A double-blind, placebo-controlled study to assess tolerability, pharmacokinetics and preliminary pharmacodynamics of single escalating doses of Z13752A, a novel dual inhibitor of the metalloproteases ACE and NEP, in healthy volunteers

Abstract

Aims

The objective of this study was to evaluate the tolerability of a novel dual ACE-NEP inhibitor, Z13752A, after the oral administration of rising single doses in healthy volunteers. This study was also a preliminarily investigation of Z13752A pharmacodynamics (PD) and pharmacokinetics (PK).

Methods

In this randomized, placebo-controlled, sequential study, two alternating panels of eight healthy male volunteers each (six subjects receiving the active treatment + two subjects receiving placebo) were treated with increasing oral doses of Z13752A: 10, 50, 200, and 600 mg were given to panel I and 20, 100, 400 and 800 mg were given to panel II. The study was double-blind relative to placebo or active treatment, and was open with respect to the dose levels. The same volunteer received placebo only once.

Results

Single oral doses of Z13752A, as high as 800 mg, were well tolerated. Only six mild-to-moderate adverse events mainly headache, were reported and appeared to be of little clinical relevance. After administration of 200, 400, 600 and 800 mg of Z13752A, a nonsignificant fall in diastolic blood pressure was detected, in both the standing and supine position. After single oral doses of Z13752A, ACE inhibition appeared to be significant at all the doses tested, linearly correlated with the dose and was almost complete at doses ≥ 100–200 mg. NEP inhibition was indicated by elevation of ANP and cGMP plasma concentrations in almost all subjects. In the 200–800 mg dose range, Z13752A produced a 50–100% increase of plasma cGMP levels and a 50–80% elevation in urinary cGMP concentrations. Detectable plasma levels of Z13752A were found in all the treated subjects. Z13752A was well and rapidly absorbed, with peak concentrations reached approximately 2.5 h after administration. The mean apparent elimination half-life from plasma was approximately 12 h. The pharmacokinetics of Z13752A after single oral doses were characterized by low intersubject variability and appeared to be dose-independent.

Conclusions

Z13752A showed a good single dose tolerability profile at doses up to 800 mg. The pharmacokinetic data indicate that Z13752A administered orally is rapidly absorbed and available to the systemic circulation in humans. The relatively slow clearance indicates that a once-a-day dose regimen could be considered for Z13752A.

Introduction

The renin-angiotensin-aldosterone system (RAS) and the natriuretic peptide system have contrasting actions on blood pressure and volume homeostasis. Alterations in both systems are involved in the pathogenesis of hypertension and congestive heart failure (CHF).

Angiotensin-converting enzyme (ACE, E.C. 3.4.15.1), is responsible for the conversion of angiotensin I (AI) to the biologically active angiotensin II (AII). AII raises blood pressure by both increasing vascular resistance and by triggering the production of aldosterone. Neutral endopeptidase (NEP, E.C. 3.4.24.11) is the enzyme mainly responsible for the degradation and inactivation of atrial natriuretic peptide (ANP). This peptide, via cyclic guanosine monophosphate (cGMP) formation, plays a role in modulating cardiovascular homeostasis, leading to vasodilatation, natriuresis, diuresis, and inhibition of aldosterone formation. Since the hormonal actions of AII and ANP are functionally opposed, it is expected that attenuation of AII with concurrent potentiation of ANP will lead to a beneficial additive effect [1]. In agreement with this, the association of selective inhibitors of ACE and NEP resulted in a potentiation of their respective effects in various experimental models of hypertension and CHF [2–5]. A clinical trial in hypertensive patients with a combination therapy (captopril + sinorphan) confirmed these experimental findings [6]. The development of single chemical entities that possess the ability to inhibit both the zinc metalloproteases ACE and NEP has been the objective of recent efforts in cardiovascular research. The safety and efficacy of the combination therapy, and of novel compound acting as inhibitors of both ACE and NEP, was recently evaluated in both healthy volunteers and patients [7–11].

Z13752A (N-[(s)-2(mercaptomethyl)-1-oxo-3-phenylpropyl]-4-(2-thiazolyl)-l-phenyl-alanine) (Figure 1) is a novel potent and specific dual inhibitor of ACE and NEP, as demonstrated both in vitro and in vivo. In vitro studies have demonstrated that this compound inhibits rabbit lung ACE (IC₅₀ = 3.2 nm) and rat renal NEP (IC₅₀ = 1.8 nm), being nearly equipotent to the selective ACE inhibitor captopril and to the selective NEP inhibitor SQ28603 [12]. In an ex vivo model, i.v. administration of Z13752A to spontaneously hypertensive rats (SHR) strongly reduced ACE (ED₅₀ = 1.4 mg kg⁻¹) and NEP activities (ED₅₀ = 0.6 mg kg⁻¹). Z13752A, administered orally at the dose of 13 mg kg⁻¹, produced inhibition of plasma (90%) and lung (60%) ACE activity 30 min post dosing. This inhibition was still present 6 h post dosing. A similar inhibitory profile was obtained for renal NEP (50% at 30 min) [13]. In other studies, oral Z13752A produced a significant reduction in blood pressure both in SHR and DOCA-salt rats [14]. Captopril was able to reduce blood pressure in SHR, but not in DOCA-salt rats. In addition, the ACE inhibition caused by captopril evoked a more evident rise in plasma renin activity than after Z13752A. Z13752A is characterized by a low acute toxicity when given either orally or intravenously.

