Yoshiaki Kiso

Isolation of a novel analgesic pentapeptide, neo-kyotorphin, from bovine brain

Abstract A novel peptide isolated from the bovine brain was found to contain the Tyr-Arg (kyotorphin) unit at the C-terminal portion. This peptide in the methanol soluble fraction was isolated by gel filtration and cation exchange chromatography. We termed this peptide “neo-kyotorphin” and the amino acid sequence is Thr-Ser-Lys-Tyr-Arg. The synthetically prepared neo-kyotorphin proved to have a dose-dependent analgesic effect in mice.

Isolation from bovine brain of a novel analgesic pentapeptide, neo-kyotorphin, containing The Tyr-Arg (kyotorphin) unit

A novel analgesic peptide, isolated from the bovine brain, was found to contain the Tyr-Arg (kyotorphin) unit at the C-terminal portion. This peptide, in the methanol-soluble fraction, was isolated by gel filtration and cation exchange chromatography. This peptide was termed neo-kyotorphin and the amino acid sequence is Thr-Ser-Lys-Tyr-Arg. The synthetically prepared neo-kyotorphin proved to have a dose-dependent analgesic effect in mice. The median analgesic dose, ED50 of neo-kyotorphin was 195 nmol/mouse (intracisternal injection), with the tail pinch test, and the potency was approximately equal that of Leu-enkephalin.

Tripeptides acting on opioid receptors in rat colon

The tripeptides SD-34 and SD-25 induced atropine-, guanethidine-, antihistaminics-resistant but naloxone-sensitive contractions of isolated rat distal colon. They appeared to act on an opioid receptor, probably of the mu subtype, distinct from those for methionine enkephalin and morphine, because the pA2 values of naloxone for the peptides were similar to those for mu-agonists but different from those for methionine enkephalin and morphine, and because the peptides caused contractions of colon that had been desensitized to morphine. Mr 2266, a supposed kappa-antagonist, inhibited the actions of the peptides, ethylketocyclazocine and dynorphin at concentrations much lower than those inhibiting the actions of methionine enkephalin and morphine. Thus these peptides seem to act on the mu- and/or kappa-receptors. The actions of the tripeptides were inhibited by methysergide and methylergometrine, but not by the 5-HT2 antagonist ketanserin, and were not affected by 5-HT or substance P autodesensitization . Thus their actions do not seem to involve 5-HT, histamine, ACh or substance P. It seems likely that the tripeptides, through opioid receptors, directly activate the muscle, or remove some inhibitory modulation of myogenic activity, thus causing contractions.

Syndyphalin SD-25: a higly selective ligand for μ opiate receptors

After the discovery of the enkephalin [ 11, it was reported [2] that the order of potency for morphine and the enkephalins was inversed in the mouse vas deferens (in comparison with the guinea pig ileum), thus suggesting the existence of various class of opiate receptors. However, a major problem in the precise characterization of the different receptors has been the absence of very selective ligands for one peculiar subclass of opiate receptors. In [3] morphiceptin, the amide of a fragment of the milk protein fl-casein, was suggested to represent a highly selective ligand for the p opiate receptor. In [4] the enkephalin analogue, Tyr-D-SerGly-Phe-Leu-Thr was proposed as a selective ligand for the 6 opiate receptor. Synthesis of various enkephalin analogues with potent activity in the guinea pig ileum bioassay as well as significant analgesic activity after subcutaneous administration was reported in [5-71. One of these analogues, TyrD-Met-Gly-MePheol, named ‘syndyphalin (SD)25’ was 23 300-times and g-times as active as morphine in the guinea pig ileum bioassay and in the tail flick analgesic test after subcutaneous administration, respectively [7]. Here, we report the potency of this analogue on the p, 6 and K opiate receptors using [ 3H] dihydromorphine (DHM), D[ 3H] Ala’,DLeu’enkephalin (DADLEU) and [ 3H] ethylketocyclazocine (EKC) as their respective ligands. Our results show that SD-25 is a highly specific and selective ligand for the /J opiate receptor.

Neo‐kyotorphin (Thr—Ser—Lys—Tyr—Arg), a new analgesic peptide

The amino acid sequence of a newly isolated pentapeptide, neo‐kyotorphin from bovine brain was synthetically verified to be Thr‐Ser‐Lys‐Tyr‐Arg corresponding to the C‐terminal portion of hemoglobin α‐chain. The synthetic neo‐kyotorphin showed the dose‐dependent analgesia in mice which was approximately equal to that of Leu‐enkephalin.

Syndyphalin-33, a synthetic tripeptide alkylamide with prolonged analgesic activity

Pentapeptides, Metenkephalin (ENK) and LeuENK [l] with opiate agonist activity produce only weak and short-lived analgesia even after intracerebroventricular (i.c.v.) administration [2,3] and no analgesia follows their subcutaneous (s.c.) administration. This is due to their rapid enzymatic degradation, and various attempts have been made to obtain ENK derivatives with high and prolonged analgesic activity by protecting the Nand C-terminals and the peptide bond of the molecule [4]. in SD-20 also produce analgesia after S.C. administration [8]. These findings indicate that all 5 amino acids in ENK are not necessary for S.C. analgesic activity. We therefore sought the ‘minimal segment’ of ENK required for S.C. analgesia and could find a tripeptide alkylamide with S.C. analgesia [ 121. Here, we report the synthesis of a simple tripeptide Tyr-D Met(O)-Gly-MPA named ‘syndyphalin (SD)33’ (fig.1) which exhibits potent opioid activity in vitro

Analgesic and other pharmacological activities of an enkephalin analogue, syndyphalin (SD)-25.

The pharmacological actions of Tyr-D-Met(O)-Gly-MePheol (syndyphalin (SD)-25) were compared with those of morphine after systemic administration. The analgesic potency of SD-25 was about 4 times that of morphine when administered s.c. to rats. SD-25 did not exhibit any narcotic antagonist activity. Subcutaneous administration of SD-25 produced a dose-dependent suppression of morphine withdrawal signs in morphine-dependent rats, typical morphine-like jumping in the mouse jumping test, and an increase in spontaneous locomotor activity in mice. These activities were 2-5 times those of morphine. In the anaesthetized dog, intravenous administration of SD-25 produced a 100-1000 times stronger increase in the amplitude of contractions of the jejunum than did morphine, a weaker depression in respiration than morphine, and a slight increase in blood pressure. These effects were reversed by naloxone. These results indicate that SD-25 possesses potent central nervous system actions closely similar to those of morphine, but its effect on blood pressure and respiration was weaker than that of morphine.

“MINIMAL SEGMENT” OF ENKEPHALIN FOR ANALGESIA: SYNDYPHALIN (SD)-33, A SIMPLE TRIPEPTIDE ALKYLAMIDE WITH PROLONGED SUBCUTANEOUS ANALGESIC ACTIVITY

SUMMARY A synthetic tripeptide alkylamide, Tyr-D-Met(0)-Gly-MPA (MPA=N-methylphenethylamide) named “syndyphalin (SD)-33” was approximately comparable to morphine in analgesic activity after subcutaneous(s.c.) administration.1 SD-33 is the only tripeptide exhibiting substantial analgesia after s.c. administration and may be the “minimal segment” of opioid peptides for s.c. analgesia.