Discovery of novel, orally active dual NK1/NK2 antagonists.

Exploration of the SAR around selective NK2 antagonists, SR48968 and ZD7944, led to the discovery that naphth-1-amide analogues provide potent dual NK1 and NK2 antagonists. ZD6021 inhibited binding of [3H]-NKA or [3H]-SP to human NK1 and NK2 receptors, with high-affinity (K(i)=0.12 and 0.62nM, respectively). In functional assays ZD6021 had, at 10(-7)M, in human pulmonary artery pK(B)=8.9 and in human bronchus pK(B)=7.3, for NK1 and NK2, respectively. Oral administration of ZD6021 to guinea pigs dose-dependently attenuated ASMSP induced extravasation of plasma proteins, ED(50)=0.5mg/kg, and NK2 mediated bronchoconstriction, ED(50)=13mg/kg.

Pharmacological characterization of ZD6021: a novel, orally active antagonist of the tachykinin receptors.

The tachykinins, substance P, neurokinin A, and neurokinin B, have been implicated in many diseases. The present study evaluated the pharmacological properties of a novel tachykinin antagonist ZD6021 [3-cyano-N-((2S)-2-(3,4-dichlorophenyl)-4-[4-[2-(methyl-(S)-sulfinyl)-phenyl]piperidino]butyl)-N-methyl-]-napthamide]. The affinity (K(i)) of ZD6021 for the cloned human neurokinin (NK)1, NK2, and NK3 receptors was 0.12 +/- 0.01, 0.64 +/- 0.08, and 74 +/- 13 nM, respectively. Mucin secretion by Chinese hamster ovary cells transfected with the human NK1 receptor was dose dependently inhibited by ZD6021: pIC(50) = 7.6 +/- 0.1. For NK1 and NK2 receptors, the agonist concentration-response curves using isolated tissues were displaced rightward in the presence of ZD6021: rabbit pulmonary artery, pA2 = 8.7 and 8.5; human pulmonary artery and bronchus, pKB = 8.9 +/- 0.4 and 7.5 +/- 0.2, at 10(-7) M, respectively. Senktide-induced contractions of isolated guinea pig ileum were also blocked by low concentrations of ZD6021. Oral administration of ZD6021 to guinea pigs dose dependently attenuated tracheal extravasation of plasma proteins induced by the NK1 receptor agonist Ac-[Arg6,Sar9,Met(O2)11]-SP(6-11), ED50 = 0.8 micromol/kg, and bronchoconstriction, elicited by the NK2 receptor agonist [beta-Ala8]-NKA(4-10), ED50 = 20 micromol/kg. Potency was unaffected by feeding. After oral administration of ZD6021, the time to peak activity was 150 min for the NK1 receptor and 60 min for the NK2 receptor with pharmacodynamic half-lives of 280 and 458 min, respectively. These data indicate that ZD6021 is a potent, orally active antagonist of all three tachykinin receptors. This compound may be useful for future studies of tachykinin-related pathology such as asthma.

4-Alkylpiperidines related to SR-48968: potent antagonists of the neurokinin-2 (NK2) receptor.

A series of 4-alkylpiperidine derivatives related to the potent neurokinin-2 (NK2) receptor antagonist SR-48968 (1) is described. Simple aliphatic derivatives were found to be poorly active, but appropriate placement of an alcohol functional group afforded compounds that were of similar activity to 1. Several representatives in this series, such as the 4-(1-hydroxy-1-ethylpropyl)piperidine (14), were found to exhibit oral activity in a model of labored abdominal breathing in guinea pigs. These results expand the latitude of substituents available in this region of this series of NK2 receptor antagonists.

Differential blockade by tachykinin NK1 and NK2 receptor antagonists of bronchoconstriction induced by direct-acting agonists and the indirect-acting mimetics capsaicin, serotonin and 2-methyl-serotonin in the anesthetized guinea pig.

