Andrea A. Seymour

Cardiorenal actions of neutral endopeptidase inhibition in experimental congestive heart failure.

The present studies were designed to determine the action of neutral endopeptidase inhibition (NEP-I), an inhibitor of the degradation of atrial natriuretic factor (ANF), in congestive heart failure (CHF). Studies were conducted in two groups of anesthetized dogs with CHF induced by 8 days of rapid right ventricular pacing. Group 1 (n = 5) received a specific NEP-I (SQ 28,603) at two doses administered sequentially -30 mg/kg followed by a 60 mg/kg i.v. bolus. Group 2 (n = 5) received intravenous infusion of exogenous ANF (100 ng/kg/min) to achieve increases in plasma ANF concentration as observed in group 1. NEP-I resulted in a diuresis and natriuresis (p less than 0.05) with increases in the fractional excretion of sodium and fractional excretion of lithium, the latter a marker for proximal tubule sodium delivery. Such tubular actions occurred in the absence of increases in glomerular filtration rate or renal blood flow but were associated with significant increases in urinary ANF and urinary cyclic GMP. Plasma ANF increased after the 30 mg/kg NEP-I dose. In contrast, in group 2 with exogenous ANF and despite a marked increase in plasma ANF, no natriuresis was observed. Arterial pressure did not change in either group. These studies demonstrate for the first time in CHF that NEP-I may potentiate the natriuretic action of endogenous ANF by a mechanism that is independent of systemic or renal hemodynamics and does not parallel increases in plasma ANF. These studies support an important therapeutic role for NEP-I in CHF.

Antihypertensive Activity During Inhibition of Neutral Endopeptidase and Angiotensin Converting Enzyme

The specific neutral endopeptidase (NEP) inhibitor, SQ 29,072 (7-{2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino}heptanoic acid), was studied in conscious spontaneously hypertensive rats (SHRs) and in DOCA/ salt hypertensive rats during inhibition of angiotensin-converting enzyme (ACE) activity with captopril or SQ 27,519 (the free acid of fosinopril). In the SHR, the maximal depressor responses to the combination of SQ 29,072 and captopril (−62 ± 9 mm Hg) or the combination of SQ 29,072 and SQ 27,519 (−44 ± 4 mm Hg) were greater than the responses to any of the inhibitors given alone (− 26 ± 5, −40 ± 10, and −28 ± 6 mm Hg for SQ 29,072. captopril, and SQ 27,519, respectively). In contrast, the maximal antihypertensive activities of SQ 29,072 were the same in conscious DOCA/salt hypertensive rats infused with saline, captopril, or SQ 27,519 (− 54 ± 10, − 51 ± 8, and −58 ± 11 mm Hg, respectively), indicating a lack of synergism in this model. In agreement. SQ 28,133 {N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-L-leucine}, a compound that inhibits both NEP and ACE, elicited significant depressor activities in both SHR and DOCA/salt hypertensive rats. In conclusion, a selective NEP inhibitor enhanced the depressor activity of ACE inhibitors in the conscious SHR, indicating that these agents may he effectively combined for treatment of some types of hypertension.

Antihypertensive and renal activity of SQ 28,603, an inhibitor of neutral endopeptidase

