Spironolactone and potassium canrenoate in normal man The pharmacological activity of single doses of the two aldosterone antagonists, potassium canrenoate and spironolactone, was examined in two studies in healthy volunteers. Both drugs were active in reversing urinary electrolyte changes induced by fludrocortisone in periods 2 to 16 hr after treatment. Potassium canrenoate was significantly less potent than spironolactone on a weight or molar basis, with best estimates of the relative potency potassium canrenoate: spironolactone of approximately 0.3 : 1. On a weight basis the two drugs yielded plasma levels of the metabolite canrenone which were approximately equivalent. The results indicate that canrenone is not the principal pharmacologically active metabolite of spironolactone. Our study suggests that a major part of the renal anti mineralocorticoid activity of spironolactone may be attributable to minor sulfur-containing metabolites or their precursors having a high renal clearance that affords access to their site of activity via the renal tubular fluid.
Lawrence Ramsay, M.R.C.P.(U.K.), John Shelton, B.Sc., Ian Harrison, B.Sc., Michael Tidd, B.Sc., M.B., B.S., and Michael Asbury, M.R.C.P.(U.K.), High Wycombe, Bucks, England Division of Scientific Affairs, G. D. Searle & Co. Ltd.
Spironolactone (Aldactone) and potassium canrenoate (Soldactone)* are steroid compounds (Fig. I) that have properties consistent with specific competitive antagonism of aldosterone and other mineralocorticoids. 3, 11, 14 Both drugs are used clinically in conditions such as congestive heart failure, hepatic ascites, primary aldosteronism, and essential hypertension. 17 There are certain points of similarity in the pharmacokinetics of these compounds. When spironolactone is administered to man it is rapidly metabolized and the parent drug does not appear in plasma or urine in measurable quantities. The 7 a-acetylthio substituent is Received for publication March 4, 1976. Accepted for publication March 22, 1976. Reprint requests to: Lawrence Ramsay, Department of Medicine, Gardiner Institute, Western Infirmary. Glasgow Gil 6NT, United Kingdom. 'Other names, canrenoate-K, SC-14266.
eliminated from 80% of the administered dose24 to yield canrenone* (Fig. I) as the principal unconjugated metabolite. When potassium canreno ate is given to man the major metabolites in plasma are canrenoate (Fig. 1) and canrenone, with the former predominant. 16 Spironolactone and potassium canrenoate are currently thought to be equipotent as mineralocorticoid antagonists in man. 18 , 23 Sactee, Dagcioglu, and Schroder23 showed that the two drugs gave similar levels of canrenone in plasma when administered to man in equimolar dosage. Since canrenone is active as a mineralocorticoid antagonist in animals,13 they suggested that it was the principal active agent of both drugs and, since plasma levels were similar, that the drugs were equipotent. They have in fact been shown to be equipotent in rats.14 Sactee and 'Other names, aldadiene. SC-9376.
167
168
Ramsay et al.
Clinical Pharmacology and Therapeutics
_,m
,COOK OH
O~cx;r-
POTASSIUM CANRENOATE
n
.0\ I..
r
coo-
0.CJ5D ' o.COXS CANRENONE
OH
CANRENOATE
Fig. 1. Structures of compounds referred to in text. co-workers have pointed out more recently that equipotency of these drugs in man was an assumption,25 and there is unequivocal evidence that canrenone is not the major active metabolite of spironolactone in either rats or dogs. 13 We have recently described methods for accurate quantitative comparison of the pharmacologic activity of single doses of aldosterone antagonists in healthy subjects,19, 20 and in the studies described here have used these methods to re-evaluate spironolactone and potassium canrenoate. The precise objectives were to determine whether the drugs are equipotent in man as related to renal antimineralocorticoid activity, and whether the pharmacologic activity of each drug can be attributed to canrenone. Subjects and methods
Outline. The methods used in the two studies have been described previouslyl9, 20 and are based on the observations of ROSS21 that a single dose of the synthetic mineralocorticoid fludrocortisone administered to healthy subjects in the evening causes reproducible changes in the electrolyte excretion in overnight urine, and that aldosterone antagonists reverse the fludrocortisone-induced urinary electrolyte changes. Activity of aldosterone antagonists is manifest as increased sodium excretion, elevation of the urine Na/K ratio, and, usually, decreased potassium excretion. In the present studies, a further dose of fludrocortisone was given on the
following morning and a second urine collection was obtained, a modification introduced to allow examination of the later phases of activity of the two antagonists. Subjects. Twenty-four healthy male subjects (12 in each study) were admitted to the studies after medical history, physical examination, and laboratory screening. All medication, and specifically aspirin, which may block the renal activity of spironolactone ,26 was prohibited from one week before the study until its completion, and alcohol was not allowed during each phase of the studies. Design of studies. Study 1 was a 3-phase crossover study, balanced for order of medication, involving 12 subjects. The three treatments compared were spironolactone, potassium canrenoate (each at a single dose level), and placebo. Study 2 was a 6-phase crossover study with a balanced design, consisting of two William's squares,2 using 12 subjects. The six