Kinin B1 receptor antagonists containing alpha-methyl-L-phenylalanine: in vitro and in vivo antagonistic activities.

-To protect from metabolism and to improve potency of the AcLys-[D-betaNal7,Ile8]desArg9-bradykinin (BK) (R 715), we prepared and tested 3 analogues containing alpha-methyl-L-Phe ([alphaMe]Phe) in position 5: these are the AcLys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 892), Lys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 913), and AcLys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 914). The new compounds were tested against the contractile effect induced by desArg9BK on 2 B1 receptor bioassays, the human umbilical vein, and the rabbit aorta. Their antagonistic activities were compared with those of the early prototypes (Lys-[Leu8]desArg9BK and [Leu8]desArg9BK) and with other recently described peptide antagonists. The 3 (alphaMe)Phe analogues showed high antagonistic potencies (pA2) at both the human (8.8, 7.7, and 8. 7, respectively) and rabbit (8.6, 7.8, and 8.6, respectively) B1 receptors. No antagonistic effects (pA2<5) were observed on the B2 receptors that mediate the contractile effects of BK on the human umbilical vein, the rabbit jugular vein, and the guinea pig ileum. Moreover, these new B1 antagonists were found to be resistant to in vitro degradation by purified angiotensin-converting enzyme from rabbit lung. The Nalpha-acetylated forms, R 892 and R 914, were resistant to aminopeptidases from human plasma. In vivo antagonistic potencies (ID50) of B1 receptor antagonists were evaluated in anesthetized lipopolysaccharide-treated (for B1 receptor) and nontreated (for B2 receptor) rabbits against the hypotensive effects of exogenous desArg9BK and BK. R 892 efficiently inhibited (ID50 2.8 nmol/kg IV) hypotension induced by desArg9BK without affecting that evoked by BK (ID50 >600 nmol/kg IV). Conversely, the peptide antagonists Lys-Lys-[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK (B 9858) and DArg-[Hyp3,Thi5,D-Tic7,Oic8] desArg9BK (S 0765) showed dual B1/B2 receptor antagonism in vitro and in vivo. It is concluded that R 892 and congeners provide selective, highly potent, and metabolically stable B1 kinin receptor antagonists that can be useful for the assessment of the physiological and pathological roles of kinin B1 receptors.

Structure-activity studies of B1 receptor-related peptides. Antagonists.

We tested several peptides related to des-Arg9-bradykinin as stimulants or inhibitors of B1 (rabbit aorta, human umbilical vein) and B2 (rabbit jugular vein, guinea pig ileum, human umbilical vein) receptors. We also incubated the compounds with purified angiotensin-converting enzyme from rabbit lung to test their resistance to degradation. We evaluated apparent affinities (in terms of the affinity constant pA2) of compounds and their potential residual agonistic activities (alpha E). Bradykinin and des-Arg9-bradykinin were used as agonists for the B2 and B1 receptors, respectively. Degradation of peptides by the angiotensin-converting enzyme was prevented in the presence of a D-residue in position 7 of des-Arg9-bradykinin. Replacement of Pro7 with D-Tic combined with Leu, Ile, Ala, or D-Tic in position 8 led to weak B1 receptor antagonists, some of which had strong residual agonistic activities on the B2 receptor preparations. The use of D-beta Nal in position 7, combined with Ile in position 8 and AcLys at the N-terminal (eg, AcLys[D-beta Nal7, Ile8]des-Arg9-bradykinin) gave the most active B1 receptor antagonist (pA2 of 8.5 on rabbit aorta and human umbilical vein), which is also partially resistant to enzymatic degradation. Extension of the N-terminal end by Sar-Tyr-epsilon Ahx (used for labeling purposes) and even cold-labeling of Tyr with iodine were compatible with high, selective, and specific antagonism of the B1 receptors. We compared some compounds with some already known B1 receptor antagonists to underline the novelty of new peptidic compounds.

In vitro and in vivo characterization of bradykinin B2 receptors in the rabbit and the guinea pig.

A comparative study has been performed in isolated organs and in anesthetized animals, rabbits, and guinea pigs, to evaluated the myotropic responses (in the organs) and the blood pressure changes (in the animals) induced by bradykinin (BK) and related peptides. Antagonist affinities have also been estimated in vitro in terms of PA2 and in vivo in terms of ID50, to characterize the kinin B2 receptors in the two species. Differences have been found both in the order of potency of agonists and in the affinity of antagonists: in fact, in the rabbit, [Hyp3]BK > [Aib7]BK, is the opposite order of what is found in the guinea pig, namely, [Aib7]BK < [Hyp3]BK, both in vitro and in vivo. Results obtained with antagonists also show important differences between the two species, since DArg[Hyp3, DPhe7, Leu8]BK is more active in the rabbit than in the guinea pig, while WIN-64338 is fairly active in the guinea pig and almost inactive in the rabbit. HOE-140, the long-acting antagonist of the B2 receptor, shows similar affinities in vitro in the two species. In another series of experiments, peptide degradation by angiotensin converting enzyme (ACE) has been investigated to see whether the differences of potency observed between certain peptides interacting with the B2 receptor were due to metabolic degradation. When incubated in the presence of pure ACE from rabbit lung, BK,[Hyp3]BK, and des Arg9BK are readily degraded, while [Aib7]BK, HOE-140, and DArg[Hyp3, DPhe7, Leu8]BK are not. When applied intravenously (i.v.), to obtain degradation by the lung, and intraarterially (i.a.), to avoid such degradation, the effect of BK (i.v.) is markedly reduced (compared with the effect i.a.), while no difference is observed for [Aib7]BK. Thus, despite its resistance to degradation by ACE, [Aib7]BK shows very little activity in the rabbit, suggesting that the major cause in the variation of affinities observed between kinin analogs is related to their pharmacodynamic properties. Taken together, the results speak strongly in favor of the existence of B2 receptor subtypes in the peripheral circulation of the rabbit and the guinea pig. Results obtained in vivo, both in pharmacological and biochemical experiments, are in accord with the findings obtained in isolated organs and with purified ACE enzyme.