Development of orally active nonpeptidic inhibitors of human neutrophil elastase.

5-Amino-2-phenylpyrimidin-6-ones, some of their desamino derivatives, and miscellaneous derivatives were synthesized and biologically evaluated on both in vitro activity and oral activity in an acute hemorrhagic assay. These compounds contained an alpha-keto-1,3,4-oxadiazole moiety to bind covalently to the Ser-195 hydroxy group of human neutrophil elastase (HNE). Among those tested, compounds 11a-c,e,i-l(F), 11d,e,k(H), 21d,e,k(F), and 21d,e(H) showed a good oral profile. RS-Mixture 3(H) was selected for clinical evaluation based on its oral potency, duration of action, enzyme selectivity, safety profile, and ease of synthesis. Structure-activity relationships (SARs) are discussed.

Biochemical characterization of alpha-ketooxadiazole inhibitors of elastases.

A series of alpha-ketooxadiazole compounds was prepared and evaluated in vitro as potential inhibitors of human neutrophil elastase (HNE), proteinase-3 (PR-3), and porcine pancreatic elastase (PPE). Several compounds have been found to be very potent, fast, reversible, and selective inhibitors of HNE with Ki values below 100 pM. The highest kon value exceeded 10(7) M(-1) s(-1). Some alpha-ketooxadiazoles were also very effective against PR-3 and PPE with Ki values in the range of 5(-10) nM and 0.1(-2) nM, respectively. The two rings, 1,2,4- and 1,3,4-oxadiazole, are amenable to substitutions, extending the P' side of the inhibitor and allowing additional binding interactions at S' subsites of the enzyme. Nonpeptidic HNE inhibitors containing the oxadiazole heterocycle displayed promising oral bioavailability.

Proteolytic destruction of functional proteins by phagocytes in human peritonitis.

BACKGROUND: Besides phagocyte-derived oxidative autoaggression, proteolytic destruction of functional proteins in the peritoneal cavity may also be involved in the pathomechanism of secondary peritonitis. To evaluate the pattern of proteolysis, 43 patients undergoing initial operation for acute peritonitis (n = 30) or scheduled abdominal lavages (Etappenlavage) for resolution of persistent peritonitis (n = 13) and 16 surgical patients with abdominal exudation without peritonitis were enrolled in our study. MATERIALS AND METHODS: Thirty blood samples and purulent exudates were taken simultaneously in each peritonitis group at the surgical interventions. Sixteen clear exudates were obtained from patients with post-operative non-infectious irritations. The following parameters were measured: (a) elastase (from neutrophils) and cathepsin B (from monocytes/macrophages); (b) alpha1-proteinase inhibitor (alpha1PI) and overall cysteine proteinase inhibitor capacity; and (c) opsonic activity and degradation products of fibrinogen, complement C3 and immunoglobulin IgG. RESULTS: Circulating levels of phagocyte proteinases and of alpha1PI were significantly elevated, whereas antigen concentrations and opsonic activity of C3 and IgG were slightly reduced in peritonitis patients compared to healthy volunteers. No degradation products were detectable in patients' blood. Discharge of phagocyte proteinases was even more pronounced in both types of peritonitis exudates. Although most of the elastase was complexed with alpha1PI, active elastase and its specific fibrinogen split product was found along with significantly reduced inhibitory capacity for elastase and cysteine proteinases. Local opsonic activity was dramatically diminished because of proteolytic degradation of C3 and IgG. Despite some phagocyte proteinase release, no destruction of functional proteins was seen in clear exudates. CONCLUSIONS: Higher values of extracellularly released phagocyte proteinases concomitant with lower opsonin activity in exudates from patients with persistent peritonitis can be taken as a further hint of the involvement of local proteolysis-induced pathomechanisms in the development of lethal multiple organ failure, which occurred more frequently in patients with persistent peritonitis (54%) than in those with acute peritonitis (27%).

Endogenous B1 receptor mediated hypotension produced by contact system activation in the presence of endotoxemia.

