Rasmus P Clausen and Harald S Hansen

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CONTENTS

19.1 Opioid Receptors 313

19.1.1 Opioid Receptor Subtypes and Effector Mechanism 314

19.1.2 Endogenous Opioid Receptor Ligands 314

19.1.3 Nonendogenous Opioid Receptor Ligands 316

19.1.4 Therapeutic Applications and Prospects 320

19.2 Cannabinoid Receptors 321

19.2.1 Endocannabinoid System 321

19.2.2 Cannabinoid Receptor 1 323

19.2.3 Cannabinoid Receptor 2 324

19.2.4 Other Cannabinoid Receptors 324

19.2.5 Therapeutic Use and Potential 324

19.2.6 FAAH-Inhibitors and Anandamide Uptake Inhibitors 326

19.2.7 CBj-Receptor Agonist/Antagonist 326

19.2.8 CB2-Receptor Agonist/Antagonist 326

19.2.9 Diacylglycerol Lipase Inhibitors 327

19.2.10 Monoacylglycerol Lipase Inhibitors 327

Further Readings 328

19.1 OPIOID RECEPTORS

Presently she cast a drug into the wine of which they drank to lull all pain and anger and bring forget-fulness of every sorrow.

The Odyssey, Homer (ninth century BC)

The history of opioids and its receptors spans several millennia. The first evidences of uses of the seed pods of Papaver somniferum dates back to 4200 BC and numerous findings and descriptions through out the history witness the use of different parts of this plant in food, anesthesia, and ritual purposes. Opium (from opos, the Greek word for juice) refers to the liquid that appears on the unripe seed capsule, when it is notched. This liquid contains as much as 16% of morphine, a compound that was isolated already in 1806 as the major active ingredient in opium (Figure 19.1). A few years, later codeine was also isolated. Morphine could now be produced and applied in its pure form for the treatment of pain and as an adjunct to general anesthetics, but it was quickly realized that morphine had the same potential of abuse as opium. In 1898, heroin was synthesized and claimed to be a safer, more efficacious, and nonaddicting opiate as were several other analogues around that time; however, they all proved not to be safer later on. Heroin is an early example of a

Monoacetyl Morphine Metabolism

Heroin

FIGURE 19.1 Chemical structures of morphine, codeine, and heroin. 3D-structure of morphine.

Heroin

FIGURE 19.1 Chemical structures of morphine, codeine, and heroin. 3D-structure of morphine.

prodrug since the highly potent analgesic properties can be attributed to the rapid metabolism to 6-monoacetylmorphine and morphine, combined with higher blood-brain barrier penetration due to better lipid solubility compared to morphine.

19.1.1 Opioid Receptor Subtypes and Effector Mechanism

The idea that morphine and other opioids caused analgesia by interacting with a specific receptor arose around the 1950s. The observation 40 years earlier that the N-allyl analogue of codeine antagonized the respiratory action of morphine was actually an evidence of such a proposal. However, it was first fully realized, when similar N-allyl analogue of morphine (nalorphine) was shown to antagonize the analgesic effects of morphine.

Today, it is known that all of the opioid receptors are G-protein coupled receptors (GPCRs) belonging to family A (Figure 19.2) that mediates its effects through Gi/Go proteins. So far, four different opioid receptor subtypes have been cloned sharing more than 60% sequence homology. These are termed m, k, and 8 receptors (corresponding to MOR, KOR, and DOR, respectively) and an "orphan" receptor termed ORL1, which was the first orphan GPCR to be cloned.

The different effects mediated by each receptor type (m-euphoria versus K-dysphoria; m-supraspinal analgesia versus ORL1-supraspinal antagonism of opioid analgesia) in the intact animal are the result of different anatomical localizations and not due to different cellular responses. Each receptor type has been further subdivided into m1/m2, K1/K2, and 81/82 receptors based on pharmacological and radioligand studies. However, the origin of this subdivision is not genetically based, and it is not known whether it arises from posttranslational modification, cellular localization, or interactions with other proteins; however, it was recently shown that heterodimerization of the receptors could be important for some of these pharmacological differences.

Morphine has the ability to both excite and inhibit single neurons. Opioid inhibition of neuronal excitability occurs largely by the ability of opioid receptors to activate various potassium channels. Another well-established mechanism of action is the inhibition of neurotransmitter release. The observation in 1917 that morphine inhibited the peristaltic reflex in the guinea-pig ileum (giving rise to constipation, one of the side effects of morphine) was 40 years later shown to result from the inhibition of acetylcholine release. Also glutamate, GABA, and glycine release throughout the central nervous system (CNS) can be inhibited by opioid receptor activation. In general, the CNS effects of opioids are inhibitory, but certain CNS effects (such as euphoria) result from excitatory effects (Table 19.1).

