Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Angiotensin-converting enzyme (EC 3.4.25.1) and neutral endopeptidase 24.11 (EC 3.4.24.11) are two zinc-containing metalloendopeptidases involved in the metabolism of a variety of biological peptides (Erdos, 1990; Roques et al., 1993). ACE converts the inactive Ang I to Ang II, and NEP metabolizes ANP, Ang II and Ang I. Both enzymes metabolize BK-(1-9) to BK-(1-7) (Erdos, 1990; Roques et al., 1993). Thus, inhibition of both ACE and NEP would be predicted to decrease Ang II formation and to potentiate the actions of ANP and BK-(1-9).

ACE inhibitors are clinically useful for the treatment of hypertension and cardiac failure (Hansson et al., 1993; Crozier et al., 1993). Moreover, NEP inhibitors have diuretic and natriuretic effects (Richards et al., 1990; Schmitt et al., 1994) and have beneficial effects in animal models of heart failure (Rademaker et al., 1996a, b; Willenbrock et al., 1996). Thus, inhibition of both ACE and NEP offers the possibility of improved therapy for hypertension and cardiac failure (Flynn et al., 1995; Marguilies et al., 1991; Fournié-Zaluski et al., 1994a, b; Trippodo et al., 1995a, b). Several dual inhibitors of ACE and NEP have been developed (Fournié-Zaluski et al., 1994a, b; Flynn et al., 1993; Gros et al., 1991; Kirk and Wilkins, 1996; Trippodo et al., 1995b), but little information exists concerning the dose-related effects of these compounds on ACE and NEP activity in vivo. Changes in angiotensin and bradykinin peptide levels may mediate in part the effects of these dual inhibitors, and there are no previous studies of the effects of these inhibitors on circulating and tissue levels of angiotensin and bradykinin peptides.

S 21402-1 is a sulfhydryl-containing potent inhibitor of both ACE and NEP (Fournié-Zaluski et al., 1994a, b; Vera et al., 1995; Gonzalez et al., 1996a) which has therapeutic potential for the treatment of hypertension and congestive cardiac failure (Gonzalez et al., 1996a, b). S 21402-1, previously called RB105, inhibits ACE with a K_i of 4.5 nM and NEP with a K_i of 1.7 nM (Fournié-Zaluski et al., 1994a, b). We investigated the dose-related effects of S 21402-1 on circulating levels of Ang II and Ang I, renal levels of Ang II, Ang I, BK-(1-7) and BK-(1-9) and urinary levels of BK-(1-7), BK-(1-8) and BK-(1-9), and compared the effects of S 21402-1 with those of the sulfhydryl-containing ACE inhibitor captopril, which has a K_i of 1.7 nM (Cushman et al., 1977). Contrary to the similar potency of inhibition of ACE and NEP by S 21402-1 in vitro (Fournié-Zaluski et al., 1994a, b), we found that in vivo inhibition of ACE by S 21402-1 required doses at least 1000-fold higher than those required to inhibit NEP.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Animals. Male Sprague-Dawley rats (~250 g) were fed GR 2+ pellets (Clarke King & Co., Gladesville, Australia) and tap water ad libitum. This study was performed in accordance with the guidelines of the Animal Experimentation Ethics Committee of St. Vincent's Hospital. S 21402-1 initially was dissolved in ethanol, then diluted to 5% ethanol in 0.3 M sodium phosphate buffer, pH 7.4. Captopril was dissolved in 0.3 M sodium phosphate buffer, pH 7.4. Each drug was administered to rats in a volume of 2 ml by intraperitoneal injection. A water load of 20 ml/kg was administered by gavage, and the rats were placed in metabolic cages for 4 h. Urine was collected in containers cooled to the temperature of dry ice and stored at 80°C until assay for sodium, potassium, creatinine, cyclic GMP and bradykinin peptides. At the end of the 4-h urine collection rats were sacrificed by decapitation, trunk blood was collected for the measurement of plasma levels of renin, angiotensinogen, NEP and angiotensin peptides, the left kidney was homogenized immediately in 4 M guanidine thiocyanate, 1% trifluoroacetic acid (GTC/TFA) for the measurement of tissue levels of angiotensin and bradykinin peptides, and the right kidney was frozen in isopentane cooled to the temperature of dry ice for in vitro autoradiography.