Previous studies have shown that an intravenous infusion of dextran sulfate (DXS) causes arterial hypotension via release of bradykinin (BK) and stimulation of bradykinin B2 receptors in pigs. The bradykinin B1 receptor is not physiologically present but its expression can be induced by bacterial lipopolysaccharide (LPS). This study was designed to assess the relative roles of bradykinin B2 and B1 receptors in the hypotensive response produced by DXS in LPS-treated pigs. In LPS-treated pigs a continuous infusion of DXS produced a progressive drop in blood pressure that peaked at approximately 30 min after onset of the infusion and returned to baseline after another 30 min. In animals receiving the selective B2 receptor antagonist Hoe-140 a significant attenuation of the peak fall in blood pressure to DXS was observed. In pigs treated with Hoe-140 and the selective B1 receptor antagonist CP-0298 (Lys(0)-Leu(8)-des-Arg(9)-bradykinin) DXS infusion had no effect on blood pressure. This is the first demonstration in vivo that following activation of the contact system both B2 and B1 receptors are involved in the resulting hypotensive response. This would be consistent with the production of BK (which stimulates B2 receptors) that is subsequently converted to the biologically active metabolite des-Arg(9)-BK in sufficient concentrations to activate B 1 receptors. The significance of these observations to pathophysiology remains to be determined.

B1 kinin receptor activity in pigs is associated with pre-existing infection.

Bradykinin (BK) and related kinins are potent inflammatory mediators produced during acute and chronic inflammation. The effects of these kinins are mediated via the stimulation of either a B2 or a B1 receptor. The B1 receptor is not normally present but its expression can be induced within 4 h by a variety of noxious stimuli, specifically, gram-negative bacteria or bacterial lipopolysaccharide (LPS) given to healthy animals. This study compared the cardiovascular response of healthy pigs and pigs diagnosed with a pre-existing spontaneously acquired infection to BK, a B2 receptor agonist, and des-Arg9-BK, a B1 receptor agonist. Eighty-eight percent of the animals diagnosed with an established infection based on a standardized clinical evaluation demonstrated increased sensitivity and responsiveness to des-Arg9-BK but normal responsiveness to BK and acetylcholine. In contrast, only 15% of healthy animals showed elevated responses to des-Arg9-BK. The response to des-Arg9-BK and BK in each group was characterised as B1 and B2, respectively, using the selective B1 and B2 antagonists Lys0-Leu8-des-Arg9-BK and Hoe 140, respectively. This study demonstrates the existence and function of the B1 receptor in animals with a previously acquired infection. These observations lend validity to animal experiments with LPS infusion in order to model bacterial inflammation.

Oxidative autoaggression by phagocytes in human peritonitis.

In 60 abdominal exudates from patients with the diagnosis of either acute or persistent (chronic) peritonitis, indicators of phagocyte-derived oxidative systems (myeloperoxidase, chemiluminescence) and proteases (elastase) were measured. These exudates reveal a picture of maximal inflammatory activation. Both types of exudates (30 each) showed a significant influx of inflammatory cells, with the mean leucocyte count being 73,000 microL and 32,000 microL-1 respectively. Local myeloperoxidase concentrations were approximately 1000-fold greater than that of normal plasma. Spontaneous and elicitable chemiluminescence--indicators of phagocyte respiratory burst activity--were dramatically increased. In addition, levels of extracellularly released elastase (from neutrophils) were found to be up to about 1000-fold that of normal plasma values. Although most of the elastase detected in the exudates was complexed with alpha 1-proteinase inhibitor (alpha 1 PI), enzymatically active elastase could be measured in approximately 60% of the samples being investigated. As there was an excess of immunoreactive alpha 1 PI in these exudates, the free elastase activity implies that much of the alpha 1 PI was inactive, presumably subjected to oxidative destruction. Moreover, a trypsin-inhibitory activity to antigen ratio below 1 (mean = 0.81) in 75% of the purulent exudates indicated also partial proteolytic degradation of alpha 1 PI. In contrast, 16 clear exudates (no bacteria, white cell count below 500 microL-1) taken from the non-infected peritoneal cavity of patients undergoing intra-abdominal surgery revealed a similar permeability increase of the peritoneum but did not show relevant oxidative and proteolytic activity or destruction of alpha 1 PI compared with purulent specimens. Thus, only the inflammatory process of peritonitis appears to result in an overwhelming local phagocytic activity that initiates and maintains protease inhibitor consumption and/or inactivation. The tremendous oxidative potential found in purulent exudates may cause destruction in a wide variety of defence systems.