19.1.2 Endogenous Opioid Receptor Ligands

It was proposed in the early 1970s, that the physiological role of opioid receptors was not to be target for opium alkaloids, but that endogenous agonists might exist as mediators of the opioid system.

Opioid Receptor Mor Pdb Structure

FIGURE 19.2 Structure of opioid receptors. (Left) Serpentine model of the opioid receptor. Each transmembrane helix is labeled with a roman number. The white empty circles represent nonconserved amino acids and white circles with a letter represent identical amino acids among the four opioid receptors. Violet circles represent further identity between the MOR, DOR, and KOR. Green circles highlight the highly conserved fingerprint residues of family A receptors. Yellow circles depict the two conserved cysteines in EL loops 1 and 2, likely forming a disulfide-bridge. IL, intracellular loop; EL, extracellular loop. (Right) Proposed arrangement of the seven transmembrane helices of opioid receptors as viewed from the top (extracellular side). (From Waldhoer, M. et al., Ann. Rev. Biochein.. 73, 953, 2004.)

FIGURE 19.2 Structure of opioid receptors. (Left) Serpentine model of the opioid receptor. Each transmembrane helix is labeled with a roman number. The white empty circles represent nonconserved amino acids and white circles with a letter represent identical amino acids among the four opioid receptors. Violet circles represent further identity between the MOR, DOR, and KOR. Green circles highlight the highly conserved fingerprint residues of family A receptors. Yellow circles depict the two conserved cysteines in EL loops 1 and 2, likely forming a disulfide-bridge. IL, intracellular loop; EL, extracellular loop. (Right) Proposed arrangement of the seven transmembrane helices of opioid receptors as viewed from the top (extracellular side). (From Waldhoer, M. et al., Ann. Rev. Biochein.. 73, 953, 2004.)

TABLE 19.1

Opioid Receptor Ligands

Receptor

Agonist

Morphiceptin DAGO Normorphine Sufentanyl

Deltorphin

DPDPE

DADLE

U 50,488

Antagonist

Naloxone

ICI 154,126 ICI 174,864

MR2266

Agonist Effect(s)

Analgesia

Respiratory depression Miosis

Reduced gastrointestinal motility

Nausea

Vomiting

Euphoria

Supraspinal analgesia

Analgesia (spinal level) Trifluadom miosis (weak) Respiratory depression (weak) Dysphoria

Note: DAGO, Tyr-D-Ala-Gly-MePhe-Gly-ol; DPDPE, [D-Pen2, D-Pen5]enkephalin; Pen, penicillamine; DADLE, [D-Ala2, D-Leu5]enkephalin; deltorphin II, Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2; morphiceptin, b-casomorphin-(1-4)-amide or Tyr-Pro-Phe-Pro-NH2.

At that time, there were no hints of what kind of compounds to look for. After 2 years of collecting extracts from pig brain and applying them in a functional bioassay, Kosterlitz and coworkers in 1975 identified two closely related endogenous pentapeptide opioids (Table 19.2).

The amino acid sequences are YGGFM and YGGFL and termed as [Met]- and [Leu]-enkephalin, respectively. Since then, many other peptide opioids of varying lengths have been identified. They are all cleavage products of longer peptides and can be divided into four families based on their precursors. Three of these families all start with the [Met]- and [Leu]-enkephalin. The endogenous opioid peptides have varying affinities for the opioid receptor subtypes; however, none of them are specific for a single subtype, although the neuropeptide nociceptin is the endogenous ligand specific for ORL1. The precursors are often made up of repeating copies of the opioid peptide products.

High affinity opioid receptor peptides (dermorphins and deltorphins) have been isolated from frog skins and are quite unusual in having D-amino acids in the sequence. Also milk-derived casomorphins, hemorphins from hemoglobin, and cytochrophins (fragments of cytochrome B), have low affinity for the opioid receptors.

Besides the endogenous peptides, it has been shown that morphine is present in various tissues and body fluids and SH-SY5Y human neuroblastoma cells are capable of producing morphine. The biosynthetic route is similar to that found in Papaver somniferum.

