Cardiovascular Drug ReviewsVolume 18, Issue 1 p. 1-24 Free Access Fasidotril: The First Dual Inhibitor of Neprilysin and ACE J. Bralet, Corresponding Author J. Bralet Laboratoire de Pharmacodynamie, Faculté de Pharmacie, DijonAddress correspondence and reprint requests to Dr. J. Bralet, Laboratoire Bioprojet, 9 rue Rameau, 75002 Paris, France.Search for more papers by this authorC. Marie, C. Marie Laboratoire de Pharmacodynamie, Faculté de Pharmacie, DijonSearch for more papers by this authorC. Gros, C. Gros Unité de Neurobiologie et Pharmacologie (U 109) de l'INSERM, ParisSearch for more papers by this authorJ. C. Schwartz, J. C. Schwartz Unité de Neurobiologie et Pharmacologie (U 109) de l'INSERM, ParisSearch for more papers by this authorJ. M. Lecomte, J. M. Lecomte Laboratoire Bioprojet, Paris, France.Search for more papers by this author J. Bralet, Corresponding Author J. Bralet Laboratoire de Pharmacodynamie, Faculté de Pharmacie, DijonAddress correspondence and reprint requests to Dr. J. Bralet, Laboratoire Bioprojet, 9 rue Rameau, 75002 Paris, France.Search for more papers by this authorC. Marie, C. Marie Laboratoire de Pharmacodynamie, Faculté de Pharmacie, DijonSearch for more papers by this authorC. Gros, C. Gros Unité de Neurobiologie et Pharmacologie (U 109) de l'INSERM, ParisSearch for more papers by this authorJ. C. Schwartz, J. C. Schwartz Unité de Neurobiologie et Pharmacologie (U 109) de l'INSERM, ParisSearch for more papers by this authorJ. M. Lecomte, J. M. Lecomte Laboratoire Bioprojet, Paris, France.Search for more papers by this author First published: 07 June 2006 https://doi.org/10.1111/j.1527-3466.2000.tb00030.xCitations: 6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume18, Issue1March 2000Pages 1-24 ReferencesRelatedInformation

We have designed two radioactive substrates, hippuryl-L-[3H]phenylalanine and 3-(p-hydroxy, m-[125I]phenyl)propionic acid ([125I]Bolton reagent) derivative of L-arginyl-L-phenylalanine, i.e. [125I]BRF, for a highly sensitive assay of carboxypeptidase A (CPA) activity. After cleavage of the C-terminal phenylalanine residue by CPA, the radioactive product of the reaction was conveniently separated by polystyrene bead chromatography. Using [125I]BRF, typical CPA activity inhibited by 1 microM 2-benzyl-3-mercaptopropanoic acid was detected in extracts from rat pancreas or intestine. In brain and some other tissues, however, [125I]BRF-hydrolyzing activity was only inhibited by this compound in 1000-fold higher concentration, suggesting the participation of a metallopeptidase distinct from CPA.

The preferred conformation of thiorphan during the inhibitor-neprilysin docking process was investigated. A series of achiral inhibitors were tested. This study led to the design of a potent inhibitor, in which the ethylenic bond bears the aryl residue of P′1.

