Marked difference between angiotensin-converting enzyme and neutral endopeptidase inhibition in vivo by a dual inhibitor of both enzymes

Dual inhibition of neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE) offers the potential for improved therapy of hypertension and cardiac failure. S 21402-1 [(2S)-2-[(2S,3R)-2-thiomethyl-3-phenylbutanamido] propionic acid] is a sulfhydryl-containing potent inhibitor of both NEP (Ki = 1.7 nM) and ACE (Ki = 4.5 nM). S 21402-1 and the sulfhydryl-containing ACE inhibitor captopril were administered to rats by intraperitoneal injection (0, 0.3, 3, 30, 300 mg/kg). Urine was collected for 4 h; then plasma and kidneys were collected. The difference in NEP and ACE inhibition by S 21402-1 in vivo was greater than 1000-fold. All doses of S 21402-1 inhibited NEP, as indicated by plasma NEP activity, radioinhibitor binding to kidney sections, urinary sodium excretion and bradykinin-(1-7)/bradykinin-(1-9) ratio. However, only 300 mg/kg S 21402-1 inhibited ACE, as indicated by plasma angiotensin II/angiotensin I ratio, renin and angiotensinogen levels. Although S 21402-1 (30 and 300 mg/kg) inhibited renal NEP, as indicated by the bradykinin-(1-7)/bradykinin-(1-9) ratio in kidney, S 21402-1 had no effect on renal ACE, as indicated by the angiotensin II/angiotensin I ratio in kidney. Moreover, captopril was greater than 10-fold more potent than S 21402-1 as an ACE inhibitor in vivo. In separate experiments, the pressor response of anesthetized rats to angiotensin I showed more rapid decay in ACE inhibition by S 21402-1 than by captopril. These studies indicated that in vivo modification of S 21402-1 caused a much greater decrease in potency of ACE inhibition than NEP inhibition. Consequently, effective ACE inhibition by S 21402-1 required doses much higher than those required for NEP inhibition.     

Optimal recognition of neutral endopeptidase and angiotensin-converting enzyme active sites by mercaptoacyldipeptides as a means to design potent dual inhibitors

An interesting approach for the treatment of congestive heart failure and chronic hypertension could be to avoid the formation of angiotensin II by inhibiting angiotensin converting enzyme (ACE) and to protect atrial natriuretic factor by blocking neutral endopeptidase 24.11 (NEP). This is supported by recent results obtained with potent dual inhibitors of the two zinc metallopeptidases, such as RB 105, HSCH2CH(CH3)PhCONHCH(CH3)COOH (Fournié-Zaluski et al. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4072-4076), which reduces blood pressure in experimental models of hypertension, independently of the salt and renin angiotensin system status. In order to develop new dual inhibitors with improved affinities, long duration of action, and/or better bioavailabilities, various series of mercaptoacyldipeptides corresponding to the general formula HSCH(R1)CONHCH(R1')CON(R)CH(R2')COOH have been synthesized. The introduction of well-selected beta-branched chains in positions R1 and R1', associated with a tyrosine or a cyclic amino acid in the C-terminal position, led to potent dual inhibitors of NEP and ACE such as 21 [N-[(2S)-2-mercapto-3-methylbutanoyl]-Ile-Tyr] and 22 [N-[(2S)-2-mercapto-3-phenylpropanoyl]Ala-Pro] which have IC50 values in the nanomolar range for NEP and subnanomolar range for ACE. These compounds could have different modes of binding to the two peptidases. In NEP, the dual inhibitors seem to interact only with the S1' and S2' subsites, whereas additional interactions with the S1 binding subsite of ACE probably account for their subnanomolar inhibitory potencies for this enzyme. The localization of the Pro residue of 22 outside the NEP active site is supported by biochemical data using (Arg102,Glu)NEP and molecular modeling studies with thermolysin used as model of NEP. One hour after oral administration in mice of a single dose (2.7 x 10(-5) mol/kg), 21 inhibited 80% and 36% of kidney NEP and lung ACE, respectively, while 22 inhibited 40% of kidney NEP and 56% of lung ACE.     

