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Abstract
Renin is the main determinant of angiotensin (Ang) II levels. It, therefore, always appeared desirable to reduce Ang II levels by direct inhibition of renin. So far, specific renin inhibitors lacked potency and/or oral availability. We tested the new orally active nonpeptidic renin inhibitor SPP100 (Aliskiren, an octanamide with a 50% inhibitory concentration [IC50] in the low nanomolar range) in 18 healthy volunteers on a constant 100 mmol/d sodium diet using a double-blind, 3-way crossover protocol. In 3 periods of 8 days, separated by wash-outs of 6 days, each volunteer received 2 dosage levels of Aliskiren (low before high; 40 and 80 or 160 and 640 mg/d) and randomized placebo or 20 mg enalapril. Aliskiren was well tolerated. Not surprisingly, blood pressure and heart rate remained unchanged in these normotensive subjects. There was a dose-dependent decrease in plasma renin activity, Ang I, and Ang II following single doses of Aliskiren starting with 40 mg. Inhibition was still marked and significant after repeated dosing with maximal decreases in Ang II levels by 89% and 75% on Days 1 and 8, respectively, when the highest dose of Aliskiren was compared with placebo. At the same time, mean plasma active renin was increased 16- and 34-fold at the highest dose of Aliskiren. Plasma drug levels of Aliskiren were dose-dependent with maximal concentrations reached between 3 to 6 hours after administration; steady state was reached between 5 and 8 days after multiple dosing. Less than 1% of dose was excreted in the urine. Plasma and urinary aldosterone levels were decreased after doses of Aliskiren ≥80 mg and after enalapril. Aliskiren at 160 and 640 mg enhanced natriuresis on Day 1 by +45% and +62%, respectively, compared with placebo (100%, ie, 87±11 mmol/24h) and enalapril (+54%); kaliuresis remained unchanged. In conclusion, the renin inhibitor Aliskiren dose-dependently decreases Ang II levels in humans following oral administration. The effect is long-lasting and, at a dose of 160 mg, is equivalent to that of 20 mg enalapril. Aliskiren has the potential to become the first orally active renin inhibitor that provides a true alternative to ACE-inhibitors and Ang II receptor antagonists in therapy for hypertension and other cardiovascular and renal diseases.
- renin
- angiotensin I
- angiotensin-converting enzyme
- aldosterone
- blood pressure
- hypertension, essential
- hypertension, renovascular
- heart failure
Over the last 3 decades, inhibition of the renin-angiotensin system was successfully used in the treatment of hypertension and heart failure.1–4⇓⇓⇓ Reduced activation of the Ang II receptor appears to be a key event to counteract increased blood pressure and sympathetic tone as well as harmful cardiovascular hypertrophy and renal lesions. Pharmacological blockade of the Ang II receptors of the AT1-subtype so far turns out to be clinically equally effective to the less specific ACE-inhibition,5 but the generation of Ang II remains unopposed during AT1-blockade and leaves the potential for stimulation of other Ang II receptor subtypes.6 Specific inhibitors of renin reduce Ang II generation,7 but unlike ACE-inhibitors they do not cumulate other peptides like substance P or bradykinin and consequently untoward drug effects like cough and angioedema are not to be expected. Several specific renin inhibitors were synthesized as of the 1970s, but low efficacy or the lack of oral availability or high cost of synthesis always prevented renin inhibitors from becoming successful drugs.8–22⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓ The present paper introduces the new orally active nonpeptidic renin inhibitor Aliskiren. The aim of the study was to investigate in healthy volunteers the effect of repeated ingestion of four oral doses of Aliskiren on the renin-angiotensin-aldosterone system and on blood pressure, heart rate, and plasma and urinary drug levels and in comparison to the ACE-inhibitor enalapril.
Subjects and Methods
Two groups of 9 healthy male volunteers aged 20 to 34 years participated in the study. All of the subjects were fully informed of the goals and potential risks of the investigation and gave written informed consent. The institutional ethical committee approved the protocol.