Endothelial cell associated anti-elastolytic activity.

Addition of cultured and then carefully-washed bovine pulmonary artery endothelial cells (EC) decreased (p < 0.05) human neutrophil elastase activity (HNE) in vitro. HNE activity was also decreased (p < 0.05) by addition of histone or protamine treated EC. However, addition of papain or trypsin treated EC decreased HNE activity less than addition of untreated cells suggesting that a protein rather than a difference in cell surface charge was responsible. Other observations suggest that EC anti-elastolytic activity was not due to binding of antiprotease from culture media but was dependent on EC protein synthesis. First, addition of EC grown previously in serum-free media decreased HNE activity the same (p < 0.05) as addition of EC cultured in media containing serum. Second, addition of EC treated beforehand with cycloheximide decreased HNE activity less than (p < 0.05) addition of untreated control EC. We conclude that EC most likely make and have anti-elastolytic activity on their surfaces and speculate that EC associated anti-elastolytic activity may modulate inflammatory, repair and other biologic processes involving neutrophil elastase.

Effect of combined B1 and B2 kinin receptor blockade in porcine endotoxin shock.

In order to investigate the contribution of kinin receptor antagonism in the treatment of LPS-induced shock we conducted a randomized study with anaesthetized piglets. Before randomization the animals were stratified according to predetermined health criteria under baseline conditions. One group of control animals received LPS from S. abortus equi (2 micrograms/kg/h i.v. for 8 h) and saline (Group 1). Another group received LPS and the B2 antagonist CP-0127 (3 micrograms/kg/min), beginning 1 h after LPS (Group 2). Group 3 received LPS and the B2 antagonist in the aforementioned doses, and the B1 antagonist Leu9-des-Arg10-kallidin (3 micrograms/kg/min), also beginning 1 h after LPS. Overall survival figures after 8 h of LPS infusion were: Group 1, 10/22 (45%); Group 2, 10/17 (59%); Group 3, 10/28 (36%). Fifty percent (29/58) of animals that were healthy at baseline survived, but only 11% (1/9) of sick animals survived (Log Rank p = 0.0001). In the subset of healthy animals, survival rates for Groups 2 and 3 were 77% and 38%, respectively (p = 0.0519). It appears, therefore, that B2 blockade attenuates LPS-induced mortality whereas additional B1 blockade seems to reverse these beneficial effects. This suggests that in this animal model the B1 receptor does not serve the same purpose as the B2 receptor, and that up-regulation of B1 receptors during LPS shock may be an important mechanism of host defence.

Dextran sulfate activates contact system and mediates arterial hypotension via B2 kinin receptors.

To define some of the mechanisms underlying dextran sulfate (DXS)-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor des-Pro2-[Arg15] aprotinin (BAY x 4620) or the specific bradykinin B2-receptor antagonist Hoe-140 on the hypotensive response to DXS. In the first study, anesthetized miniature pigs were given DXS alone, DXS plus BAY x 4620 in various doses, or saline. As expected, DXS alone produced a profound but transient systemic arterial hypotension with a concomitant reduction in kininogen. Circulating kinin levels, complement fragment des-Arg-C3a, and fibrin monomer were all increased. Treatment with BAY x 4620 produced a dose-dependent attenuation of these effects with complete blockade of the hypotension as well as the observed biochemical changes at the highest dose (360 mg). In a second study, two groups of pigs were given either DXS alone or DXS plus Hoe-140. DXS-induced hypotension was completely blocked by Hoe-140 pretreatment; however, kininogen was again depleted. We conclude, therefore, that DXS-induced hypotension is produced by activation of plasma kallikrein that results in the production of bradykinin and that liberation of bradykinin and its action on B2 receptors in the vasculature are both necessary and sufficient to produce the observed effects on circulatory pressure.