Figure 1.

Chemical structure of Z13752A(N-[(s)-2(mercaptomethyl)-1-oxo-3-phenylpropyl]-4-(2-thiazolyl)-l-phenyl-alanine).

This phase I study was designed to evaluate the tolerability of Z13752A after rising single oral doses of this dual ACE-NEP inhibitor in healthy volunteers. This study also provided a preliminary investigation of Z13752A pharmacodynamics and pharmacokinetics.

The oral doses administered were selected on the basis of the pharmacological and toxicological studies carried out in rats and dogs. In subchronic toxicity studies (repeated oral administration for 4 weeks), the no-observed-adverse-effect-level (NOAEL) in both species was 80 mg kg⁻¹ day⁻¹. The starting dose selected for the healthy volunteer study was much less than one-twentieth of the NOAEL and the dose-range chosen included those doses that were found to be pharmacologically active in animals.

Methods

Subjects

Sixteen healthy male Caucasian volunteers, aged 19–36 years (mean ± s.d. = 25 ± 4 years) weighing 59–82 kg (mean ± s.d. = 72 ± 7 kg) gave their written informed consent to participate in the study, which was approved by the Medical Ethics Committee (CCPPRB) of Paris-Boucicaut, France. All the subjects included were nonsmokers, within 20% of ideal body weight (based on height and age). Their 24h creatinine clearance values (mean ± s.d.) were 105 ± 12 ml min⁻¹.

These subjects had no abnormalities at prestudy physical and clinical examination, laboratory investigations or 12-lead ECG (defined ranges: PR duration interval 120–200 ms, QRS duration interval < 105 ms and QTc duration interval 360–430 ms). All subjects were normotensives, defined as having a supine systolic blood pressure (SBP) between 110 and 145 mmHg and a supine diastolic blood pressure (DBP) between 65 and 90 mmHg.

The intake of coffee/tea/xanthine, or quinine beverage, or grapefruit juice, and alcohol consumption was stopped 48 h before each drug administration.

No medications or over-the-counter drugs were allowed for 1 week prior to and throughout the study period. However, paracetamol was allowed up to 48 h before each study period.

Study design

This was a randomized, placebo-controlled, rising single oral doses, sequential study, using two alternating panels of eight healthy male volunteers each (six subjects receiving the active treatment and two subjects receiving placebo). The study was double-blind relative to placebo or the active treatment in each panel, and was open with respect to the dose levels.

Each subject received four increasing oral doses of Z13752A: 10, 50, 200, and 600 mg in panel I and 20, 100, 400 and 800 mg in panel II. Randomization was in the ratio of 1 placebo to 3 active treatments (2 : 6) for each dose level investigated; the same volunteer received placebo only once. A 7 day washout period separated two consecutive administrations.

The study was performed at ASTER, rue E. Millon 3 and 5, Paris, France and the pharmacodynamic analyses were carried out at Aster Biotechnologies, Les Nertières, Allèe H. Pintus 06610, La Gaude, France. Pharmacokinetic and statistical analyses were performed at Zambon Group S.p.A., via L. del Duca 10, Bresso, Italy.

Subjects were required to remain at the study centre from 12 h before to 48 h after each dose administration. Fasting conditions were maintained from 12 h before to 4 h post dosing. Water and salt intake were controlled during the hospitalization period by administering salt deficient food and providing known quantities of salt, and by monitoring water intake. The total NaCl intake per day was approximately 7 g for all the subjects studied. Protein intake was 1 g kg⁻¹ body weight/day with a total of 1500–2000 kcal/day. Finally, mean water intake during the stay of the subjects at the centre was approximately 4.5 l (range: 4,1–4,8 l). No differences in water intake between the dose administration periods were seen.

A complete physical examination was carried out at study entry, at completion and before each study period. Laboratory assessments (haematology, biochemistry and urinalysis) was performed at screening, before each administration and 24h post dosing.

Tolerability assessments

Blood pressure was measured immediately before and at 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360 min post dose, every 1 h up to 12 h post dosing and at 24 and 48 h post dose. Each measurement was made in the same arm, with the subject firstly in the supine position (after 3 min rest) and then in the standing position (after 1 min in the erect position) using an automatic device (DATEX). Blood pressure measurements were always carried out before blood sampling. Readings were made to the nearest 2 mmHg and using Korotkoff phase V for the diastolic blood pressure.

Heart rate Cardiac telemetric monitoring was used to observe the electrical activity of the heart and to obtain measurements of heart rate pre-dose (starting from 10 min before drug administration) and up to 4 h post dose. Readings were taken in the supine and erect position at the same time intervals as for BP up to 4 h post dose.

Pulse rate The radial pulse rate was measured immediately before and at 15, 30, 45, 60, 90, 120, 180, 240 min post dose, every 1 h up to 12 h post dose and at 24 and 48 h post dose.