From 5 days before to the study and throughout the 6-week study period, the volunteers maintained a 100 mmol/d sodium diet. Compliance was verified by measurements of 24-hour urinary sodium and creatinine excretion. Following a randomized, parallel-group, double-blind 3-way crossover protocol, each volunteer participated in 3 periods of 8 days, separated by wash-outs of 6 days. Each volunteer received either placebo or 20 mg enalapril and 2 oral dosage levels of Aliskiren (SPP100) once a day (40 and 80 mg, or 160 and 640 mg). Aliskiren or placebo (powder in bottles) were dissolved in 200 mL of water containing Bitrex to mask the taste. Enalapril tablets were ingested with 200 mL water.
On the day of the study, the subjects came to the hospital ward at 7:00 am after an overnight fast and were placed in a supine position. An indwelling cannula (Venflon, Viggo AG) was inserted into a forearm vein for blood sampling. The volunteers remained in the supine position for at least 60 minutes before blood sampling. The ECG was monitored with an HP Model 7380 (Hewlett-Packard Co) at 0, 4, and 24 hours after dosing on Days 1 and 8. Upright and supine blood pressure and heart rate were determined sphygmomanometrically by an automatic blood pressure measuring device (Dinamap SXP, Critikon) before every drug intake and in the supine position at subsequent blood sampling times. On Day 1, blood samples were obtained at times 0, 0.5, 1, 3, 6, 10, and 24 hours after dosing for the measurement of plasma renin activity (PRA),23 active renin (immunoreactive renin),24 Ang I,25 Ang II,26 aldosterone,27 and drug levels using antiserum and radiolabel of a previously published assay for Aliskiren.28On Day 8, additional drug levels were obtained at 32, 48, and 96 hours. Trough blood samples were taken before dosing on Days 3 and 5. Twenty-four–hour urine samples were collected immediately before and after dosing for the measurement of drug and aldosterone excretion.29 The volunteers received meals at 7 and 11 hours after dosing on collection days. They then went home and returned, having fasted, to the clinical study facility for the 24-hour measurements and for the subsequent drug intake. All measurements after the first dosing (Day 1) were repeated after the last dosing (Day 8).
Aliskiren (SPP100, Speedel Pharma) is a potent (IC50 0.6 nmol/L), low-molecular-weight (MW 610, free base MW 552), nonpeptidic renin inhibitor that is readily soluble in water and consists of 2(S),4(S),5(S),7(S)-N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy-)phenyl]-octanamid hemifumarate salt (Figure 1). The free base is a third generation competitive transition state analog and specific inhibitor of human renin.
Figure 1. Structural formula of the renin inhibitor SPP100 (Aliskiren) or 2(S),4(S),5(S),7(S)-N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy-)phenyl]-octanamid hemifumarate.
Determinations of the Aliskiren in plasma and urine were performed by a direct liquid phase radioimmunoassay using antiserum and radiolabel of a previously published solid phase assay.28 Twenty-five microliters of unknown plasma diluted 1:10 or 25 μL of unknown urine diluted 1:100 in PBS (0.1 N, pH 7.4), 25 μL PBS to compensate for the volume of the standard samples, and 75 μL [125]-iodinated Aliskiren and 100 μL specific rabbit antiserum against Aliskiren (diluted 1:500 000 in PBS) were incubated for 24 hours at 4°C. For standard slopes, increasing amounts of Aliskiren (0.38 to 192 pg) were dissolved in 25 μL PBS, and 25 μL diluted pool plasma/urine without Aliskiren was added instead of the unknown plasma/urine. Antibody-bound and free Aliskiren were separated by the dextran-coated charcoal method.30 The detection limit was 0.15 ng/mL in plasma and 1.5 ng/mL in urine. Recoveries of various amounts of Aliskiren added to plasma and urine samples ranged from 94% to 105%. Intra- and interassay coefficients of variation were consistently below 15%.
