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.     

Contractile responses of aortae from WKY and SHR to vasoconstrictors

Aortae taken from spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats aged 4, 8 and 16 weeks were prepared as rings and used to measure the effects of five vasoconstrictors. The endothelium was removed in order to measure selectively the contractile responses induced by potassium chloride (KCl), phenylephrine (PHE), angiotensin-II (Ang II), endothelin-1 (ET-1) and human urotensin-II (U-II). These responses were assumed to derive from the activation of specific receptors (namely alpha1, AT1, ETA and UT-II) or from depolarization of the smooth muscle fibers by KCl. Specific antagonists prazosin, losartan, BQ-123 and [Orn8]-UII were used at various concentrations for a pharmacological characterization of these latter receptor systems. The primary purpose of the study was to explore mechanisms or factors that may intervene in the development and maintenance of high blood pressure in SHR. Results indicate that isolated aortae of SHR and WKY contain contractile sites (receptors) whose pharmacological profiles (pEC50 for agonists, pA2 for antagonists) are very similar to those of other biological systems and should be considered as typical for the alpha1, AT1, ETA and UT-II receptor types. Aortae taken from SHR 4 (non hypertensive), 8 and 16 weeks old (hypertensive) responded to the vasoconstrictors with reduced maximal contractions compared to those of age-matched WKY. These unexpected reduced responses of aortae, observed with the five vasoconstrictors, may be attributed to a non specific lesions. Maximal contraction of aortae from SHR increased from 4 to 16 weeks for KCI, PHE and U-II, decreased for Ang II, and remained stable for ET-1. There was also an age-dependent increase of maximal contraction induced by U-II in WKY. It is suggested that aortae from SHR undergo early remodelling that leads to reduced contractility in vitro and possibly to vessel rigidity in vivo. The factors involved in this process appear to be of genetic origin since they are present before hypertension: they may contribute to modify aortic compliance and perhaps vascular resistance in hypertensive animals and thus being the cause and not the consequence of high blood pressure.     

Nphe(1)]-Nociceptin (1-13)-NH(2), a nociceptin receptor antagonist, reverses nociceptin-induced spatial memory impairments in the Morris water maze task in rats

1. The present study was undertaken to investigate the effects of the novel nociceptin receptor antagonist, [Nphe(1)]-Nociceptin (1-13)-NH(2) (bilateral intrahippocampal injection, 50 nmole rat(-1)) on purported nociceptin-induced (bilateral intrahippocampal injection, 5 nmole rat(-1)) deficits in spatial learning in the rat Morris water maze task. In addition, experiments were performed in an 'open field' to investigate possible peptide-induced changes in exploratory behaviour. 2. Nociceptin significantly impaired the ability of the animal to locate the hidden platform throughout training (P<0.001 versus control group). 3. Pretreatment with [Nphe(1)]-Nociceptin (1-13)-NH(2) significantly blocked nociceptin-induced impairment of spatial learning (P<0.001 versus nociceptin group). 4. A probe trial revealed that vehicle-treated animals spent more time in the quadrant that had previously contained the hidden platform, whereas nociceptin-treated animals did not spend more time in any one quadrant. 5. Learning impairments were not attributable to non-specific deficits in motor performance or change in exploratory behaviour. 6. Taken together, our results reveal that [Nphe(1)]-Nociceptin (1-13)-NH(2) represents an effective and useful in vivo antagonist at the nociceptin receptors involved in learning and memory.     

Nphe(1)]nociceptin-(1-13)NH(2) selectively antagonizes nociceptin effects in the rabbit isolated ileum

