Kelatorphan

Effects of kelatorphan and other peptidase inhibitors on the in vitro and in vivo release of methionine-enkephalin-like material from the rat spinal cord

The effects of the novel mixed peptidase inhibitor, kelatorphan [N-(R)-3-(N-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl)-L-alanine], were compared to those of a combination of the potent "enkephalinase" inhibitor thiorphan and the nonselective aminopeptidase inhibitor bestatin, on the catabolism of [3H]Met-enkephalin and on the release of endogenous Met-enkephalin by the rat spinal cord in vitro and in vivo. At 20 microM, kelatorphan almost prevented completely the degradation of exogenous [3H] Met-enkephalin by slices of the dorsal zone of the lumbar enlargement. Similarly, the addition of 20 microM kelatorphan to a [3H] Met-enkephalin-containing artificial cerebrospinal fluid superfusing the whole spinal cord of halothane-anesthetized rats efficiently protected the exogenous peptide from enzymatic degradation. In contrast, in the same in vitro and in vivo models, thiorphan (1 microM) or bestatin (20 microM) alone was inactive, and only their combination induced a significant protection of the exogenous peptide. In vitro and in vivo, kelatorphan (20 microM) increased markedly the spontaneous outflow of endogenous Met-enkephalin-like material as well as the peptide overflow due to K+-induced depolarization (in vitro and in vivo) or noxious stimulation (in vivo). Under similar conditions, thiorphan (1 microM) plus bestatin (20 microM) also enhanced the efflux of Met-enkephalin-like material, but generally to a lower extent than kelatorphan. Compared to thiorphan plus bestatin, kelatorphan exerts additional inhibitory effects on dipeptidylaminopeptidase activity and the present results could indicate that this enzyme also may be involved in the inactivation of extracellular Met-enkephalin at the spinal level in rats.

In vitro and in vivo effects of kelatorphan on enkephalin metabolism in rodent brain

Biologically relevant assays were used to compare the potency of kelatorphan (N-[3(R)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]-L-alanine) as inhibitor of the peptidase-induced metabolism of enkephalins to that of bestatin, a non-specific inhibitor of aminopeptidase and thiorphan, a highly potent blocker of the neutral endopeptidase (EC 3.4.24.11) designated as enkephalinase. Kelatorphan almost completely inhibited the formation of the three metabolites [3H]Tyr, [3H]Tyr-Gly and [3H]Tyr-Gly-Gly produced by incubation of [3H][Tyr1,Met5]enkephalin with rat striatal slices. Co-administered with [Met5]enkephalin in mouse brain, kelatorphan was able to prevent by 80% the degradation of the exogenous peptide. Moreover, a mixture of thiorphan (1 microM) and bestatin (20 microM) or kelatorphan alone (20 microM) induced a 2.2 to 2.5-fold increase in endogenous [Met5]enkephalin overflow after evoked depolarization of superfused rat striatal slices. In this assay, kelatorphan was the only compound to increase by 63% the basal level of released [Met5]enkephalin. Kelatorphan was about 100 times less potent than bestatin to inhibit the total rat striatal aminopeptidases, but as efficient (IC50 = 4 X 10(-7) M) as bestatin to inhibit a minor aminopeptidase activity resembling aminopeptidase M. Therefore the reported enhanced analgesic potency of kelatorphan with regard to the association of bestatin and thiorphan is very likely related to its ability to almost completely inhibit enkephalin-degrading enzymes (including the Tyr-Gly releasing peptidase) and to its better selectivity for the biologically relevant aminopeptidase M. Kelatorphan would be a valuable probe, preferable to the association of bestatin and thiorphan, to investigate the physiological functions regulated by a phasic enkephalinergic activity.

Potent antinociceptive effects of kelatorphan (a highly efficient inhibitor of multiple enkephalin-degrading enzymes) systemically administered in normal and arthritic rats

The effects of various i.v. doses (2.5, 5, 10 and 15 mg/kg) of the highly efficient inhibitor of multiple enkephalin-degrading enzymes, Kelatorphan, were evaluated on the vocalization threshold to paw pressure in normal rats and in rats with Freund's adjuvant-induced arthritis. In normal rats, Kelatorphan at doses as low as 2.5 mg/kg i.v. at which the enkephalinase inhibitor acetorphan was ineffective, produced potent antinociceptive effects, comparable to that induced by 1 mg/kg i.v. morphine. In contrast, for the higher doses used (5, 10, 15 mg/kg i.v.), the effects of Kelatorphan were not more pronounced than that of acetorphan. Unlike acetorphan, Kelatorphan was found to be much more effective in arthritic than in normal rats in raising the vocalization threshold, even at the lower concentration, 2.5 mg/kg i.v.: 244% in arthritic vs 144% in normal rats. The effects of Kelatorphan were prevented by naloxone at the dose of 0.5 mg/kg i.v. The enhanced potency of Kelatorphan is discussed in relation with the increase in peptidase-sensitive dynorphin fragments in arthritic rats.

Effects of the potent analgesic enkephalin-catabolizing enzyme inhibitors RB101 and kelatorphan on respiration

We investigated whether the enkephalin-catabolizing enzyme inhibitors RB101 and kelatorphan, which have been shown to be potent analgesics, depress respiration as do opioid analgesics. Ventilation was measured in cats and rodents by the barometric method, in the awake state and during anesthesia. Tissue distribution of the inhibitors was either generalized (RB101, 40-160 mg/kg i.p.), largely restricted by the blood-brain barrier to the periphery (kelatorphan, 0.7-20 mg/kg i.v.), or restricted to the brainstem (i.c.v. injection of RB101 in the fourth ventricle). RB101 did not affect ventilation in any condition tested, and large doses of kelatorphan produced a naloxone-reversible increase in ventilation and breathing frequency. Thus endogenous opioids released during conditions of normal ventilation do not exert any depressant neuromodulatory effect on this function, even when their extracellular concentrations are increased by peptidase inhibitors. The differential effect of these inhibitors on ventilation and nociception is discussed. We conclude that kelatorphan and RB101 are devoid of respiratory-depressant effects and might be interesting pharmacological alternatives to morphine and other opioid agonists.

Effects of Kelatorphan and morphine before and after noxious stimulation on immediate-early gene expression in rat spinal cord neurons

Expression of the immediate-early genes (IEG) c-FOS, NGF1-A and c-JUN was induced by noxious thermal stimulation in neurons of the rat spinal cord dorsal horn. Intravenous injection of Kelatorphan (5, 10 and 20 mg/kg), an inhibitor of multiple enkephalin-degrading enzymes, 20 min before noxious stimulation reduced the overall number of dorsal horn neurons expressing c-FOS and NGF1-A by up to 20-30%. While c-FOS expression was suppressed in superficial and deep laminae of the spinal cord, NGF1-A and c-JUN was only suppressed in superficial laminae. Morphine (5, 7.5 and 10 mg/kg) produced a dose-dependent reduction of c-FOS expression by up to 70% only when injected before noxious stimulation. Morphine injected 10 min after the noxious treatment was virtually ineffective. The depressant effect of Kelatorphan and morphine could be prevented by prior application of the opioid antagonist naloxone. Naloxone itself slightly increased the overall number of c-FOS-positive neurons in all laminae of the spinal cord. The present data support the existence of a tonic release of endogenous opioid peptides at the spinal level and show that inhibition of their peptidase-induced degradation modulates IEG expression in dorsal horn neurons of the rat. The finding that opioid agonists were ineffective when applied after stimulation underline the necessity of pre-emptive analgesia to prevent long-term activity-dependent changes in spinal cord neurons.