Results are presented as mean±SEM. Significance of differences between means of plasma hormones was evaluated by two-way analysis of variance for comparison of the effect of placebo and that of the different doses of the drugs. When overall statistical significance was achieved (P<0.05) a posteriori testing was used according to Bonferroni.31 The parametric testing was done after logarithmic transformation of the data to adapt for skewed data. Changes in urinary aldosterone and electrolyte excretion were tested for significance between pretreatment (Day −1) and acute (Day 1) and sustained (Day 8) drug effects by one-way analysis of variance followed by Bonferroni’s multiple comparison test.
Results
The renin inhibitor Aliskiren (SPP100) was generally well tolerated. Adverse events were similar in all treatment groups, including placebo and enalapril. Orthostatic hypotension and incidents of dizziness were reported on the days of blood sampling on rising, after volunteers had been lying down for the first 6 hours of the blood collection periods. There was a tendency to a higher incidence of dizziness with the 640 mg Aliskiren dose. No ECG abnormalities were observed throughout the study. Trough serum potassium was unchanged. Blood pressure and heart rate remained unchanged in all subjects.
The time courses of plasma renin activity, Ang I, Ang II, and active renin are depicted in Figures 2A to 2D. Table 1⇓ presents means±SEM values for plasma hormones including aldosterone and drug levels together with statistical significance levels of differences between drug and placebo effects.
Figure 2. Time profiles of mean plasma renin activity (A), angiotensin I (B), angiotensin II (C), and active renin (D) in 9 healthy men after acute (Day 1) and sustained (Day 8) inhibition of the renin-angiotensin system by the renin inhibitor SPP100 (Aliskiren) and by enalapril. B, The ordinate is interrupted and extended by a logarithmic scale to enable plotting of the very high angiotensin I concentrations.
Plasma Renin Activity, Angiotensin II, Angiotensin I, Active Renin, Aldosterone, and Drug Levels After Oral Administration of SPP100 (Aliskiren)
Continued
Aliskiren significantly inhibited the renin-angiotensin system in a dose-dependent manner. On Day 1, Ang II was maximally reduced within one hour of Aliskiren administration, whereas maximal reduction was reached with enalapril only after 6 hours. The diurnal rhythm of Ang II plasma levels is shown on the placebo curve. Ang II levels were significantly decreased up to 6 hours by Aliskiren at all doses except the 40-mg dose on Day 1. Similar observations were made on Day 8: there was an inhibition of Ang II synthesis for up to 6 hours. The percent inhibition versus baseline of Ang II by Aliskiren at doses of 160 mg (56%) or 640 mg (76%) were similar to or greater than inhibition by enalapril 20 mg (57%).
PRA and Ang I inhibition paralleled the changes in Ang II following Aliskiren. PRA and Ang I were increased by enalapril >15-fold on Day 1 and even further on Day 8 (Figures 2A and 2B).
Aliskiren increased active renin in a dose-dependent manner up to 20-fold higher than placebo following administration of a 640-mg dose. Enalapril increased active renin approximately 10-fold compared with placebo; the values were similar to those obtained with 160 mg of Aliskiren. Active renin was further increased on Day 8 in all dose groups, including enalapril.
Plasma aldosterone was decreased 3 hours postdrug on Day 1 with 80, 160, and 640 mg Aliskiren and with enalapril, and this effect lasted up to 6 hours with enalapril and up to 24 hours following 640 mg Aliskiren. The lower doses of Aliskiren and enalapril did not significantly decrease plasma aldosterone at 24 hours (Table 1⇑). The 40-mg dose of Aliskiren had no effect on plasma aldosterone. On Day 8, plasma aldosterone was no longer significantly reduced (Table 1⇑). Mean urinary aldosterone excretion was decreased up to 56% with Aliskiren, particularly on Day 1, and remained 31% inhibited on Day 8 (Figure 3 and Table 2). The 40 mg Aliskiren reduced aldosterone excretion only after repeated dosing, but all the higher doses of the renin inhibitor decreased aldosterone excretion already on Day 1, and this effect was still apparent but sometimes attenuated (80 mg, 640 mg) on Day 8. Comparable decreases in mean aldosterone excretion were observed with enalapril although they did not reach significance (Table 2).
