC or camtabinoid receptor agonists (29,30) while others have found that lower doses (l-5 mg/kg) of naloxone partially antagonize and higher dose (10 mgikg) fully block the antinociception effect of A’-THC (3 1). Presumably, this high dose of naloxone may be acting at u-, K-, and 6- opioid receptors. By using more selective opioid antagonists such as P-funaltrexamine (a u-opioid selective antagonist) or nor-binaltorphimine (a K-opioid selective antagonist), several studies have demonstrated that u- and preferentially K- but not &opioid receptors are involved in the antinociceptive action of A’-THC ($3 1,32,33). Furthermore, cross-tolerance to analgesia has been reported between u-opioid receptors (34) or K- opioid receptors (5,6,35,36) and A’-THC.

The administration of the CB, receptor antagonist SR 141,716A (17) blocked the antinociception induced by A’-THC or by the synthetic cannabinoid receptor agonist CP-55,940 (31,37). In addition to the central antinociceptive action of camrabinoids, recently, Calignano et al., (38) have also demonstrated the involvement of peripheral CB,-like and CB,-like receptors in the control of pain, since administration of anandamide and palmitoylethanolamide reduce pain response induced by tissue damage 1 OO-fold than does each compound alone, and that SR- 14 1,716A and CB, antagonist SR-144,528 prolong and enhance the pain.

In summary, a new system of nociception has been defined by the demonstration of the existence of a central cannabinoid receptor that may be stimulated by endogenous agonists such as am&amide. As an important evidence, the effect of cannabinoids on pain is prevented by a selective CB, receptor antagonist. At variance, opioid receptor antagonists produced inconsistent effects on antinociception-induced by A’-THC and other CB, receptor agonists.

3. Therapeutical prospects for the enhancement of cannabinoid antinociception by endogenous or exogenous opioids.

The potentiation between A’-THC and morphine was first described by Welch and Stevens (32) after i.t. administration of both drugs. In our experience, i.v. administration of A’-THC and morphine (Figure 3 ) also elicited potent antinociceptive responses. This, suggested a greater than additive interaction between cannabinoids and opioids, since inactive doses of these drugs induced antinociception when given in combination to the same animal. The antagonism induced by the opioid receptor antagonist naloxone as well as by the cannabinoid receptor blocker SR- 14 1,716A indicates the involvement of both the two different type of receptors in this antinociceptive response. This combined effect of morphine and A’-THC was also antagonized by the K-opioid receptor antagonist nor-binaltorphimine when given i.t. but not i.c.v.. The K-opioid component of the potentiation between morphine and A’-THC is probably due to the cannabinoid rather than to morphine as the involvement of K-opioid binding sites in the antinociceptive effect of morphine is not well established. The implication of this tc-opioid receptor in the antinociception induced by cannabis is supported both by the fact that nor-binaltorphimine blocks A’-THC antinociception after peripheral or i.t. administration (3 1,32) and also by results that showed prevention of A’-THC-induced antinociception by i.t. administration of an antiserum against dynorphin A-(1-8) (31). It is pertinent to point out that as an indication of supraspinal u-opioid receptors in the morphine potentiating effect on A’-THC antinociception, the effect was blocked by i.c.v. P-funaltmxamine administration. This implication of u-opioid supraspinal receptors was confirmed by using subeffective doses of the u-selective opioid

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Fig.4 Effect of the u-opioid receptor agonist DAMGG on the analgesic effect of A’- tetrahydrocannabinol . DAMGG was administered icv 15 min before the test. Tail-flick (white-columns) and hot-plate (jumping; hatched columns) test. Percentage of analgesia corresponds to ((test latency-control latency)/(cutt-off time-control latency)) x 100. Values on the graph are expressed as means + SEM and correspond to values for groups of 7-9 mice. *, P< 0.05; !*, PcO.01. Newman-Keuls’test.

receptor agonist D-Ala2, IV-Me-Phe4, Gl$-ol)enkephalin (DAMGO; Figure 4) administered by i.c.v. route. This selective agonist, like peripheral morphine, also potentiated the effect of A’-THC (33). On these bases, it appears that both cannabinoid and p-supraspinal opioid receptors activate similar descending inhibitory pathways regulating, at a spinal level, the release of neurotransmitters involved in nociceptive transmission. Thus, a combination of both A’-THC and morphine may result in the sequential activation of spinal and supraspinal mechanisms leading to nociception.

