Alessandra Tavani

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.

Differential postnatal development of μ-, δ- and χ-opioid binding sites in mouse brain

: By selective labelling techniques together with analysis of saturation curves it is shown that the concentrations of the mu-, delta- and chi-binding sites increase during postnatal development particularly in the first few weeks after birth. There are little or no changes in affinity. The rate of development is different for each type of site. The most striking finding is that the development of mu- and chi-sites precedes that of delta-sites.

Interaction of U-69,593 with μ-, ∂- and k-opioid binding sites and its analgesic and intestinal effects in rats

The k-opioid compound U-69,593 was studied in rats in vitro in binding assays to assess its selectivity at the single types of opioid sites and in vivo to assess its analgesic activity and effect on intestinal propulsion. In vitro the U-69,593 inhibition curve of (/sup 3/H)-(-)-bremazocine binding suppressed at ..mu..- and delta-sites was biphasic and the inhibition constant (K/sub l/) at the high-affinity site (10-18nM) was two orders of magnitude smaller the K/sub l/ at the low-affinity site. The K/sub l/ at ..mu..- and delta-sites were respectively 3.3 and 8.5 ..mu..M. Thus (/sup 3/H)-(-)-bremazocine, suppressed at ..mu..- and delta-sites, may still bind more than one site, which U-69,593 might distinguish. In vivo U-69,593 i.p. prolonged the reaction time of rats on a 55/sup 0/C hot-plate and the dose of naloxone required to antagonize this effect was 40 times the dose that antagonized morphine-induced antinociception, suggesting the involvement of the k-receptor. In the intestinal transit test U-69,593 at doses between 0.5 and 15 mg/kg i.p. only slightly slowed intestinal transit of a charcoal meal in rats with no dose-relation; it partly but significantly antagonized morphine-induced constipation. These results suggest that the k-type of opioid receptor, with which U-69,593 interacts may inducemorexa0» analgesia, but has no appreciable role in the mechanisms of opioid-induced inhibition of intestinal transit in rats.«xa0less

Regional variations in binding capacities at μ-, δ- and κ-opioid sites in membrane suspensions from rabbit brain

Abstract The highest maximum binding capacities at the μ-sites of rabbit brain are in the striatum, with intermediate levels in the diencephalon, mesencephalon, cerebellum and cortex and low level in the pons-medulla and hippocampus. For the δ-site the highest maximum binding capacity is also in the striatum; there are almost equally low levels in the other brain regions. At the κ-sites the maximum binding capacities are highest in the diencephalon; there are intermediate levels in the cortex and striatum, and low levels in the mesencephalon, cerebellum, hippocampus and pons-medulla. The K D values lack reproducibility; there are no regional variations at the κ-site as estimated with [ 3 H](−)-bremazocine, but the possibility cannot be excluded that there are regional variations in the K D values for [ 3 H][D-Ala 2 , MePhe 4 , Gly-ol 5 ]enkephalin at the μ-site or for [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin at the δ-site. It may be important to use saturation analysis in future investigations of the distributions of the binding sites.

Antagonism by N-methyl levallorphan-methane sulphonate (SR 58002 C) of morphine-elicited acute and chronic central and peripheral effects

The peripheral activity of the quaternary narcotic antagonist N-methyl levallorphan-methane sulphonate (SR 58002 C) at opioid sites located in the periphery and in the central nervous system (CNS), was studied by different approaches in rats after subcutaneous injection (s.c.). Pretreatment with SR 58002 C 2,8 or 32 mg/kg s.c. 10, 50 or 110 min before buprenorphine consistently reduced buprenorphine in vivo binding only in the small intestinal longitudinal muscle with attached myenteric plexus (MP), whereas naloxone (1 mg/kg s.c.) 10 min before buprenorphine lowered buprenorphine binding in MP and brain (without cerebellum). Plasma levels were not altered by SR 58002 C or naloxone. The same doses of SR 58002 C injected 10, 50 or 110 min before morphine selectively antagonized the inhibition of transit of a charcoal meal along the small intestine (mainly a peripheral effect) induced by the agonist, but did not antagonize morphine-elicited analgesia in the hot-plate test (central effect). Naloxone (1 mg/kg s.c.) injected 10 min before morphine antagonized both agonist effects simultaneously. In morphine-dependent rats SR 58002 C (0.25, 1, 4 and 32 mg/kg s.c.) induced diarrhea, dose-dependently, in most animals within the first 30 min, while jumping, measured in the same rats, occurred in some animals, not dose-dependently, from 60 min on. Naloxone (1 mg/kg s.c.) induced both effects in most rats. These findings suggest that, although SR 58002 C probably penetrates the blood-brain barrier in some morphine-dependent rats, it discriminates peripheral and CNS opioid effects.

