D. L. Larson

The irreversible narcotic antagonistic and reversible agonistic properties of the fumaramate methyl ester derivative of naltrexone

The fumaramate methyl ester derivatives of naltrexone (beta-FNA) and oxymorphone (beta-FOA) were both found to be reversible agonists on the guinea pig ileal longitudinal muscle preparation. In addition, beta-FNA possessed on irreversible antagonistic effect against morphine whereas beta-FOA had no such capacity. Analysis by pA2 values revealed that beta-FOA resembled pure agonists like morphine and enkephalin while beta-FNA resembled the mixed agonist-antagonists like nalorphine and pentazocine. The antagonism by beta-FNA was very selective in that it antagonized pure agonists but had little or no effect on the effects of either mixed agonist-antagonists, ethylketocyclazocine or other non-opiate-type agonists like norepinephrine.

Opioid agonist and antagonist bivalent ligands as receptor probes

Bivalent ligands are molecules which contain two pharmacophores linked by a connecting chain (spanner). The present report describes the use of oxymorphamine (Oxy) and naltrexamine (Nal) as the opioid agonist and antagonist pharmacophores separated by a variable length spanner composed of succinyl-bis-oligoglycine. The agonist series, [CH2CO(Gly)nOxy]2, and antagonist series, [CH2CO(Gly)nNal]2, were synthesized (n = 0-4) and tested on the electrically stimulated GPI. All of the antagonist bivalent ligands (Nal) antagonized the effects of morphine, with the greatest potency enhancement (60 x) residing with the succinyl (n = 0) congener. A dramatically different SAR profile was observed in the agonist (Oxy) series where the greatest potency enhancement (17 x) occurs when n = 2. By contrast with the antagonist series the agonist bivalent ligand with n = 0 is equipotent to its monovalent agonist analogue. The significance of these results with respect to the possibility of discrete opioid agonist and antagonist recognition sites are discussed.

Opioid receptor binding characteristics of the non-equilibrium μ antagonist, β-funaltrexamine (β-FNA)

Abstract β-Funaltrexamine (β-FNA) bound to mouse brain membranes in a reversible and an irreversible (not removed by washing of the membrane) manner, and a portion of each type of binding was opioid-specific. Addition of 100 mM NaCl to the incubating medium enhanced the binding of β-FNA to membranes. Using membranes preincubated with β-FNA (1 μM) and then washed three times, the maximum number of binding sites available to [3H]morphine was markedly diminished whereas the affinity of morphine for binding sites was not significantly altered. The binding of [3H]naltrexone was also reduced markedly by β-FNA pretreatment. In similarly pretreated membranes, the binding of [3H]methionine enkephalin [3H][D-Ala2,D-Leu5]enkephalin (DADLE) or [3H]ethylketazocine was reduced to a smaller extent. Using brain membranes from mice pretreated with a single subcutaneous injection of β-FNA (100 mg/kg) 48 h prior to use, the binding of [3H]methionine enkephalin was unaffected whereas the number of binding sites available to [3H]morphine was significantly reduced. The inhibition by various ligands of the reversible binding of [3H]β-FNA resembled the relative ability of the same ligands to inhibit the binding of [3H]ethylketazocine. It was concluded that the irreversible portion of the binding of β-FNA demonstrates a selectivity for μ over δ binding sites, and that the reversible portion of the binding of β-FNA demonstrates a selectivity for κ binding sites over μ or δ binding sites. As such, the binding characteristics of β-FNA are consistent with its profile in vivo and in isolated tiussue studies in vitro.

Inhibition of substance P release from spinal cord tissue after pretreatment with capsaicin does not mediate the antinociceptive effect of capsaicin in adult mice

Abstract Substance P (SP) is released from primary afferent fibers in response to nociceptive stimuli. Capsaicin, which produces an initial hyperalgesic response followed by persistent antinociception, also elicits release of SP from primary afferent fibers. Capsaicin pretreatment decreases the content and release of SP from primary afferent fibers. This effect on SP has been hypothesized to mediate the antinociceptive effect of capsaicin. To test this hypothesis, mice were injected intrathecally (i.t.) with antinociceptive doses of capsaicin or SP(1–7) before superfusion of spinal cord tissue with 3 &mgr;M capsaicin 24, 48, 96 or 168 h later. N‐terminal metabolic fragments of SP that accumulate after capsaicin‐induced SP release and are involved in the antinociceptive effect of capsaicin, were also tested. Like capsaicin SP(1–3), SP(1–4) and SP(1–7) were each antinociceptive when injected 24 h before nociceptive testing. However, at this time there was no decrease in capsaicin‐evoked release of SP in tissue from capsaicin‐ and SP(1–7)‐pretreated animals compared to those injected with vehicle. In contrast, capsaicin‐evoked SP release decreased significantly in tissue from mice pretreated with capsaicin or SP(1–7) 48 h prior to testing. d‐Substance P(1–7), which prevents antinociception, blocked capsaicin‐ and SP(1–7)‐induced decreases in SP release, indicating that these effects are mediated by SP N‐terminal activity. Total spinal cord content of SP did not differ amongst treatment groups. These data indicate that antinociception does not appear to depend on decreases in SP release or content as antinociception precedes decreases in SP release.