Figure 3. Urinary aldosterone excretion in 9 healthy men after acute (Day 1) and sustained (Day 8) inhibition of the renin-angiotensin-aldosterone system by the renin inhibitor SPP100 (Aliskiren) and by enalapril.
Urinary Excretion of Aldosterone, Sodium, and Potassium After Oral Administration of SPP100 (Aliskiren)
Plasma concentrations of Aliskiren were measured in all subjects at all doses of Aliskiren (Figure 4A). There was a dose-dependent increase in drug levels, with maximal concentrations reached 3 to 6 hours after administration of 80 to 640 mg of Aliskiren. Plasma levels of 0.26±0.05 ng/mL were detected after 24 hours at the lowest dose (40 mg). Steady state was reached between 5 and 8 days after multiple dosing. Mostly, less than 1% of dose was excreted in the urine in a dose-dependent manner (Figure 4B).
Figure 4. Time profile of plasma concentrations (A) of the renin inhibitor SPP100 and urinary excretion (B) of SPP100 (Aliskiren) in 9 healthy men after acute (Day 1) and sustained (Day 8) inhibition of the renin-angiotensin-aldosterone system by the renin inhibitor SPP100 (Aliskiren) and by enalapril.
Urinary sodium and potassium excretion, as well as urinary aldosterone and Aliskiren excretion, are summarized in Table 2. On Day 1, a significant natriuretic effect was observed after administration of 640 mg Aliskiren (+91%) and after 20 mg enalapril (+54%). A trend to natriuresis was already present with Aliskiren at smaller doses, but not with placebo (Table 2). On Day 8, this enhancement of natriuresis had disappeared, and the volunteers excreted sodium according to the dietary intake. Potassium excretion remained unchanged throughout the study. Urinary excretion of Aliskiren was 0.1 to 1.1% of the administered dose at steady state. Traces of Aliskiren were found in the urine of several volunteers on enalapril or placebo who had received Aliskiren in a previous session. This indicates that the wash-out period of 6 days may not have been adequate for Aliskiren’s half-life of 24 hours, particularly following the higher doses.
Discussion
The new specific renin inhibitor Aliskiren (SPP100), unlike any of the previous renin inhibitors described over the last 3 decades, induces in human subjects, after oral administration, a significant and sustained decrease in plasma renin activity, Ang I and Ang II plasma levels. The concentration of active renin in plasma clearly increased dose-dependently in response to the fall in Ang II levels and, hence, as a consequence of the reduced feed-back restriction of renal renin secretion. The decreases in plasma and urinary aldosterone levels, and an increased natriuresis, further document the efficient blockade of the renin-angiotensin-aldosterone system.
These favorable properties of Aliskiren may be due to several causes. First, the high affinity of the compound to human renin (IC50 in the low nanomolar range, data not shown), which is the rate-limiting enzyme in the production of Ang II in man, places Aliskiren among the most potent of such anti-Ang II molecules. Second, Aliskiren is the first specific renin inhibitor to be shown to elicit a marked and sustained blockade of the renin-angiotensin system in man after oral administration. Third, Aliskiren has a plasma half-life of approximately 24 hours, which appears to be ideal for once-a-day dosing. Using Michaelis-Menten kinetics, we have previously calculated for the renin inhibitor Remikiren that 0.05 to 0.5 μmol/L plasma drug concentrations would be needed for significant Ang II suppression, and we doubted that Remikiren could become a useful cardiovascular drug.21 The same calculations allow a more optimistic prognosis for Aliskiren. Given the high affinity of Aliskiren for renin, the improved bioavailability, and early results in patients,32 one might indeed predict effective antihypertensive properties of this new renin inhibitor.