Extraction and RIA of angiotensin peptides from plasma. Plasma levels of Ang II and Ang I were measured as described previously (Campbell et al., 1993b). Trunk blood (2-3 ml) was collected rapidly into tubes containing 0.5 ml inhibitor solution (1 mM renin inhibitor acetyl-His-Pro-Phe-Val-Sta-Leu-Phe-NH₂ (Hui et al., 1988), 146 µM pepstatin, 50 mM 1,10-phenanthroline, 125 mM ethylenediaminetetraacetate, 2 g/l neomycin sulfate, 2% dimethyl sulfoxide and 2% ethanol in water) at 4°C. The blood was centrifuged and the plasma (1-2 ml) was extracted immediately with Sep-Pak C₁₈ cartridges (Waters Chromatography Division, Milford, MA). Angiotensin peptides were acetylated and piperidine-treated before HPLC and assay of HPLC fractions by N-terminal directed RIA (Campbell et al., 1993b, 1995). Data were corrected for recovery as reported elsewhere (Campbell et al., 1993b).

Extraction and RIA of angiotensin and bradykinin peptides from kidney. The left kidney was removed rapidly, weighed and homogenized immediately in GTC/TFA and then processed as described previously (Campbell et al., 1993a) before acetylation and piperidine treatment, HPLC and measurement of angiotensin and bradykinin peptides by N-terminal directed RIA (Campbell et al., 1993a, b). Data were corrected for recovery as reported elsewhere (Campbell et al., 1993a, b).

Measurement of sodium, potassium, creatinine, cyclic GMP and bradykinin peptides in urine. Urinary sodium, potassium and creatinine were measured by autoanalyzer by the Department of Chemical Pathology, St. Vincent's Hospital. Cyclic GMP was measured by RIA with reagents from Amersham International, Buckinghamshire, UK. For the measurement of bradykinin peptides, 1 ml freshly thawed urine was added to 10 ml GTC/TFA and extracted with Sep-Pak C₁₈ cartridges. Urine extracts were acetylated and treated with piperidine by a modification of previously described methods. Extracts were acetylated by addition of 1 ml water, 100 µl triethylamine and 50 µl acetic anhydride for 5 min at room temperature, then evaporated to dryness under vacuum before piperidine treatment with 100 µl piperidine in 1 ml water for 60 min at room temperature. The extracts were evaporated to dryness, then run on HPLC and bradykinin peptides measured by RIA as described above. Recoveries from urine were 64 ± 26% (mean ± S.D., n = 8) for BK-(1-7), 77 ± 13% for BK-(1-8) and 76 ± 18% for BK-(1-9). Data were corrected for recovery.

Measurement of renin, angiotensinogen and NEP in plasma. Trunk blood for measurement of renin, angiotensinogen and NEP was collected into heparinized tubes on ice, then centrifuged and the plasma rapidly frozen on dry ice and stored at 80°C. The plasma concentrations of active renin and angiotensinogen were measured as described previously (Campbell et al., 1991). NEP enzymatic activity was measured as described by Yandle et al. (1992), with succinyl-Ala-Ala-Phe-amidomethylcoumarin as substrate; further incubation with aminopeptidase M released free amidomethylcoumarin which was measured fluorometrically.

In vitro autoradiography. Cryostat sections of kidney (20 µm) were cut in an atmosphere of carbon dioxide to prevent oxidation of sulfhydryl groups and were mounted on gelatin-coated slides. In vitro autoradiography was performed immediately or the following day, after storage of the sections at 80°C. Precautions were taken to protect the slides from light at all stages.

10

Statistical analysis. Data are presented as means ± S.E. Data were analyzed by one-way analysis of variance and comparisons with control were made by Dunnett's test. Logarithmic transformation of data was performed where appropriate to obtain similar variances among groups. Statistical analyses were performed by SuperANOVA (Abacus Concepts, Inc., Berkeley, CA).