When suspended in vitro in isolated organ baths, segments of the rabbit ileum show a fairly strong and stable spontaneous activity, which derives from the continuous release of acetylcholine and the activation of muscarinic receptors, since the activity is completely eliminated by atropine. Dynorphin A (pEC(50): 8.6+/-0.07), neuropeptide Y and its congener human pancreatic polypeptide (pEC(50): 9.40+/-0.10), and nociceptin (pEC(50): 8.08+/-0.12) dose-dependently inhibit the spontaneous activity through the activation of receptors, which are specifically antagonised respectively by naloxone (pA(2): 7.17+/-0.12), 2-(naphtalen-1-ylamino)-3-phenylpropionitrile (JCF 104; pA(2): 5. 80+/-0.10), and [Nphe(1)]nociceptin-(1-13)NH(2) (pA(2): 6.17+/-0.19). This last compound, a selective nociceptin-receptor (OP(4)) antagonist, inhibits the effect of nociceptin in a competitive manner, as demonstrated by Schild analysis. [Nphe(1)]nociceptin-(1-13)NH(2) also antagonizes the effects of other OP(4) receptor ligands such as the full agonist, nociceptin-(1-13)-NH(2), and the partial agonists, [Phe(1)psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)-NH(2) (intrinsic activity (alpha(E))=0.5) and Ac-RYYWK-NH(2) (alpha(E)=0.5), with pA(2) values ranged from 5.8 to 6.2. These results indicate that the functional site mediating the inhibitory effect of nociceptin in the rabbit ileum, is pharmacologically identical to the OP(4) sites of other species (mouse, rat, guinea pig, man), since the potencies (pA(2) values) of the pure and competitive antagonist [Nphe(1)]nociceptin-(1-13)NH(2) is very similar to the values obtained in the other species. Moreover, the rabbit ileum is one of the few isolated organs that allow classifying compounds, which interact with OP(4) receptors as full agonists, partial agonists, or pure antagonists.     

In vitro characterization of J-113397, a non-peptide nociceptin/orphanin FQ receptor antagonist

The lack of availability of a selective, highly potent, competitive antagonist for the nociceptin receptor (OP4) devoid of residual agonistic activity has hampered studies in this area. We report here the in vitro pharmacological properties of the novel non-peptide OP4 antagonist, J-113397, which was recently discovered by Banyu Pharmaceutical investigators. The compound was synthesized as a racemic mixture in our laboratories. J-113397 was shown to antagonize (pA2 7.52) the nociceptin-induced inhibition of cAMP formation in cells expressing the recombinant human OP4 receptor (CHOhOP4) and to displace [125I]Tyr14nociceptin from CHOhOP4 membranes with a pKi of 8.56. It also competitively antagonized the contractile actions of nociceptin in the mouse colon (pA2 8.07) and the inhibitory effect of nociceptin in electrically stimulated preparations such as the mouse vas deferens (pA2 7.85), the guinea pig ileum (7.75), and the rat vas deferens (7.77). At high concentrations (10 microM), the compound was devoid of agonist activity in the mouse vas deferens and CHOhOP4, while it contracted the mouse colon and increased the twitch response of the rat vas deferens, and produced a naloxone-sensitive inhibition of the electrically evoked twitches in the guinea pig ileum. pA2 values for the new antagonist against deltorphin I in the mouse vas deferens (OP1 receptors), or against dermorphin in the guinea pig ileum (OP3 receptors), etorphine in the rat vas deferens (OP receptors), U69593 in the rabbit vas deferens (OP2 receptors) and endomorphin 1 in the mouse colon (OP3 receptors) were lower than 6. Taken together, these data indicate that J-113397 is a high-affinity, selective and competitive antagonist of the OP4 receptor; this novel pharmacological tool will be of great value in studies directed at evaluating the physiological roles of the nociceptin/OP4 system.     

Total oxyradical scavenging capacity (TOSC) in polluted and translocated mussels: a predictive biomarker of oxidative stress

Several classes of environmental pollutants are known to enhance the intracellular formation of reactive oxygen species in marine invertebrates with different consequences on their antioxidant system. Despite variations in the endogenous levels of antioxidants may reveal biological effects induced by pollutants, the overall efficiency of antioxidant system is not evaluated from such data. The total oxyradical scavenging capacity (TOSC) assay measures the biological resistance to various kinds of oxyradicals, thus providing useful indications to predict oxyradical-mediated adverse effects on the physiological condition of the organisms. In the present work, the capability to neutralize three potent cellular oxidants (peroxyl radicals, hydroxyl radicals and peroxynitrite) was compared in two natural populations of mussels from a clean and a metal polluted area, respectively, and in control organisms translocated for 8 weeks to the contaminated site. Within each group of mussels, the relative efficiency towards the various forms of oxyradicals, showed peroxyl radicals and hydroxyl radicals as the less and the most difficult species, respectively, to neutralize by cellular antioxidants. When the two populations were compared, polluted organisms revealed a significantly higher susceptibility to oxidative stress as indicated by their lower TOSC values. A significant reduction of antioxidant capacity was observed also in translocated organisms where only a moderate recovery was evident at the longer exposure time. Separation of soluble antioxidants from the protein fraction, suggested a greater depletion of low molecular weight molecules during the first phase of exposure to pollutants. The pollutant-induced impairment of antioxidant capability precludes the appearance of oxyradical toxicity. In the present study a high correlation was obtained between reduced TOSC values and lower Neutral Red retention time in lysosomal compartment of circulating haemocytes. The TOSC towards different forms of oxyradicals is confirmed an useful biomarker with predictive validity at the organism level.     

