Steps in Pain Perception

Peripheral Nerves

Nociceptors (pain receptors) are present in the skin and most other tissues. They respond to mechanical, thermal or chemical stimulation. The chemical stimulation is due to a variety of substances released into damaged tissues, for example prostaglandins and bradykinin. Sensation is carried to the spinal cord either by "fast" fibres, which detect sharp, localised, short-lived pain, or by "slow" fibres, which carry signals of diffuse, ongoing pain.

Wind Up

Wind up is a normal process in which peripheral and central neurones become sensitised, leading to amplification of signals. The painful area may become hypersensitive to touch. The understanding of this process is still being worked out but it involves a complex chain of neurochemical events.

Transmission

Pain is transmitted up the spinal cord into the brain. This invokes an interaction of arousal, perception, emotion, interpretation and memory. It also triggers physiological changes. The transmission of pain signals across millions of neurones is mediated by neuropeptides, including beta-endorphin, enkephalin, dynorphine, serotonin and other catecholamines, these enhance or inhibit transmission.

Descending Inhibition

There is a descending system of nerves through the spinal cord back to the dorsal horn cells which can inhibit or enhance the pain perceived. Various neurotransmitters are involved. Descending inhibition damps down incoming pain impulses, providing analgesia. It operates when, for example, someone is injured but feels no pain until away from the site of danger. Inhibitory signals travel from the brain down the spinal cord and "damp down" incoming pain impulses. Similarly pain may be increased. This is the mechanism by which for example, happiness or distraction will reduce pain, whilst depression, anxiety or sleeplessness will aggravate it.

Gate Theory

The concept of the "gate" was introduced in 1966 by Melzak and Wall to explain the processing of pain in the dorsal horn of the spinal cord. The wider the gate is open, the more signals are transmitted. The most important control of the gate comes from the brain itself, mediated through the descending pathways described above. From the periphery, touch can be used to close the gate (e.g. transcutaneous nerve stimulation, rubbing, massage). However, in the acute situation touch may have the opposite effect and intensify pain perception.

Neuroplasticity

The nervous system is plastic in both acute and chronic pain. Nerve cells can change the quantity and type of transmitter that they release, receptors can change their activity and new synapses can develop.

Cutting or Damaging a Nerve

Damage to a peripheral nerve can cause major changes in cell function at all levels through to the cerebral cortex. These changes may be permanent, so the idea that a nerve can simply be cut to control pain is incorrect.

ANALGESICS AND HOW THEY WORK

Analgesics affect the transmission of pain in a wide variety of ways and places and are categorised in the following way:

NSAIDs (non-steroidal anti-inflammatory drugs) such as aspirin, ibuprofen and indomethacin, have anti-inflammatory activity inhibiting the formation of prostaglandins from arachidonic acid via the cyclo-oxygenase pathway. Their main site of action is in the periphery at the site of tissue damage but there may also be an effect within the spinal cord. Paracetamol is a para-aminophenol derivative with analgesic and antipyretic activity but no anti-inflammatory activity, with the spinal cord as its site of probable action. Opioid analgesics such as morphine and pethidine act on specific receptors on the descending pathways to inhibit pain. The main mode of action is by pre—and post-synaptic inhibition thereby preventing transmission of neural signals to the brain.

Antidepressants modulate the response to pain within the brain and spinal cord. It has been suggested that the analgesic action of tricyclic antidepressants and monoamine oxidase inhibitors is mediated by their action on central neurotransmitter functions; particularly serotonin and noradrenaline pathways. Anticonvulsants affect the abnormal triggering and transmission of pain along nerve fibres by acting as membrane stabilisers. Carbamazepine is the most often prescribed, although sodium valproate, clonazepam and clobazam are also used.

A wide range of agents are being explored nowadays, targeted on the various steps in the complex pathway of the transmission of pain. Agents such as clonidine, baclofen and ketamine are being used and their roles evaluated.

It is against this background that the pharmacological and clinical investigations of the cannabinoids will now be discussed.

NEUROTRANSMITTERS INVOLVED WITH CANNABINOID ACTION

Cannabis is a complex mixture of cannabinoid molecules (over 61 have been identified) and other chemicals (of which 400 have been identified); with THC as the main active cannabinoid responsible for the psychotropic effects. All these chemicals may have a wide variety of mechanisms of action and that of their metabolites may well be different again. So far, studies have concentrated on THC and a number of synthetic analogues, revealing a number of possible mechanisms of action.