19.1.3 Nonendogenous Opioid Receptor Ligands

The synthetic efforts in the opioid field over the last century have mainly been stimulated by the search for a safer alternative to morphine that maintained the analgesic effects but was devoid of respiratory depression and abuse potential. Different medicinal chemistry approaches have been followed in the development of opioid receptor ligands:

TABLE 19.2

Endogenous Opioid Peptides

Precursor

Pro-enkephalin

Pro-opiomelanocortin Prodynorphin

Pronociceptin/orphanin-FQ

Prodermorphin and prodeltorphina

Opioid Peptide Product

[Met]-enkephalin [Leu]-enkephalin

Peptide E BAM 22P Metorphamide

ß-Endorphin

Dynorphin A Dynorphin A(1-8) Dynorphin B a-Neoendorphin ß-Neoendorphin

Nociceptin/orphanin-FQ

Endomorphin-1

Endomorphin-2

Dermorphin Deltorphin Deltorphin I Deltorphin II

Amino Acid Sequence

YGGFM YGGFL YGGFMRF YGGFMRGL

YGGFMRRVGRPEWWMDYQKRYGGFM

YGGFMRRVGRPEWWMDYQKRYG

YGGFMRRVNH2

YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE

YGGFLRRIRPKLKWDNQ

YGGFLRRI

YGGFLRRQFKVVT

YGGFLRKYPK

YGGFLRKYP

FGGFTGARKSARKLANQ YPWF-NH2

ypff-nh2

Y(D)AFGYPS-NH2 Y(D)MFHLMD-NH2 y(d)afdvvg-nh2 Y(d)afevvg-nh2

Note: The pentapeptide sequences corresponding to [Met]- and [Leu]-enkephalin contained in other opioid peptides are shown in bold. Note that b-endorphin and most of the opioid peptides derived from proenkephalin contain [Met]-enkephalin at their N-termini, whereas the sequence of [Leu]-enkephalin is present in those peptides derived from prodynorphin.

a Dermorphin and deltorphins are derived from multiple precursors and all have a naturally occurring D-amino acid in position 2.

• Chemical modification of morphine and related structures (opiates)

• Simplification of the morphine structure

• Dimerization (bivalent ligands)

• Peptides and peptidomimetics

The early development was focused on the first two approaches. Examples of opiates that display similar affinity to all subtypes are shown in the upper part of Figure 19.3. Introduction of bulky sub-stituents to the morphine structure generally yields antagonists, and naloxone and naltrexone are unse-lective antagonists. N-Allyl analogue nalorphine is an example of a mixed agonist-antagonist. It was originally characterized as an antagonist, but later shown to have antagonist activity at MOR but agonist activity at KOR. Nalorphine was one of the first compounds to be extensively tested in the clinic in combination with morphine to find an ideal agonist-antagonist ratio for maximizing analgesics properties and minimizing adverse effects. Buprenorphine is a potent analgesic and partial agonist at the MOR and antagonist at DOR and KOR. Diprenorphine is reported as an unselective antagonist.

An increasing number of subtype selective ligands has been reported and a few examples are shown in the lower part of Figure 19.3. Compounds that are m-selective include morphine that is

Unselective opiates

O OH

Morphine R = Me Nalorphine

O OH

Morphine R = Me Nalorphine

Selective opiates

H OH

Selective opiates

H OH

Naltrexone R = Naloxone R= "irC-""5:::;.

Naltrexone R = Naloxone R= "irC-""5:::;.

O OH

Diprenorphine R=Me Buprenorphine R = f-Bu

O OH

Diprenorphine R=Me Buprenorphine R = f-Bu

OH O^OMe

7-Spiroindanyloxymorphone (SIOM)

7-Spiroindanyloxymorphone (SIOM)

OH O^OMe

FIGURE 19.3 Chemical structures of classical unselective opiates (except morphine, which is m-selective) based on the morphine scaffold and chemical structures of selective opiates. Morphine and b-FNA are m-selective agonist and irreversible antagonist, respectively. SIOM and NTI are examples of S-selective antagonists, whereas the introduction of charged guanidinium group converts NTI into the k-selective antagonist gNTI.

an agonist and the irreversible antagonist b-funaltrexamine (b-FNA). 7-Spiroindanyloxymorphone (SIOM) and naltrindole (NTI) are examples of S-selective antagonists, and guanidinyl-NTI (gNTI) represents a k-selective antagonist. Interestingly, gNTI was recently shown to have higher affinity toward HEK-293 cells expressing KOR together with DOR or MOR compared to cells expressing only KOR, and the agonist effect of gNTI at the heterodimers KOR/DOR and KOR/MOR could be blocked by antagonists selective against DOR and MOR, respectively. This underlines the importance of heterodimerization and the fact that gNTI is analgesic when injected into the spinal cord but not when injected into the brain, could arise from different heteromeric populations.

The development of selective opiates has followed the "message-address" concept. This states that the amino and the aromatic group in the morphine determine the activity (the "message") of the opiates, whereas the lipophilic region around the allylic alcohol confers selectivity (the "address"). This is demonstrated by the conversion of NTI from a S-selective antagonist into the k-selective antagonist gNTI by the introduction of charged guanidinium group. Already in the 1960s, a 3D-pharmacophore model was conceived stating the importance of the spatial placement of the amine, the aromatic group, and the lipophilic region for ligand affinity. The successive breakdown of the morphine structure has led to a number of simpler nonopiate structures obeying this early and simple 3D-pharmacophore model. This breakdown is shown schematically in Figure 19.4 defining a structural classes of opioid receptor ligands developed over the last century.