Structure-activity studies on nociceptin/orphanin FQ: from full agonist, to partial agonist, to pure antagonist

A heptadecapeptide (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln) was identified from rat brain and from porcine brain as a ligand for OP4, a new G-protein coupled receptor that is similar in sequence to opioid receptors. The OP4 receptor is widely expressed in the nervous system where it mediates a broad range of physiological functions. The new peptide, nociceptin (NC), has a primary sequence recalling that of opioid peptides. Despite the homologies (a) of the OP4 receptor with known opioid receptors, especially the OP2 (kappa) receptor, and (b) of NC with opioid peptides, particularly dynorphin A, the two biological systems have different anatomical locations and chemical requirements for activation. NC does not bind to opioid receptors, and mammalian opioid peptides do not interact with the OP4 receptor. The presence of Phe in position 1 and Arg in position 8, appear to be instrumental to exclude NC from interacting with the opioid receptors. Contrary to opioid peptides which strikly require Tyr in position 1, the active core that activates the OP4 appears to be towards the centre of the peptide molecule and includes Phe4. Based on the message/address model, several changes have been made in the N-terminal tetrapeptide Phe-Gly-Gly-Phe (message) and a few also in the C-terminal of the template NC(1-13)-NH2, a fragment that acts as a full agonist both in vitro and in vivo. Subtle changes of the N-terminal sequence, especially at Phe1, led to the discovery of peptide antagonists ([Phe1 psi (CH2-NH)Gly2[-NC(1-13)-NH2 and [Nphe1[-NC(1-13)-NH2). The first compound has been widely used to characterize NC actions in the periphery and in the central nervous system. It has been shown to act mainly as an antagonist outside the brain and as an agonist in the central nervous system. [Nphe1[-NC(1-13)-NH2- on the contrary, acts as antagonist both in the periphery and in the brain. These first peptide prototypes may soon be followed by non-peptide compounds, some of which, are already described in patient literature.     

Involvement of bradykinin B1 and B2 receptors in pulmonary leukocyte accumulation induced by Sephadex beads in guinea pigs.

The effects of selected bradykinin receptor antagonists on leukocyte infiltration into the lungs were studied in a model of guinea pig lung inflammation induced by the intravenous injection of Sephadex beads. The bradykinin B1 receptor antagonist, [Leu8]desArg9-BK (40 mg kg(-1) 24 h(-1)) and the bradykinin B2 receptor antagonist, DArg[Hyp3,Thi5,DTic7,Oic8]BK (code name HOE 140; 4 mg kg(-1) 24 h(-1)), administered intravenously by osmotic pumps, significantly reduced eosinophil counts by 33% and 42% in bronchoalveolar fluid, respectively. HOE 140 decreased neutrophil counts by 35%. LysLys[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK+ ++ (code name B 9858), a newly described bradykinin B1 receptor antagonist, administered intraperitoneally (1 mg kg(-1)), decreased eosinophil and neutrophil counts by 45% in bronchoalveolar fluid. D-Arg[Hyp3,Igl5,D-Igl7,Oic8]BK (code name B 9430), a non-selective bradykinin B1/B2 receptor antagonist, also administered intraperitoneally (1 mg kg(-1)), decreased eosinophil and macrophage counts by 62% and 80% in bronchoalveolar fluid. These results suggest that bradykinin B1 and B2 receptors are involved in leukocyte recruitment in our model of lung inflammation.