The central nervous system (CNS) transmitters that modulate the perceptions of pain include noradrenaline, serotonin (5HT), acetylcholine, GABA, the opioid peptides and the prostaglandins. Reports suggest that the analgesic effects seen with the cannabinoids involve prostaglandins, noradrenaline, 5HT and the opioid peptides, but not GABA or acetylcholine. The involvement of the prostaglandins is complex. The cannabinoids are stimulators of phospholipase A2, promoting the production of prostaglandins, but also inhibitors of cycloxygenase therefore also inhibiting production. The scene is further complicated by the fact that prostaglandins oppose pain centrally but cause pain at peripheral sites (Bhattacharya, 1986). This may explain why in some tests involving cutaneous electrical pain stimulation to the finger tips in human subjects, cannabis increased sensitivity to both painful and nonpainful stimulation and reduced tolerance to pain (Hill et al., 1974).

The mechanism of the anti-inflammatory effect of THC has been investigated by Burstein et al. (1973). They explain that THC inhibited prostaglandin synthesis in an in-vitro system by reducing the conversion of arachidonic acid to prostaglandin E2. It was also found to be an inhibitor of the formation of prostaglandin E1. Cannabidiol was found to be far more active than THC in this test suggesting a structural relationship between analgesic and anti-inflammatory activity among the cannabinoids. It is also proposed that the cannabinoids interfere with prostaglandin action on adenylate cyclase which is reported to mediate pain perception. Levonantradol, a cannabinoid derivative from Pfizer Laboratories also inhibits prostaglandin induced diarrhoea in animals (Mine et al., 1981).

The involvement of 5HT as a mediator for analgesia with the cannabinoids is debatable. Analgesia is potentiated in the mouse tail flick test by 5-hydroxytryptophan (the precursor of 5HT) and imipramine (a 5HT re-uptake inhibitor) and the cannabinoids are known to affect 5HT. However intrathecally injected methysergide (a 5HT antagonist) has no effect on THC induced analgesia.

The noradrenergic system is a likely mechanism for cannabinoid induced analgesia, as the effects are reduced when yohimbine (an alpha-2 adrenoceptor antagonist) is injected into the lumbar region of the spinal cord. The alpha-1 noradrenergic antagonist, phenoxybenzamine, fails to block cannabinoid induced analgesia. Although the cannabinoids do not act at opiate sites, the effects of both drug classes may be mediated through a common descending noradrenergic mechanism. Analgesia produced by injecting morphine in the periaqueductal grey matter is also blocked by intrathecally injected noradrenergic antagonists (Lichtman and Martin, 1991).

Rats or mice rendered tolerant to the analgesic effects of morphine show a tolerance to cannabinoid induced analgesia (Bloom et al., 1978; Chesher, 1980). Naloxone can decrease the analgesic effects of cannabis in the tail-flick test, the phenylquinone abdominal stretch test, and the hot plate test, but at high doses only. Doses of naloxone known to reverse the analgesic effects of pethidine and morphine in the hot plate and abdominal stretch test do not reverse the analgesic effects of cannabis. After oral administration, THC and morphine produce dose dependent depressions of the passage of a charcoal meal through the gut of mice. THC works out to be about five times less potent than morphine in constipating effect (Chesher et al., 1973). These results tend to suggest that cannabinoids do have an involvement with opioid receptors but that the relationship is not straight forward.

It has been reported (Lichtman et al., 1991) that the kappa opioid antagonist, nor-binaltophimine (nor-BNI) effectively blocks the analgesic effects of the cannabinoids, which is compelling evidence for a link between opioid and cannabinoid analgesic systems. The opioid delta antagonist, ICI 174864 and low doses of naloxone are incapable of blocking cannabinoid induced analgesia and there is evidence of cross tolerance between THC and U50488, a kappa agonist. This suggests that only the opioid kappa receptors are involved. Nor-BNI does not affect the behavioural effects of cannabinoids in mice which raises the possibility of developing a cannabinoid derivative with only the analgesic properties. Both THC and morphine analgesic effects are blocked by potassium channel blockers; however the cannabinoids seem to be blocked by calcium-gated potassium channels via apamin, while morphine interacts with ATP-gated potassium channels. It may be that the potassium channel modulation may explain in part the profound cannabinoid/opioid synergism seen in some pain assessment tests. (International Cannabis Research Society Meeting, Keystone, 1992).