The presence of carboxypeptidase A (EC 3.4.17.1; CPA) gene transcripts and corresponding catalytic activity was investigated in brain and other extradigestive rat tissues in which presence of the pancreatic enzyme had not been reported so far. Transcripts of two known CPA genes, CPA1 and CPA2, were identified in extremely low abundance in brain and several other extrapancreatic tissues using Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Whereas the CPA1 gene transcripts in brain, heart, stomach, or colon had a size similar to that in pancreas (1.35 kilobases), the CPA2 gene transcripts in brain, testis, or lung were of a smaller size (1.1 kilobases). Northern blot analysis using various probes, RT-PCR, and 5'-rapid amplification of cDNA 5'-end (5' RACE analysis) all indicated that this smaller size of the brain transcript was attributable to production by alternative splicing of the pro-mRNA. This process corresponds to deletion of the first four exons, leading to a mRNA encoding a protein in which the signal peptide and activation peptide of prepro-CPA2 are absent but the active site remains. The prediction that the shorter CPA2 isoform, designated CPA2(S), should correspond to a cytoplasmic metallopeptidase that does not require tryptic activation was verified by characterization of the recombinant protein and comparing it with the native CPA-like activity in brain. Both recombinant CPA2(S) generated in Escherichia coli and a soluble protein from brain displayed similar sizes on Western blots (32 kDa to be compared to 34 kDa for pancreatic CPA2). Recombinant CPA2(S) and a soluble CPA-like activity from brain displayed similar sensitivity to a series of inhibitors, contrasting with that of the pancreatic enzyme. It is concluded that alternative splicing produces a truncated CPA2 with distinct subcellular localization and modified catalytic activity. In spite of the presence of the CPA1 mRNA, no corresponding CPA activity could be detected in brain extracts, even after tryptic activation. This apparent discrepancy seems attributable to the presence of an endogenous peptide inhibitor which remains to be identified.

The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the presence of a soluble endogenous protein inhibitor. This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain. Peptides were obtained by partial proteolysis of purified TCI, a protein of approximately 30 kDa, and starting from their sequences, a full-length cDNA encoding a 223-amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library. Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and display characteristic CPA-inhibiting activity. TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki approximately 3 nM) and mast-cell CPA (Ki = 16 nM) and inactive on various other proteases. This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites. The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation.

The aim of the study was to compare, in a rat model of congestive heart failure, the effect of captopril, a selective angiotensin-converting enzyme (ACE; EC 3.4.15.1) inhibitor, to that of alatriopril, a mixed inhibitor of ACE and atriopeptidase (EC 3.4.24.11), an enzyme implicated in the degradation of atrial natriuretic factor (ANF). Myocardial infarction was induced by ligation of the left coronary artery. Groups of rats received orally twice daily captopril (10 mg/kg), alatriopril (100 mg/kg) or vehicle. Treatments were started 18 to 20 h after ligation and continued for 4 weeks. Hypertrophic and hormonal changes reflecting congestive heart failure were assessed in rats with large infarcts by measuring the relative weight of cardiac tissues as well as by assaying ANF in heart and plasma and by measuring renin activity in plasma. Both treatments significantly reduced cardiac hypertrophy, but alatriopril showed a greater efficacy than captopril--the increase in relative heart weight reaching 38% with captopril and only 22% with alatriopril (P < .05). The hypertrophy of right ventricle was reduced by 47% with alatriopril and by 35% with captopril (N.S.), whereas the corresponding reductions for atria were 47% vs. 21% (P < .05). Both treatments prevented the ligation-induced increase of ANF level in the right ventricle. In contrast, plasma ANF level was significantly reduced after captopril but not after alatriopril treatment, a difference that probably reflects the protection of endogenous ANF in circulation resulting from atriopeptidase inhibition. Plasma renin was increased by 36-fold after captopril but only by 1.6-fold after alatriopril, a difference that presumably reflects the inhibition of renal renin secretion by endogenous ANF after alatriopril. These data suggest that enhancement of ANF levels in circulation via atriopeptidase inhibition magnifies the capacity of ACE inhibitors to prevent cardiac hypertrophy, and they show the potential therapeutic value of mixed ACE-atriopeptidase inhibitors in congestive heart failure.