Electrocardiogram (ECG 12-lead) was recorded with the subject in the supine position at screening, immediately before dosing, at the following intervals post dose: 30, 60, 180, 360 min, at 12, 24 and 48 h and at the study end.

Respiratory rate was monitored by visual count at screening, before dosing and at the following intervals post dose: 60, 120, 180, 360 min, 12, 24 and 48 h.TemperatureOral body temperature was assessed at screening, before dosing, 24 and 48 h postdosing.Tolerability was assessed by the spontaneous reporting of adverse events and was also elicited by asking the volunteer the general question ‘how are you feeling?’ 1, 4, 8, 12, 24 and 48 h post dosing. All untoward effects occurring during the study were recorded, together with their duration, severity and measures taken.

Drug formulation and administration

Z13752A was provided as 10 mg, 50 mg, 100 mg and 200 mg tablet or matching placebo by Zambon Group S.p.A., Vicenza, Italy. At each dose level the eight subjects in the panel received a single oral dose of Z13752A or placebo, after an overnight fast, according to the randomization list. The study medication was administered with 150 ml of tap water between 08.00 h and 09.01 h.

Blood samples for PK

To estimate the main pharmacokinetic parameters of Z13752A, 16 venous blood samples (approximately 2 ml each) were collected in each session, before dosing (time 0) and 5, 15, 30, 45, 60, 90, 120 min, 3, 4, 6, 8, 12, 16, 24 and 48 h postdosing. Blood was collected into chilled tubes containing 10 µl ml⁻¹ of blood of a 200 mg ml⁻¹ solution of ascorbic acid in 0.5 m K₃EDTA. Plasma was obtained by centrifugation at 800 × g for 10 min at 4 °C, within 10 min after collection. Plasma samples were stored at −80 °C until assayed.

Blood samples for PD

Samples for ACE activity determination were collected through an intravenous catheter placed in the upper limbs or by venous punction prior to dosing (time 0) and 5, 15, 30, 45, 60, 90 min, 2, 3, 4, 6, 8, 12, 16, 24 and 48 h postdosing

Blood was collected into chilled tubes and within 10 min from the collection time, serum was separated by centrifugation (800 g for 10 min at 4 °C) and stored at −20 °C pending analysis. Samples for ANP and cGMP determination were collected as described above prior to dosing (time 0) and 30, 60, 120 min, 4, 8, 16, and 24h postdosing.

For atrial natriuretic peptide (ANP) determination, blood was collected into chilled tubes containing 7.5 mm K₃EDTA and a cocktail of protease inhibitors (aprotinin: 500 KIU ml⁻¹ plasma, plus soybean trypsin inhibitor: 50 BAEE U ml⁻¹ plasma). Within 10 min from collection, plasma was separated by centrifugation (800 × g for 10 min at 4 °C) and then stored at −20 °C until analysis. For cyclic guanosine monophosphate (cGMP) evaluation, blood was collected into chilled tubes containing 7.5 mm K₃EDTA only. Plasma samples were then processed as for ANP.

Urine samples

Cyclic guanosine monophosphate (cGMP) concentrations were also determined in urine. A urine sample was collected before the study drug administration, and 0–2 h, 2–4 h, 4–8 h, 8–12 h and 12–24h postdosing. The volume and pH of each sample was measured and samples were stored at −20 °C pending assay.

Sample analysis for PK

Z13752A concentrations were determined in plasma using a validated h.p.l.c. method with fluorescence detection [15]. For quality control samples, intra-and interassay coefficients of variation were below 10%. The limit of quantification was set at 50 ng ml⁻¹.

Sample analysis for PD

Serum ACE activity was determined using a standard commercial kit (Sigma 305–10). The limit of quantification of the assay was set at 7 U l⁻¹ with intra-and interassay coefficients of variation below 10%.

Plasma levels of cGMP were measured using a commercially available radioimmunoassay kit (RPA 525, Amersham). The limit of quantification of the assay was set at 44 fmol ml⁻¹, with intra-and interassay coefficients of variation below 20%.

Plasma levels of ANP were measured using a commercially available radioimmunoassay kit (RPA 512, Amersham). The limit of quantification of the assay was set at 8 fmol ml⁻¹ with intra-and interassay coefficients of variation below 20%.

Urinary levels of cGMP were measured using a commercially available radioimmunoassay kit (RPA 525, Amersham). The limit of quantification of the assay was set at 44 fmol ml⁻¹ with intra-and interassay coefficients of variation below 20%.

Pharmacokinetic analysis

Non-compartmental methods were used for the Z13752A pharmacokinetic analysis (Kinetica™ software, SIMED, release 1.1). The maximum plasma concentration C_max and the corresponding time t_max were read as the co-ordinates of the highest data point in the plasma concentration vs time curve. The area under this curve was calculated using the linear trapezoidal rule up to the last measurable concentration (AUC(0,t)) and beyond that time by extrapolation from time t to infinity (AUC) assuming monoexponential decay. The terminal elimination half-life (t_½) was estimated by linear regression analysis of natural log concentration against time, t_½ = ln2/slope. The choice of the terminal points was based primarily on graphic judgement by eye. Apparent plasma clearance (CL/F) was calculated as CL/F = dose/AUC. Not quantifiable concentration (n.q.) were set to zero in the calculations of both means and of the pharmacokinetic parameters.