This study has examined the abilities of (+/-)-CP96345 and (+/-)-SR48968, nonpeptide antagonists selective for the tachykinin NK1 and NK2 receptors, respectively, to block bronchoconstriction caused by intravenous administration of direct-acting receptor agonists and the indirect-acting mimetics capsaicin, serotonin and 2-methyl-serotonin in the anesthetized guinea pig. The NK1 antagonist (+/-)-CP96345 was found to cause, at a maximally tolerated dose of 9 mumol/kg, an approximate 10-fold rightward shift of the dose-response curves for selective NK1 agonists substance P (SP), [Sar9,Met(O2)11]SP and Ac-[Arg6,Sar9,Met(O2)11]SP6-11 without altering responses to selective NK2 agonists neurokinin A (NKA), [Nle10]NKA4-10 or [beta-Ala8]NKA4-10. The NK2 antagonist (+/-)-SR48968 caused dose-dependent rightward shifts of the dose-response curves for the NK2 but not the NK1 agonists. Results using combinations of the receptor antagonists indicate that the NK2 agonists could cause bronchoconstriction by acting on the NK1 receptors at large doses relative to those used without antagonists. Of the agonists used here, [beta-Ala8]NKA4-10 appeared to be the most selective for the NK2 receptors. When used alone, only (+/-)-SR48968 was found to block bronchoconstriction caused by capsaicin, serotonin (after blockade of 5-HT2 receptors by LY53857) and 2-methyl-serotonin. When (+/-)-CP96345 was also given, larger additional blockade was seen with capsaicin than with serotonin or 2-methyl-serotonin as mimetic substance. Atropine caused small and variable degrees of blockade of serotonin and 2-methyl-serotonin but not of capsaicin after combinations of the two antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonadrenergic, noncholinergic contractile responses of the guinea pig hilar bronchus involve the preferential activation of tachykinin neurokinin2 receptors.

Experiments were designed to characterize receptor(s) that mediate nonadrenergic, noncholinergic contractions of the guinea pig hilar bronchus using selective neurokinin (NK)1 (CP 96,345) and NK2 (R396 and MEN 10,376) tachykinin receptor antagonists. Left and right hilar bronchi were studied as pairs in the presence of atropine, propranolol, phentolamine; indomethacin and thiorphan. (2S, 3S)-cis-2-(diphenylmethyl)-N-(2-methoxyphe nyl)-1-azabicyclo[2,2,2]octan-3-amine (CP 96,345) selectively antagonized contractions of the bronchus to the NK1 agonist Ac-[Arg6,Sar9,Met(O2)11]-SP(6-11) with a -log molar KB value of about 8.0. Similarly, Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2 (R396) and [Tyr5, D-Trp6,8,9, Lys10]-NKA(4-10) (MEN 10,376) selectively antagonized contractions to the NK2 agonist [beta Ala8]-NKA(4-10) with -log molar KB values of about 5.5 and 6.7, respectively. CP 96,345 (3 x 10(-7) M) had no effect on contractions evoked by transmural electrical stimulation (TES). However, both R396 (1 x 10(-5) to 1 x 10(-4) M) and MEN 10,376 (1 x 10(-6) to 1 x 10(-5) M) caused blockade of responses to TES. CP 96,345 (3 x 10(-7) M) antagonized TES-induced contractions only when studied after substantial blockade by R396 or MEN 10,376. Contractions to TES were not abolished by R396, MEN 10,376 or a combination of these antagonists with CP 96,345. R396 (1 x 10(-6) M) increased the maximum contraction to TES and potentiated the frequency-response curve in bronchi treated with MEN 10,376 (1 x 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Recovery of rat nasal mucosa from the effects of aminopeptidase inhibitors.

Aminopeptidase inhibitors may be useful for improving the systemic bioavailability of peptide drugs administered nasally or by other routes. Preferably, their effects would be rapidly reversible. The recovery of peptide hydrolytic activity after exposure of the rat nasal cavity to various aminopeptidase inhibitors was evaluated. Leucine enkephalin (0.1 mM) was used as a model peptide which is predominantly metabolized by aminopeptidases nasally. All experiments involved in situ perfusion of the rat nasal cavity with leucine enkephalin and the inhibitor for 90 min, followed by a washout with saline, and finally a second experimental phase of perfusion with leucine enkephalin but no inhibitor. Boroleucine (0.1 microM) was a potent inhibitor of leucine enkephalin metabolism, and, after its removal, the leucine enkephalin metabolism rate returned to control levels. Much higher concentrations of bestatin (0.1 mM) and puromycin (1 mM) did not inhibit leucine enkephalin metabolism as much as did boroleucine. Furthermore, when these inhibitors were washed out, the rates of leucine enkephalin disappearance rebounded to higher than control levels. With bestatin this could have been partially due to membrane disruption, as evidenced by significantly increased protein concentrations in the perfusates. However, protein release was much lower than that caused by sodium glycocholate, a nasal membrane permeation enhancer. In considering the use of peptidase inhibitors as pharmaceutical adjuvants for peptide drugs, the aminoboronic acid derivatives, including boroleucine, have the advantages of efficacy at very low concentrations and reversibility of effects.

Inhibition of leucine enkephalin metabolism in rat blood, plasma and tissues in vitro by an aminoboronic acid derivative.

