ABSTRACT The effect of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) inhibition on microvascular plasma leakage (extravasation) was evaluated in a rat model. Progressive inhibition of ACE using captopril caused increased extravasation when lung ACE was inhibited by Ͼ55%. In contrast, the selective inhibition of renal NEP by Ͼ90% using ecadotril did not increase extravasation. In NEP-inhibited rats, extravasation produced by the ACE inhibitors captopril and lisinopril was markedly enhanced. The dual ACE and NEP inhibitor omapatrilat, at oral doses of 0.03, 0.1, and 0.3 mg/kg, selectively inhibited lung ACE by 19, 61, and 76%, respectively, and did not cause significant extravasation. Doses of 1 and 10 mg/kg omapatrilat, which produced Ͼ90% inhibition of ACE and also inhibited renal NEP by 54 and 78%, respectively, significantly increased extravasation. In this model, bradykinin and substance P produced extravasation that could be abolished by the bradykinin 2 (B2) receptor antagonist Hoe 140 (icatibant) or the neurokinin1 (NK1) antagonist CP99994 [(ϩ)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine], respectively. Bradykinin induced extravasation was also partially (ϳ40%) inhibited by CP99994, indicating that a portion of the response involves B2 receptor-mediated release of substance P. In conclusion, this study is the first to relate the degree of ACE and/or NEP inhibition to extravasation liability in the rat model. Our data clearly demonstrate that ACE inhibitor-induced plasma extravasation is enhanced by concomitant inhibition of NEP. In addition, this study provides further evidence for the role for B2 and NK1 receptors in mediating plasma extravasation in the rat. Since their introduction nearly three decades ago, the angiotensin I-converting enzyme inhibitors (ACEIs) have become one of the more effective and highly used treatments for hypertension and heart failure. The therapeutic efficacy of these agents is derived in large part from their ability to inhibit the conversion of angiotensin I to angiotensin II, a vasoactive peptide whose direct vasoconstrictor and aldosterone-releasing actions promote increased blood pressure. There are some data to suggest that part of the therapeutic effect of these agents may be due to decreased breakdown of bradykinin (BK), which is also a substrate for ACE 1141 esterase inhibitor deficiency in humans leads to angioedema Materials and Methods Experimental Preparation(s). Male Sprague-Dawley strain rats weighing between 250 and 350 g were used in these studies. The procedures involving the use of rats in these experiments were reviewed and approved by the Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines (NIH publication 85-23). The animals were housed two per cage with free access to food and water and a 12-h light/dark cycle. To determine the role of ACE and/or NEP inhibition on plasma extravasation, groups of rats received drug treatment orally using an 18-gauge dosing needle and 5-ml syringe. Approximately 50 to 55 min after dosing, the rats were anesthetized via intraperitoneal injection of 70 mg/kg sodium pentobarbital. When anesthesia was achieved (typically less than 10 min), Evans blue dye (30 mg/kg) in heparinized saline (30 U/ml) was administered intravenously at a dose volume of 0.2 ml/100 g b.wt. via the tail vein using a 26-gauge, 1.5-inch-long needle. Five minutes post-Evans blue injection, the thoracic and peritoneal cavities were opened via a single midline incision. A 0.8 to 1.0 cc blood sample was obtained by cardiac puncture using a heparinized 1-ml syringe and 23-gauge needle and placed on ice. The tip of the right atrium was then cut and a steel cannula, attached by latex tubing to a peristaltic pump (Harvard Apparatus Inc., Holliston, MA), was inserted into the heart at the bottom of the left ventricle and was slid up through ventricle until the tip of the cannula was visible in the aortic arch. The cannula was manually held in place using forceps clamped across the heart. The pump was then started and the vascular system was perfused with 120 ml of saline delivered at a rate of 40 ml/min, which results in a perfusion pressure pulse of 80 to 100 mm Hg. This procedure is similar to that described by others For determination of extravasation liability of proinflammatory peptides, rats were anesthetized by intraperitoneal injection of 70 mg/kg pentobarbital sodium. The rats then received an intravenous dose of 30 mg/kg Evans blue dye in saline containing 30 U/ml heparin administered at a dose volume of 200 l/100 g b.wt. via tail vein injection. The Evans blue injection was followed immediately by intravenous injection of bradykinin, des-Arg9-bradykinin, or substance P. In some studies, the effect of selective B2 receptor blockade with Hoe 140 (10 g/kg i.v.) or selective NK1 receptor blockade with CP99994 (3 mg/kg i.v.) on bradykinin and substance P-mediated extravasation was also determined. In those studies, the selective antagonists were administered 3 to 5 min before Evans Blue injection. Five minutes after bradykinin, des-Arg9-bradykinin, or substance P injection, the thoracic and peritoneal cavities were opened via a single midline incision, and the rat was perfused as described above. For evaluation of plasma ex vivo ACE activity, the blood