Brian I. Knapp

Opioids Activate Brain Analgesic Circuits Through Cytochrome P450/Epoxygenase Signaling

To assess the importance of brain cytochrome P450 (P450) activity in μ opioid analgesic action, we generated a mutant mouse with brain neuron–specific reductions in P450 activity; these mice showed highly attenuated morphine antinociception compared with controls. Pharmacological inhibition of brain P450 arachidonate epoxygenases also blocked morphine antinociception in mice and rats. Our findings indicate that a neuronal P450 epoxygenase mediates the pain-relieving properties of morphine.

Glycosylation Improves the Central Effects of DAMGO

A series of μ‐agonist DAMGO analogs were synthesized and pharmacologically characterized to test the ‘biousian’ hypothesis of membrane hopping. DAMGO was altered by incorporating moieties of increasing water solubility into the C‐terminus via carboxamide and simple glycoside additions. The hydrophilic C‐terminal moieties were varied from glycinol in DAMGO (1) to l‐serine amide (2), l‐serine amide β‐d‐xyloside (3), l‐serine amide β‐d‐glucoside (4), and finally to l‐serine amide β‐lactoside (5). Opioid binding and mouse tail‐flick studies were performed. Antinociceptive potency (intravenous) increased, passing through a maximum (A50 ≈ 0.2 μmol/kg) for 2 and 3 as membrane affinity versus water solubility became optimal, and dropped off (A50 ≈ 1.0 μmol/kg) for 4 and 5 as water solubility dominated molecular behavior. Intravenous A50 values were plotted versus hydrodynamic values (glucose units, g.u.) for the glycoside moieties, or the hydrophilic/hydrophobic Connolly surface areas (A50 versus e−Awater/Alipid), and provided either a V‐shaped or a U‐shaped curve, as predicted by the ‘biousian’ hypothesis. The μ‐selective receptor profile was maintained (Ki’s = 0.66–1.3 nm) upon modifications at the C‐terminus. The optimal ‘degree of glycosylation’ for the DAMGO peptide message appears to be between 1.25 and 1.75 g.u. (hydrodynamic g.u.), or 0.75 and 0.90 in terms of the surface‐derived amphipathicity values.

Synthesis and opioid receptor binding affinities of 2-substituted and 3-aminomorphinans: ligands for mu, kappa, and delta opioid receptors.

The phenolic group of the potent mu and kappa opioid morphinan agonist/antagonists cyclorphan and butorphan was replaced by phenylamino and benzylamino groups including compounds with para-substituents in the benzene ring. These compounds are highly potent mu and kappa ligands, e.g., p-methoxyphenylaminocyclorphan showing a K(i) of 0.026 nM at the mu receptor and a K(i) of 0.03 nM at the kappa receptor. Phenyl carbamates and phenylureas were synthesized and investigated. Selective o-formylation of butorphan and levorphanol was achieved. This reaction opened the way to a large set of 2-substituted 3-hydroxymorphinans, including 2-hydroxymethyl-, 2-aminomethyl-, and N-substituted 2-aminomethyl-3-hydroxymorphinans. Bivalent ligands bridged in the 2-position were also synthesized and connected with secondary and tertiary aminomethyl groups, amide bonds, and hydroxymethylene groups, respectively. Although most of the 2-substituted morphinans showed considerably lower affinities compared to their parent compounds, the bivalent ligand approach led to significantly higher affinities compared to the univalent 2-substituted morphinans.

Univalent and Bivalent Ligands of Butorphan: Characteristics of the Linking Chain Determine the Affinity and Potency of Such Opioid Ligands†

Bivalent morphinan compounds containing ester linkers were synthesized and their binding affinities at the mu, delta, and kappa opioid receptors determined. Addition of methyl groups adjacent to the hydrolytically labile ester linkage increased stability while only partially affecting binding affinity. The resulting bivalent ligands with optimized spacer length and structure show potent binding profiles with the most potent compound (4b), having K(i) values of 0.47 nM for both the mu and kappa opioid receptors, and 4a, having K(i) values of 0.95 and 0.62 nM for the mu and kappa receptors, respectively. Both 4a and 4b were partial agonists at the kappa and micro receptors in the [(35)S]GTPgammaS binding assay.

Brain P450 epoxygenase activity is required for the antinociceptive effects of improgan, a nonopioid analgesic

&NA; The search for the mechanism of action of improgan (a nonopioid analgesic) led to the recent discovery of CC12, a compound that blocks improgan antinociception. Because CC12 is a cytochrome P450 inhibitor, and brain P450 mechanisms were recently shown to be required in opioid analgesic signaling, pharmacological and transgenic studies were performed in rodents to test the hypothesis that improgan antinociception requires brain P450 epoxygenase activity. Intracerebroventricular (ICV) administration of the P450 inhibitors miconazole and fluconazole, and the arachidonic acid (AA) epoxygenase inhibitor N‐methylsulfonyl‐6‐(2‐propargyloxyphenyl)hexanamide (MS‐PPOH) potently inhibited improgan antinociception in rats at doses that were inactive alone. MW06‐25, a new P450 inhibitor that combines chemical features of CC12 and miconazole, also potently blocked improgan antinociception. Although miconazole and CC12 were weakly active at opioid and histamine H3 receptors, MW06‐25 showed no activity at these sites, yet retained potent P450‐inhibiting properties. The P450 hypothesis was also tested in Cprlow mice, a viable knock‐in model with dramatically reduced brain P450 activity. Improgan (145 nmol, ICV) antinociception was reduced by 37% to 59% in Cprlow mice, as compared with control mice. Moreover, CC12 pretreatment (200 nmol, ICV) abolished improgan action (70% to 91%) in control mice, but had no significant effect in Cprlow mice. Thus, improgan’s activation of bulbospinal nonopioid analgesic circuits requires brain P450 epoxygenase activity. A model is proposed in which (1) improgan activates an unknown receptor to trigger downstream P450 activity, and (2) brainstem epoxygenase activity is a point of convergence for opioid and nonopioid analgesic signaling. The present study found that the nonopioid analgesic drug improgan utilizes cytochrome P450 epoxygenase enzymes in the brain to produce its pain‐relieving actions.