Reactivity of glutathione with α,β-unsaturated ketone flavouring substances

Abstract The relative reactivities of a number of α,β-unsaturated ketones used as flavourings were determined using glutathione as the nucleophile. Monosubstitution at the β-position of the α,β-unsaturated system impeded nucleophilic addition by approximately 1000 times. β-Disubstitution reduced reactivity by more than 100,000 times. Endocyclic α,β-unsaturated ketones were generally less reactive than alicyclic analogues. By way of comparison, the most reactive flavouring investigated, 2-octene-4-one, was consumed by glutathione about 700 times less rapidly than was methylvinyl ketone. Methylvinyl ketone was found to condense with guanylic acid 240,000 times more slowly than with glutathione. It is concluded that α,β-unsaturated ketones used as flavourings generally possess low electrophilicity.

Isolation of selective 3H-chlornaltrexamine-bound complexes, possible opioid receptor components in brains of mice

Abstract The nonequilibrium narcotic antagonist, chlornaltrexamine (CNA) was used to bind selectively and covalently pioid specific sites on brain membrane preparations. Selective binding of [ 3 H]CNA occured with a saturation maximum of 185 fmol/mg protein. Bound [ 3 H]CNA was extracted with Triton X-100, dialyzed against Brij 36T, precipitated with trichloroacetic acid and chromatographed on an ultrogel AcA 22 column. The elution profile suggests that this extract contains a minimum of four selective [ 3 H]CNA complexes. At least two of these complexes migrate in a single large peak. Column calibration showed that this peak eluted at 590,000 daltons. One of these specific [ 3 H]CNA complexes elutes at the elution volume of the column and is dialyzable. Finally, putative aggregate of these complexes elutes with the void volume.

Long-acting agonist and antagonist activities of naltrexamine bivalent ligands in mice☆

A series of naltrexamine bivalent ligands, compounds with two naltrexamine pharmacophores separated by a spacer which contains a variable number of glycyl units flanking a succinyl group, were synthesized and evaluated in vivo in mice. These compounds possessed long-acting agonist and especially antagonist activities. The bivalent ligands, 2 and 3 displayed antinociceptive activity that lasted greater than 4 h. Compound 1, a bivalent ligand and 4, the monomer, antagonized the antinociceptive effect of morphine for a week after a single injection i.c.v. The long duration of action may be due to entrapment of these ligands in the central nervous system. These compounds may give future insights into the design of long-acting agonists and antagonists.

Transfer of fatty acyl groups from membrane phosphatides to opiate ligands

Investigation of the persistent opioid receptor binding of hydrazine-containing opiate ligands to brain membranes has revealed that it is related to conversion of these ligands to fatty acylhydrazone or fatty acylhydrazide derivatives. The fatty acyl group was found to be derived from membrane phosphatides. Persistent binding does not occur when this pool of phosphatides is removed by extensive washing, and it is restored upon addition of phosphatide to the membranes. In washed membranes, synthetically derived fatty acylhydrazones exhibited persistent binding similar to that found when naltrexonazine was incubated with brain membranes. It is suggested that the persistent binding is a consequence of the localization of fatty acylnaltrexazone derived from naltrexonazine in a membrane lipid bilayer that interfaces with the opioid receptor system rather than the persistence of naltrexonazine itself.

STRUCTURE-ACTIVITY RELATIONSHIP STUDY OF AFFINITY LABELS THAT ARE SPECIFIC FOR μ OPIOID RECEPTORS

Naltrexamine derivatives which contain electrophilic groups have been synthesized and tested in the electrically stimulated guinea pig ileum. The 6β epimers irreversibly and specifically block μ opioid receptors, whereas the corresponding compounds in the 6α series are reversibly acting agents.