Design, synthesis, and in vitro activity of bis(succinimido)hexane peptide heterodimers with combined B1 and B2 antagonist activity.

We have developed a series of peptide heterodimers based on the B2 antagonist D-Arg0-[Hyp3,D-Phe7,Leu8]-BK (1) and the B1 antagonist Lys0-[Leu8,des-Arg9]-BK (7) that are potent antagonists of both B1 and B2 receptors. From this series, compound 50 (alternatively, CP-0364), the 1,6-bis(succinimido)hexane heterodimer of D-Arg0-[Hyp3,Cys6,D-Phe7,Leu8]-BK (2), and D-Arg0-[Cys1,Hyp3,Leu8,des-Arg9]-BK (6), was found to be the most active both in vitro and in vivo. Compound 50 has a pA2 of 8.3 when measured against bradykinin (BK)-induced rat uterine smooth muscle contraction and an IC50 of approximately 10(-8) M against [des-Arg9]-BK-induced rabbit aorta smooth muscle contraction in vitro. Compounds such as 50 may be useful in the treatment of both subacute and chronic inflammatory disorders wherein both B2 and B1 receptors appear to contribute to the clinical manifestations of the disease.

Purification and characterization of a novel zinc-proteinase from cultures of Aeromonas hydrophila.

While searching for an enzyme capable of breaking epsilon-(gamma-Glu)-Lys isopeptide bonds cross-linking protein chains, we purified a metallo-proteinase which mimics the action of an isopeptidase on the gamma-chain dimers of cross-linked fibrin. The enzyme is present in the growth medium of the bacterium Aeromonas hydrophila, isolated from the intestinal tract of the leech Hirudo medicinalis. It is a 19-kDa protein which specifically hydrolyzes the Gly-Ala peptide bond within the Gly-Gly-Ala sequence, located near the cross-link site in the gamma-chain dimer of fibrin. Substrate specificity studies with a number of synthetic peptides suggest that the enzyme prefers Gly-Gly or acetyl-Gly in the P2 and P1 positions, respectively (Schecter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162). Nonpolar amino acid residues seem to be favored in the P1' and P2' positions. The enzyme contains one atom of zinc and is inhibited by 1,10-phenanthroline, but not by EDTA. Iodoacetate, leupeptin, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, pepstatin, and alpha 2-macroglobulin have no effect on enzyme activity. Disulfide reducing reagents, such as dithiothreitol or 2-mercaptoethanol, inactivate the enzyme completely. The partial amino-terminal sequence shows 46% identity with a zinc metallo-proteinase from a strain of Lysobacter enzymogenes and 69% identity with the LasA protein from Pseudomonas aeruginosa.

A new class of bradykinin antagonists: synthesis and in vitro activity of bissuccinimidoalkane peptide dimers.