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Inhibition of NEP in vivo. In vitro autoradiography of kidney sections showed that all doses of S 21402 produced 86 to 91% occupancy of renal NEP, as determined by binding of ¹²⁵I-RB104 (fig. 1). This result was confirmed by measurement of plasma NEP activity, where all doses of S 21402-1 inhibited plasma NEP (fig. 1).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Inhibition of NEP in vivo. Bar graphs show effects of S 21402-1 administration on 125I-RB104 binding to kidney sections in vitro (upper panel, n = 5 rats per group, with four sections per rat), and plasma NEP activity (lower panel, n = 7-8 per group). (mean ± S.E. **P < .01, compared with control).

Plasma renin, angiotensinogen and angiotensin peptides. S 21402-1 at 300 mg/kg increased plasma renin by 4-fold, associated with a 40% decrease in plasma angiotensinogen levels (fig. 2). By contrast, captopril doses of 3 to 300 mg/kg increased plasma renin levels 1.8- to 28-fold, associated with decreases in plasma angiotensinogen of 40 to 57% at captopril doses of 30 and 300 mg/kg (fig. 2).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Bar graphs show effects of S 21402-1 and captopril administration on plasma levels of renin (upper panels) and angiotensinogen (lower panels). (mean ± S.E. *P < .05, **P < .01, compared with control, n = 8 per group).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Bar graphs show the effects of S 21402-1 and captopril administration on plasma Ang II, Ang I and the Ang II/Ang I ratio. (mean ± S.E. **P < .01, compared with control, n = 7-8 per group).

Kidney angiotensin and bradykinin peptides. S 21402-1 at 30 and 300 mg/kg decreased kidney Ang II levels by 30% and 38%, respectively, associated with a 38% decrease in kidney Ang I levels at 300 mg/kg, and with no change in the Ang II/Ang I ratio (fig. 4). Captopril reduced kidney Ang II levels by 46% and 48% at 30 and 300 mg/kg. However, in contrast to S 21402-1, captopril increased kidney Ang I levels 2.7-fold at 300 mg/kg, associated with a marked decrease in the Ang II/Ang I ratio of 50% and 75% at 30 and 300 mg/kg, respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Bar graphs show effects of S 21402-1 and captopril administration on kidney Ang II, Ang I and the Ang II/Ang I ratio. (mean ± S.E. *P < .05, **P < .01, compared with control, n = 7-9 per group).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Bar graphs show effects of S 21402-1 and captopril administration on kidney BK-(1-7), BK-(1-9) and BK-(1-7)/BK-(1-9) ratio. (mean ± S.E. *P < .05, **P < .01, compared with control, n = 7-9 per group).

Urinary electrolytes, cyclic GMP and bradykinin peptides. S 21402-1 had no effect on urine volume, although captopril reduced urine volume at 3 mg/kg (table 1). S 21402-1 increased urine sodium excretion by approximately 30%. Although the increase in urine sodium excretion was not statistically significant for any of the individual doses of S 21402-1, contrast analysis of all S 21402-1 doses versus vehicle showed that the increase was statistically significant (fig. 6). S 21402-1 doubled cyclic GMP excretion at 30 and 300 mg/kg (fig. 6). Captopril reduced urinary sodium excretion at 300 mg/kg and had no effect on cyclic GMP excretion (fig. 6). Neither compound affected urinary potassium excretion (table 1). All doses of S 21402-1 decreased the urine BK-(1-7)/BK-(1-9) ratio (fig. 6) and 300 mg/kg S 21402-1 increased urinary BK-(1-8) and BK-(1-9) excretion (table 1). Captopril had no effect on the urine BK-(1-7)/BK-(1-9) ratio (fig. 6), but BK-(1-8) levels increased at 30 and 300 mg/kg captopril (table 1).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Bar graphs show effects of S 21402-1 and captopril administration on urinary excretion of sodium, cyclic GMP and the urine BK-(1-7)/BK-(1-9) ratio. (mean ± S.E. *P < .05, **P < .01, compared with control, n = 7-8 per group).