1. The acute effects of a single oral dose of sinorphan (100 mg), an inhibitor of neutral endopeptidase, on the plasma atrial natriuretic factor level and the fractional excretion of sodium were examined in 12 patients with severe chronic renal failure who were not on maintenance haemodialysis and who ingested a normal sodium diet. The drug was administered against placebo by a double-blind cross-over protocol. 2. Basal plasma atrial natriuretic factor level and fractional excretion of sodium were high (23.2 +/- 3.7 pmol/l and 2.64 +/- 0.38%, respectively). Sinorphan inhibited plasma neutral endopeptidase activity by 68-75% 30 min after ingestion. This effect persisted for at least 4 h. There were simultaneously increases in plasma atrial natriuretic factor and cyclic GMP levels to 1.9 and 1.4 times the basal values, respectively. Fractional excretion of sodium increased during the second and third hour periods after ingestion of the drug with a peak of 1.9 times the basal value in the second period. Changes in fractional excretion of sodium were significantly correlated with those in plasma atrial natriuretic factor and cyclic GMP levels. Plasma aldosterone level, creatinine clearance and mean blood pressure were unchanged, whereas plasma renin activity increased slightly. An increase in urinary cyclic GMP excretion was observed in parallel with the increase in plasma cyclic GMP level.(ABSTRACT TRUNCATED AT 250 WORDS)

We examined the acute effects of sinorphan, an inhibitor of enkephalinase, on plasma atrial natriuretic factor (ANF) and urinary sodium excretion in cirrhotic patients with ascites. A single oral dose of sinorphan (100 or 30 mg in 11 and 5 patients, respectively) was administered against placebo according to a double blind cross-over protocol. Basal plasma ANF levels varied over a large range between 2.6-79 pmol/L. Sinorphan, at a dose of 100 mg, inhibited 70% of plasma enkephalinase activity 60 min after ingestion and elicited simultaneously an increase in plasma ANF and cGMP levels 1.8 and 1.5 times basal values, respectively. There was a transient increase in sodium urinary output without a change in creatinine clearance over the initial 2-h period following drug administration. An increase in urinary cGMP was also observed on a longer period of 6 h. Plasma aldosterone decreased significantly, but the lowest concentration was reached 1 h later than the peak of plasma ANF. Mean blood pressure and PRA were unmodified. The effects of 30 mg sinorphan on plasma ANF, cGMP, and aldosterone were also significant, but less marked than those of the higher dose. Therefore, enkephalinase inhibition transiently increases sodium urinary excretion in cirrhotic patients with ascites via a mechanism that is likely to imply reduction of ANF catabolism. These results suggest that ANF could play a role in the control of sodium homeostasis in liver cirrhosis with ascites.

The interaction of the potent histamine H3-receptor ligands i.e. (R)α-methylhistamine, an agonist, and thioperamide, an antagonist, with the three classes of cerebral histamine receptors was studied in vitro and in vivo. The histamine-induced stimulation of 3',5'-cyclic AMP accumulation in slices of the guinea-pig hippocampus was not modified by thioperamide (up to 0.1 mM) and (R)α-methylhiistamine stimulated cyclic AMP accumulation only at millimolar concentrations. Hence, both (R)α-methylhistamine and thioperamide were at least 100 000-fold more potent at H3- than at H1- or H2-receptors in brain. In vivo, the turnover of histamine in rat cerebral cortex, as determined from its depletion elicited by α-fluoromethylhistidine in a synaptosomal fraction was not nmodified by mepyramine and zolantidine but was markiedly enhanced by thioperamide at a (ED50 = 2 mg/kg). Thiopermide also elicited a long-lasting decrease in synaptosomal histamine and increase radioimmunoassayable Nτ-methylhistamine. In contrast, (R)α-methylhistamine markedley reduced cortical [3H]histamine synthesis (ED50 = 5 mg/kg). This long-lasting action was accompanied by an increase in synaptosomal histamine and a decrease in Nτ-methylahistamine levels. These changes were compared with those in plasma drug levels. Hence the two H3-receptor ligands appear to modify the activity of cerebral histamine neurons markedly and in a long-lasting and opposite manner.

