European Journal of Pharmacology, 127 (1986) 187-195 187 Elsevier METABOLICALLY STABLE ANALOGUES OF SUBSTANCE P: PERSISTENT ACTION OF PARTIALLY MODIFIED RETRO-INVERSO ANALOGUES OF SUBSTANCE P ON RAT PAROTID AND HYPOTHALAMIC SLICES MICHAEL CHOREV *, ELI RUBINI *, YO'AV HART **, CHAIM GILON *, UR1 WORMSER ** and ZV1 SELINGER **'a * Department of Pharmaceutical Chemistry, School of Pharmacy, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120, ~ Department of Organic Chemistry and ** Department of Biological Chemistry, Faculty of Science, The Hebrew University of Jerusalem, Jer~valem 91904, Israel Received 2 January 1986, revised MS received 1 May 1986, accepted 20 May 1986 M. CHOREV, E. RUBINI, Y. HART, C. GILON, U. WORMSER and Z. SELINGER, Metabolically stable analogues of substance P: persistent action of partially modified retro-inoerso analogues of substance P on rat parotid and hypothalamic slices, European J. Pharmacol. 127 (1986) 187-195. In a search for metabolically stable analogues of substance P (SP) the hexapeptide [pGIu6]SP-(6-11) was modified by reversal of the direction of a single amide bond. This novel peptide modification reverses the direction of the amide bonds at the peptide backbone but attempt to retain the topology of the amino acid side-chains at the peptide surface. The partial retro-inverso modification was successfully applied in a previous study for enkephalin analogues which were found to have potent and protracted morphinomimetic activity both in vivo and in vitro. The partially modified retro-inverso analogues: [pGiu6~(NH-CO)(RS)-PheTISP-(6-11 ) (analogue II) and [pGlu6,Pheg~(NH-CO)Glyg]sP-(6 - 11) (analogue III) were tested on guinea-pig ileum and for K ÷ release from rat parotid slices. Metabolic stability of the analogues was measured by their ability to produce persistent K ÷ release from parotid slices, their half life time (t~/2) in the rat parotid and hypothalamic slice systems and their resistance to proteolytic cleavage by chymotrypsin, pepsin, papain and pronase. Analogue II was devoid of biological activity and was slowly degraded in the parotid system and by several proteases. Analogue III was a full agonist of the SP-P receptor with a potency of 22 and 15% of the parent compound I, in the guinea-pig ileum and parotid slice system respectively. Pretreatment of the guinea-pig ileum with atropine (0.3/~M) had no effect on the potency of analogue III. On the other hand, when tested on rat vas deferens (an SP-E system), analogue 1II was about 20-fold more potent than the parent compound I. Analogue I11 was not degraded by rat parotid or hypothalamic slices and was resistant to proteolytic cleavage by chymotrypsin, pepsin, thermolysin and pronase, and only degraded at a slow rate by papain (tl/2 - 2.5 h). Partially modified retro-inverso Rat parotid slices Metabolically stable analogues Guinea-pig ileum Rat hypothalamic slices Substance P 1. Introduction Substance P, a hypotensive and smooth muscle contracting agent (Von Euler and Gaddum, 1931), is now known to be an undecapeptide which has the following sequence of amino acids: H-Arg- Pro- Lys- Pro-Gin-Gln-Phe-Phe-Gly- Leu-Met-N H 2 a To whom all correspondence should be addressed: Depart- ment of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. (Chang et al., 1971). Several lines of evidence indicate that substance P is a transmitter candi- date in the central and peripheral nervous system (Nicoll et al., 1980). The suggestion that substance P might function as a neurotransmitter has raised considerable interest in its metabolism. While sub- stance P is degraded by various brain preparations (Berger et al., 1979; Blumberg and Teichberg, 1979; Lee et al., 1979; Marks, 1977), membrane- bound enzyme was only recently purified to near homogeneity from human brain and was shown to 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.