Inhibition of the pressor response to Ang I. Captopril (1 mg/kg) produced complete inhibition of the pressor response to Ang I at 2 min, with an approximate linear recovery of pressor response over time, reaching 50% recovery at 180 to 240 min (fig. 7). By contrast, 10 mg/kg S 21402-1 was required for complete inhibition of Ang I response at 2 min, with an approximate linear recovery of pressor response over time reaching 50% recovery at 120 min. For 1 mg/kg S 21402-1, inhibition at 2 min was only 90%, and the decay in inhibition was much more rapid than that observed for 10 mg/kg S 21402-1 (fig. 7). Similar results were obtained after oral administration of captopril and S 21402-1 (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Line graphs show the time course of the change in pressor response to Ang I after intravenous administration of either vehicle () captopril (, 1 mg/kg) or S 21402-1 (, 1 mg/kg; , 10 mg/kg). Percentage inhibition was calculated from the amount of Ang II required to produce a pressor response equal to that seen with Ang I. (mean ± S.E., n = 4 per group).

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

A major finding of this study was the greater than 1000-fold difference in ACE and NEP inhibition by S 21402-1 in vivo, despite only a 2.6-fold difference in K_i in vitro as reported by Fournié-Zaluski et al. (1994a, b). We also studied the inhibition of ACE and NEP by S 21402-1 in vitro with rat lung membranes and observed a similar 2.7-fold difference in K_i for ACE and NEP inhibition (F. Anastasopoulos, R. Lueng, T. A. Briscoe, T. P. Gorski and D. J. Campbell, unpublished data from this laboratory). Previous studies showed a discrepancy between ACE and NEP inhibition by S 21402-1 in vivo. Vera et al. (1995) found that intravenous infusion of S 21402-1 produced near-maximal increases in urinary sodium and immunoreactive ANP excretion at the lowest infusion rate of S 21402-1 studied (2.5 mg/kg per h), whereas increases in plasma renin required infusion of 25 mg/kg per h or higher, which suggests that NEP inhibition was achieved at doses much lower than those required to achieve ACE inhibition. Studies of the in vivo potency of orally administered S 21402-1 used a prodrug with a benzoyl group protecting the sulfhydryl of S 21402-1 (mixanpril) (Fournié-Zaluski et al., 1994a, b). Oral administration of mixanpril to mice demonstrated approximately 50% occupancy of NEP and ACE with 0.7 and 7 mg/kg, respectively (Fournié-Zaluski et al., 1994b). Fournié-Zaluski et al. (1994a) reported that administration of 10 mg/kg mixanpril to mice produced prolonged and complete inhibition of kidney NEP with 93% inhibition at 8 h, whereas inhibition of lung ACE was incomplete and only 40% at 4 h.

S 21402-1 was a potent NEP inhibitor. All doses of S 21402-1 inhibited NEP, as indicated by plasma NEP activity, radioinhibitor binding to kidney sections, urinary sodium excretion and the BK-(1-7)/BK-(1-9) ratio. The natriuresis and increase in urinary cyclic GMP excretion in S 21402-1-treated rats were consistent with potentiation of the actions of endogenous ANP and BK-(1-9). The BK-(1-7)/BK-(1-9) ratio was a much more sensitive indicator of NEP inhibition than the absolute levels of bradykinin peptides in kidney and urine. Much higher doses of S 21402-1 were required to reduce the BK-(1-7)/BK-(1-9) ratio in kidney than in urine. The different responses of the BK-(1-7)/BK-(1-9) ratio in kidney and urine may reflect the relative roles of NEP in BK-(1-9) metabolism in different compartments of the kidney. Although NEP has a major role in the metabolism of BK-(1-9) in urine, it may have a lesser role in BK-(1-9) metabolism in the renal interstitium. It is also possible that higher levels of S 21402-1 were achieved in urine than in the renal interstitium.