Antihypertensive and natriuretic effects of CGS 30440, a dual inhibitor of angiotensin-converting enzyme and neutral endopeptidase 24.11

Dual angiotensin-converting enzyme (ACE)/neutral endopeptidase (NEP) inhibitors, by decreasing angiotensin-II production and by preventing the degradation of atrial natriuretic peptide (ANP), may be useful for the treatment of hypertension and congestive heart failure. The thiol dipeptide CGS 30440 (prodrug of CGS 30008, IC50: ACE/NEP = 19/2 nM) administered to rats (10 mg/kg p.o.) inhibited lung tissue ACE activity by 98% and 61% at 1 and 24 hr (P < .001) and inhibited the angiotensin-I pressor response by 75 to 90% for more than 6 hr. Renal tissue NEP activity was reduced by 80% at 1 hr and 73% at 24 hr (P < .001). In rats supplemented with exogenous ANP, CGS 30440 (1 mg/kg p.o.) elevated the concentration of circulating ANP (133%, P < .025) for 4 hr and increased the excretion of urine (300%, P < .001), sodium (194%, P < .025) and cyclic GMP (238%, P < .005). CGS 30440 (10 mg/kg p.o.) administered to hypertensive rats with aortic ligation between the renal arteries (mean arterial blood pressure, 209 +/- 4 mm Hg) produced a 48 mm Hg blood pressure reduction (P < .001) within 4 hr. CGS 30440 given to cynomolgus monkeys at 2 mg/kg inhibited plasma ACE activity by 96% within 1 hr (P < .001), and this inhibition was maintained for 7 and 21 days in monkeys receiving the compound orally at 2.5 mg/kg b.i.d. These studies demonstrate that CGS 30440 is an orally active agent which produces tissue ACE and NEP inhibition in rats and plasma ACE inhibition in primates and suggest that the compound may be useful in the treatment of hypertension and congestive heart failure.     

Synthetic inhibitors of endopeptidase EC 3.4.24.15: potency and stability in vitro and in vivo

1. The role of the metalloendopeptidase EC 3.4.24.15 (EP 24.15) in peptide metabolism in vivo is unknown, in part reflecting the lack of a stable enzyme inhibitor. The most commonly used inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP-AAY-pAB, Ki = 16 nM), although selective in vitro, is rapidly degraded in the circulation to cFP-Ala-Ala, an angiotensin converting enzyme (ACE) inhibitor. This metabolite is thought to be generated by neutral endopeptidase (NEP; EC 3.4.24.11), as the Ala-Tyr bond of cFP-AAY-pAB is cleaved by NEP in vitro. In the present study, we have examined the role of NEP in the metabolism of cFP-AAY-pAB in vivo, and have tested a series of inhibitor analogues, substituted at the second alanine, for both potency and stability relative to the parent compound. 2. Analogues were screened for inhibition of fluorescent substrate cleavage by recombinant rat testes EP 24.15. D-Ala or Asp substitution abolished inhibitory activity, while Val-, Ser- and Leu-substituted analogues retained activity, albeit at a reduced potency. A relative potency order of Ala (1) > Val (0.3) > Ser (0.16) > Leu (0.06) was observed. Resistance to cleavage by NEP was assessed by incubation of the analogues with rabbit kidney membranes. The parent compound was readily degraded, but the analogues were twice (Ser) and greater than 10 fold (Leu and Val) more resistant to cleavage. 3. Metabolism of cFP-AAY-pAB and the Val-substituted analogue was also examined in conscious rabbits. A bolus injection of cFP-AAY-pAB (5 mg kg-1, i.v.) significantly reduced the blood pressure response to angiotensin I, indicating ACE inhibition. Pretreatment with NEP inhibitors, SCH 39370 or phosphoramidon, slowed the loss of cFP-AAY-pAB from the plasma, but did not prevent inhibition of ACE. Injection of 1 mg kg-1 inhibitor resulted in plasma concentrations at 10 s of 23.5 microM (cFP-AAY-pAB) and 18.0 microM (cFP-AVY-pAB), which fell 100 fold over 5 min. Co-injection of 125I-labelled inhibitor revealed that 80-85% of the radioactivity had disappeared from the circulation within 5 min, and h.p.l.c. analysis demonstrated that only 25-30% of the radiolabel remained as intact inhibitor at this time. Both analogues were cleared from the circulation at the same rate, and both inhibitors blunted the pressor response to angiotensin I, indicative of ACE inhibition. 4. These results suggest that both NEP and other clearance/degradation mechanisms severely limit the usefulness of peptide-based inhibitors such as cFP-AAY-pAB. To examine further EP 24.15 function in vivo, more stable inhibitors, preferably non-peptide, must be developed, for which these peptide-based inhibitors may serve as useful molecular templates.     

