MU OPIOID RECEPTORS IN PAIN MANAGEMENT

Abstract

Most of the potent analgesics currently in use act through the mu opioid receptor. Although they are classified as mu opioids, clinical experience suggests differences among them. The relative potencies of the agents can vary from patient to patient, as well as the side-effect profiles. These observations, coupled with pharmacological approaches in preclinical models, led to the suggestion of multiple subtypes of mu receptors. The explosion in molecular biology has led to the identification of a single gene encoding mu opioid receptors. It now appears that this gene undergoes extensive splicing, in which a single gene can generate multiple proteins. Evidence now suggests that these splice variants may help explain the clinical variability in responses among patients.

Introduction

Opiates have been used to manage pain for centuries. Opium, which contains a number of alkaloids including morphine and codeine, was eaten, smoked and used as a tincture. Following isolation, morphine and codeine, are used individually. Thousands of analogs have been generated in an effort to avoid some of the difficulties encountered with opiate use, such as respiratory depression, sedation and constipation. Most of these agents act through mu opiate receptors, as defined by the selectivity of their binding for the three classes of opioid receptors that have been cloned. This is to be expected since the structure of morphine is used as a template for the synthesis of most of them. Clinicians have long observed that patients can have markedly varying responses to mu opioids. Some patients may encounter severe side effects such as nausea and vomiting with one agent, while having no difficulties at all with the other. For example, it is not uncommon to find patients with severe nausea/vomiting from morphine tolerating methadone without problems. In addition, the analgesic activity of the opioids also can vary among patients. One patient may find one mu opioid to be quite effective while a different drug may not work anywhere near as well. However, the same drug may not be the best for all patients. This variability among patients has led to accepted paradigm that analgesics need to be individualized. Opioid Rotation also illustrates differences among the mu opioids. In Opioid Rotation patients highly tolerant to one opioid a switch to a different opioid, often restors the analgesic effectiveness and sensitivity.^1,2 While all mu drugs display tolerance with prolonged dosing, cross tolerance is incomplete. That is, the tolerance to the second drug may not be as pronounced as the first, explaining why the switch is effective. All these features have raised questions about the mechanisms of these drugs, specifically how a single mu receptor could mediate these actions.

EARLY STUDIES OF MU OPIATE RECEPTOR AND MULTIPLICITY

The first opioid receptor identified in binding assays in 1973^3-5 were mu receptors. A receptor for the opiates had been predicted years earlier from a variety of traditional pharmacological approaches and the rigid structure activity studies of morphine and related chemical scaffolds.^6-8 Since the studies used morphine-related probes, it is not surprising that the receptors were mu. However, several other families of opioid receptors have also been identified (Table 1). In vivo studies led Martin to propose the existence of kappa receptors, in addition to the mu receptors.^9,10 Delta receptors, which are selective for the enkephalins, were identified¹¹ after the isolation and identification of endogenous opioids.^12-14 Indeed, it turns out that the endogenous ligand for the kappa receptors is another endogenous opioid, dynorphin A.^15,16 Work is in progress developing new selective delta and kappa opioids for clinical use, but for the most part the drugs available clinically are mu. There are several mixed agonists/antagonists that interact with both mu and kappa receptors, but they have limited use.

Table 1.

Classification of the opioid receptor families

Receptor		Drugs	Analgesia	Physical dependence	Dysphoria	Respiratory Depression	Gene
Mu		Morphine	Yes	Yes	No	Yes	MOR-1
Kappa	Kappa1	Dynorphin A U50,488H	Yes	No	Yes	No	KOR-1
	Kappa2	Ethylketocyclazocine	Yes	No	Yes	No	KOR-1/DOR-1
	Kappa3	Naloxone Benzoylhydrazone Levorphanol	Yes	No	No	No	See below
Delta		Enkephalin DPDPE	Yes	No	?	No	DOR-1

Three major families of opioid receptors have been proposed. Subtypes of all have been proposed using pharmacological and molecular biological approaches. The genes for all have been established, except for kappa₃. This subtype, which is important in the analgesic actions of a variety of opioid analgesics, including levorphanol and nalbuphine, is most likely a heterodimer involving one of the truncated 6TM MOR-1 splice variants and an unknown second receptor.⁶⁴ However, this remains to be confirmed.