Examples of these classes are m-selective agonist fentanyl (piperidine), ethyl-ketocyclazine (benzomorphane), methadone (phenylpropylamine), and meperidine (piperidine) (Figure 19.5). However, other structural classes have appeared, such as S-selective agonist SNC-80 and k-selective agonist U50,488, and more recently several new scaffolds come from screening compound libraries of the cloned opioid receptors including heteromeric combinations.

"Address"

H

uN^

-\az/r~oh \

1 jo ry

"Message" \

Piperidines Benzomorphans Morphinans

Phenylpropylamines Piperidines

h2n h2n

Spiro[benzofuran]- Methylenoxy- Morphines piperidines benzazocines

FIGURE 19.4 The message-address concept of the development of opiates is shown schematically in the box. The message region defines the activity of the compounds whereas the address region defines the selectivity of the compounds. The structural development in the progressive simplification of the morphine scaffold via morphinans and benzomorphans to piperidines, but also via benzazocines, spiropiperidines to piperidines and phenylpropylamines.

Sufentanil R = CH2OMe, Ar =

Ethyl-ketocyclazine (EKC)

O

Methadone

Meperidine

Meperidine

U50,488

FIGURE 19.5 Examples of different structural classes of opioid receptor ligands.

The dimerization of ligands is a popular strategy in medicinal chemistry to alter the pharmacological properties of a monomeric ligand. This strategy was advanced in the early 1980s by Portoghese and coworkers using opiates. Initially, the idea was to develop such bivalent ligands with a spacer of optimal length that would exhibit a potency that is greater than that derived from the sum of its two monovalent pharmacophores. This would provide an evidence that the receptors existed as dimers. One of the first series where compounds 19.1 (Figure 19.6, n = 0, 2, 4, 6, 8), dimerizing a naltrexone analogue. The optimal spacer length was shown to be n = 4 giving the highest activity.

FIGURE 19.6 Examples of dimeric or bivalent opioid receptor ligands.

Endomorphin-2

FIGURE 19.7 Examples of peptidomimetic opioid receptor ligands.

19.4

nh oh oh o

19.4

ho ho o

Today there is a substantial evidence to show that G-protein coupled receptors exist as dim-ers. The concept of making bivalent ligands has been shown to be applicable in many other areas wherein it is possible to modulate other pharmacological properties of a ligand such as degradation, uptake, etc. The concept has also been used to target heterodimeric receptor populations. For example, the dimerization of the analogues of naltrexone and NTI yields a series of heterobivalent ligands 19.2 (n = 2-7), where tolerance and dependence are significantly reduced with increasing linker length, whereas agonist potency is increased. It is hypothesized that 8-k heterodimers are targeted specifically with longer linkers. Also, ligands that selectively target 8-k heterodimers, which are localized in the spinal cord, have been developed.

The last approach that will be mentioned here is the use of peptides and peptidomimetics. New agonists and antagonists of opioid receptors have been obtained by making large combinatorial libraries of d- and L-amino acids including mix libraries and screening these compounds against MOR, KOR, and DOR. The sequences span from tetra- to decapeptides. In this way, potent and selective peptides have been obtained that differ from the endogenous peptides. Furthermore, the modification of the peptide backbone has yielded potent peptidomimetics. The modifications include minor modifications such as backbone amide alkylation. But examples of more extensive modifications are the use of a polyamine backbone as in compound 19.3, or compound 19.4, which is a peptidomimetic analogue of endomorphin-2, a potent agonist of MOR with high selectivity for MOR over DOR and KOR (Figure 19.7).

19.1.4 Therapeutic Applications and Prospects

Although the development of opiates has been spurred primarily by the search for efficient analgesics with few side effects, other clinical applications of opioid receptor agonists and antagonists are known. Agonists are primarily applied as analgesic, anesthetic, antitussive, and in the treatment of diarrhea. Morphine and codeine are mostly used as analgesics. Fentanyl is a very potent analgesic and used in anesthesia. Meperidine is used for acute pain. Methadone is applied to control the withdrawal of heroin from addicts. Antagonists are used for the reversal of some of the effects induced by agonists. Thus, naloxone has been used to reverse coma and respiratory depression of opioid overdose (methadone and heroin). It is also indicated as an adjunct agent to increase blood pressure under septic shock. Naltrexone has been approved as an adjunctive therapy in the treatment of alcohol dependence and the treatment of narcotic addiction to opioids. However, there are also potential indications including obesity, obsessive compulsive disorder, and schizophrenia.

