Dynorphin A (1-13) amide

Dynorphin A(1-13), a tridecapeptide of the endogenous opioid peptides, has modest effects in reducing mild opiate withdrawal in humans. Previous studies revealed that dynorphin also potentiates the analgesic effect of morphine in morphine-tolerant rats and mice. The therapeutic potential of dynorphin A(1-13) is limited due to extensive metabolism by human metabolic enzymes resulting in an in vivo half-life of less than one minute. Chemical modifications of dynorphin A(1-13), such as N-methylation of Tyr1 and amidation of the C-terminus have been shown to be effective in protecting against the proteolytic enzymes in human plasma. This article is a general review of the metabolism of dynorphin A(1-13) in human plasma and CSF.

The effect of dynorphin A (Dyn A)-related peptides and nociceptin on the binding of the D2 receptor antagonist, [(3)H]raclopride, was examined in membrane preparations of rat heart. Non-linear regression saturation binding analysis of [(3)H]raclopride binding revealed the presence of a single high-affinity binding site with a K(d)of 4.1 n M and a B(max)of 220 fmol/mg protein. The D2 stereospecificity of [(3)H]raclopride binding was demonstrated by competition experiments using two enantiomer pairs of antagonists. (+)-Butaclamol (IC(50): 8.0 n M) and (-)-sulpiride (IC(50): 112.3 n M) were 27 000 and 24 times more potent than (-)-butaclamol (IC(50): >100 microm) and (+)-sulpiride (IC(50): 2666 n M), respectively. Nociceptin and Dyn A-(1-13) were also potent inhibitors of the binding of [3H]raclopride with shallow inhibition curves that fitted best with two sites model. Their order of potency on the low affinity site [alpha -Neo-endorphin>nociceptin>Dyn A-(2-13)>Dyn A-(1-13)>Dyn B>Dyn A-(6-10)] correlated well with their ability to inhibit the binding of [3H]nociceptin (r=0.82). The indirect nature of the inhibitory effects of the peptides on the D2 receptor was demonstrated by their inability to inhibit [(3)H]raclopride binding to a membrane preparation (Sf9 cells transfected with the human D2(long)receptor) that does not contain the ORL(1)receptor and the lack of effect of raclopride (0.1 n M-10 microm) on both [(3)H]nociceptin and [(3)H]Dyn A-(1-13) binding. Isolated cardiac mitochondrial-synaptosomal fractions submitted to ischemic conditions (1 m M iodoacetate +2 m M NaCN, 5 min at 37 degrees C) released 10.9% of their content in preloaded [(3)H]noradrenaline ([(3)H]NA). Dyn A-(1-13) (10 microm), nociceptin (10 microm) and the selective D2 receptor agonist, quinpirole (10 microm) were potent blockers of the release of [(3)H]NA evoked by the ischemic conditions. The inhibitory effect of Dyn A-(1-13), nociceptin and quinpirole were antagonized by the selective D2 receptor antagonist, raclopride (10 microm); whereas naloxone, at a concentration (1 microm) known to affect the ORL(1)receptor, blocked the effects of the peptides but not those of quinpirole. The results demonstrate the presence of D2 receptors in rat heart and suggest that Dyn A-(1-13) and nociceptin modulate ischemia-induced NA release by a mechanism that involves the participation of both ORL(1)and D2 receptors.

A series of 2-substituted dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues was prepared by solid phase peptide synthesis and evaluated for opioid receptor affinities in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI) assay. Amino acid substitution at the 2 position produced marked differences in both opioid receptor affinities and potency in the GPI assay; Ki values for the analogues in the radioligand binding assays and IC50 values in the GPI assay varied over three to four orders of magnitude. The parent peptide, Dyn A-(1-13)NH2, exhibited the greatest affinity and selectivity for kappa receptors and was the most potent peptide examined in the GPI assay. The most important determinant of opioid receptor selectivity and opioid potency for the synthetic analogues was the stereochemistry of the amino acid at the 2 position. Except for [D-Lys2]Dyn A-(1-13)NH2 in the kappa receptor binding assay, the analogues containing a D-amino acid at position 2 were much more potent in all of the assays than their corresponding isomers containing an L-amino acid at this position. The L-amino acid-substituted analogues generally retained some selectivity for kappa opioid receptors. The more potent derivatives with a D-amino acid in position 2, however, preferentially interacted with mu opioid receptors. Introduction of a positively charged amino acid into the 2 position generally decreased opioid receptor affinities and potency in the GPI assay.