Renal and depressor activities of inhibitors of neutral endopeptidase and angiotensin converting enzyme in monkeys infused with angiotensin II

In a previous study, the depressor activity of combined selective inhibitors of neutral endopeptidase EC 3.4.24.11 (NEP) and angiotensin-converting enzyme (ACE) depended on the level of ACE inhibition, whereas the renal responses were determined by NEP inhibition. Our study confirmed that a mixed NEP/ACE inhibitor BMS-182657 ([S-(R*,R*)]-2,3,4,5-tetrahydro-3-[(2-mercapto-1-oxo-3- phenylpropyl)amino]-2-oxo-1H-benzazepine-1-acetic acid) reduced mean arterial pressure (MAP) when renin release was reduced by a sodium load, suggesting that the depressor response did not require suppression of endogenous angiotensin II generation. Furthermore, a pressor dose of 30 ng/min of angiotensin II was required to block the depressor response to BMS-182657 in the presence or absence of exogenous human atrial natriuretic peptide (hANP 99-126). Thirty ng/min of angiotensin II also significantly enhanced the natriuresis induced by hANP 99-126 after BMS-182657 administration. In contrast, a nonpressor dose of angiotensin II (3 ng/min) reduced basal sodium excretion and the natriuretic responses to exogenous hANP 99-126 in the presence or absence of BMS-182657. The potentiation of the urinary ANP and cyclic guanosine monophosphate (cGMP) responses to hANP 99-126 by BMS-182657 was similar for all doses of angiotensin II; therefore angiotensin did not alter the effects of BMS-182657 on ANP metabolism or cGMP accumulation in the kidney. In summary, the renal responses to mixed metalloprotease inhibitors were apparently mediated by ANP potentiation and were modulated by angiotensin II. The depressor activity depended on ACE inhibition but was not mediated solely by reductions in endogenous angiotensin II levels.     

Degradation of bradykinin in semen of ram and boar

The pattern of bradykinin (BK; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9)-inact iva ting peptidases in semen of boar and ram was investigated. The degradation of BK in semen was completely abolished by the metalloprotease inhibitors EDTA and o-phenanthroline. Inhibitors of angiotensin-converting enzyme (ACE; EC 3.4.15.1) and phosphoramidon, an inhibitor of neutral metalloendopeptidase (NEP; EC 3.4.24.11), were only partially effective in preventing BK degradation in semen. An additive effect was seen with simultaneous inhibition of both enzymes, resulting in complete abolition of BK degradation. HPLC analysis demonstrated that exogenous BK in semen is cleaved at Gly4-Phe5, Phe5-Ser6 and Pro7-Phe8. These results indicate that NEP and ACE are the main peptidases responsible for rapid BK inactivation in semen. The involvement of other peptidases known to be responsible for BK cleavage in other tissues and body fluids, namely carboxypeptidase N (EC 3.4.12.7), post proline cleaving enzyme (EC 3.4.21.26) and aminopeptidase P (EC 3.4.11.9) was excluded. NEP and ACE were shown to be localized mainly in seminal plasma and to a lesser extent on sperm cells.     