Pharmacodynamic assessments

Due to the intersubject variability of the parameters (both of physiological and analytical origin), for calculation purposes data below the limit of quantification (LOQ) of the method were set at one-half the LOQ. This approach reduced the bias given by those samples containing analyte concentrations below the LOQ of the assay, and provided a more conservative estimate of the pharmacodynamic parameters estimated.

The following parameters were calculated from the estimated plasma and urinary concentrations:

1. ACE activity in plasma:

time course of mean values of ACE activity (expressed as percentage of predose values) and the corresponding area under the curve estimated in the time interval 0–24h [AUC_ACE]

2. ANP concentration in plasma:

time course of mean values expressed as percentage changes from predose values, and the corresponding area under the curve estimated in the time interval 0–24h [AUC_ANP]

3. cGMP concentration in plasma:

time course of mean values expressed as percentage changes from predose values, and the corresponding area under the curve estimated in the time interval 0–24h [AUC_cGMP]

4. cGMP concentration in urine:

cumulative excretion data in the time intervals 0–2, 0–4, 0–8, 0–12, 0–24h after drug administration;

% variation with respect to placebo of the 0–24h cumulative excretion

5. Urine volume:

urinary recovery was assessed in the time intervals 0–12 h, 12–24, 24–36, and 36–48 h after drug administration.

Statistics

Demographic data, laboratory tests and raw data obtained on vital signs (blood pressure, heart rate and pulse rate, respiratory rate, temperature), as well as changes from predose assessment and the maximum decrease from baseline of blood pressures were summarized by descriptive statistics (means and s.d.).

Moreover, the area under the curve (calculated using the trapezoidal rule) of the changes in blood pressure from baseline vs time (Δ mmHg × min) in the time interval 0–12 h, for diastolic (AUC_DBP) and systolic (AUC_SBP) blood pressure were calculated after each dose and were then compared by analysis of variance (anova). This analysis was followed by a Dunnett's t-test to compare each dose of Z13752A with placebo. In addition, the same statistical analysis was performed on the maximum decrease in blood pressure (BP_min), irrespective of time, in the time interval 0–12 h. The area under the curve of changes from baseline in pulse rate vs time (Δ mmHg × min) in the time interval 0–12 h (AUC_HR) was also calculated and analysed as described above.

The pharmacokinetic parameters calculated after each dose were compared by anova, followed by a Tukey's test for pairwise comparisons. t_max values were compared nonparametrically (Kruskall-Wallis). C_{max and} AUC values were adjusted to the dose of 1 mg (C_max/dose, AUC/dose) before the analysis. Finally, for C_{max and} AUC a linear regression analysis (C_maxvs dose, AUC vs dose) was also performed.

ACE activity in plasma: a least square regression analysis was applied to study the relationship between dose and [AUC_ACE]; In addition, a comparison among treatment groups was carried out by anova followed by a Dunnett's t-test to compare each Z13752A dose to placebo.

ANP concentration in plasma:[AUC_ANP] were submitted to anova followed by a Dunnett's t-test to compare each Z13752A dose to placebo.

In addition, a linear regression analysis of the AUC vs dose was performed.

cGMP concentration in plasma:[AUC_cGMP] were submitted to anova followed by a Dunnett's t-test to compare each Z13752A dose to placebo. In addition, a linear regression analysis of the AUC vs dose relationship was performed.

cGMP concentration in urine: a relationship between dose and percent variation (with respect to placebo) of the 0–24h cumulative excretion data were evaluated using a least square regression analysis; in addition, a comparison of cumulative excretion data (in the time intervals 0–2 h, 0–4 h, 0–8 h, 0–12 h and 0–24h) was carried out by anova followed by a Dunnett's t-test to compare each Z13752A dose to placebo.

Urine volume: An overall comparison of cumulative urinary recovery in the time intervals 0–12 h, 12–24h, 24–36 h and 36–48 h was performed by anova, followed by a Dunnett's t-test to compare each dose of Z13752A with placebo.

Confidence intervals (95%) for differences of all dose levels with placebo were calculated. All tests were carried out at the 5% α-level.

Results

Clinical observations

Compliance with the study protocol was 100%. To complete the study, it was necessary to recruit an additional volunteer, as one of the subjects receiving placebo was withdrawn because of a serious, not study-related, adverse event (wounded hand at home, treated surgically) that took place after the first study period. No other serious adverse events occurred. Up to the maximal dose assessed (800 mg), only 9 adverse events were experienced during all the dosing sessions, by 5 of the 16 subjects; one subject reported 5 events. The most frequently reported adverse event was headache (5/9). Two mild episodes occurred after placebo. Three mild to moderate headaches occurred after administration of 50 and 200 mg Z13752A.

No clinically relevant changes in laboratory parameters or ECG modifications were observed. In addition, no significant difference was evidenced among treatments with regard to the body temperature (BT) and the respiratory rate (RR).