The success of ACE-inhibitors and Ang II receptor antagonists in the treatment of hypertension and heart failure is mainly attributed to an anti-Ang II effect of these drugs.2–5⇓⇓⇓ Renin inhibitors should provide similar therapeutic effects, but, unlike ACE-inhibitors, side-effects like cough or angioedemas33 should be unlikely. In addition, unlike Ang II receptor antagonists, renin inhibitors should chronically decrease, rather than increase, circulating Ang II levels. Therefore, the overstimulation of unblocked Ang II receptors of any subtype should become irrelevant. Thus, whereas Ang II receptor blockers increase circulating Ang I and II and, hence, concentrations of the prothrombotic plasminogen activator inhibitor 1 (PAI-1), the renin inhibitors can be expected to decrease PAI-1, because they decrease Ang levels like ACE-inhibitors.34,35⇓
Presently, many authors advocate the coadministration of small doses of ACE-inhibitors and angiotensin II receptor antagonists to achieve greater blockade of the renin-angiotensin system. This has the obvious disadvantage that it introduces the side effects inherent to the ACE-inhibitors used in this therapy. In the future, the combination of a renin inhibitor with an angiotensin II receptor antagonist—if such a combination is at all needed—could be much more appropriate because this combination can be expected to be devoid of any major side effects.
The present study showed significant renin blocking effects of even the lowest dose of SPP100 (40 mg/d). During placebo treatment, a clear circadian activity pattern of renin, Ang I, and Ang II, as well as aldosterone, could be observed with lower activity in the evening (10 hours post-drug). The first sign of renin inhibition appears to be a flattening of the meridian rise in plasma renin activity and Ang I and Ang II concentrations. With higher doses of the renin inhibitor, the blockade became stronger and lasted longer. The comparison to a daily dose of 20 mg enalapril indicated nearly equipotent reduction at doses of ≥160 mg of Aliskiren in Ang II plasma levels, in plasma and urinary aldosterone and in the reactive rise in plasma active renin levels. Interestingly, the decrease in urinary aldosterone excretion was significant with all doses of Aliskiren. The long half-life of the renin inhibitor may contribute to reduced aldosterone excretion. Plasma potassium was monitored at the end of each session of hormone profiling and was found unchanged. The effect of the enalapril may be slightly overestimated on Day 1 because of the possible carryover of Aliskiren from previous sessions. However, this carryover should not have affected the actions of enalapril on Day 8 of dosing, although the variation of results increased because of the findings in two subjects.
Blood pressure and heart rate were not significantly changed, either by Aliskiren or by enalapril. This is not surprising in healthy men on a constant 100 mmol/d sodium diet and in keeping with earlier observations. The urinary sodium excretion during placebo and before drug treatment (Day −1) confirmed a rather good adherence of the volunteers to the diet. A small natriuretic effect of Aliskiren and of enalapril could be demonstrated together with a slight decrease in aldosterone concentrations on the first day of inhibition of the renin-angiotensin-aldosterone system. These effects were no longer significant with sustained drug administration (Day 8). Aliskiren was generally well tolerated. Episodes of orthostatic hypotension or dizziness were comparable in all treatment groups, including the enalapril group.
In conclusion, the new specific renin inhibitor Aliskiren (SPP100) dose-dependently decreases Ang II concentrations in human subjects after oral administration. The effect is long-lasting and, at ≥160 mg doses, equivalent to that of 20 mg enalapril. SPP100 has the potential to become the first renin inhibitor that provides a true alternative to ACE-inhibitors and Ang II receptor antagonists in therapy for hypertension and other cardiovascular and renal diseases.
Acknowledgments
The authors thank Catherine Amstutz, Françoise Nicoud Favre, Sylvie Novelli, and Yolande Parisod for technical assistance and Monique Salvi for clinical and administrative assistance.
Footnotes
-
These data were partially presented at the 54th Annual Fall Conference of the Council for High Blood Pressure Research in Washington, DC, October 24–27, 2000.
- Received October 5, 2001.
- Revision received October 18, 2001.
- Accepted November 15, 2001.
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- Angiotensin II Suppression in Humans by the Orally Active Renin Inhibitor Aliskiren (SPP100)
