endomorphin-2 and 2-aminocyclobutanecarboxylic-acid

This study reports on the in vivo effects of four endomorphin-2 (EM-2) derivatives (EMD1-4) containing unnatural amino acids, i.e. 2-aminocyclohexanecarboxylic acid (Achc2), para-fluorophenylalanine (pFPhe4), β-methylphenylalanine (βMePhe4) and/or 2',6'-dimethyltyrosine (Dmt1). After induction of osteoarthritis by monosodium iodoacetate into the ankle joint of male Wistar rats, a chronic intrathecal catheter was inserted for spinal drug delivery. The mechanical threshold was assessed by a dynamic aesthesiometer. Intrathecal injection of the original EM-2 and the ligands (0.3-10 μg) caused dose-dependent antiallodynic effects. The comparison of the different substances revealed that EMD3 and EMD4 showed more prolonged antinociception than EM-2, and the effects of the highest dose of EMD4 were comparable to morphine, while EMD3 caused paralysis at this dose. The potency of the different ligands did not differ from EM-2. The results show that the derivatives of EM-2 have similar in vivo potency to the original ligand, but their effects were more prolonged suggesting that these structural modifications may play a role in the development of novel endomorphin analogues with increased therapeutic potential.

Topics: Aminobutyrates; Analgesics, Opioid; Animals; Arthralgia; Carboxylic Acids; Chronic Pain; Cyclobutanes; Disease Models, Animal; Drug Design; Edema; Hyperalgesia; Injections, Spinal; Male; Nociceptors; Oligopeptides; p-Fluorophenylalanine; Rats; Rats, Wistar; Tyrosine

As part of ongoing work aimed at generating proteolytically stable, readily applicable, radiolabeled endomorphin-2 (EM-2) analogs for elucidation of the topological requirements of peptide binding to μ-opioid receptors, we report here on the synthesis, radiolabeling, binding kinetics and binding site distribution of an EM-2 analog in which Pro(2) is replaced by 2-aminocyclohexanecarboxylic acid, ACHC. [(3)H][(1S,2R)ACHC](2)EM-2 (specific activity 63.49Ci × mmol(-1)) bound specifically to its binding sites with high affinity (K(D) = 0.55 ± 0.06 nM) and saturably, yielding a receptor density, B(max) of 151 ± 4 fmol × mg protein(-1) in rat brain membranes. A similar affinity value was obtained in kinetic assays. Both Na(+) and Gpp(NH)p decreased the affinity, proving the agonist character of the radioligand. Specific μ-opioid ligands displaced the radioligand with much higher affinities than did δ- and κ-ligands. The autoradiographic distribution of the binding sites of [(3)H][(1S,2R)ACHC](2)EM-2 agreed well with the known locations of the μ-opioid receptors in the rat brain. In consequence of its high affinity, selectivity and enzymatic resistance [19], the new radioligand will be a good tool in studies of the topographical requirements of μ-opioid-specific peptide binding.

Topics: Animals; Autoradiography; Carboxylic Acids; Cyclobutanes; Kinetics; Molecular Mimicry; Oligopeptides; Proline; Radioligand Assay; Rats