A slight not significant reduction of both supine and standing blood pressure compared with placebo was seen at the highest doses administered (200 and 600 mg in group I, 400 and 800 mg in group II). A graph showing the time-course of the mean values of the changes from predose assessment in SBP and DBP after administration of placebo and the highest dose in each of the groups tested (600 mg in group I, 800 mg group II) is given in Figure 2. The comparison of (BP_min), in the standing and supine positions (data not shown) indicates no statistically significant differences between treatment groups, with the only exception of the 50 mg group for standing systolic BP (−23.3 mmHg for Z13752A compared with 9.9 mmHg for placebo). With regard to (AUC_HR), no significant differences were seen from placebo after Z13752A administration at all the doses, with the only exception of the 200 mg dose in the standing position (+ 12.9 beats min⁻¹ for Z13752A compared with + 1.8 beats min⁻¹ for placebo).

Figure 2.

Time course of systolic (SBP) and diastolic (DBP) blood pressure, recorded in the supine position, over the first 12 h after administration of placebo (▪ group 1; • group 2) or after the highest administered doses of Z13752A (▴ 600 mg, group 1; ♦ 800 mg, Group 2. Values are expressed as changes from baseline (▵ mmHg). The mean curves for six subjects are shown (eight for placebo).

Pharmacokinetics

The mean Z13752A plasma concentration-time curves (after 10–800 mg oral administration) are shown in Figure 3. From the lowest administered dose of Z13752A (10 mg), detectable plasma levels of Z13752A were found in all the treated subjects. Therefore, the noncompartmental pharmacokinetic analysis was possible in all the subjects investigated.

Figure 3.

Mean plasma concentration-time profiles after oral administration of Z13752A tablet at eight dose levels (▪ 10 mg; □ 20 mg; ▾ 50 mg; ▿ 100 mg; ♦ 200 mg; ◊ 400 mg; • 600 mg; ○ 800 mg) in healthy volunteers.

The plasma concentration after oral administration of Z13752A rose rapidly, with the first detectable levels appearing between 5 and 15 min. Peak plasma concentrations were reached between 1.7 and 2.8 h after drug intake.

A visual inspection of the individual plasma concentration-time curves of Z13752A in the treated subjects indicated that at all the tested doses a very low intersubject variability was found. The individual pharmacokinetic parameters of Z13752A, calculated by means of noncompartmental analysis after oral administration of the test compound at the doses of 10–800 mg, are shown in Table 1.

Table 1.

Mean pharmacokinetic parameters of Z13752A ± s.d.

Z13752A dose	Cmax (µg ml−1)	tmax (h)	AUC (µg ml−1 h)	t½ (h)
10 mg	0.9 ± 0.2	2.2 ± 0.9	8.1 ± 2.6	8.6 ± 5.2
20 mg	2.2 ± 0.7	2.4 ± 0.7	16.9 ± 4.8	8.5 ± 3.8
50 mg	7.4 ± 1.7	1.7 ± 0.7	57.8 ± 9.0	11.2 ± 2.5
100 mg	15.6 ± 3.4	2.0 ± 0.5	137.1 ± 31.8	10.6 ± 1.5
200 mg	26.9 ± 8.2	2.3 ± 0.5	253.3 ± 62.8	11.0 ± 2.3
400 mg	58.1 ± 7.7	2.8 ± 0.8	545.6 ± 85.3	13.0 ± 5.2
600 mg	95.9 ± 20.9	2.7 ± 0.5	889.7 ± 113.6	11.0 ± 1.4
800 mg	107.7 ± 13.3	2.8 ± 0.8	1058.2 ± 194.9	11.0 ± 1.6

The mean ± s.d. of maximum plasma concentration (C_max) after the dose of 10 mg was 0.91 ± 0.18 µg ml⁻¹, and the corresponding time (t_max) was 2.2 ± 0.9 h. At doses ≥ 20 mg, the C_max and AUC increased in a dose-independent fashion. At all the tested doses the percentage of the total AUC which was extrapolated never exceeded 12% (mean value 3.0 ± 2.2%).

The t_max values (± s.d.) after Z13752A administration at all doses averaged 2.4 ± 0.7 h (median 2 h; range 1–4 h). The mean apparent elimination half-life (t_½) from plasma was 10.6 ± 3.4 h (range 4.4–23.3 h). No statistically significant difference among treatments for either t_max or t_½ was found.

The relationship between C_max, AUC and the administered dose is shown in Figure 4a and b, respectively. A significant linear correlation (P < 0.0001) was found, with high values of r² (0.94 and 0.95 for C_max and AUC, respectively).

Figure 4.

Relationship between Z13752A dose and individual C_max (a) or AUC (b) values at the eight doses tested. The best-fitted linear regression line (solid line) and the 95% confidence region (dotted lines) are also shown.

No statistically significant differences were seen with regard to the dose-adjusted C_max values, with the only exception of the values calculated after the 10 mg dose, that resulted significantly different from the 50, 100 and 600 mg doses. For AUC, significant differences were observed for the dose-adjusted values only after the dose of 10 mg (vs 100, 200, 400, 600, 800 mg) and the dose of 20 mg (vs 100, 400, 600 and 800 mg).