Captopril was greater than 10-fold more potent than S 21402-1 as an ACE inhibitor in vivo. Inhibition of ACE was seen with only 300 mg/kg S 21402-1, whereas 3 to 300 mg/kg captopril caused ACE inhibition, as assessed by plasma Ang II/Ang I ratio and renin and angiotensinogen levels. Captopril inhibited renal ACE, as indicated by the decrease in Ang II levels, increase in Ang I levels and decrease in the Ang II/Ang I ratio. By contrast, S 21402-1 reduced both Ang II and Ang I levels in kidney, with no change in the Ang II/Ang I ratio. The lack of change in the renal Ang II/Ang I ratio is evidence against inhibition of renal ACE by S 21402-1. However, we cannot explain the decline in renal Ang II and Ang I levels in S 21402-1-treated rats, especially given the increase in plasma renin levels in these animals.

In contrast to this study, Gonzalez et al. (1996b) reported that captopril and S 21402-1 were approximately equipotent in the inhibition of the enzymatic activity of plasma ACE after oral administration to rats; however, the method of measuring plasma ACE enzymatic activity was not described by Gonzalez et al. (1996b). This study is the first to report the effects of a dual inhibitor of ACE and NEP on plasma angiotensin peptide levels, and renal tissue levels of angiotensin and bradykinin peptides. We previously showed that the plasma Ang II/Ang I ratio is the most sensitive index of ACE inhibition in vivo (Campbell et al., 1994). Moreover, the Ang II/Ang I ratio avoids difficulties associated with ACE enzymatic assay where inhibition by sulfhydryl-containing compounds may be underestimated because of oxidation during sample processing or storage (Boomsma et al., 1981).

The present results for inhibition of the pressor response to Ang I by captopril agreed closely with previous studies by Ondetti et al. (1977). Comparison of the effects of S 21402-1 and captopril on the pressor response to Ang I confirmed that captopril was greater than 10-fold more potent than S 21402-1. Moreover, the more rapid decay in ACE inhibition by S 21402-1 than captopril was consistent with either more rapid clearance or more rapid modification of S 21402-1 than captopril. NEP inhibition was maintained by the lowest dose of S 21402-1 for at least 4 h, which indicates that the decay in ACE inhibition was not caused solely by drug clearance but also represented, at least in part, modification of S 21402-1 in vivo, such that ACE inhibitory potency was lost but NEP inhibitory potency was maintained.

No information is currently available concerning the metabolism of S 21402-1. In preliminary studies we found that exposure of S 21402-1 to plasma caused an increase in IC₅₀ for ACE inhibition of greater than 100-fold, whereas the IC₅₀ for NEP inhibition increased by only 3-fold (T. A. Briscoe and D. J. Campbell, unpublished data from this laboratory). Evidence that this effect of plasma was caused by the free sulfhydryl of S 21402-1 was the failure of plasma to influence the IC₅₀ for ACE inhibition by the non-sulfhydryl-containing ACE inhibitor lisinopril (unpublished data from this laboratory). Sulfhydryl-containing drugs rapidly form mixed disulfide conjugates in vivo (Drummer and Jarrott, 1986; Friedman, 1977; Mannervik, 1982). The major metabolites of captopril that appear in blood and urine are mixed disulfide conjugates either with low molecular weight endogenous thiols (glutathione, cysteine) or with proteins (Drummer and Jarrott, 1986). Plasma concentrations of mixed disulfide conjugates are much higher than those of free captopril and may accumulate with chronic therapy (Cody et al., 1982). Further studies are required to reveal the mechanism of the marked difference between ACE and NEP inhibition by S 21402-1 in vivo. However, our study emphasizes that caution must be exercised in the extrapolation of the relative inhibitory potencies of a dual enzyme inhibitor in vitro to its ability to inhibit the same two enzymes in vivo, in that modification of the dual inhibitor in vivo may result in relative inhibitory potencies which differ markedly from those of the parent compound. Consequently, effective inhibition of ACE by a dual inhibitor of ACE and NEP may require doses much higher than those required for NEP inhibition.