Opioid Bifunctional Ligands from Morphine and the Opioid Pharmacophore Dmt-Tic

Bifunctional ligands containing an ester linkage between morphine and the δ-selective pharmacophore Dmt-Tic were synthesized, and their binding affinity and functional bioactivity at the μ, δ and κ opioid receptors determined. Bifunctional ligands containing or not a spacer of β-alanine between the two pharmacophores lose the μ agonism deriving from morphine becoming partial μ agonists 4 or μ antagonists 5. Partial κ agonism is evidenced only for compound 4. Finally, both compounds showed potent δ antagonism.

Preliminary pharmacological evaluation of enantiomeric morphinans.

A series of levo- and dextromorphinan pairs have been synthesized and evaluated for their affinities to the mu, kappa, and delta opioid receptors, the N-methyl-D-aspartate (NMDA) channel, and sigma 1 and 2 receptors. It was found that levo isomers tended to have higher affinities at the opioid receptors and moderate to high affinities to the NMDA and sigma receptors, while dextro isomers tended to have lower affinities to the opioid receptors but comparatively higher affinities to the NMDA and sigma receptors. This series of compounds have interesting and complex pharmacological profiles, and merit further investigation as potential therapies for drug abuse treatment.

Synthesis and pharmacological evaluation of aminothiazolomorphinans at the mu and kappa opioid receptors.

Previous studies with aminothiazolomorphinans suggested that this class of opioid ligands may be useful as a potential pharmacotherapeutic to decrease drug abuse. Novel aminothiazole derivatives of cyclorphan were prepared to evaluate a series of aminothiazolomorphinans with varying pharmacological properties at the κ opioid receptor (KOR) and μ opioid receptor (MOR). This study was focused on exploring the regioisomeric analogs with the aminothiazole on the C-ring of the morphinan skeleton. Receptor binding and [(35)S]GTPγS binding assays were used to characterize the affinity and pharmacological properties of the aminothiazolomorphinans. Intracranial self-stimulation (ICSS) was used to compare the effects of a representative aminothiazolomorphinan with the morphinan mixed-KOR/MOR agonist butorphan (MCL-101) on brain-stimulation reward.

Phosphorylation of Enkephalins: NMR and CD Studies in Aqueous and Membrane‐Mimicking Environments

Phosphorylation of l‐serine‐containing enkephalin analogs has been explored as an alternative to glycosylation in an effort to increase blood–brain barrier permeability and CNS bioavailability of peptide pharmacophores. Two enkephalin‐based peptides were modified for these studies, a set related to DTLES, a mixed μ/δ‐agonist, and one related to DAMGO, a highly selective μ‐agonist. Each unglycosylated peptide was compared to its phosphate, its mono‐benzylphosphate ester, and its β‐d‐glucoside. Binding was characterized in membrane preparations from Chinese hamster ovary cells expressing human μ, δ and κ‐opiate receptors. Antinociception was measured in mice using the 55 °C tail‐flick assay. To estimate bioavailability, the antinociceptive effect of each opioid agonist was evaluated after intracerebroventricular (i.c.v.) or intravenous administration (i.v.) of the peptides. Circular dichroism methods and high‐field nuclear magnetic resonance were used in the presence and absence of sodium dodecylsulfate to understand how the presence of a membrane might influence the peptide conformations.

Structural Requirements for CNS Active Opioid Glycopeptides

Glycopeptides related to β-endorphin penetrate the blood-brain barrier (BBB) of mice to produce antinociception. Two series of glycopeptides were assessed for opioid receptor binding affinity. Attempts to alter the mu-selectivity of [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO)-related glycopeptides by altering the charged residues of the amphipathic helical address were unsuccessful. A series of pan-agonists was evaluated for antinociceptive activity (55 °C tail flick) in mice. A flexible linker was required to maintain antinociceptive activity. Circular dichroism (CD) in H2O, trifluoroethanol (TFE), and SDS micelles confirmed the importance of the amphipathic helices (11s → 11sG → 11) for antinociception. The glycosylated analogues showed only nascent helices and random coil conformations in H2O. Chemical shift indices (CSI) and nuclear Overhauser effects (NOE) with 600 MHz NMR and CD confirmed helical structures in micelles, which were rationalized by molecular dynamics calculations. Antinociceptive studies with mice confirm that these glycosylated endorphin analogues are potential drug candidates that penetrate the BBB to produce potent central effects.