Kinin-mediated coronary nitric oxide production contributes to the therapeutic action of angiotensin-converting enzyme and neutral endopeptidase inhibitors and amlodipine in the treatment in heart failure

Increasing evidence suggests that angiotensin-converting enzyme (ACE) inhibitors can increase vascular nitric oxide (NO) production. Recent studies have found that combined inhibition of ACE and neutral endopeptidase (NEP) may have a greater beneficial effect in the treatment of heart failure than inhibition of ACE alone. Amlodipine, a calcium channel antagonist, has also been reported to have a favorable effect in the treatment of patients with cardiac dysfunction. The purpose of this study was to determine whether and the extent to which all of these agents used in the treatment of heart failure stimulate vascular NO production. Heart failure was induced by rapid ventricular pacing in conscious dogs. Coronary microvessels were isolated from normal and failing dog hearts. Nitrite, the stable metabolite of NO, was measured by the Griess reaction. ACE and NEP inhibitors and amlodipine significantly increased nitrite production from coronary microvessels in both normal and failing dog hearts. However, nitrite release was reduced after heart failure. For instance, the highest concentration of enalaprilat, thiorphan, and amlodipine increased nitrite release from 85 +/- 4 to 156 +/- 9, 82 +/- 7 to 139 +/- 8, and 74 +/- 4 to 134 +/-10 pmol/mg (all *p <.01 versus control), respectively, in normal dog hearts. Nitrite release in response to the highest concentration of these two inhibitors and amlodipine was reduced by 41% and 31% and 32% (all #p <.01 versus normal), respectively, in microvessels after heart failure. The increase in nitrite induced by either ACE or NEP inhibitors or amlodipine was entirely abolished by Nw-nitro-L-arginine methyl ester, HOE 140 (a B2-kinin receptor antagonist), and dichloroisocoumarin (a serine protease inhibitor) in both groups. Our results indicate that: 1) there is an impaired endothelial NO production after pacing-induced heart failure; 2) both ACE and NEP are largely responsible for the metabolism of kinins and modulate canine coronary NO production in normal and failing heart; and 3) amlodipine releases NO even after heart failure and this may be partly responsible for the favorable effect of amlodipine in the treatment of heart failure. Thus, the restoration of reduced coronary vascular NO production may contribute to the beneficial effects of these agents in the treatment of heart failure.     

Up-regulation of neutral endopeptidase (CALLA) in human neutrophils by granulocyte-macrophage colony-stimulating factor

Neutral endopeptidase 24.11 (NEP/CALLA/CD10), an enzyme expressed on early lymphoid progenitors, neutrophils, and various other cell types, inactivates many biologically active peptides, including the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Inhibition of CD10/NEP on the surface of human neutrophils (PMNs) in vitro inhibits migration toward this chemotaxin, suggesting that enzymatic inactivation by NEP regulates the neutrophil response to fMLP. Because PMNs in inflammatory sites are exposed to various cytokines, we evaluated the effects of selected cytokines on CD10/NEP activity in vitro. Of five cytokines tested--interleukin-1 (IL-1), IL-6, and IL-8, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor (GM-CSF)--GM-CSF provided the most consistent increase in surface NEP activity. Low concentrations (10(-9)-10(-7) M) of GM-CSF increased NEP activity in a time- and concentration-dependent manner to more than 225% that of control (phosphate-buffered saline-treated) cells. Cytofluorometry of cells stained with a fluorescent antibody to CD10 indicated that GM-CSF increased expression of surface CD10/NEP antigen in a similar manner. The effect of GM-CSF on NEP activity was enhanced still further by simultaneous exposure to IL-1, suggesting that combinations of cytokines may direct and regulate the neutrophil response within an inflammatory site. Rapid upregulation of CD10/NEP underscores the importance of this enzyme for control of peptide mediators of inflammation.     