Some synergism must also exist in the mechanisms for mu or delta opioid analgesia with cannabinoid analgesia, because intrathecal pre-treatment of mice with subeffective doses of THC or several other cannabimimetic compounds was able to shift the dose response curve to the left for intrathecal morphine in the tail-flick test; i.e. increase the potency of the morphine (Welch et al., 1992). The exact interaction the cannabinoids have with these neurotransmitters to cause an effect is not clearly known. It is possible that the effects seen are brought about allosterically via the cannabinoid receptor; a mechanism that would allow some sort of selectivity and action only where there was a link between the two types of receptor. It could be by affecting absorption, distribution or fate of a transmitter or even synthesis, storage and release. Some actions of the cannabinoids could be explained by an effect on drug metabolism like cannabidiol which is a known potent inhibitor of drug metabolism (Narimatsu et al., 1990). There is also a report of cannabis increasing the permeability of the blood brain barrier (Agrawal et al., 1989).

In summary, it is likely that in regard to analgesic effects, the cannabinoids have more than one action on any particular system.

CANNABINOID RECEPTORS

So far, two types of cannabinoid receptor, CB1 and CB2, have been identified. The CNS responses to the cannabinoids are likely to be via the CB1 receptor, as evidence for the presence of the CB2 receptor has only been found in the spleen. The CB1 receptor was the first to be identified and has since been cloned. It has been found in rat brain, with the greatest abundance being in the cortex, cerebellum, hippocampus and striatum, with a lesser concentration in the brain stem and spinal cord (Bidaut-Russell et al., 1990).

Certain of the in-vitro effects seen with the cannabinoids may not be mediated by a receptor mechanism. The lipophilic nature of the cannabinoid compounds results in significant changes in the "fluidity" of phospholipid containing membranes and this may be the property responsible for the altered responses of membrane-associated enzymes and proteins. The mechanism of action is comparable to the steroid anaesthetic, alphaxolone and the volatile anaesthetic halothane. However THC produces considerably less fluidization than alphaxolone, thus explaining the lack of clinical anaesthesia. The psychotropically inactive, cannabidiol produces an opposite effect: a decrease in the molecular disorder of the lipid bilayer. Apart from the evidence that cannabinoids can alter the physical properties of membranes there is also evidence that THC can alter the composition of the membranes within the brain and affect the biosynthesis of membrane lipids (Pertwee, 1988).

MEDICINAL CANNABINOID PROTOTYPES

The structure-activity relationships of cannabinoids have been investigated in considerable depth (Razdan, 1986). Minor changes in structure have been shown to cause major changes in activity. For example 2-methyl, delta-8-THC is a potent cannabimimetic, but 4-methyl, delta-8-THC is inactive. Such major changes as a result of relatively small chemical modifications are characteristics seen with compounds which act via receptors. Reports that the 11-hydroxy metabolites of D9 THC (Figure 1(a)) and D8 THC (Figure 1(b)) were more potent in the mouse hot plate tests than the parent compounds led to the development of HHC (9-nor-9 beta—hydroxy hexahydrocannabinol); see Figure 1(c).

Pfizer Inc. examined the structure—activity relationships for analgesia based on HHC, determining that the c-3 alkyl side chain could be optimised by making it longer and that the phenolic hydroxyl was critical for activity. However, because the pyran ring could be modified without extensive loss of potency, the analgesic and antiemetic drug nantradol was developed by replacing the pyran oxygen with nitrogen and removing the axial methyl substituent. The levo enantiomer of nantradol was found to have twice the potency of the dextro enantiomer. In the battery of animal model tests for analgesia (see below), levonantradol (Figure 1(d)) was found to be up to 100 times more potent than THC (Milne et al., 1980). In a controlled study in humans with acute moderate to severe post-operative pain, levonantradol was significantly superior to placebo in terms of analgesic activity (Jain et al., 1981). Drowsiness was the most reported side effect (40% of responses); fewer than 10% reported other effects such as dry mouth, dizziness, strange dreams, nervousness, headache, hallucinations and dysphoria.

Nabilone (Figure 1(e)) is a successful outgrowth of a cannabinoid research program at Lilly Laboratories. Using the usual approach of pharmaceutical industry, the plan was to discover new therapeutic drugs through synthesis and pharmacological evaluation in animals of hundreds of new chemical entities. Nabilone is a non-THC cannabinoid (i.e., a 9-keto analogue of (+/-)-hexahyrocannabinol-dimethylheptyl), albeit with a spectrum of activity closely related to that of (-)-THC.