collected by cardiac puncture was spun for 2 min at maximum speed in a Microfuge. Plasma was collected from the top. Thirty-five microliters of plasma was added to a conical-bottomed 96-well plate with 5 l of 1 M KCl, 0.5 M sodium borate, pH 8.3, and 3 M zinc sulfate. Ten microliters of 12.5 mM hippuryl-his-leu substrate For determination of lung ACE activity and kidney NEP activity, approximately 250 mg of tissue was homogenized in 6 volumes of 0.1 M KH 2 PO 4 , pH 8.3, 0.3 M NaCl, and 1 M ZnSO 4 using a Teflonglass motor-driven pestle. For lung ACE activity, 40 l of homogenate was added to conical-bottomed 96-well plates and warmed to 37°C for 5 min. Ten microliters of 7.5 mM hippuryl-his-leu (1.5 mM final) was added to each sample and incubated for 10 min at 37°C. One hundred microliters of 10% TCA was added to each well, and the plates were centrifuged to pellet precipitated proteins. Fifty microliters of supernatant was added to 100 l of 2 mg/ml o-phthaldialdehyde in 10% ethanol and 50 l of 1 N NaOH in a black fluorometric plate. After 60 min, the plate was read in a fluorometer at 390-nm excitation and 460-nm emission. Standard curves were generated using his-leu. Kidney NEP activity was measured by adding 35 l of homogenate to wells containing 5 l of buffer or 10 M phosphoramidon. Plates were warmed to 37°C for 5 min. Ten microliters of 2.5 mM N-dansyl-D-ala-gly-p-nitrophe-gly substrate Drugs. The selective ACE inhibitors captopril and lisinopril were dissolved in water and administered orally at doses of 0.3, 1, 3, 10, and 30 mg/kg (captopril) or 1 and 3 mg/kg (lisinopril) at a dose volume of 1.0 ml/100 g b.wt. The dual ACE/NEP inhibitor omapatrilat was dissolved in a 30% polyethylene glycol200/70% of 25% cyclodextran vehicle and was administered orally as described above at doses of 0.03, 0.1, 0.3, 1, or 10 mg/kg. The selective NEP inhibitor ecadotril was dissolved in the polyethylene glycol200/cyclodextrin vehicle and was administered orally at doses of 3, 10, neutral endopeptidase or 30 mg/kg. Sulpizio et al. In some studies, inhibition of both ACE and NEP was produced by the oral administration of various doses of the selective ACE inhibitor captopril or lisinopril in rats pretreated orally with the selective NEP inhibitor ecadotril. Statistics. All data are expressed as the mean Ϯ S.E.M. The analysis of the differences in the extravasation measurement values between levels of treatments regimes used two-sample Wilcoxon tests. Based on the assumption of increasing extravasation with larger drug doses, one-sided tests were appropriate for the identification of the lowest dosage level with a significant increase in the extravasation values. The identification of these lowest dosages applied a fixed sequence test strategy All other comparisons between dosage levels also had predetermined directions of changes in the extravasation values and used single-sided values from two-sample Wilcoxon tests. The reported p values for these comparisons were all less than 0.05 (Bonferroni adjusted p values were reported for the four comparisons of the captopril and ecadotril combinations and the two comparisons of the bradykinin and CP99994 combinations). The trend comparison for the bradykinin and Hoe combinations used the single-sided Jonckheere-Terpstra trend test. All statistical tests used SAS System Release 8.01 as the analysis software (proc npar1way for the exact Wilcoxon tests and proc freq for the Jonckheere-Terpstra test). An unpaired Student's t test was used to test the effect of drug treatment on enzyme activity. Absolute enzyme rates of drug-treated rats were compared with enzyme rates of the vehicle-treated (control) rats. Acceptance of a significant difference between the groups was at the 0.05 p value level. Results Basal extravasation of Evans blue into the trachea of 29 vehicle-treated rats accumulated over the course of these studies was 8.3 Ϯ 0.43 ng/mg tissue. Baseline plasma ACE, lung ACE, and renal NEP enzyme activity in vehicle-treated rats was 32.7 Ϯ 1.7 nmol/ml/min and 6.5 Ϯ 1.8 and 2.6 Ϯ 0.2 nmol/mg protein/min, respectively. Treatment with increasing oral doses of captopril produced dose-related inhibition of plasma and lung ACE activity and was without effect on renal NEP. The reductions in ACE activity were associated with increased extravasation as measured by tracheal Evans blue concentration Oral doses of 0.03, 0.1, and 0.3 mg/kg of the dual ACE/NEP inhibitor omapatrilat produced dramatic, dose-related inhibition of both plasma and lung ACE with no inhibition of renal NEP Effect of Increasing Lung ACE Inhibition in NEPInhibited Rats. The data with ompatrilat did not clearly define the role that NEP inhibition played in the extravasation of Evans blue into the trachea because the degree of both ACE and NEP inhibition varied with dose. To define the role of NEP in extravasation further, rats were treated with 3.0 mg/kg ecadotril, which resulted in a relatively consistent ACE/NEP Inhibition and Plasma Extravasation in Rat 1143 background inhibition of ϳ74% in renal NEP The selective ACE inhibitor lisinopril was also tested alone and in combination with ecadotril. Doses of 1 and 3 mg/kg lisinopril alone inhibited lung ACE by 63 and 83%, respectively, and did not increase tracheal Evans blue extravasatio