Angiotensin-converting enzyme activity in stools of healthy subjects and patients with celiac disease

Angiotensin-converting enzyme (ACE) is a dipeptidylcarboxypeptidase that occurs in three types of cells: endothelial, epithelial, and neuroepithelial. ACE activity is present in plasma, urine, and vascular endothelium. High levels of ACE are found in the brush border of human small bowel. The aim of this study was to evaluate ACE activity in human stools and to find a correlation with the intestinal loss of epithelial cells. Fifteen healthy subjects (HS) (8 males, 7 females; age range 6-56 years), 20 patients with celiac disease (CD) (11 males, 9 females; age range 15-53 years), and 18 patients with CD in remission after a gluten-free diet (CD-GFD) (8 males, 10 females; age range 14-54 years) were enrolled in the study. The fecal ACE activity was measured in all groups. Fecal samples were kept at -20 degrees C for a subsequent test. In HS, fecal ACE activity was 21.03 +/- 16.17 nmol/min/100 g (mean +/- SD). In patients with CD with subtotal mucosa atrophy, ACE activity was significantly higher (113 +/- 88.94) than in HS and CD on GFD (36.65 +/- 23.9). We have demonstrated ACE activity in human stools. ACE activity in stools seems to derive from the microvilli of the intestinal mucosa, thus suggesting the potential usefulness of ACE determination as an index of enterocyte damage.     

Characterisation of the processing by human neutral endopeptidase 24.11 of GLP-1(7-36) amide and comparison of the substrate specificity of the enzyme for other glucagon-like peptides

The post-secretory processing of the potent insulinotropic peptide hormone, GLP-1(7-36)amide, probably involves one or more of a small group of membrane-bound ectopeptidases. Reported here, is the characterisation of the endoproteolysis of human GLP-1(7-36)amide by the recombinant human form of neutral endopeptidase (NEP) 24.11, which is one of the best characterised and widely-distributed of ectopeptidases and is involved in the processing of other peptide hormones. The products of the limited endoproteolysis were characterised by mass and primary structure following fractionation using high performance liquid chromatography. The rate of this endoproteolysis by NEP 24.11 was estimated and compared to that of GLP-1(7-36)amide-related peptides. GLP-1(7-36)amide appears to be good substrate for NEP 24.11 with most, but not all potential target bonds being cleaved. Also, the structurally-related peptides, secretin and glucagon appear to be good substrates whereas GIP and exendin-4 are very poor substrates. That the GLP-1(7-36)amide super-agonist, exendin-4 is a poor substrate for NEP 24.11 is significant for the possible use of this peptide as a prototype for the development of clinically-useful peptide agonists. Further studies should reveal whether NEP 24.11 is important for the metabolic clearance of GLP-1(7-36)amide and will be highly relevant for the attempts to realise the suggested therapeutic value of GLP-1(7-36)amide.     

Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

Two intramolecularly quenched fluorogenic peptides containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-DArg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were K(m) = 2.8 microM, kcat = 5.3 min-1, kcat/K(m) = 2 min-1 microM-1 and K(m) = 5.0 microM, kcat = 7.0 min-1, kcat/K(m) = 1.4 min-1 microM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE; EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by alpha-chymotrypsin. The high specificity of these substrates suggests their use for specific NEP assays in crude enzyme preparations.     

Inhibition of angiotensin converting enzyme and potentiation of bradykinin by retro-inverso analogues of short peptides and sequences related to angiotensin I and bradykinin

There is pharmacological evidence indicating that, in addition to the inhibition of angiotensin converting enzyme (ACE; EC 3.4.15.1), the potentiation of bradykinin (BK) responses may also involve the BK receptor or some binding site in the structures involved in the contractile response to this peptide. Dipeptides such as Val-Trp and some of its analogues as well as tripeptide homologues, including total and partial retro-inverso peptides, were synthesized and assayed for their ability to inhibit purified guinea pig plasma ACE and to potentiate the action of BK on the isolated ileum of the same species. The peptides containing the P2-P1, P1-P'1, and P'1-P'2 inverted amide bonds inhibited ACE, were resistant to hydrolysis, and, depending on the amino acid composition, some of them potentiated the contractile response to BK while others did not. Des-[Arg1]-BK, which has an intrinsic activity at concentrations higher than 10(-5) M, and the very dissimilar angiotensin I (AI) analogue [Cys5-Cys10]-angiotensin-I-(5-10)-amide, which has no detectable contractile activity, were able to inhibit ACE and potentiate BK. In contrast to these peptides, BPP5a and BPP9a from Bothrops jararaca venom, and Potentiators B and C from Agkistrodon halys blomhoffi venom were more effective as BK potentiators than as ACE inhibitors. In conclusion, we have synthesized and assayed compounds that preferentially inhibit ACE, e.g. retro-inverso tripeptides, or potentiate the response of smooth muscle to BK, e.g. snake venom peptides.     