D-optimal design applied to binding saturation curves of an enkephalin analog in rat brain

The D-optimal design, a minimal sample design that minimizes the volume of the joint confidence region for the parameters, was used to evaluate binding parameters in a saturation curve with a view to reducing the number of experimental points without loosing accuracy in binding parameter estimates. Binding saturation experiments were performed in rat brain crude membrane preparations with the opioid mu-selective ligand [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO), using a sequential procedure. The first experiment consisted of a wide-range saturation curve, which confirmed that [3H]-DAGO binds only one class of specific sites and non-specific sites, and gave information on the experimental range and a first estimate of binding affinity (Ka), capacity (Bmax) and non-specific constant (k). On this basis the D-optimal design was computed and sequential experiments were performed each covering a wide-range traditional saturation curve, the D-optimal design and a splitting of the D-optimal design with the addition of 2 points (+/- 15% of the central point). No appreciable differences were obtained with these designs in parameter estimates and their accuracy. Thus sequential experiments based on D-optimal design seem a valid method for accurate determination of binding parameters, using far fewer points with no loss in parameter estimation accuracy.

Characterization of Opioid Binding Sites in Rat Spinal Cord

Binding sites were characterized in rat whole spinal cord crude membrane preparations using selective labelling techniques with multiple methods of mathematical analysis of experimental curves. Mathematical analysis of single [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (DAGO) saturation curves suggested binding of the [3H]-ligand at one site, while displacement curves of low concentrations of [3H]-DAGO with selective mu-ligands indicated the presence of high- and low-affinity sites. All the [3H]-DAGO curves processed simultaneously by LIGAND analysis showed the presence of high (27%) and low (73%) affinity components, with a total Bmax of 3.19 pmol/g tissue. Eighty percent of [3H]-[D-Ala2,D-Leu5] enkephalin (DADLE) binding was displaced by DAGO with high affinity, indicating that a high percentage of [3H]-DADLE binding was at mu-sites. Saturation curves of [3H]-DADLE after inhibition of mu-sites by unlabelled DAGO (delta-sites) were monophasic with non-linear fitting analysis and the Bmax was 0.90 pmol/g tissue. Most mathematical analysis of single saturation curves of [3H]-(-)-bremazocine indicated binding at more than one site. DAGO, DADLE, U-69,593 and PD 117302 displaced 0.15 nM of [3H]-(-)-bremazocine biphasically: the percentages of displacement calculated with the non-linear fitting program were respectively 50 (mu-sites), 64 ((mu + delta)-sites), 18 and 25 (kappa-sites). Haloperidol displaced [3H]-(-)-bremazocine only at microM concentrations. suggesting no binding at sigma-sites. In the presence of 225 nM of DAGO, DADLE displaced only 21% of [3H]-(-)-bremazocine 0.15 nM binding (delta-sites). Most mathematical analysis of saturation curves of [3H]-(-)-bremazocine, after inhibition of binding at mu- and delta-sites by DAGO and DADLE, still indicated binding at more than one site and the selective kappa-ligands U-69,593 and PD 117302 displaced [3H]-(-)-bremazocine in these experimental conditions. LIGAND analysis of saturation and inhibition curves of [3H]-(-)-bremazocine by U-69,593 and PD 117302 showed the presence of high (43%) and low (57%) affinity components, with a total Bmax of 2,73 pmol/g tissue. Thus in rat spinal cord there are at least two mu-sites bound by [3H]-DAGO which amount together to approximately 47% of total opioid sites, delta-sites bound by [3H]-DADLE amounting to approximately 13%, kappa-sites and other unknown sites (possibly a kappa-subtype) bound by [3H]-(-)-bremazocine, which together are approximately 40% of total opioid sites.

Binding Capacities and Affinities AT μ-, ∂- And k-Opioid Sites in Membrane Suspensions from Guinea-Pig Brain Regions

AbstractBinding capacities and affinities (KD) of ligands at μ-, ∂- and k-opioid binding sites were determined by selective labelling techniques together with analysis of saturation curves in seven regions of the guinea-pig brain. The k-sites predominated over the other sites in most regions and were 90% of the total in the cerebellum; binding capacities at ∂-sites were highest in the cortex, intermediate in the cerebellum, striatum and mesencephalon and lowest in the diencephalon, hippocampus and pons-medulla. At the μ-sites, binding capacities were highest in the diencephalon and mesencephalon, with intermediate levels in the pons-medulla, cortex and striatum, and low levels in the hippocampus and cerebellum. The highest binding capacity at the ∂-sites was in the striatum, intermediate in the cortex, diencephalon and hippocampus, low in the mesencephalon and pons-medulla and not detectable in the cerebellum. No regional differences in binding affinities were found at μ-, ∂- and k-sites with [3H]-[D-Ala2...

THE INTERACTION OF OPIOID PEPTIDES AND ALKALOID OPIATES WITH μ-, δ- AND κ-BINDING SITES

Under suitable conditions, the μ-, δ- and κ-binding sites can be selectively labelled. The spectrum of activity of opioid peptides and alkaloid opiates at the three binding sites can thus be determined and it should be possible to develop agonists and antagonists which exhibit a greater selectivity for the individual sub-types than those currently available. Such selective compounds are required before the physiological functions of the sub-types of the opiate receptor can be investigated.