Boroleucine, an aminoboronic acid derivative, is a potent inhibitor of aminopeptidases. Its effects on leucine enkephalin metabolism in rat whole blood, plasma and skeletal muscle and brain homogenates were determined in vitro. In the absence of the inhibitor, leucine enkephalin disappeared very rapidly from each medium. The rank order of metabolism rates was brain greater than muscle greater than whole blood approximately plasma. Des-tyrosyl-leucine enkephalin rapidly appeared as a result of metabolism by aminopeptidases. In whole blood and in the muscle and brain homogenates the aminopeptidase metabolite could account for all of the lost leucine enkephalin. In plasma, however, it appeared that another metabolic route contributed. Boroleucine prolonged the leucine enkephalin degradation half-life 1.6-1.9-fold in each medium at 0.1 microM concentrations, 1/1000th the concentration of leucine enkephalin. At 1.0 microM boroleucine concentrations the half-lives were prolonged 4.0-6.4-fold. Boroleucine also inhibited the degradation of des-tyrosyl-leucine enkephalin added to whole blood, but did not inhibit its degradation in muscle or brain homogenates. Boroleucine and other aminoboronic acid derivatives may be useful tools for studying peptide metabolism, and as pharmaceutical adjuvants to inhibit the degradation of peptide drugs metabolized by aminopeptidases.

An aminoboronic acid derivative inhibits thymopentin metabolism by mucosal membrane aminopeptidases.

Thymopentin is a pentapeptide with immunomodulatory activity. Transmucosal delivery may offer advantages over other routes, but published data have shown relatively poor efficacy when dosed nasally. Metabolism of thymopentin by the rat nasal mucosa and the effects of an aminoboronic acid aminopeptidase inhibitor, boroleucine, were evaluated. Thymopentin concentrations in a solution perfused through the isolated nasal cavity decayed with a first-order half-life of 12 minutes. Thymopentin was metabolized primarily by aminopeptidases, based on the amount of tetrapeptide metabolite formed. In the presence of boroleucine, at an inhibitor/substrate molar concentration ratio of 0.015/1, the degradation half-life was prolonged to 37 minutes. Appearance of the tetrapeptide metabolite of aminopeptidases was delayed. Boroleucine and other aminoboronic acid peptidase inhibitors may be useful for improving thymopentin delivery.

The use of alpha-aminoboronic acid derivatives to stabilize peptide drugs during their intranasal absorption.

alpha-Aminoboronic acid derivatives, potent and reversible inhibitors of aminopeptidases, were tested nasally in situ in rats for stabilizing externally administered peptides. These inhibitors, at nanomolar concentrations, were found to inhibit greatly the degradation of the model peptide, leucine-enkephalin (Leu-enk) in the nasal perfusate. Enzyme inhibition was greater with boroleucine and borovaline than that observed with boroalanine. Boroleucine was more than 100 times more effective in enzyme inhibition than bestatin and more than 1000 times more effective than puromycin.

Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes.

15N NMR spectroscopy was used to examine the active-site histidyl residue of alpha-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. With the better inhibitors of the class this arrangement is stable over the pH range 4.0-10.5. The results are consistent with a putative tetrahedral intermediate like complex involving a negatively charged, tetrahedral boron atom covalently bonded to O gamma of the active-site serine. (2) Boronic acids that are not substrate analogues form complexes in which N epsilon 2 of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N delta 1 of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. Benzeneboronic acid, which falls in this category, forms an adduct with histidine. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field 1H resonances and the 15N-H spin couplings. These results, coupled with the kinetic data of the preceding paper [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry (preceding paper in this issue)], indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed.

Interaction of peptide boronic acids with elastase: circular dichroism studies.

Boronic acid derivatives of good peptide substrates of the serine proteases cause slow-binding inhibition, manifested as biphasic binding (Kettner and Shenvi: J. Biol Chem. 259:15106-15114, 1984). These inhibitors are thought to act as reaction-intermediate analogs. Three peptide boronic acids--Ac-Pro-boro-Val-OH, DNS-Ala-Pro-boro-Val-OH, and Ac-Ala-Ala-Pro-boro-Val-OH--were chosen for far-ultraviolet circular dichroism (CD) studies in order to determine whether the second phase involves a conformational change of pancreatic elastase. The dipeptide is a simple competitive inhibitor (Ki = 0.27 microM) and the latter are slow-binding inhibitors (Ki = 16.4 and 0.25 nM, respectively). Spectral deconvolution and correction for the formation of antiparallel beta-sheet by the peptide inhibitor itself indicate that there is no significant change in the secondary structure of the enzyme in either the initial or final inhibitor complex. A kinetic experiment confirmed that the slow-binding step was not associated with a CD spectral change, and that therefore a protein conformational change was not responsible for the slow binding.

Serine protease mechanism: structure of an inhibitory complex of alpha-lytic protease and a tightly bound peptide boronic acid.