SQ 28,603 is a potent and selective inhibitor of neutral endopeptidase 3.4.24.11 (NEP), an enzyme that degrades atrial natriuretic peptide (ANP). In conscious deoxycorticosterone acetate (DOCA)/salt hypertensive rats, 300 μmol/kg, i.v., of SQ 28,603 significantly lowered mean arterial pressure (MAP) from 177 ± 12 to 154 ± 8 mm Hg and increased urinary cyclic guanosine monophosphate (GMP) excretion from 204 ± 70 to 1,068 ± 326 pmol/kg/min within 2 h. Urinary sodium excretion increased within 20 min from a control 51.2 ± 17.3 to 102.1 ± 26.7 μEq/kg/min. Infusion of SQ 28,603 (3.7 μmol/kg/min) for 6 h in a separate group of conscious DOCA/salt hypetensive rats gradually reduced MAP from 180 ± 7 to 142 ± 7 mm Hg and increased urinary cyclic GMP excretion from 182 ± 36 pmol/kg/min to 1.009 ± 394 pmol/kg/min. Despite the continuous infusion of the inhibitor, the natriuretic response peaked during the first hour of treatment at 128 ± 18 μEq/kg/min (vehicle = 54 ± 10 μEq/min). Plasma ANP was significantly greater in the rats infused with SQ 28,603 than in those receiving vehicle (333 ± 108 and 98 ± 14 fmol/ml, respectively), SQ 28,603 also significnatly reduced NEP activity by 95% in the kidneys (1.28 ± 0.08 vs. 18.35 ± 0.61 μmol/min after SQ 28,603 and vehicle respectively) and by 77% in the lungs (0.29 ± 0.03 vs. 0.92 ± 0.14 μmol/kg after SQ 28,603 and vehicle, respectively). In conclusion, inhibition of NEP activity by SQ 28,603 significantly decreased MAP and increased plasma ANP concentrations and urinary excretion of cyclic GMP in conscious DOCA/salt hypertensive rats.

Potentiation of the depressor responses to atrial natriuretic peptides in conscious SHR by an inhibitor of neutral endopeptidase.

In previous studies, neutral endopeptidase (NEP) hydrolyzed the Cys105-Phe106 bond of atrial natriuretic peptides (ANP) in vitro. Three such ring-opened peptides derived from ANP 99–126, 103–126, and 103–123 were inactive in conscious rats. In conscious spontaneously hypertensive rats (SHR) in the present study, 100 μmol/kg, intravenously (i.v.) of the NEP inhibitor, SQ 29,072 (7-[[2-(mercaptomethyl)-1-oxo-3-phenyl-propyl]amino]heptanoic acid), significantly increased the area over the curve (AOC) of the depressor response to 3 nmol/kg of ANP 103–126 from 165 ± 36 to 792 ± 350, 1,515 ± 374, and 828 ± 164 mm Hg min at 15, 30, and 60 min after inhibitor treatment. Thirty minutes after 3, 10, 30, and 100 u-mol/kg of SQ 29,072, the AOC of 3 μmol/kg of ANP 99–126 increased from 175 ± 59 mm Hg · min in vehicle-treated rats to 296 ± 100, 318 ± 34, 632 ± 194 (p < 0.05) and 656 ± 151 (p < 0.05) mm Hg · min. Furthermore, 100 μmol/kg of SQ 29,072 potentiated the AOC of human ANP 99–126 and 105–126 and rat ANP 99–126, 103–126, and 103–123, suggesting that the exocyclic N-terminal residues and the C-terminal tri-peptide did not influence ANP potentiation by SQ 29,072. In contrast, inhibitors of aminopeptidase, angiotensin-converting enzyme (ACE), and serine protease and an arginine vasopressin (AVP) antagonist did not substantially affect the AOC of 3 nmol/kg ANP 99–126. Finally, SQ 29,072 did not alter the activities of bradykinin, AVP, or angiotensin I or II. In conclusion, NEP may inactivate ANP in vivo by cleavage of susceptible bonds within the ANP ring.

Renal and Depressor Effects of SQ 29,072, a Neutral Endopeptidase Inhibitor, in Conscious Hypertensive Rats