New azole antifungals. 3. Synthesis and antifungal activity of 3-substituted-4(3H)-quinazolinones

A series of 92 azole antifungals containing an amido alcohol unit was synthesized. The nature and substitution of the amide portion was systematically modified in search of improved antifungal activity, especially against filamentous fungi. The compounds were tested in vitro against a variety of clinically important pathogens and in vivo (po) in a murine candidosis model. Thiazole and thiophene carboxamides carrying both a substituted phenyl ring and a small alkyl group were best suited for activity against filamentous fungi. In a subset of these compounds, the amide portion was conformationally locked by means of a pyrimidone ring and it was proven that only an orthogonal orientation of the phenyl ring yields bioactive products. A tendency to display long plasma elimination half-lives was observed in both series. Two compounds, 74 and 107, representative of the open and cyclic amides, respectively, were chosen for further studies, based on their excellent activity in in vivo murine models of candidosis and aspergillosis. This work describes the SARs found within this series. The next paper displays the results obtained in a related series of compounds, the quinazolinones.     

Inhibition of neutral endopeptidase causes vasoconstriction of human resistance vessels in vivo

BACKGROUND: Neutral endopeptidase (NEP) degrades vasoactive peptides, including the natriuretic peptides, angiotensin II, and endothelin-1. Systemic inhibition of NEP does not consistently lower blood pressure, even though it increases natriuretic peptide concentrations and causes natriuresis and diuresis. We therefore investigated the direct effects of local inhibition of NEP on forearm resistance vessel tone. METHODS AND RESULTS: Four separate studies were performed, each with 90-minute drug infusions. In the first study, 10 healthy subjects received a brachial artery infusion of the NEP inhibitor candoxatrilat (125 nmol/min), which caused a slowly progressive forearm vasoconstriction (12+/-2%; P=0.001). In a second two-phase study, 6 healthy subjects received, 4 hours after enalapril (20 mg) or placebo, an intra-arterial infusion of the NEP inhibitor thiorphan (30 nmol/min). Thiorphan caused similar degrees of local forearm vasoconstriction (P=0.6) after pretreatment with both placebo (13+/-1%, P=0.006) and enalapril (17+/-6%, P=0.05). In a third three-phase study, 8 healthy subjects received intra-arterial thiorphan (30 nmol/min), the endothelin ETA antagonist BQ-123 (100 nmol/min), and both combined. Thiorphan caused local forearm vasoconstriction (13+/-1%, P=0.0001); BQ-123 caused local vasodilatation (33+/-3%, P=0.0001). Combined thiorphan and BQ-123 caused vasodilatation (32+/-1%, P=0.0001) similar to BQ-123 alone (P=0.98). In a fourth study, 6 hypertensive patients (blood pressure >160/100 mm Hg) received intra-arterial thiorphan (30 nmol/min). Thiorphan caused a slowly progressive forearm vasoconstriction (10+/-2%, P=0.0001). CONCLUSIONS: Inhibition of local NEP causes vasoconstriction in forearm resistance vessels of both healthy volunteers and patients with hypertension. The lack of effect of ACE inhibition on the vasoconstriction produced by thiorphan and its absence during concomitant ETA receptor blockade suggest that it is mediated by endothelin-1 and not angiotensin II. These findings may help to explain the failure of systemic NEP inhibition to lower blood pressure.     