As a further strategy to explore the potentiation between opioids and cannabinoids, enzyme inhibitors of’opioid degradation were i.c.v. injected in mice. The rationale in this case was that by so doing activation of opioid receptors by endogenous agonists would potentiate the antinociceptive action of CI3, receptor agonists. For this purpose, the opioid degrading enzyme inhibitor bestatin (a preferential aminopeptidase blocker) and tiorphan (a selective inhibitor of neutral endopeptidase (NEP); EC3.4.24.11, also called enkephalinase (39)) were used.

As illustrated in Figure 5, i.c.v. administration of thiorphan (T) + bestatin (B) shifted the dose- response curve of i.v. A’-THC to the left in the three responses studied of the hot-plate test. A number of receptor specific inhibitors were used to characterize this antinociceptive effect. The data obtained

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Fig.5 Dose-response curves for the antinociceptive activity of A’-tetrahydrocannabinol (A9- THC) alone Q and in animals that received an ineffective dose thiorphan + bestatin (0). Antinociceptive responses were evaluated in tha tail-flick (A) and hot plate (B: jumping; C: paw-lick) test. A9-THC was given i.v. 20 min prior to the test. Thiorphan + bestatin were given i.c.v. 10 min before the test. % MPE corresponds to ((test latency-control latency)/(cutt-off time-control latency)) x 100. Values on the graph are expressed as means + SEM and correspond to values for groups of 8- 10 mice. *, P< 0.05; **, PcO.01. Newman-Keuls’test.

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indicated that the action of T+B on A’-THC-induced antinociception was blunted by blockade of the CB, receptor with SR- 14 1,7 16 A. Moreover, a pharmacological analysis‘of the central region and the opioid receptor type mediating this effect was carried out. The use of p-funaltrexamine, nor- binaltorphimine and naltrindole given by i.c.v. or i.t. routes showed that supraspinal p- and spinal K- opioid receptors were implicated in the interaction of both opioid and cannabmoid systems regulating nociception in mice.

Endogenous peptides of the dynorphin family are known to be substrates of both puromycin- sensitive arninopeptidase and aminopeptidase M (40). Thus, the combination of bestatin plus thiorphan may produce an increase of both enkephalin and dynorphin family peptides. Our data together with results published by other investigators suggest the existence of complex neuronal circuits in which cannabinoids receptors activate endogenous opioid pathways, leading to K-opioid receptor occupation after cannabinoid-induced release of their endogenous agonists (3). Therefore, cannabinoids may act both at the supraspinal level (PAG) of the descending inhibitory pathways and at the level of the dorsal horn of the spinal cord where intrinsic intemeurons containing dynorphin peptides may regulate transmission between primary afferent fibers and spinothalamic tract transmission neurons.

The possible therapeutic effects of cannabinoids are currently entangled iu the debate about prohibition of recreational drug abuse (41). Nevertheless, these drugs have already found a place in therapeutics and now are used in the treatment of emesis and in the enhancement of appetite in some conditions. The fact that their antinociceptive action is enhanced by opioids may be a step forward for the use of cannabmoids as analgesics as their use could be carried out at doses below those that produce adverse effects, mainly psychotomimetic and motor actions.

This work was supported by the Universidad Complutense of Madrid grant PR294/95-6189, Concerted Action from the European Union grant BMHl-CT-94-1108 and Spanish Ministry of Education grant DGICYT UE95-0017. 3. Corchero is a Predoctoral Fellow supported by the “Comunidad Autonoma de Madrid” and J. Manzanares is a Senior Fellow supported by the Spanish Ministry of Education.

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