19.2 CANNABINOID RECEPTORS

Different parts of the plant Cannabis sativa has for millennia been used for recreational and medicinal purposes, as it can be seen from old Chinese, Assyrian, and Roman literature. However, it was first in 1964 that the active principle causing the psychoactive effects was isolated and found to be D9-tetrahydrocannabinol (THC) (Figure 19.8). Originally, it was thought that THC due to its lipophi-licity somehow acted through fluidizing the cellular membranes, but in the early 1990s it was discovered that THC activates two receptors, cannabinoid receptor-1 (CBj-receptor) and cannabinoid receptor-2 (CB2-receptor). Cannabinoid effect in rodents is characterized by the so-called tetrad test. In this test, measurement of spontaneous activity, thermal pain sensation, catalepsy, and rectal temperature are made, and compounds with cannabinoid activity should produce hypomotility, analgesia, catalepsy, and hypothermia. Shortly after the discovery of the receptors, two endogenous compounds were identified that could activate these receptors, i.e., anandamide (A-arachidonoylethanolamine or arachidonoylethanolamide) and 2-arachidonoylglycerol (2-AG) (Figure 19.9), and they are called endocannabinoids. Both the endocannabinoids are lipids and thus not very water soluble. They may associate with albumin or lipoproteins in the extracellular space and endocannabinoids function in an autocrine and paracrine fashion where they are formed "on demand" and then degraded, i.e., they are not stored in vesicles like neurotransmitters or peptide hormones. Tissue levels of anandamide and 2-AG are usually in the pmol/g and nmol/g tissue, respectively, but it is not clear whether these levels represent the ligand concentration available to the receptors. However, it is generally considered that there is an endogenous tone of endocannabinoid level in most tissues. Endocannabinoids are also found in very small concentrations in plasma where they are thought to represent spill over from the tissues.

19.2.1 Endocannabinoid System

The endocannabinoid system comprises the two cannabinoid receptors, the two endocannabinoids and the enzymes that synthesize and degrade the endocannabinoids. Biosynthesis of anandamide is complex, but it is formed from an unusual phospholipid having three fatty acids, A -arachidonoyl-phosphatidylethanolamine that again is formed from phosphatidylethanolamine by the acyla-tion of the amino group catalyzed by a poorly known calcium-stimulated A -acyltransferase.

FIGURE 19.8 Plant cannabinoid (THC) and two endocannabinoids

FIGURE 19.9 Biosynthesis of anandamide. The precursor phospholipids (NArPE) is generated from phos-phatidylethanolamine by a N-acyltransferase (NAT). It can then be hydrolyzed by a phospholipase C (PLC), by N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD), or by alpha-beta-hydrolase 4 (Abh4). Other acylethanolamides may be formed by the same enzymes. (R = fatty acids). The enzymes "X" and "Y" are not well characterized yet.

FIGURE 19.9 Biosynthesis of anandamide. The precursor phospholipids (NArPE) is generated from phos-phatidylethanolamine by a N-acyltransferase (NAT). It can then be hydrolyzed by a phospholipase C (PLC), by N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD), or by alpha-beta-hydrolase 4 (Abh4). Other acylethanolamides may be formed by the same enzymes. (R = fatty acids). The enzymes "X" and "Y" are not well characterized yet.

This enzyme uses acyl groups (e.g., arachidonoyl) from the Sn-1 position of phosphatidylcholine as substrate in the acylation process, and it will use whatever fatty acid is present. Thus, a number of N-acylphosphatidylethanolamines are always formed having different fatty acids in the N-acyl position of which N-arachidonoyl is only a minor component and those with palmitic acid, stearic acid, or oleic acid are much more abundant. From this precursor phospholipid, N-acyl-phosphatidylethanolamine, a number of different enzyme-catalyzed pathways can result in the generation of acylethanolamides including anandamide that usually amounts to less than 5% of the acylethanolamides (Figure 19.9). Which pathway is most relevant for a particular tissue or a particular physiological/pathophysiological setting is not known at present. The cellular localization of anandamide formation is not known and several of the involved enzymes have not been cloned yet. The different acylethanolamides have a number of more or less specific biological activities, e.g., palmitoylethanolamide is anti-inflammatory and oleoylethanolamide has anorexic and neuroprotective activity that may be mediated via the activation of a transcription factor PPARa, and/ or an orphan receptor GPR119. Other acylethanolamides do not bind to the cannabinoid receptors. Anandamide is a partial agonist for the cannabinoid receptors but it can also activate vanilloid receptor and several different ion channels, but it is uncertain to what degree it does this in vivo. All acylethanolamides are degraded by a fatty acylethanolamide hydrolase (FAAH) and FAAH-knock out mice have increased levels of ananadmide and other acylethanolamides and increased pain threshold. Acylethanolamides can also be degraded by some other hydrolases. Endogenous levels of anandamide and other acylethanolamides are low and can be increased several fold during tissue injury. It has been suggested that there exists an anandamide transporter responsible for the uptake of anandamide into cells before it is degraded by the FAAH enzyme that is located in the endoplasmic reticulum. However, a transporter protein has not been characterized and the concept of an uptake transporter for a lipophilic molecule is disputed.

