S.J. Paterson

The binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties

SummaryFour groups of narcotic analgesic drugs have been assessed for their opiate activities by using three binding assays and three pharmacological bioassays. In the binding assays, their inhibition constants (KI, nM) were determined against the binding of the μ-ligand, [3H]-[d-Ala2,MePhe4, Gly-ol5]enkephalin, of the δ-ligand, [3H]-[d-Ala2,d-Leu5]enkephalin and of the ϰ-ligand, [3H]-(±)-ethylketazocine after suppression of μ- and δ-binding by 100 nM of the unlabelled μ-ligand and 100 nM of the unlabelled δ-ligand. The pharmacological agonist or antagonist activities were assayed on the guinea-pig ileum, mouse vas deferens and rat vas deferens.The first group of compounds were pure agonists in all three pharmacological bioassays. The majority of the compounds showed preference to μ-binding but phenazocine and particularly etorphine had also high affinities to the δ- and ϰ-binding sites.The second group consisted of N-allyl and N-cyclopropylmethyl homologues of the morphine, 3-hydroxymorphinan and normetazocine series which had agonist and antagonist activities in the guinea-pig ileum and mouse vas deferens but were pure antagonists in the rat vas deferens. In the binding assays, μ-binding and ϰ-binding were prominent.The third group was made up by the ketazocine-like compounds which in the guinea-pig ileum and mouse vas deferens were pure agonists and in the rat vas deferens pure antagonists. The binding spectrum showed particularly high binding to the ϰ-binding site.The fourth group was the antagonists which were devoid of agonist activity with the exception of diprenorphine and Mr 2266 which had retained some agonism. The binding spectrum showed considerable variation, naloxone in low concentration being a selective μ-antagonist, Mr 2266 having high affinities to the μ- and ϰ-binding sites and diprenorphine having considerable affinities to the μ-, δ- and ϰ-binding sites.Since each of the four groups of compounds, whether pure agonists, agonist-antagonists, ketazocine-like drugs or pure antagonists, shows independent varittions in the affinities to the μ- and ϰ-binding sites, their different pharmacological behaviour cannot be solely due to difference in the binding spectra.

CHARACTERIZATION OF THE k‐SUBTYPE OF THE OPIATE RECEPTOR IN THE GUINEA‐PIG BRAIN

1 In homogenates of guinea‐pig brain, the characteristics of the binding of [3H]‐ethylketazocine, an agonist for the putative k‐receptor, were determined by estimation of the affinity and capacity of binding, by competitive inhibition for the binding site by unlabelled ligands and by selective protection of the binding site from alkylation by phenoxybenzamine. 2 At 25°C the maximum number of binding sites for [3H]‐ethylketazocine was about 14pmol/g fresh brain, of which about 50% were high affinity sites. 3 In competition experiments, the high affinity binding of [3H]‐ethylketazocine to the k‐binding site was readily displaced by several k‐agonists but not by the selective μ‐ligand, D‐Ala2, MePhe4, Glyol5‐enkephalin or the selective δ‐ligand, D‐Ala2, D‐Leu5‐enkephalin. In contrast, the k‐agonists tested so far exhibit a high degree of cross‐reactivity with the μ‐binding site but somewhat less with the δ‐binding site. Similar specificities were observed in protection experiments. 4 The approximate proportions of the three subtypes of opiate receptor in the guinea‐pig brain are 25% μ‐binding sites, 45% δ‐binding sites and 30% k‐binding sites. 5 The endogenous opioids, Met‐enkephalin, Leu‐enkephalin and porcine β‐endorphin have only a low affinity for the k‐binding site.

Selectivities of opioid peptide analogues as agonists and antagonists at the δ-receptor

1 The endogenous opioid ligands interact with more than one of the μ‐, δ‐ and κ‐binding sites. By the use of binding assays and bioassays, enkephalin analogues have been assessed for their selectivity for binding at the (5‐binding site and for their agonist and antagonist activities at the δ‐receptor. The electrically‐induced contractions of myenteric plexus‐longitudinal muscle preparations of the guinea‐pig ileum were inhibited by μ‐ and κ‐receptor ligands. Inhibitions were seen with μ‐, δ‐ and κ‐receptor ligands in the mouse vas deferens, mainly with μ‐receptor ligands in the rat vas deferens and only with κ‐receptor ligands in the rabbit vas deferens. 2 From observations on a considerable number of [Leu5] enkephalin analogues, it has been concluded that [d‐Pen2, d‐Pen5] enkephalin and [d‐Pen2, l‐Pen5] enkephalin are the most selective δ‐agonists and that N,N‐diallyl‐Tyr‐Aib‐Aib‐Phe‐Leu‐OH is the most selective antagonist (Aib = α‐aminoisobutyric acid). The binding of these peptides at the δ‐site is 99% of the total binding. As to potency, the agonists are superior to the antagonists.