Biological markers of ACE and NEP activity

ACE In the subjects receiving placebo, serum ACE activity remained stable over time, with variation not exceeding ± 20% of predose values in the majority of the cases. In those subjects receiving Z13752A a dose-related inhibition of ACE activity was observed, starting approximately 15 min postdosing, reaching a peak after 1–2 h and being still marked (40–60% inhibition), 12 h after the 100, 200, 400, 600 and 800 mg doses (Figure 5a). At doses ≥ 200 mg, the maximal inhibition of ACE activity was reached and maintained for 6–12 h approximately, depending on the Z13752A dose administered.

Figure 5.

(a) Time course of inhibition of serum ACE activity over 48 h after single oral administration of placebo (▪ group 1; □ group 2) or Z13752A (• 10 mg; ○ 20 mg; ▴ 50 mg; ▵ 100 mg; ▾ 200 mg; ▿ 400 mg; ♦ 600 mg; ◊ 800 mg).Values are expressed as percentage changes from predose values. The mean curves for six subjects are shown (eight for placebo). (b) Relationship between mean Z13752A plasma concentration at C_max and mean ACE activity inhibition after single oral administration of Z13752A at eight dose levels in healthy volunteers.Standard deviation of mean ACE activity and Z13752A concentration are shown by vertical and horizontal bars, respectively.

At 48 h, the last sampling time, the inhibition of ACE activity was still approximately 15% (400 mg dose) and 25% (800 mg dose) from baseline.

Marked ACE inhibition (minimum value reached in the interval 0–24h: 33% ± 5% from baseline) was seen even at a dose of 50 mg Z13752A.

Mean ± s.d. values of the AUC_ACE were 105% ± 35% and 114% ± 26% ACE activity × min for placebo panel I and II, respectively. After administration of the highest dose in each of the group tested (600 mg in group I, 800 mg in group II) AUC_ACE was 34% ± 2% and 29% ± 5% ACE activity × min, respectively.

At all Z13752A doses except 10 mg and 20 mg AUC_ACE values were significantly different from placebo (median values and 95% CI for differences from placebo are reported in Table 2). Moreover, there was a statistically significant linear correlation (P < 0.001; r² = 0.66), between AUC_ACE and the Z13752A dose. The mean ACE activity inhibition detected in serum 2 h (maximum effect) after administration of the eight doses tested, was highly correlated (P < 0.001) with the log-transformed C_max values, according to a sigmoidal function (Figure 5b).

Table 2.

Median values and 95% confidence intervals for all significant (S) and non significant (NS) differences between Z13752A doses and placebo.

Biomarkers of ACE and NEP activity	10 mg	20 mg	50 mg	100 mg	200 mg	400 mg	600 mg	800 mg
Serum ACE (% of predose values h−1)	−10.3	−17.7	−33.6	−54.1	−59.4	−78.6	−71.5	−84.9
	(5.4, −22.3)	(10.2, −41.6)	(−4.3, −52.5)	(−8.5, −87.6)	(32.8, −79.6)	(−64.3, −92.2)	(−64.0, −80.1)	(−72.8, −97.8)
	NS	NS	S	S	S	S	S	S
Plasma ANP (% changes of predose values h−1)	1457	−122	468	1165	775	450	2710	1838
	(−1336, 4249)	(−2792, 2547)	(−2607, 3543)	(−1505, 3835)	(−1834, 3384)	(−2220, 3120)	(231, 5189)	(−831, 4509)
	NS	NS	NS	NS	NS	NS	S	NS
Plasma cGMP (% changes of predose values h−1)	19.2	15.3	39.8	20.3	33.5	43.9	76.7	21.6
	(−10.1, 48.5)	(−10.4, 41.0)	(10.5, 69.1)	(−4.2, 47.3)	(4.2, 62.8)	(18.2, 69.6)	(47.4, 106.0)	(−4.2, 47.3)
	NS	NS	S	NS	S	S	S	NS
24h urinary cGMP (mmol ml−1)	52	172	364	367	609	879	466	494
	(−321, 425)	(−355, 700)	(−9, 738)	(−160, 895)	(237, 983)	(351, 1407)	(93, 840)	(−33, 1022)
	NS	NS	NS	NS	S	S	S	NS

ANP In subjects receiving placebo, ANP plasma concentrations remained stable over time, with variation not exceeding ± 40% from baseline in the majority of the cases. Mean ± s.d. baseline values were 10.3 ± 5.2 fmol ml⁻¹ and 8.7 ± 5.3 fmol ml⁻¹ for panels I and II, respectively. Mean baseline values for all the treatment periods were in the range of 5.4–12.6 fmol ml⁻¹. In subjects receiving Z13752A, a dose-related increase in plasma ANP concentrations was found, starting approximately at 1 h, reaching a peak after 2–4 h and being still marked (two-fold higher than placebo) after 8 h for doses of 100, 400, 600 and 800 mg (Figure 6).

Figure 6.