2-AG is formed primarily from diacylglycerol, e.g., 1-stearoyl-2-arachidonoyl-glycerol, catalyzed by a Sn-1 specific diacylglycerol lipase and it is degraded by a monoacylglycerol lipase. The precursor, diacylglycerol, is known to be formed during receptor-stimulated turnover of inositol phospholipids where inositol-1,4,5-trisphosphate is also formed. Thus, this diacylglycerol formation occurs in the cell membrane where the diacylglycerol lipase also is located. It is generally accepted that 2-AG is formed in postsynaptic neurons upon activation of neuronal inositol phospho-lipids whose turnover depends on phospholipase Cb, whereupon it activates, in a retrograde fashion, the presynaptic CB1-receptor that subsequently results in the inhibition of neurotransmitter release (Figure 19.10). It is not exactly known how 2-AG travels through the aqueous fluid to reach the presynaptic neuron. This retrograde signaling can decrease the release of glutamate, GABA, acetyl-choline and other neurotransmitters. 2-AG is degraded by a monoacylglycerol lipase that is located in the presynaptic neuron. In this way 2-AG and the CB1-receptors may contribute to homosynaptic plasticity of excitatory synapses and heterosynaptic plasticity between excitatory and inhibitory contacts that is part of the basic mechanism in learning and memory. This retrograde control is also called as the depolarisation-induced suppression of inhibition (DSI) and depolarisation-induced suppression of excitation (DSE) for GABAergic and glutamatergic synapses, respectively.

Tetrahydrocannabinol Mechanism Action
FIGURE 19.10 Synaptic endocannabinoid formation during neurotransmitter release. PLC, phospholipase C; DAG, diacylglycerol; DAGL, diacylglycerol lipase; 2-AG, 2-arachidonoyl glycerol; CBj, cannabinoid receptor-1.

19.2.2 Cannabinoid Receptor 1

CBj-receptor belongs to the Class A rhodopsin-like family of GPCRs and it couples through Gi/o proteins negatively to adenylate cyclase and positively to mitogen-activated protein kinase. In addition, CB1-receptor can also couple to ion channels through the same G-proteins, positively to A-type and inwardly rectifying K+-channels and negatively to N-type and P/Q type Ca2+-channels. CBj-receptor is primarily localized in the brain where it is particularly abundant in cortex, hippocampus, amygdale, basal ganglia, cerebellum, and the emetic centers of the brain stem. CB1-receptor can also be found in lower abundance in spleen, tonsils, white blood cells, gastrointestinal tissue, urinary bladder, adrenal gland, heart, lung, and reproductive organs. CBj-receptor may form homodimeric complexes, and heterodimeric complexes with m-opioid receptor or dopamine D2 receptor. Anandamide and 2-AG may reach the receptor from the lipid phase of the membrane and not from the aqueous site.

In vitro CBj-receptor seems to have constitutive activity or it may be under endocannabinoid stimulatory tone. Several antagonists have also been shown to be inverse agonists but it is unclear whether this has any in vivo significance.

Knock out mice that are lacking the receptor protein have been generated. They are generally healthy and fertile with no apparent gross anatomical defects. However, they do have a number of abnormalities, e.g., the dysregulation of the hypothalamus-pituitary-adrenal axis suggesting a role of endocannabinoids in modulating neuroendocrine functions. Furthermore, they have a lighter and leaner body phenotype and seem to have higher energy expenditure. In a number of experimental settings these mice do also behave differently, e.g., in studies of alcohol dependence. CBj-receptor knock out mice do not show hypothermia, hypoalgesia, and hypoactivity in response to THC. There is evidence of splice variation of CBj-receptor of very low abundance but their biological significance is unclear. Activation of GPR55 can be blocked by the CB receptor antagonist Rimonabant (refer to Figure 19.12), but not by the CB2-receptor antagonist SR 144528 (refer to Figure 19.13).