The first suggestion that there may be more than one class of mu receptors coming from receptor binding studies in the mid 1970s that revealed a novel site with unusual characteristics.¹⁷ Although mu drugs like morphine labeled it with high potency, confirming its classification as a mu receptor, its ability to bind other drugs clearly distinguished it from the original mu receptor seen in receptor binding assays. The new site was termed mu₁ while the original morphine-selective site was termed mu₂.¹⁸ Understanding the pharmacological significance of these two mu receptors was facilitated by the development of selective antagonists that could selectively block the mu₁ site and not the mu₂ site.^19-21 The naloxonazine-sensitive site corresponds to the mu₁ site while the naloxonzine-insensitive mu site is mu₂. When given in vivo, these antagonists blocked morphine analgesia, but not respiratory depression or the inhibition of gastrointestinal transit or many of the signs of physical dependence.^22-27 Together, these studies implied that drugs such as morphine were acting through more than one mu receptor.

MOLECULAR BIOLOGY OF MU OPIATE RECEPTORS

The opiate receptors were first cloned in 1992 with the isolation of the delta receptor DOR-1 by two independent groups.^28,29 This initial report confirmed the existence of the receptors within the G-protein coupled receptor family, one of the largest families of receptors known. Based upon these structures, the other members of the opiate receptor family were quickly cloned, including the mu^30-32 and the kappa₁ receptors.^33-35 Soon after, yet another receptor was identified with a strong homology to the opiate receptors^36-41 but which was subsequently found to have a previously unknown peptide as its endogenous ligand, orphanan FQ/nociceptin.^42,43

The most important class of opiate receptors that clinically remains is the class of mu receptors. Most of the drugs used clinically act through these receptors and understanding their actions is important in defining their pharmacology. Pharmacological studies suggested more than one class of mu opiate receptor. This has now been confirmed at the molecular level. Although only a single mu receptor gene has been reported, the receptor undergoes extensive alternative splicing to generate a host of splice variants (Fig 1).

Figure 1. Schematic of the human mu opioid receptor (OPRM1) gene structure and alternative splicing.

A. Gene structure of the human OPRM1 gene. Exons and introns are showed by boxes and horizontal lines, respectively. Intron size is indicated below the introns as kilobases (kb). Promoters are showed by arrows.

B. Alternatively spliced variants of the human OPRM1 gene. Exon composition for each alternatively spliced variant was showed by appropriate exon boxes. The lines between exons are introns that are spliced out during splicing. Translation start and stop points are shown by bars below and above exon boxes, respectively. From the literature.⁶⁸

Following the cloning of MOR1 much effort was placed on confirming its pharmacological relevance. The first studies involved using an antisense approach in which very short sequences of antisense targeted specific sequences of the mRNA, leading to its degradation.^44,45 These initial reports quickly established that MOR-1 was critical in the production of morphine analgesia. However, the antisense approach can be used more extensively to perform antisense mapping to assess the presence of specific exons within the mRNA responsible for making a protein.^46-48 Certain exon targets were important in morphine analgesia, including exons 1 and 4. However, antisense probes targeting exons 2 and 3 had little effect on morphine even though they significantly diminished the activity of heroin and morphine-6β-glucuronide. Thus, at the molecular level there does appear to be receptor differences for these mu opiates.

The association of MOR-1 with morphine analgesia was further confirmed by the generation of several different knock-out animals in which the MOR-1 gene was disrupted. In all of these different knockout animals, which targeted exons 1, 2 and/or 3, morphine lost all activity.^49-52 However, in one exon1 knockout mouse, heroin and M6G were still analgesic. This was quite confusing and it was initially suggested that these drugs were activating alternative opioid receptors, such as delta or kappa₁. This possibility was eliminated with the finding that the residual heroin and morphine-6β-glucuronide analgesia remained in a triple knockout mouse, which had the same disruption of exon 1 or MOR-1 and additional disruptions of the delta (DOR-1) and kappa₁ (KOR-1) receptors. What made this particular knockout mouse interesting was the continuous presence of a number of MOR-1 splice variants.⁵² Some of these, under the control of the exon 11 pomoter, do not contain exon 1, perhaps explaining their continuous expression.