A systematic study on the dimerization of the bradykinin (BK) antagonist D-Arg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-D-Phe 7-Leu8-Arg9 has been performed. The first part of this study involved compounds wherein dimerization was carried out by sequentially replacing each amino acid with cysteine and cross-linking with bismaleimidohexane. The second part of this study utilized a series of bissuccinimidoalkane dimers wherein the intervening methylene chain was varied systematically from n = 2 to n = 12 while the point of dimerization was held constant at position 6. The biological activities of these dimers were then evaluated on BK-induced smooth muscle contraction in two different isolated tissue preparations: guinea pig ileum (GPI) and rat uterus (RU). Several of the dimeric BK antagonists displayed remarkable activities and long durations of action. In addition, dimerization at position 4, 7, 8, or 9 produced dimeric analogues with markedly reduced potency. Rank order of antagonist potency as a function of dimerization position is as follows: rat uterus, 6 greater than 5 greater than 0 greater than 2 greater than 1 greater than 3 much greater than 4, 7, 8, 9; guinea pig ileum, 6 greater than 5 greater than 3 greater than 2 greater than 1 greater than 0 much greater than 4, 7, 8, 9. Evaluation of the linker length as represented by the number of methylene units indicated an optimal distance between the two monomeric peptides of six to eight methylene moieties. These studies also revealed that the carbon-chain length significantly affected the duration of action in vitro and resulted in partial agonism effects when n greater than 8. The optimum activity in vitro was achieved with dimerization at position 6 and n = 6 (designated herein as compound 25; alternatively, CP-0127). Similar effects in potency were also seen when the monomeric antagonist D-Arg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-D-Phe 7-Phe8-Arg9 (NPC-567) was dimerized using similar chemistry. These results suggest that the development of BK antagonists of significant therapeutic potential may be possible using a dimerization strategy that can overcome the heretofore limiting problems of potency and in vivo duration of action found with many of the BK antagonists in the literature.

CP-0127, a novel potent bradykinin antagonist, increases survival in rat and rabbit models of endotoxin shock.

The bradykinin antagonist dimer CP-0127 was found to be a potent and selective inhibitor of the depressor response to bradykinin in the anaesthetized rat and rabbit. When given as a single dose s.c. (3.6 mumol/kg), the depressor response to bradykinin was blocked for the duration of the experiment (4 hours). In anaesthetized control rats, LPS from E. coli produced a profound and immediate hypotensive response, while in rats infused with CP-0127, the response to LPS was almost totally reversed. In addition, CP-0127 given as a single subcutaneous dose (3.6 mumol/kg) to rats 1 hour before LPS challenge produced a 93% survival rate, compared to 14% in control animals. Finally, a survival rate of 86% was achieved in rabbits infused with CP-0127 at 0.36 nmol/kg/min i.v., compared to 45.5% in saline-infused control animals given LPS (500 micrograms/kg i.v.). The results of these experiments provide evidence for a significant role for the kallikrein-kinin system in these models of endotoxic shock, and indicate the therapeutic potential of a bradykinin antagonist such as CP-0127 for treating this disorder in man.

Bradykinin antagonists: synthesis and in vitro activity of bissuccinimidoalkane peptide dimers.

A systematic study on dimerization of the bradykinin (BK) antagonist D-Arg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-D-Phe 7-Leu8-Arg9 has been performed. Several of the dimeric BK antagonists displayed remarkable activities and long durations of action. Rank order of antagonist potency as a function of dimerization position is as follows: rat uterus 6 > 5 > 0 > 2 > 1 > 3 >> 4,7,8,9; guinea pig ileum 6 > 5 > 3 > 2 > 1 > 0 >> 4,7,8,9. These results suggest that the development of BK antagonists of significant therapeutic potential may be possible using a dimerization strategy that can overcome the heretofore limiting problems of potency and in vivo duration of action.

Inhibition of human leucocyte elastase by ursolic acid. Evidence for a binding site for pentacyclic triterpenes.