19.2.3 Cannabinoid Receptor 2

CB2-receptor also belongs to the 7TM-receptors and the human CB2-receptor has 44% homology with human CB1-receptor. It seems to couple to the same G-proteins and signaling pathways as does CB1-receptor. However, CB2-receptor is found primarily in the spleen, immune cells, tonsils, and brain microglial cells where its expression can be induced by transformation of microglia to mac-rophage-like cells. Furthermore, CB2-receptor is found in osteoblasts, osteocytes, and osteoclast where it plays a critical role in the maintenance of normal bone mass. CB2-receptor knock out mice appear healthy and fertile, but they have low-bone mass. In animal models of pain and inflammation, these CB2-receptor knock out mice have indicated a clear role for this receptor in modulating acute pain, chronic inflammatory pain, postsurgical pain, cancer pain, and pain associated with nerve injury.

19.2.4 Other Cannabinoid Receptors

Since 2-AG and especially anandamide have a number of pharmacological effects that cannot be fully explained by activation of the known cannabinoid receptors, it has been suggested that there may exist more cannabinoid-like receptors. One such receptor may be the recently described GPR55 that is found in the brain and that can potently be activated by both THC, 2-AG, anandamide and the cannabinoid receptor agonist CP 55,940 (refer to Figure 19.12). Another cannabinoid receptor agonist, WIN 55,212-2, can however not activate GPR55. A number of agonists (e.g., CP 55,940, WIN 55,212-2, O-1812) and antagonists (e.g., Rimonabant, AM251, LY 320125) (refer to Figure 19.12) have been developed. The biological significance of GPR55 is at present not known. Especially anandamide can activate a number of other receptors and ion channels as mentioned above and this may add to the confusion regarding the existence of additional cannabinoid receptors.

19.2.5 Therapeutic Use and Potential

THC in capsules (Marinol, Solway Pharmaceutical) is used for treatment of nausea and vomiting that are common side effects of chemotherapy, and for stimulation of appetite in AIDS patients. In Canada, THC in the form of an extract of cannabis sativa called Sativex (GW Pharmaceuticals) is provided as a mouth spray for multiple sclerosis patients who can use it to alleviate neuropathic pain and spasticity. Sativex also contains other cannabinoids including cannabidiol that may add to its function. Medicinal cannabis, i.e., marihuana or hashish prescribed by a doctor for increased well being and alleviation of pain, spasticity, or loss of appetite by patients having AIDS, cancer, and multiple sclerosis has been approved in the Netherlands and there is a strong lobby for approval in certain states of United States.

Sanofi-Aventis has a CBj-antagonist (SR141716A, Rimonabant, trade name Acomplia, refer to Figure 19.12) on the European market for the treatment of obesity (body mass index above 30). Large clinical trials have shown that Rimonabant induces a weight loss of ~10% of initial body weight within 1 year. The discontinuation of Rimonabant treatment results in the regain of lost weight. It is not clear how large a fraction of the weight loss effect of rimonabant is mediated by CB1-receptors in the CNS relative to CBj-receptors in the peripheral organs like the liver and adipose tissue. Side effects are increased frequency of nausea and mood disturbances like depression. Rimonabant is not approved in United States. A CBj-receptor antagonist that does not cross the blood-brain barrier may possibly also have beneficial effects on energy metabolism.

Emerging evidence points to a possible participation of the endocannabinoid system in the regulation of the relapsing phenomenon of drug abuse in animal models. CB1-receptor seems to be important in drug as well as cue-induced reinstatement of drug seeking behavior. Stimulation may elicit relapse not only to cannabinoid seeking but also to cocaine, heroin, alcohol, and metham-phetamine, and this effect is significantly attenuated in animal experiments by pretreatment with CB1-receptor antagonists.

Potential clinical application involves drugs that can increase or decrease endocannabinoid levels (i.e., FAAH-inhibitors and monoacylglycerol-lipase inhibitors or diacylglycerol-lipase inhibitors, respectively) or serve as agonists/antagonists for the two cannabinoid receptors (Table 19.3). Thus the potential is large but so is also the risk for side effects since the endocannabinoid system appears to be so ubiquitous.

TABLE 19.3

Potential Clinical Applications of the Endocannabinoid System

Therapeutic Target

Clinical Conditions

Alzheimer's disease

AIDS and Cancer Appetite stimulation and inhibition of nausea and vomiting

Cancer

Inhibition of growth, angiogenesis, and metastasis

Inflammatory bowel diseases Stimulation of gastrointestinal mobility and reduction of inflammation

Multiple sclerosis Inhibition of tremors and spasticity

Obesity X

Weight loss

Osteoporosis Inhibition of bone loss

Parkinson's disease

Pain

Chronic, inflammatory, and neuropathic

CBrAgonists CB2-Agonists CB,-Antagonists FAAH-Inhibitors

Note: X, based on preclinical data from corresponding animal models; #, based on data from human studies or clinical trial.

19.2.6 FAAH-Inhibitors and Anandamide Uptake Inhibitors

As discussed above it is not clear whether an anandamide transporter exists and several compounds believed to be uptake inhibitors have turned out to be inhibitors of FAAH. URB597 is shown as an example of a experimental FAAH-inhibitor (Figure 19.11).