Time course of plasma ANP variation over 24h after single oral administration of placebo (a) ▪ group 1; b) □ group 2) or Z13752A (▴ 10 mg; ▾ 50 mg; • 200 mg; ♦ 600 mg; ▵ 20 mg; ▿ 100 mg; ○ 400 mg; ◊ 800 mg). Values are expressed as percentage changes from predose values. The mean curves for six subjects/panel are shown (eight for placebo).

A high intersubject variability in AUC_ANP was found and no statistically significant differences with respect to placebo were found, except the 600 mg dose (see Table 2). The linear regression analysis gave results that were not statistically significant (P = 0.07; r² = 0.08). However, the mean changes in plasma ANP concentrations from predose values detected 2 h after the administration of the 8 doses tested correlated significantly (r² = 0.57) with the C_max values, according to a linear function.

cGMP in plasma cGMP plasma concentrations in subjects receiving placebo were stable over time, with variation not exceeding ± 20% of predose values in the majority of the cases. Mean ± s.d. baseline values were 3.5 ± 1.4 pmol ml⁻¹ and 3.7 ± 0.9 pmol ml⁻¹ for group I and II, respectively.

In those subjects receiving Z13752A a dose-related rise in plasma cGMP concentrations was found, starting approximately at 1–2 h and reaching a peak 8 h post dosing. This increase was still marked 24h after the 200 (6.2 vs 3.2 pmol ml⁻¹), 400 (5.3 vs 3.7 pmol ml⁻¹), 600 (6.3 vs 3.2 pmol ml⁻¹) and 800 mg doses (6.2 vs 3.7 pmol ml⁻¹) vs placebo. The time-course of the plasma cGMP concentration over the 24h postdosing period is reported in Figure 7.

Figure 7.

Time course of plasma cGMP variation over 24h after single oral administration of placebo (a) ▪ group 1; b) □ group 2) or Z13752A (▴ 10 mg; ▾ 50 mg; • 200 mg; ♦ 600 mg; ▵ 20 mg; ▿ 100 mg; ○ 400 mg; ◊ 800 mg). Values are expressed as percentage changes from predose values. The mean curves for six subjects/panel are shown (eight for placebo).

For AUC_cGMP a statistically significant difference from placebo was found for the 50, 200 400 and 600 doses (see Table 2). A high intersubject variability in this parameter was found for the 100 and 800 mg doses. No significant linear relationship was found between AUC_cGMP and the Z13752A doses.

cGMP in urine In subjects receiving placebo, the mean ± s.d. values of cumulative cGMP urinary excretion in the time interval 0–24h were 586 ± 159 and 778 ± 423 µmol ml⁻¹ for group I and II, respectively. During the same time period, the 50 and 100 mg doses caused a raise in cGMP excretion of approximately 50%. cGMP excretion almost doubled compared with placebo after administration of Z13752A at the doses of 200 (1196 ± 754 µmol ml⁻¹), 400 (1668 ± 1098 µmol ml⁻¹), 600 (1053 ± 258 µmol ml⁻¹) and 800 mg (1272 ± 528 µmol ml⁻¹).

The total cGMP cumulative excretion was significantly different from placebo after 200 mg, 400 mg, and 600 mg doses (Table 2). A regression analysis carried out on the percentage variation vs placebo in the time-interval 0–24h, showed a statistically significant linear correlation (P < 0.01, r² = 0.16).

Urinary volume

During the first 12 h post dosing, urinary excretion was 1.49 and 1.55 l for the placebo group I and group II, respectively. A slight increase in urinary volume, although not statistically significant, was found after Z13752A administration during the same time interval, starting from the 50 mg dose and upwards (data not shown).

Discussion

ACE inhibitors are widely used therapeutic agents, commonly prescribed for the management of hypertension and congestive heart failure. Efficacy is greater in those patients having a high renin activity [16, 17]. NEP inhibitors could be effective in those subjects characterized by a low renin activity, by increasing the vascular, tissue and renal effects of ANP and bradykinin, producing diuresis and natriuresis without kaliuresis. However, their real efficacy is still under evaluation since they seems to have little or no sustained blood pressure lowering activity in hypertensive patients [18–21].

Therefore, dual ACE and NEP inhibitors are promising candidates, potentially more efficacious than the currently available therapies for the treatment of a variety of cardiovascular disorders [22–25]. As an example, they should prevent escape phenomena and the activation of the RAS during long-term therapy with ACE inhibitors.

The results of this study show that single oral doses of Z13752A, as high as 800 mg, are well tolerated in healthy volunteers. The few adverse events reported appeared to be of little clinical relevance, with only six mild-to-moderate adverse events, mainly headache, occurring during the entire course of the study. No relationship of these adverse events with either the dose or the plasma concentrations of Z13752A was seen in either of the groups of subjects treated.

After administration of 200, 400, 600 and 800 mg Z13752A, a slight, not significant reduction in diastolic blood pressure was detected, in both the standing and supine positions. This finding was expected since the present study was conducted in healthy volunteers [26–28]. A slight decrease of the AUC_DBP occurred at the higher doses in both panels of the volunteers. This trend was also noted with the diastolic BP_min values. It is possible that, using a model of mild sodium depletion in these healthy subjects, a greater fall in blood pressure could have been obtained.