CP 55940 (Figure 1(f)) is another prototype developed by Pfizer with a spectrum of activity similar to levonantradol but about three times more potent. In comparison to morphine CP55940 has between 8 and 25 times the potency of morphine in a variety of animal analgesic tests (Razdan, 1986). HU-210 (Figure 1(g)) is a dimethylheptyl analogue of 11 hydroxy, delta-8-THC which also has a spectrum of activity similar to levonantradol. Win 55212-2 (Figure 1(h)) is a prototype of a novel series of atninoalkylindole analgesics, synthesised by the research group at Sterling Drug Inc. This compound and its congeners are structurally different from all other known cannabinoids. Nevertheless, studies with tritium labelled Win 552122 indicate that this compound binds very strongly to the cannabinoid receptor.

THE ENDOGENOUS CANNABINOID—ANANDAMIDE

Arachidonyl ethanolamine amide, generically named anandamide (Figure 1(i)), is an eicosanoid derivative that was initially isolated from porcine brain. It was independently isolated from calf brain and identified as a regulator of L-type calcium channels. Subsequently, other ethanolamine amides were identified in porcine brain having the same affinity for CB1 as the ethanolamine amide of arachidonic acid. Mechoulam and colleagues proposed that the family of unsaturated fatty acid ethanolamine amides that bind to the cannabinoid receptor be referred to collectively as anandamides (Mechoulam et al., 1995). Anandamide shows analgesic activity in the hot plate test (Fride and Mechoulam, 1993) and has tranquillising effects in animals (Musty et al., 1995). This does support the theory of endogenous cannabinoids having a role in the control of pain and anxiety.

It has been proposed that anandamide is produced and released from neurones in a calcium ion-dependent manner, when they are stimulated with membrane depolarising agents. Devane and Axelrod (1994) propose that anandamide is formed by an enzymatically catalysed condensation reaction between arachidonic acid and ethanolamine. But because endogenous levels of free arachidonic acid and ethanolamine are very low in the brain, Di Marzo et al. (1994) propose that the formation of anandamide occurs through a phosphodiesterase mediated cleavage of a novel phospholipid precursor and that the degradation of anandamide involves hydrolysis to ethanolamine and arachidonic acid.

Anandamide possesses cis double bonds at carbons 5, 8, 11, and 14 and is structurally different from other cannabinoid receptor agonists, such as THC, CP-55940 and HU-210. Anandamide, like other cannabinoids, inhibits forskolin-stimulated cAMP production in cells expressing the cannabinoid receptor and inhibits N-type calcium currents.

Anandamide mimics many of the pharmacological properties of THC, but has a shorter duration of action. Following IV administration of anandamide, the pharmacological effects, with the exception of analgesia, are almost completely dissipated within 30 minutes (Smith et al., 1994). In contrast THC has a long half life and produces effects for hours. Vela et al. (1995) have shown that anandamide, like THC, can decrease naloxoneprecipitated withdrawal signs in mice chronically treated with morphine. This further supports the role of anandamide as an endogenous cannabinoid agonist and provides additional support for a link between endogenous opioid and cannabinoid systems.

The discovery of the "anandamide system" is important as it may provide possibilities for new drugs to be developed and even provide targets for existing drugs. These targets may be receptors or the processes of synthesis, storage and release of anandamide itself.

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Figure 1 (a) D9 THC, (b) D8 THC, (c) HHC, (d) levonantradol, (e) nabilone, (f) CP-55940, (g) HU-210, (h) WIN 55212-2, (i) anandamide

CANNABINOID RECEPTOR ANTAGONISTS

Sanofi Recherche have made a cannabinoid antagonist, SR141716A, that displays a nanomolar affinity for CB1 but micromolar affinity for CB2 in ligand binding assays. SR141716A antagonises responses of the potent cannabinoid analogues CP-55940, WIN-55212-2 and anandamide in the mouse vas deferens and rat brain adenylate cyclase assays in vitro. When administered orally to animals, SR141716A antagonises the analgesic effects produced by WIN-55212-2.

Another antagonist AM630 (iodopravadoline) is a more potent antagonist of THC and CP55940 than of WIN 55212-2 in the mouse vas deferens; unlike SR141716A which is equipotent. This suggests the presence of more than one cannabinoid receptor in the mouse vas deferens (Pertwee et al., 1995).

LABORATORY EVIDENCE FOR CANNABINOID ANALGESIC ACTIVITY

From the large number of methods available for evaluating the effectiveness of analgesics, it is clear that the optimal tool for estimating pain and pain perception is lacking; however, a comprehensive picture can be obtained by using several testing procedures. Experiments with rats and mice have shown that some cannabinoids are effective analgesics in a number of standard tests which are used to evaluate drug analgesic activity, examples of which are:

Continue reading here: The Tail Flick Test

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