URB597

FIGURE 19.11 Inhibitor of FAAH, the enzyme degrading anandamide and other acylethanolamides.

19.2.7 CB-,-Receptor Agonist/Antagonist

Besides THC, anandamide, and 2-AG, experimentally used synthetic CBj-receptor agonists involves CP55,940 and the aminoalkylindole WIN55,212-2 that target both the cannabinoid receptors. More selective CBj-receptor agonists are arachidonic acid derivatives like O-1812. Selective CBj-receptor antagonist involves rimonabant that are in clinical use and the experimental compounds AM251 and LY 320135 (Figure 19.12).

CP 55,940

WIN 55,212-2

CP 55,940

WIN 55,212-2

o-1812

o-1812

MeO'

Rimonabant

Cl AM251

FIGURE 19.12 Agonists and antagonists for CBj-receptor.

Rimonabant

Cl AM251

FIGURE 19.12 Agonists and antagonists for CBj-receptor.

MeO'

LY 320135

19.2.8 CB2-Receptor Agonist/Antagonist

THC, 2-AG, and to a lesser extent anandamide activates the CB2-receptor. Selective synthetic agonists include JHW 133 and GW 405833 (partial agonist) that are used experimentally (Figure 19.13). SR 144528 is a selective CB2-receptor antagonist.

cl jhw 133

gw 405833

sr 144528

FIGURE 19.13 Agonists and antagonists for CB2-receptor.

cl jhw 133

gw 405833

sr 144528

FIGURE 19.13 Agonists and antagonists for CB2-receptor.

19.2.9 Diacylglycerol Lipase Inhibitors

There are no really specific inhibitors of diacylglycerol lipase, but RHC 80267, tetrahydrolipstatin (Orlistat), and O-3841 have been used in experimental settings (Figure 19.14).

FIGURE 19.14 Inhibitors of diacylglycerol lipase, the enzyme generating 2-arachidonoylglycerol.

19.2.10 Monoacylglycerol Lipase Inhibitors

URB602 (Figure 19.15) is used as an inhibitor of monoacylglycerol lipase in experimental settings, but is not a very specific inhibitor.

As can be seen from the description of the enzyme inhibitors above, this research area is at the very early stage of drug discovery, but if potent and specific inhibitors can be found, there are numerous clinical settings where such enzyme inhibitors may be alternatives to cannabinoid receptor agonists or antagonists.

FIGURE 19.15 Inhibitor of monoacylglycerol lipase, the enzyme degrading 2-arachidonoylglycerol.

FIGURE 19.14 Inhibitors of diacylglycerol lipase, the enzyme generating 2-arachidonoylglycerol.

o-3841

o-3841

FIGURE 19.15 Inhibitor of monoacylglycerol lipase, the enzyme degrading 2-arachidonoylglycerol.

urb602

urb602

FURTHER READINGS

Corbett, A. D., Henderson, G., McKnight, A. T., and Paterson, S. J. (2006) 75 years of opioid research: The exciting but vain quest for the Holy Grail. Br. J. Pharmacol. 147, S153-S162. Di Marzo, V. and Petrosino, S. (2007) Endocannabinoids and the regulation of their levels in health and disease.

Curr. Opin. Lipidol. 18, 129-140. Fattore, L., Spano, M. S., Deiana, S., Melis, V., Cossu, G., Fadda, P., and Fratta, W. (2007) An endocannabinoid mechanism in relapse to drug seeking: A review of animal studies and clinical perspectives. Brain Res. Rev. 53, 1-16.

Hansen, H. S., Petersen, G., Artmann, A., and Madsen, A. N. (2006) Endocannabinoids. Eur. J. Lipid Sci. Technol. 108, 877-889.

Kaine, B. E., Svensson, B., and Ferguson, D. M. (2006) Molecular recognition of opioid receptor ligands.

AAPS J. 8 (1), Article 15, E126-E137. Matias, I. and Di Marzo,V. (2007) Endocannabinoids and control of energy balance. Trends Endocrinol. Metab. 18, 27-37.

Pacher, P., Batkai, S., and Kunos, G. (2006) The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol. Rev. 58, 389-462. Pertwee, R. G. (editor) (2005) Cannabinoids. Handbook of Experimental Pharmacology, Vol. 168. SpringerVerlag, Berlin, Heidelberg.

Portoghese, P. S. (2001) From models to molecules: Opioid receptor dimers, bivalent ligands and selective opioid receptor probes. J. Med. Chem. 44(14), 2260-2269. Waldhoer, M., Bartlett, S. E., and Whistler, J. L. (2004) Opioid receptors. Ann. Rev. Biochem. 73, 953-990.

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