Abstract: Serum albumin conjugates of histamine or tele‐methylhistamine, a major catabolite, were prepared using 1,4‐benzoquinone as the coupling agent and used to raise polyclonal antibodies in rabbits. The same reagent was used to prepare the [125I]iodinated tracer and treat tissue extracts submitted to the radioimmunoassays. The IC50 values of prederivatized histamine and tele‐methylhistamine in the radioimmunoassays were 0.3 nM and 0.5 nM, respectively, whereas nonderivatized histamine or tele‐methylhistamine, histidine, a variety of histamine derivatives, amines, etc., had at least 1,000‐fold higher IC50 values. Application of the radioimmunoassays to nonpurified extracts of rat brain allowed the quantification of the two amine immunoreactivities in samples corresponding to less than 1 mg of hypothalamus. The tissue immunoreactivity corresponded to authentic histamine or tele‐methylhistamine, as shown by (a) the parallel 125I‐tracer displacement curves, (b) the similar elution patterns from HPLC columns, (c) the regional levels of histamine and tele‐methylhistamine in brain, similar to those obtained with other methods, and (d) the clearcut effects of treatments with inhibitors of l‐histidine decarboxylase or monoamine oxidase. The two radioimmunoassays appear as simple and sensitive tools to evaluate steady‐state levels and turnover rates of histamine and tele‐methylhistamine

Aminophenpyramine--i.e., N-(5-[2-(4-aminophenyl)ethanamidopentyl])-N'-(4-methoxybenzyl)-N-m ethyl-N'- (2-pyridinyl)-1,2-ethanediamine, a derivative of mepyramine (pyrilamine), a typical antagonist of histamine at its H1 receptor--was synthesized and converted into [125I)iodoazidophenpyramine, a potential photoaffinity probe for the H1 receptor. In the dark, reversible binding of this probe to cerebellar membranes occurred with a Kd of 1.2 x 10(-11) M and a Bmax of 240 fmol/mg of protein and was inhibited by various H1-receptor antagonists with the expected potencies. These features establish the compound as one of the most potent H1-receptor antagonists known so far. Upon UV irradiation, 5% of the bound radioactivity was covalently incorporated into cerebellar membrane polypeptides as shown by standard NaDodSO4/PAGE. Two bands of 47 and 56 kDa were consistently labeled, labeling being prevented by various H1-receptor antagonists with the expected potencies and stereoselectivity. In the presence of protease inhibitors, labeling of the 56-kDa peptide increased at the expense of the 47-kDa peptide, suggesting that the latter was produced by hydrolysis of the former under the action of membrane proteases. In the absence of 2-mercaptoethanol, a band of 350-400 kDa appeared, apparently at the expense of the lighter bands, suggesting that the latter might be linked by one or more disulfide bridges to a higher molecular mass complex. We propose that at least part of the ligand binding domain of the histamine H1 receptor resides within a subunit of apparent molecular mass 56,000.

SK & F 94461, an aminopentyl analogue of mepyramine, is a recently described H1 receptor antagonist. At variance with the other available H1 receptor ligands, SK & F 94461 offers the possibility of coupling to a protein carrier to render the molecule immunogenic. SK & F 94461 coupled to succinylated bovine serum albumin was used as an immunogen to raise polyclonal antibodies in rabbits and BALB/c mice. In parallel, spleen cells from immunized mice were used to produce hybridomas by somatic cell fusion. Thus, six different murine monoclonal antibodies sharing anti-SK & F 94461 specificity were selected for further detailed characterization of their binding properties. Pharmacologic studies of competitive inhibition using a set of 11 histaminergic agents allowed analysis of the fine specificity of anti-SK & F 94461 antibodies. Both polyclonal and monoclonal anti-SK & F 94461 antibodies showed very high affinity for the immunizing molecule (i.e., Ka values for monoclonal antibodies 8 and 12 were, respectively, 3 X 10(10) and 1.4 X 10(10) M-1). Both types of antibodies bound with high affinity (IC50 ranging from 10(-10) to 10(-12) M) to mepyramine, which has a chemical structure closely resembling that of SK & F 94461. Moreover, these antibodies displayed clear-cut stereoselectivity inasmuch as they bound the d-configuration of chlorpheniramine with significantly higher affinity than the l-form. Thus, all six monoclonal antibodies showed IC50 values 1 to 6 log units lower for d- than for l-chlorpheniramine. For some monoclonal antibodies, spectroscopic and fluorescence spectra studies showed that their different binding capacities correlated with their optical properties. Similarly, polyclonal anti-SK & F 94461 antibodies showed a 500-fold lower affinity for l- than for d-chlorpheniramine. All these results indicate that the polyclonal and the majority of monoclonal anti-SK & F 94461 antibodies recognized with high affinity structural configurations known to be important for the pharmacologic activity of H1 ligands, namely the presence of the dimethylaminoethyl side chain and, with stereochemical selectivity, the d-configuration of chlorpheniramine. These data extend for the first time to an H1 histamine receptor ligand results reported in other hormone systems.