The rate (C_max, t_max) and extent (AUC) of absorption of Z13752A appeared to increase linearly with the dose administered. The terminal elimination half-life was dose-independent.

The only exceptions were found after administration of the lower doses of Z13752A. This could be explained by the LOQ of the analytical method employed since the terminal elimination phase of the plasma concentration vs time curve profile could not be defined unambiguously after the 10 and 20 mg doses. Thus the insufficient number of data points prevented an accurate estimation of the terminal elimination rate constant from which the terminal elimination half-life is derived. The mean calculated apparent plasma clearance values (CL/F) ranged from 0.5 to 1.9 l h⁻¹ and were dose-independent in the dose range investigated. These values are considerably lower than both the hepatic blood flow (75 l h⁻¹) and the glomerular filtration rate (8 l h⁻¹), indicating that Z13752A should have a low total clearance in humans.

Detectable plasma concentrations of Z13752A were found in all the treated subjects. Z13752A was well and rapidly absorbed when administered orally as tablet, with peak concentrations reached approximately 2.5 h after administration.

The preliminary nature of this study precluded a formal investigation of the relationship between the pharmacokinetics and the pharmacodynamics and the use of models. However, the data obtained indicate that the maximum pharmacodynamic effect in healthy volunteers was obtained at the highest doses.

The ACE inhibition afforded in humans in the present study confirms the findings of animal experiments. The inhibition appeared to be significant at all the doses tested, was linearly correlated with the dose and was almost complete starting from the 100–200 mg dose. It was of early onset, remained more or less at plateau for 6–12 h (depending on the dose), and then slowly decreased. ACE inhibition was still present 24–48 h after Z13752A administration.

NEP inhibition was supported by an elevation of ANP and cGMP plasma concentrations in almost all the subjects receiving Z13752A. Increase in plasma ANP concentrations started at approximately 1 h, reached a peak after 2–4 h and was still marked 8 h after the 400 and 800 mg doses. A plateau of the effect seemed to be reached at doses ≥ 100–400 mg.

Despite a high level of intersubject variability, a similar increase in plasma cGMP (ANP second messenger) concentrations was also detected, with a peak approximately 8 h after Z13752A administration and being still marked 24h after the higher Z13752A doses. In addition, when the AUC_cGMP was plotted against AUC_ANP, a significant linear relationship (r² = 0.71, P < 0.001) between these two NEP activity markers was found.

The urinary data for cGMP also support these findings. During a 24h period, the 50 and 100 mg doses raised cGMP excretion by approximately 50% of the baseline. cGMP excretion almost doubled compared with placebo after administration of Z13752A at the doses of 200, 400 and 800 mg.

To our knowledge there are very few studies reporting the pharmacodynamics of dual ACE and NEP inhibitors in humans. The majority of these studies have been carried out in healthy subjects.

Rousso et al.[29] reported in healthy volunteers the effects of MDL 100, 240 on the vasopressor response to angiotensin I and angiotensin II, by evaluating the inhibitory properties of the compound towards ACE, but not its NEP inhibition. The same authors in a different study [30] described the effects of the same dual inhibitor administered intravenously on the GFR and renal tubular function, assessing, among other variables, plasma and urinary ANP and cGMP levels. After the administration of 25 mg MDL 100,240, urinary ANP and cGMP rose, whereas no changes in plasma ANP were detected. The effects on plasma cGMP were not reported. ACE and NEP inhibition was also investigated in healthy subjects by Vesterqvist and colleagues [31], after single oral doses of BMS-186716. ACE inhibition was demonstrated, with an accompanying rise of urinary ANP and cGMP. However, they found no increase in the plasma concentration of ANP in the dose range investigated (2.5–500 mg).

Stergiou et al.[32] reported the effects of concomitant administration of candoxatril (a pure NEP inhibitor) and lisinopril (an ACE inhibitor) in hypertensive patients, resulting in a marked reduction of supine and standing blood pressure. Two studies documented the efficacy of the combined ACE-NEP inhibitor sampatrilat in hypertensive patients. In the first one sampatrilat, given orally at doses ranging from 50 to 200 mg, did not affect the blood pressure after the first dose, but significantly lowered this parameter after 10 days of treatment at all the doses tested. ACE activity was inhibited in a dose-related fashion, with an increase in urinary cGMP excretion [33]. In a second study carried out in black hypertensive subjects, this dual inhibitor (50–100 mg daily) produced a more sustained decrease on DBP than that caused by lisinopril treatment (10–20 mg) [34].

The pharmacodynamic data gathered in the present study, although preliminary, provided the proof of concept for the dual inhibition of ACE and NEP in healthy volunteers afforded by Z13752A after oral administration as tablets. ACE inhibition was complete, long lasting and therefore comparable with that afforded by the ACE inhibitors currently used in therapy. Similar assumptions can be made for NEP inhibition. 200–800 mg of Z13752A produced an increase of 50–100% in plasma cGMP levels and of 50–80% in urinary cGMP concentrations. These values are very similar to those obtained in healthy volunteers treated with 200–800 mg of the NEP inhibitor S-thiorphan [35].