COMPARISON OF THE BINDING CHARACTERISTICS OF TRITIATED OPIATES AND OPIOID PEPTIDES

1 Binding assays on homogenates of guinea‐pig brain showed that the maximal number of binding sites was different for different tritiated ligands interacting with the opiate receptors. 2 At 25°C the binding capacity of morphine or dihydromorphine was only about 3 pmol/g fresh brain whereas etorphine and d‐Ala2‐l‐Leu5‐ and d‐Ala2‐l‐Met5‐enkephalin amide had capacities of 13 to 15 pmol/g brain. d‐Ala2‐d‐Leu5‐enkephalin had an intermediate capacity of about 6 pmol/g brain. 3 The binding capacities of the natural methionine‐ and leucine‐enkephalins measured at 0°C were 5 to 6 pmol/g brain. At this temperature, the binding capacity of dihydromorphine, d‐Ala2‐d‐Leu5‐enkephalin and of the two enkephalin amides was only slightly lower than at 25°C. 4 In assays in which unlabelled ligand competed with the same labelled ligand, the inhibition constants (K1) were equal to or not more than twice as large as the equilibrium dissociation constant (Kd) determined in saturation assays. In contrast, the K1 of unlabelled dihydromorphine against [3H]‐d‐Ala2‐d‐Leu5‐enkephalin or of unlabelled d‐Ala2‐d‐Leu5‐enkephalin against [3H]‐dihydromorphine were about 20 times higher than the respective Kd values. 5 When for a given compound the ratio of the K1 value against [3H]‐d‐Ala2‐d‐Leu5‐enkephalin to the K1 value against [3H]‐dihydromorphine (discrimination ratio) is calculated, a high value indicates selectivity in favour of the μ‐receptor and a low value selectivity in favour of the δ‐receptor. The most selective μ‐agonist known so far is normorphine with a discrimination ratio of 70 and the most selective δ‐agonist is d‐Ala2‐d‐Leu5‐enkephalin with a ratio of 0.11. The selectivity of the known antagonists is in favour of the μ‐receptor, since their discrimination ratios are larger than 1, varying between 10 for naloxone and 4 for Mr 2266.

Opioid Peptides and their Receptors

The three agonists, methionine-enkephalin, leucine-enkephalin and beta-endorphin have different pharmacological patterns. It may be of particular importance that they vary in their relative affinities to the enkephalin and naltrexone binding sites in the brain; the former are probably related to delta-receptors prevalent in the mouse vas deferens and the later to mu-receptors prevalent in the guinea-pig. It is possible that mu-receptors are more important for the mediation of analgesic effects than delta-receptors. An understanding of the pharmacokinetics of the opioid peptides will be of basic importance for the design of enkephalin analogues suitable for use as analgesics in man.

Characterization of opioid receptors in nervous tissue

The concept that endogenous opioid peptides interact with at least two different receptor sites developed from several experimental approaches. First, when the peptides were assayed by their effects on two pharmacological and two binding models, the rank order of activity differed in these four systems. Secondly, naloxone had a smaller antagonist effect on δ-receptors in the mouse vas deferens than on its μ-receptors. Thirdly, the enkephalins and morphine each occupied less than half of the total number of sites available in brain homogenates. Fourthly, cold ligands of the δ-type protected the binding of tritiated δ-agonists better than that of μ-agonists, and vice versa. Finally, tritiated ethylketazocine binds to κ-receptor sites in homogenates of guinea-pig brain. It is readily displaced by etorphine, which binds uniformly to μ-, δ- and κ-receptors, but only by very high concentrations of μ- or δ-agonists. An interesting phenomenon is the potentiation of activity when an enkephalin analogue is conjugated to tobacco mosaic virus by the group of R. Schwyzer.

The use of [3H]-[D-Pen2,D-Pen5]enkephalin as a highly selective ligand for the δ-binding site

1 The characteristics of the binding of [3H]‐[d‐Pen2,d‐Pen5]enkephalin were determined in homogenates of guinea‐pig and rat brain. 2 In the guinea‐pig, the maximum binding capacity for [3H]‐[d‐Pen2,d‐Pen5]enkephalin was 4.19 pmol g−1 and the KD 1.61 nm. In the rat, the corresponding values were 2.47 pmol g−1 and 5.42 nm. In both species, the maximum binding capacity and the affinity were not altered when μ‐binding was suppressed with [d‐Ala2, MePhe4, Gly‐ol5]enkephalin. 3 The μ‐agonists, [d‐Ala2, MePhe4, Gly‐ol5]enkephalin and morphine, displaced a small portion of the binding of [3H]‐[d‐Pen2,d‐Pen5]enkephalin with high affinities.