The structure of the complex formed between alpha-lytic protease, a serine protease secreted by Lysobacter enzymogenes, and N-tert-butyloxycarbonylalanylprolylvaline boronic acid (Ki = 0.35 nM) has been studied by X-ray crystallography to a resolution of 2.0 A. The active-site serine forms a covalent, nearly tetrahedral adduct with the boronic acid moiety of the inhibitor. The complex is stabilized by seven hydrogen bonds between the enzyme and inhibitor with additional stabilization arising from van der Waals interactions between enzyme and inhibitor side chains and the burying of 330 A2 of hydrophobic surface area. Hydrogen bonding between Asp-102 and His-57 remains intact in the enzyme-inhibitor complex, and His N epsilon 2 is well positioned to donate its hydrogen to the leaving group. Little change in the positions of protease residues was observed on complex formation (root mean square main chain deviation = 0.13 A), suggesting that in its native state the enzyme is complementary to tetrahedral reaction intermediates or to the nearly tetrahedral transition state for the reaction.

A new peptide boronic acid inhibitor of elastase-induced lung injury in hamsters.

A highly effective, reversible elastase inhibitor, MeOSuc-Ala-Ala-Pro-boroVal-OH, was tested for its ability to prevent emphysema induced by intratracheally administered elastase in hamsters. Anesthetized hamsters were given elastase intratracheally with or without the inhibitor or were given elastase intratracheally and the inhibitor intraperitoneally. Two weeks after administration, lungs were removed, and static air pressure volume curves were performed followed by intratracheal fixation and morphometric determination of mean linear intercepts. The results indicate significant preservation of structure and function whether the inhibitor is given intratracheally or intraperitoneally and suggest that this inhibitor may be useful in controlling diseases arising from aberrant proteolysis by elastolytic enzymes.

Effects of dosage and timing of administration of a peptide boronic acid inhibitor on lung mechanics and morphometrics in elastase-induced emphysema in hamsters.

The peptide boronic acid, MeOSuc-Ala-Ala-Pro-boroVal-pinacol (AAPbV), is an effective inhibitor of both pancreatic and leukocyte elastase. Initial work showed that AAPbV diminishes the effect of emphysema induced by pancreatic elastase. This initial work has been expanded to show that AAPbV provides a high degree of protection against elastase-induced increases in lung volume and mean linear intercept when given intratracheally at 200 mg/kg either 15 min before, simultaneous with, or 15 min after instilling elastase. Intraperitoneal administration, although less effective, is dose dependent and dependent on the time of treatment. We conclude that a reversible protease inhibitor can be used to prevent aberrant proteolysis in vivo.

alpha-Aminoboronic acid derivatives: effective inhibitors of aminopeptidases.

alpha-Aminoboronic acids and their derivatives have been synthesized as stable white solids. These compounds are effective inhibitors of human enkephalin degrading aminopeptidase, microsomal leucine aminopeptidase (EC 3.4.11.2), and cytosolic leucine aminopeptidase (EC 3.4.11.1) at micro- to nanomolar concentrations. The inhibition of cytosolic leucine aminopeptidase has been studied in some detail. Kinetic data correspond to the mechanism for biphasic slow-binding inhibition: E + I in equilibrium E.I in equilibrium E.I*, in which a rapid initial binding is followed by a slow transformation to a stable enzyme inhibitor complex. The initial and final binding constants are dependent on the nature of the side chain at the alpha-carbon atom but are independent of the protecting group on the boronic acid moiety and follow the trend for the hydrolysis of the corresponding amino acid amides. The first-order rate constant for the transformation of E.I to E.I* is similar for all four compounds studied. These data suggest that the slow-binding step represents the formation of tetrahedral boronate species from trigonal boronic acid.

Inhibition of the serine proteases leukocyte elastase, pancreatic elastase, cathepsin G, and chymotrypsin by peptide boronic acids.

Three alpha-aminoboronic acid-containing analogs of good peptide substrates for serine proteases were synthesized, MeO-Suc-Ala-Ala-Pro-boro-Phe-OH, MeO-Suc-Ala-Ala-Pro-boro-Ala-OH, and MeO-Suc-Ala-Ala-Pro-boro-Val-OH. They were effective inhibitors of chymotrypsin, cathepsin G, and both leukocyte and pancreatic elastase at nanomolar concentrations (0.10-20 nM). Except for cathepsin G, inhibition was not simply competitive, but showed kinetic properties corresponding to the mechanism for slow-binding inhibition, i.e. E + I in equilibrium EI in equilibrium EI*, where EI and EI* are enzyme-inhibitor complexes and EI* is more stable than EI. This type of inhibition has not been observed previously for synthetic inhibitors or serine proteases and in this study it was observed only for peptide boronic acids which satisfy the primary specificity requirements of the protease.