Several pentacyclic triterpenoid metabolites of plant origin are inhibitors of hydrolysis of both synthetic peptide substrates and elastin by human leucocyte elastase (HLE). Ursolic acid, the most potent of these compounds, has an inhibition constant of 4-6 microM for hydrolysis of peptide substrates in phosphate-buffered saline. With tripeptide and tetrapeptide substrates, the inhibition is purely competitive, whereas with a shorter dipeptide substrate the inhibition is non-competitive, suggesting that ursolic acid interacts with subsite S3 of the extended substrate-binding domain in HLE, but not with subsites S1 and S2. The carboxy group at position 28 in the pentacyclic-ring system of the triterpenes contributes to binding to HLE, since replacement of this group with a hydroxy group, as in uvaol, the alcohol analogue of ursolic acid, reduces the potency of inhibition. The inhibitory potency of ursolic acid is also reduced by addition of 1 M-NaCl, further supporting a postulated electrostatic interaction between the negative charge on the triterpene and a positively charged residue on the enzyme, which we assign to the side chain of Arg-217, located in the vicinity of subsites S4 and S5 in HLE. These observations are consistent with a binding site for ursolic acid which extends from S3 towards S4 and S5 on the enzyme. Other triterpenes, including oleanolic acid, erythrodiol, hederagenin and 18 beta-glycyrrhetic acid, can also interact with this binding site. On the basis of these results we conclude that the extended substrate-binding domain of HLE can accommodate a variety of hydrophobic ligands, including not only such molecules as fatty acids [Ashe & Zimmerman (1977) Biochem. Biophys. Res. Commun. 75, 194-199; Cook & Ternai (1988) Biol. Chem. Hoppe-Seyler 369, 629-637], but also polycyclic molecules such as the pentacyclic triterpenoids.

Association of psychiatric manifestations with antibodies to ribosomal P proteins in systemic lupus erythematosus.

PURPOSE: The goal of this study was to determine whether elevated serum levels of antibodies to ribosomal P proteins (anti-P antibodies) are associated with neuropsychiatric manifestations in patients with systemic lupus erythematosus (SLE). Additional experiments examined characteristics of these antibodies that might be associated with pathogenicity. PATIENTS AND METHODS: A large number of serum samples were collected from patients with SLE, control subjects with other rheumatic diseases, and normal individuals. At the time serum samples were obtained, patients with SLE were categorized according to the presence of psychosis, depression, and other manifestations of central nervous system (CNS) involvement. Serum anti-P antibody activity was quantitated by an enzyme-linked immunosorbent assay utilizing a synthetic peptide corresponding to the major P protein epitope. RESULTS: In a group of 79 normal individuals, mean (+/- SE) IgG anti-P activity was 0.01 +/- 0.003 and no individuals had values greater than 3 SD above the mean. Similar results were obtained measuring IgM anti-P activity. Normal levels were found in all sera from 21 patients with rheumatoid arthritis. Of 119 patients demonstrating various patterns of antinuclear and anticytoplasmic antibody activity, elevated anti-P levels were found only in patients with SLE. Overall, 19% of 269 patients with SLE demonstrated elevated levels of IgG or IgM anti-P antibodies, including 14% of 187 patients without and 29% of 82 patients with neuropsychiatric manifestations. The frequency of positive test results varied greatly depending on the nature of the CNS involvement. The frequency in patients with severe depression (n = 8) and psychosis (n = 29) was 88% and 45%, respectively, compared with only 9% in patients with nonpsychiatric neurologic disease (n = 45). For the entire SLE group, the odds ratio for the association of anti-P antibodies and severe psychiatric manifestations was 7.63 with a 95% confidence interval of 3.61 to 16.14. In a review of 187 patients with SLE originally classified as not having severe psychiatric disease, seven of 10 patients being treated with antidepressant medications had elevated levels of anti-P antibodies. In serial studies, the serum level of anti-P antibodies appeared to correlate with the activity of psychiatric disease and did not correlate with the activity of other manifestations of SLE. Anti-P antibodies in nearly all patients were IgG and directed primarily to the C-terminal 11 amino acids of the P protein. No difference in these characteristics was observed when patients with and without psychiatric manifestations were compared. Paired serum and cerebrospinal fluid (CSF) samples were also obtained from eight patients with active neuropsychiatric disease. Even when expressed as a fraction of the total IgG present, anti-P activity was markedly lower in CSF than in serum. CONCLUSIONS: Elevated levels of autoantibodies to the C-terminal region of ribosomal P proteins appear to be a specific marker for SLE, and are associated with both severe depression and psychosis in this disease. This assay is easily reproducible and may help distinguish SLE-induced psychiatric disease from that caused by other processes.