Pre-incubation of guinea-pig myenteric plexus with β-funaltrexamine: discrepancy between binding assays and bioassays

1 The acute effects of β‐funaltrexamine and the effects of pre‐incubation with this compound were examined in five in vitro assay tissues and in selective binding assays in homogenates of guinea‐pig brain and myenteric plexus. 2 In competitive displacement assays with selective ligands, β‐funaltrexamine had highest affinity for the μ‐binding site in the myenteric plexus and brain of guinea‐pig. Its affinity for the k‐site was about 15% of that for the μ‐site. 3 Pre‐incubation of the assay tissues with β‐funaltrexamine caused an increase in the IC50 values of μ‐and δ‐receptor agonists but not of k‐agonists. Although in bioassays on the myenteric plexus‐longitudinal muscle preparation of the guinea‐pig, the IC50 value of the μ‐receptor ligand [D‐Ala2, MePhe4, Gly‐ol5] enkephalin was increased up to 124 fold, its binding at the μ‐site in homogenates of the preparation was not affected by this treatment. 4 These findings indicate that the effects of pre‐incubation with β‐funaltrexamine on agonist potency of the μ‐receptor ligand are due to an interference with the coupling mechanism between the μ‐binding site and the effector system.

Radioligands for Probing Opioid Receptors

The three endogenous opioid precursors of almost 30000 Da are pro-opiocortin, proenkephalin and prodynorphin. Pro-opiocortin contains beta-endorphin, melanotropins and ACTH. Proenkephalin yields one [Leu5]enkephalin, three [Met5]enkephalins, one [Met5] enkephalyl-Arg-Arg-Val-NH2 (metorphamide or adrenorphin), one [Met5]enkephalyl-Arg-Gly-Leu and one [Met5]enkephalyl-Arg-Phe. [Leu5]enkephalin is common to all fragments of prodynorphin; its carboxyl extension by Arg-Lys leads to alpha- and beta-neo-endorphin and its carboxyl extension by Arg-Arg gives two dynorphins A and B of 17 and 13 amino acids, respectively. Another endogenous peptide is dynorphin A (1-8). The three main opioid binding sites are mu, delta and kappa. Their analysis has been facilitated by the synthesis of analogues of peptides and non-peptide compounds, which have selective agonist or antagonist action at only one site. The various physiological roles of the three types of the opiate receptor have so far not been sufficiently investigated.

Effects of cations on binding, in membrane suspensions, of various opioids at μ‐sites of rabbit cerebellum and κ‐sites of guinea‐pig cerebellum

1 At the μ‐sites of rabbit cerebellum, NaC1, LiC1, KCl, choline chloride and MnCl2 were tested for potentiation and inhibition of the binding of several opioids. Naloxone, (–)‐bremazocine and diprenorphine are μ‐antagonists in pharmacological assays and their binding is potentiated by the lower concentrations and inhibited by the higher concentrations of NaCl. The binding of the agonists [3H]‐[d‐Ala2, MePhe4, Gly‐ol5]enkephalin and [3H]‐dihydromorphine is inhibited. MnCl2/potentiates the binding of the agonist [3H]‐[d‐Ala2, MePhe4, Gly‐ol5]enkephalin but not the binding of the antagonists. The thresholds of inhibition and slopes of the dose‐response curves for inhibition by MnCl2 and LiCl vary. This finding may indicate that potentiating effects of MnCl2 and LiCl are masked by simultaneous inhibition. 2 At the κ‐sites of guinea‐pig cerebellum, NaCl, KCl and MnCl2 inhibit the binding of [3H]‐dynorphin A (1–8), [3H]‐dynorphin A (1–9), [3H]‐(–)‐bremazocine, [3H]‐tifluadom, and [3H]‐diprenorphine. NaCl also causes a small potentiation of the binding of [3H]‐diprenorphine, which is a κ‐agonist in the guinea‐pig myenteric plexus but a κ‐antagonist in the rabbit vas deferens. The slopes of the inhibitory dose‐response curves and the thresholds of inhibition vary with the different ligands. Therefore some potentiating effects may have been masked. 3 The results support the view that NaCl, and perhaps LiCl, but not KCl and choline chloride, potentiate the binding of μ‐antagonists but not the binding of μ‐agonists. It is not yet possible to decide whether, at the κ‐site, there is a similar differentiation of the binding of agonists and antagonists.