Ovarian angiotensin-converting enzyme activity in humans: relationship to estradiol, age, and uterine pathology

The present study was designed to measure angiotensin-converting enzyme (ACE) activity in the human ovary and in serum and to relate this activity to age, serum estradiol levels, and uterine and endometrial pathology. ACE activity was determined in 56 females by a radiometric assay using [3H]hippuryl-glycyl-glycine as substrate. Ovarian ACE activity, but not serum ACE, was found to increase with age (P < 0.01) and was significantly greater in postmenopausal subjects (n = 31; 1.35 +/- 0.05 nmol/mg.min) than in subjects with active ovaries (n = 21; 0.65 +/- 0.2 nmol/mg.min; P = 0.0033). Ovarian ACE activities in fertile women in the preovulatory phase (n = 14) and the postovulatory phase (n = 7) were not statistically different (0.66 +/- 0.23 and 0.63 +/- 0.17 nmol/mg.min, respectively). Serum ACE activities were similar in females with active and nonactive ovaries (87.6 +/- 5.0 vs. 81.7 +/- 5.3 nmol/mL-min, respectively). Serum estradiol levels in fertile women were significantly higher than those in postmenopausal women (P = 0.0023). Serum estradiol levels were negatively correlated with age (r = -0.46; P = 0.0041) and were not correlated with either serum ACE activity (r = 0.080; P = NS) or ovarian ACE activity. In summary, human ovarian ACE activity, but not serum ACE, is positively correlated with age. Serum estradiol levels decrease with age, but are not correlated with either ovarian or serum ACE activity. Endogenous serum estradiol levels had no apparent effect on ovarian or serum ACE activity. The presence of uterine pathology affects ovarian ACE activity. The cause of the increased ovarian ACE activity is not clear, but may be related to the aging process.     

Role of neutral endopeptidase in exercise-induced bronchoconstriction in asthmatic subjects

The membrane-bound metalloproteinase, neutral endopeptidase (NEP), is a degrading enzyme of both bronchoconstrictor and bronchodilator peptides within the airways. To examine the role of NEP in exercise-induced bronchoconstriction (EIB) in asthmatic subjects, we used inhaled thiorphan, a NEP inhibitor, as pretreatment to a 6-min standardized exercise challenge. Thirteen clinically stable asthmatic subjects participated in this double-blind, placebo-controlled, crossover study that was performed on 2 days separated by 48 h. Thiorphan was administered by two inhalations of 0.5 ml containing 1.25 mg/ml. Subsequently, exercise was performed on a bicycle ergometer at 40-50% of predicted maximal voluntary ventilation while inhaling dry air (20 degrees C, relative humidity 6%). The airway response to exercise was measured by forced expiratory volume in 1 s (FEV1) every 3 min, up to 30 min postexercise challenge, and was expressed both as the maximal percent fall in FEV1 from baseline and as the area under the time-response curve (AUC) (0-30 min). The acute effects of both pretreatments on baseline FEV1 were not different (P > 0.2), neither was there any difference in maximal percent fall in FEV1 between thiorphan and placebo (P > 0.7). However, compared with placebo, thiorphan reduced the AUC by, on average, 26% [AUC (0-30 min, +/-SE): 213.6 +/- 47.7 (thiorphan) and 288.6 +/- 46.0%fall.h (placebo); P = 0.047]. These data indicate that NEP inhibition by thiorphan reduces EIB during the recovery period. This suggests that bronchodilator NEP substrates, such as vasoactive intestinal polypeptide or atrial natriuretic peptide, modulate EIB in patients with asthma.     

Effective treatment of narcolepsy with codeine in a patient receiving hemodialysis

We evaluated the effect of endothelin-1 (ET) on the angiotensin converting enzyme (ACE) activity in rat aortic smooth muscle cells (VSMCs). ACE activity was determined by radioimmunoassay of the amount of angiotensin II generated after the addition of angiotensin I (500 pg/ml) to cultured VSMCs. The antibody used had less than 0.1% cross-reactivity with angiotensin I. ACE activity increased 1.9-fold 5 hr after the addition of 10(-6) M ET under serum-free conditions. This stimulatory effect of ET on ACE activity in VSMCs was completely inhibited by 10(-7) M captopril. Results suggested that the ACE present in SMCs is stimulated by ET.     