The splice variants associated with the mu opiate receptor can be separated into two categories based upon their promoter. The gene encoding MOR-1 is quite complex with two distinct promoters. The primary one is associated with exon 1^53,54 and generates a number of variants that are spliced at the 3’ end in mice, rats and humans (Fig 1).^55-62 These are interesting from several perspectives. They are full length seven transmembrane domain G-protein coupled receptors. Structurally, they differ only at the very tip of the intracellular C-terminus. The initial opiate receptor cloned, MOR-1, contains twelve amino acids at the very tip of the C-terminus encoded by exon 4 (Fig 2). In the other C-terminus spliced variants, these twelve amino acids are replaced with different sequences of varying lengths. However, these differences are quite limited and the basic structure of the receptor with its seven transmembrane domains and binding pocket are identical among them (Fig. 2). Thus, these splice variants share a common binding pocket and they all show high affinity and selectivity for mu opiates, as shown in receptor binding assays. Although the splicing has little effect upon the ability of the mu opioids to bind to the receptor, the small structural differences do impact the ability of mu opioids to activate the different splice variants. These differences in the activation profiles of various mu opioids for the receptor variants may help explain the subtle, but clear, differences among the various mu opioids seen clinically.

Fig 2. Schematic of the human OPRM1 carboxyl terminal splice variants’ structures and amino acid sequences predicted from downstream exons of exon 3.

The common receptor structure of all the human carboxyl terminal splice variants is indicated by heavy lines across the lipid bilayer of the cell membrane. Splice junctions between exons are indicated by arrows. The amino acid sequences predicted from different downstream exons are shown on the right along with the indicated splice variants. Potential phosphorylation sites are indicated by the italic letters and the following symbols on the top of the letters. △: cAMP- and cGMP-dependent protein kinase phosphorylation site; ○: tyrosine kinase phosphorylation site; *: casein kinase II phosphorylation site.

The second set of variants are associated with exon 11, which is located approximately 30 kilobases upstream of exon 1.^57,63 These variants include a number with quite unique structures. Unlike the exon 1-associated variants, which all encode full length, 7 transmembrane domain receptors, many of the exon 11-associated variants generate truncated variants with only 6 transmembrane domains. Despite their unique structure, there is much evidence now to suggest that they are pharmacologically important. This derives from studies on mice in which all the exon 11-containing variants are lost due to disruption of this region of the gene (Table 2).⁶⁴ Interestingly, morphine analgesia was not affected. However, the analgesic actions of heroin and the morphine metabolite morphine-6β-glucuronide were significantly reduced. Looking at the drugs, it is apparent that the analgesic actions of mu opioids can be differentiated at the molecular level.

Table 2.

Opioid analgesia in MOR-1 knockout mice

	ED50 Value (mg/kg, s.c.)
	Exon-1 KO			Exon-11 KO
Drug	WT	KO	Shift	WT	KO	Shift
Morphine	5.0	>100	>20	1.6	2.6	1.6
Methadone				1.5	1.6	1.2
M6G	3.8	8.5	2.3	0.92	19.3	21
Heroin	1.2	4.0	2.7	0.58	3.2	6
Fentanyl				0.024	0.230	10

Wildtype (WT) and knockout (KO) mice with a disruption of exon 1 of MOR-1 were tested with the indicated drug and their ED₅₀ values determined.⁵² Other groups of mice were generated with a disruption of exon 11. Wildtype (WT) and knockout (KO) mice were tested with the indicated drug and their ED₅₀ values determined.⁶⁴

THE FUTURE

Pain management remains an art, dependent upon the individualization of care. Not all patients can be managed with the same drugs. Indeed, the complexity of the clinical responses has long been evident. Many factors may play a role, but evidence is accumulating that much of this variability may result from the complexity of mu receptors. Early work from our laboratory proposed two subtypes, mu₁ and mu₂, based upon selective antagonists.^18,20,65 Studies showing differences between morphine and morphine-6β-glucuronide further implied a third subtype.^48,66,67 However, the molecular biology of the mu opioid receptor gene reveals the existence of dozens of mu receptor splice variants. Correlating these splice variants to the pharmacologically defined receptors has proven difficult. However, it seems likely that they play an important role in why patients respond so differently. As our understanding progresses, the critical question is whether or not we can utilize this knowledge to develop novel and useful analgesic lacking the detrimental actions of the currently available drugs.