The localization of two enkephalin-hydrolysing aminopeptidases i.e. aminopeptidase M (aminopeptidase N, EC 3.4.11.2) relatively insensitive to puromycin (Ki = 78 microM), and a puromycin-sensitive aminopeptidase (Ki = 1 microM) was studied in rat brain. The two aminopeptidases were differentially identified and/or localized using polyclonal anti-aminopeptidase M antibodies displaying anticatalytic activity and the inhibitors puromycin, bestatin and amastatin. Microvessels represent a major localization of cerebral aminopeptidase M as shown by the intense immunostaining of their walls in sections from various regions as well as in a fraction isolated from cerebral cortex homogenates by a sieving procedure. As compared to the starting homogenate, aminopeptidase M activity was enriched about twenty fold in this microvascular fraction. Aminopeptidase M was identified in this fraction by comparing the inhibitory potencies of antibodies and peptidase inhibitors towards the hydrolysis of [tyrosyl-3,5-3H, Met5]enkephalin to those found for the purified enzyme. A rather high aminopeptidase M activity was also localized in choroid plexuses. Following differential and gradient centrifugation analysis of cerebral cortex homogenates, aminopeptidase M activity was also enriched (by five to six fold) in fractions containing synaptic membranes. No significant soluble aminopeptidase M activity could be detected. These data suggest a dual localization of cerebral aminopeptidase M in microvessels and synaptic membranes consistent with its roles in preventing the access of circulating peptides to brain as well as in inactivating neuropeptides released from cerebral neurones. In comparison, puromycin-sensitive aminopeptidase activity, which is about 100 fold higher than aminopeptidase M activity in brain, was relatively low in microvessels and non-detectable in fractions enriched in synaptic membranes, being almost entirely restricted to soluble fractions.

This study was designed to evaluate the antinociceptive action of four dihydroxy flavone derivatives; 3,3′-dihydroxy flavone, 5,6-dihydroxy flavone, 3,7-dihydroxy flavone and 6,3′-dihydroxy flavone and to investigate the mechanisms involved.The antinociceptive effect of dihydroxy flavones was investigated in mice employing acetic acid induced abdominal constrictions, formalin-induced nociception, and hot plate assay procedures. The effects following pretreatment with naloxone, yohimbine, ondansetron, haloperidol, bicuculline and glibenclamide were also studied by acetic acid assay to reveal the involvement of opioid, adrenergic, tryptaminergic, dopaminergic, GABAergic or potassium channels respectively in the antinociceptive action of these compounds.Dihydroxy flavone derivatives significantly reduced the number of abdominal constrictions in acetic acid assay. The paw licking response time during both the early and late phases of formalin-induced nociception was reduced in a dose dependent manner by dihydroxy flavones treatment. A significant increase in reaction time was also evident in hot plate assay after dihydroxy flavones treatment.The antinociceptive effect of dihydroxy flavones in the acetic acid assay was significantly attenuated by pretreatment with either naloxone or bicuculline. However, pretreatment of animals with yohimbine, ondansetron, haloperidol, or glibenclamide did not alter the response.All the four investigated dihydroxy flavones produced dose related antinociception through mechanisms that involve an interaction with opioid and GABAergic pathways.