Design and synthesis of potent, selective inhibitors of endothelin-converting enzyme

Endothelin-1 is the most potent peptidic vasoconstrictor discovered to date. The final step of posttranslational processing of this peptide is the conversion of its precursor by endothelin-converting enzyme-1 (ECE-1), a metalloprotease which displays high amino acid sequence identity with neutral endopeptidase 24.11 (NEP) especially at the catalytic center. A series of potent and selective arylacetylene-containing ECE-1 inhibitors have been prepared. (S, S)-3-Cyclohexyl-2-[[5-(2, 4-difluorophenyl)-2-[(phosphonomethyl)amino]pent-4-ynoyl]amino] propio nic acid (47), an arylacetylene amino phosphonate dipeptide, was found to inhibit ECE-1 and NEP with IC50 values of 14 nM and 2 microM, respectively. Similarly, (S)-[[1-[(2-biphenyl-4-ylethyl)carbamoyl]-4-(2-fluorophenyl)but-3- yny l]amino]methyl]phosphonic acid (56), an arylacetylene amino phosphonate amide, had IC50's of 33 nM and 6.5 microM for ECE-1 and NEP, respectively. Slight modification of the aryl moiety was found to have dramatic effects on ECE-1/NEP selectivity. The 2-fluoro dipeptide analogue, (S, S)-2-[[5-(2-fluorophenyl)-2-[(phosphonomethyl)amino]pent-4-ynoyl]+ ++amin o]-4-methylpentanoic acid (40), showed a 72-fold selectivity for ECE-1 over NEP, while the 3-fluoro dipeptide analogue, (S, S)-2-[[5-(3-fluorophenyl)-2-[(phosphonomethyl)amino]pent-4-ynoyl]+ ++amin o]-4-methylpentanoic acid (22), was equipotent for ECE-1 and NEP. Several of these inhibitors were shown to be potent in blocking ET-1 production in vivo as demonstrated by the big ET-1-induced pressor response in rats. These potent inhibitors are the most selective for ECE-1 reported to date and are envisaged to have a variety of therapeutic applications.     

Neutral endopeptidase activity in newborn and adult rabbit tracheas

Neutral endopeptidase (NEP, E.C. 3.4.24.11), a widely distributed ectoenzyme, cleaves and inactivates a variety of biologically active peptides, including the tachykinin, substance P (SP). This study was undertaken to determine whether the modulation of SP airway smooth muscle contraction by NEP is age-dependent. We studied the contractile response of isolated tracheal rings from newborn and 120 day old New Zealand white rabbits. We measured NEP activity and determined immunoreactive NEP content in tracheal membrane preparations. NEP activity was then localized histochemically in sections of rabbit tracheas. In the presence of the NEP inhibitor, SCH 32615, the contractile response of isolated tracheal rings to SP was increased both in the newborn and 120 day old rabbits. However, the increase was greatest in the newborn animals. NEP activity in tracheal membrane preparations increased fivefold between the newborn and 120 day old rabbits. Western blot analysis also revealed a significant increase in the immunoreactive NEP content of these tracheal membrane preparations between the newborn and 120 day old rabbits. NEP activity, localized histochemically, was most intense in the epithelial region of the newborn animals, with a shift of activity to the subepithelial region with age. The prominent epithelial localization of neutral endopeptidase in the tracheas of these 1 day old rabbits, which we have shown to have relatively low neutral endopeptidase activity, suggests that the location of neutral endopeptidase in the airway, including proximity to relevant substance P receptors, may be critical to its function.     

Purification and molecular cloning of carp ovarian cystatin

Endothelin converting enzyme (ECE) from intact cells of a permanent human endothelial cell line, EA.hy926, was studied by examining the effects of phosphoramidon, an endothelin converting enzyme inhibitor, on the levels of secreted endothelin-1 and big endothelin-1. The specific ECE activity was demonstrated by a phosphoramidon dose-dependent decrease in ET-1 level with a concomitant increase in big ET-1 level. By using a specific neutral endopeptidase 24.11 (NEP 24.11) inhibitor, thiorphan, it was also shown that the phosphoramidon-sensitive ET-1 degrading activity in this cell line is due to the NEP 24.11 activity. Other serine, acid, and cysteine protease inhibitors had no effect on the endogenous synthesis of ET-1 and big ET-1 supporting the evidence that ECE is insensitive to these protease inhibitors as has been demonstrated with the isolated enzyme.