Peptides, Vol. 3, pp. 793-797, 1982. Printed in the U.S.A. Antinociceptive Potencies of /3-Casomorphin Analogs as Compared to Their Affinities Towards/z and 6 Opiate Receptor Sites in Brain and Periphery VICTOR BRANTL, 1 ANDREAS PFEIFFER, ALBERT HERZ, AGNES HENSCHEN* AND FRIEDRICH LOTTSPEICH* Department of Neuropharmacology, Max-Planck-Institut fiir Psyehiatrie Kraepelinstrasse 2, D-8000 Miinchen 40, F. R. G. and *Max-Planck-Institut fiir Biochemie, D-8033 Martinsried, F. R. G. Received 6 January 1982 BRANTL, V., A. PFEIFFER, A. HERZ, A. HENSCHEN AND F. LOTTSPEICH. Antinociceptive potencies of fl-casomorphin analogs as compared to their affinities towards tx and 6 opiate receptor sites in brain and periphery. PEPTIDES 3(5) 793-797, 1982.--fl-Casomorphins and their analogs were tested for their opioid activities in the myenteric plexus longitudinal muscle preparation of the guinea pig ileum (GPI), the isolated mouse vas deferens (MVD), and for their affinities to /z- 6- and K- binding sites in rat brain membranes. C-terminal amidation of/3-casomorphin-4 and (-5) increased opioid potency in both organ preparations (GPI, MVD) and affinity to Ix-binding sites in brain whereas binding to 6-sites was diminished. These/3-casomorphin-amides displayed a 2-3 times greater naloxone reversible antinociceptive effect than natural fl-casomorphins. Introduction of D-alanine at position 2 in the/3-casomorphin-amides increased potency in the GPI whereas activity in the MVD was only slightly changed. These compounds, however, showed a remarkable increase in binding to cS-sitesin brain with an unaffected or slightly increased binding to Ix-sites and decreased binding to K-sites. D-Ala2-/3-casomorphin-4 and (-5) amides were 10 times more potent antinociceptive agents than corresponding /3-casomorphin-amides. These results suggest firstly, that peripheral 6-receptors in the MVD are not as closely related to 6-binding sites at rat brain membranes as is the case with iz-receptors in the GPI and Ix-binding sites, and secondly, in addition to Ix-receptors, 6-receptors may be of importance in mediating antinociception. Opioid peptides /3-casomorphins /3-casomorphin analogs Multiple opiate receptors Antinociception THE existence of multiple opiate receptors has been demonstrated in both isolated organ preparations and, by the use of radio-ligand binding studies, brain tissue preparations [4, 16, 21, 25]. It has not been established, however, whether the characteristics of peripheral opiate receptors correspond to those of their counterpart in the brain. Recent experi- ments, for example, suggest that there are differences be- tween the properties of centrally- as compared to peripherally-located opiate receptors [20,25]. Moreover, it is unclear which particular type of opiate receptor is predomi- nantly involved in mediating the antinociceptive action of narcotic drugs. It has been suggested, for example, that either /z-receptors [8, 13, 22], 6-receptors [7], /z- and 6-receptors [21] or/z- and K-receptors [23] are preferentially involved in eliciting antinociception. Recently, a family of novel opioid peptides was dis- covered and named fl-casomorphins, since these peptides represent fragments of the larger bovine milk protein, /3-casein, a constituent of milk [I, 2, 3, 11, 17, 24]. We re- cently suggested that the naturaily-occurrent /3-casomor- phins (by natural, it is intended to indicate that the amino acid sequence is present in bovine/3-casein) possess preferential /z-receptor agonist activity in isolated organ preparations [3]. In addition, the fl-casomorphin-4-amide in radioligand binding studies displays a selectivity towards /z-binding sites [5, 6, 26]. On the other hand, we recently found that the introduction of D-alanine into position 2 of /3-casomorphin-5 resulted in a 12-fold increase in potency in the isolated mouse vas deferens (MVD), a preparation ex- hibiting particular sensitivity to 6-receptor agonists, whereas the potency in the myenteric plexus longitudinal muscle of the guinea-pig ileum (GPI), a preparation exhibiting particu- lar sensitivity to /z-receptor agonists, was not significantly changed [3]. In the present paper the receptor affinities of these D-Ala2-fl-casomorphin-derivatives in radioligand bind- ing studies and in isolated organ preparations are reported and compared with the antinociceptive action of these sub- stances. Moreover, the comparison between the potencies of 1New address: Boehringer Ingelheim KG, Abteilung Medizin, D-6507 Ingelheim/Rhein, F. R. G. Copyright o i982 ANKHO International Inc.--0196-9781/82/050793-05503.00/0