Summary: The depressor and renal responses to the neutral endopeptidase (NEP) inhibitor, SQ 29,072, were characterized in both the conscious spontaneously hypertensive rat (SHR) and the conscious deoxycorticosterone acetate (DOCA)/salt hypertensive rat. Inhibition of tissue NEP activity by pharmacologically active doses was also ascertained in both hypertensive models. Intravenous administration of 300 μmol/kg of SQ 29,072 significantly reduced mean arterial pressure (MAP), produced modest natriuretic and diuretic responses, and inhibited renal NEP activity by approximately 40% in conscious SHR. Doses of 100 and 300 μmol/kg of SQ 29,072 elicited greater depressor responses (−36 ± 7 and −41 ± 8 mm Hg, respectively) in DOCA/salt hypertensive rats than in SHR (−11 ± 24 and −31 ± 5 mm Hg, respectively). SQ 29,072 (300 μmol/kg, i.v.) also inhibited renal NEP activity to a greater extent (70%) in DOCA/salt hypertensive rats. Similarly, the depressor responses to exogenous ANP 99–126 (1, 3, and 10 nmol/kg, i.v.) were greater in DOCA/salt hypertensive rats (−16 ± 4, −38 ± 6, and −73 ± 6 mm Hg, respectively) than in the SHR (0 ± 6, −17 ± 3, and −24 ± 3 mm Hg, respectively). Finally, equidepressor doses of SQ 29,072 and ANP 99–126 both increased urine volume as well as sodium and cyclic GMP excretion in conscious DOCA/salt hypertensive rats. In conclusion, the profile of depressor and renal activities produced by SQ 29,072 was consistent with potentiation of endogenous ANP by inhibition of NEP in conscious SHR and DOCA/salt hypertensive rats.

In vivo pharmacology of dual neutral endopeptidase/angiotensin-converting enzyme inhibitors.

The natriuretic and depressor responses to novel dual inhibitors of neutral endopeptidase (NEP) EC 3.4.24.11 and angiotensin-converting enzyme (ACE) were used to assess their activity in conscious cynomolgus monkeys. A survey of mercaptopropanoyl inhibitors revealed that compounds containing alanylproline or certain surrogates reduced blood pressure and increased sodium excretion, indicating a desirable profile of in vivo activity. Additional compound evaluation required specific in vivo assays for NEP and ACE inhibition. Accordingly, the potency of novel inhibitors against NEP and ACE were determined in conscious monkeys by the potentiation of the natriuretic activity of exogenous human atrial natriuretic peptide and inhibition of the pressor response to angiotensin I, respectively. This strategy led to the discovery that optimal in vivo activity was achieved when the mercaptopropanoyl group was replaced with mercaptoacetyl and the C-terminal alanylproline was replaced with conformationally constrained dipeptidomimetics. This work culminated in the identification of BMS-182657 as a prototypic dual NEP/ACE inhibitor with a highly desirable profile of in vivo pharmacology.

Renal and depressor activity of C-natriuretic peptide in conscious monkeys: effects of enzyme inhibitors.

The depressor and renal responses to C-type natriuretic peptide (CNP) were determined in conscious cynomolgus monkeys treated with vehicle or inhibitors of neutral endopeptidase EC 3.4.24.11 (NEP) and angiotensin-converting enzyme (ACE). The NEP inhibitor SQ 28603 (100 mumol/kg intravenously, i.v.) significantly (p < 0.05) enhanced the depressor responses to 1 and 10 nmol/ kg i.v. CNP from -2 +/- 3 to -22 +/- 10 mm Hg and from -16 +/- 4 to -66 +/- 4 mm Hg, respectively. SQ 28603 also significantly increased the cyclic GMP responses to 1 and 10 nmol/kg CNP from 1.4 +/- 1.6 to 11.0 +/- 2.0 nmol/2 h and from 4.2 +/- 0.5 to 53.3 +/- 12.1 nmol/2 h, respectively. Furthermore, the NEP inhibitor significantly increased the natriuretic activity of 1 and 10 nmol/kg i.v. CNP from 235 +/- 99 to 760 +/- 60 microEq/2 h and from 399 +/- 208 to 1,036 +/- 79 microEq/2 h, respectively. A positive correlation between the cumulative natriuretic and cyclic GMP responses suggested a cyclic GMP-mediated mechanism. These data are consistent with the protection of CNP from degradation by renal NEP. Inhibition of ACE by 100 mumol/kg i.v. captopril did not significantly alter the depressor or renal activities of 1 nmol/kg of CNP, neither did it alter the potentiation of CNP activity by SQ 28603. The potentiation of the depressor, cyclic GMP, and natriuretic responses to CNP in nonhuman primates by SQ 28603 suggested that NEP is an important mechanism for in vivo inactivation of natriuretic peptides, including CNP.

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.