treatments tested were spironolactone, at 3 dose levels, and potassium canrenoate at 3 dose levels. This design aimed to yield a 2-drug, 3-dose parallel line bioassay. The phases of each study were separated by intervals of one week. Treatments. Study I. Single oral doses of spironolactone 100 mg (200 ml aqueous suspension) and potassium canrenoate 95 mg (200 ml aqueous solution) were compared to placebo (200 ml water). These doses of the two aldosterone antagonists are equimolar. Study 2. The six oral single-dose treatments were spironolactone 25 mg, 50 mg, and 100 mg (all as 300 ml aqueous suspension) and potassium canrenoate 100 mg, 150 mg, and 200 mg (all as 300 ml aqueous solution). These doses of potassium canrenoate were chosen in light of the results of Study 1, and the dose range was deliberately restricted to improve the chance of obtaining a log dose-response on the steep portion of the curve. 4 All treatments were dispensed open-label. Spironolactone was given as a suspension because of the difficulty of dissolving these amounts of the drug in any suitable inert and nontoxic vehicle. It was expected that the difference in dosage forms would benefit potassium canrenoate slightly as
Volume 20 Number 2
Spironolactone and canrenoate
169
Table I. Mean (SEM) results in 12 subjects (Study l)for urinary electrolyte excretion after administration of placebo, potassium canrenoate 95 mg, and spironolactone 100 mg Placebo
Potassium canrenoate 95 mg
Spironolactone 100 mg
2-10 hr Urine volume (ml) Sodium excretion (mmol) Potassium excretion (mmol) log 10 Na/K
333 10.9 21.2 0.64
(47) (2.3) (2.6) (0.08)
353 16.1 15.9 0.99
(43) (1.9) (1.6) (0.07)*
416 24.4 11.2 1.42
(50) (3.0) *t (2.3) * (0.10)*+
12-16 hr Urine volume (ml) Sodium excretion (mmo1) Potassium excretion (mmol) log 10 Na/K
160 6.3 15.6 0.52
(19) (1.5) (2.5) (0.08)
172 9.6 14.7 0.86
(20) (1.9) (2.4) (0.10)*
221 13.7 16.1 0.97
(30) (1.7) * (2.1) (0.09)*
*p < 0.01 vs placebo. tp < 0.05 vs potassium caorenoate. tp < 0.01 vs potassium canrenoate.
related to rapidity, and perhaps completeness, of absorption. The potassium content of potassium canrenoate (0.25 mmol per 100 mg) was considered too low to seriously bias the results for urinary potassium excretion. Procedure. On each study day the subjects followed normal diet and activities until 7.00 P. M., when fludrocortisone I mg was given orally. A prescribed light meal was taken, and the dose of aldosterone antagonist was given at 9.00 P.M. Water (300 ml) was taken at 11.00 P.M., and all urine passed from then until 7.00 A.M. was collected. At 7.00 A.M. a further dose of fludrocortisone (0.5 mg) was given, followed by a prescribed light breakfast. At 9.00 A.M. venous blood was obtained without stasis or forearm exercise for measurement of plasma potassium (Study 2 only), water (300 ml) was taken, and all urine passed from 9.00 A.M. to 1.00 P.M. was collected. Apart from the meals and water mentioned, the subjects took nothing by mouth. This procedure was repeated at weekly intervals. The drug responses were assessed from electrolyte excretion in the two urine collections, i.e., between 2 to 10 and 12 to 16 hr after treatment with the aldosterone antagonists. Metabolite measurements. In Study 1 venous blood for measurement of plasma canrenone and canrenoate was taken at 2, 4, 12, and 16 hr after treatments, and in Study 2 a single sample for plasma canrenone was obtained at 12 hr. Plasma was stored at -20 0 C
until analyzed. In Study 2 the urinary excretion of canrenone was also measured between 2 to 10 and 12 to 16 hr after treatments. Laboratory procedure. Urinary sodium concentration was measured by atomic absorption spectrophotometry, and plasma and urinary potassium concentration by flame photometry using lithium as the internal standard. Canrenone in plasma and urine was measured by the fluorometric method of Gochman and Gantt1 and plasma canrenoate by the method of Sadee, Dagcioglu, and Riegelman.22 The fluorometric assay in urine after spironolactone is nonspecific, measuring several metabolites,24 an aspect that will be discussed later. To ensure that fludrocortisone did not interfere with the ftuorometric assay, plasma samples after placebo treatment in Study 1 (i.e., fludrocortisone alone) were assayed for canrenone. The levels measured. (mean 0.3 ,ug/dl, range 0 to 0.7 ,ug/dl, n = 8) were similar to blank values in plasma from healthy subjects. Statistical evaluation. In each study the analysis of variance was used to compare direct treatment effects, having first isolated variance related to individual subjects and to the phase of study. In the first study the results of the three treatments were compared and where they differed significantly the Newman-Keuls procedure was used to determine which pairs of treatments differed. In the second study the variance related to treatments was partitioned to test the assumptions required for valid estimates
170
Ramsay et al.
Clinical Pharmacology and Therapeutics
Table II. Mean (SEM) results in 12 subjects (Study 2) for urinary electrolyte excretion after potassium canrenoate and spironolactone, each at 3 dose levels* Potassium canrenoate 100 mg 2 to 10 hr Urine volume (ml) Sodium excretion (mmol) Potassium excretion (mmol) Log 10 Na/K
I
150 mg
I
200 mg
I P valuet
S66 22.4 23.S 0.94
(63) (2.7) (1.4) (0.06)
46S (SS) 24.0 (1.4) 20.9 (I. I) 1.06 (0.04)
S76 32.7 23.7 1.12
(81) (4.8) (2.S) (O.OS)
NS
Lawrence Ramsay, M.R.C.P.(U.K.), John Shelton, B.Sc., Ian Harrison, B.Sc., Michael Tidd, B.Sc., M.B., B.S., and Michael Asbury, M.R.C.P.(U.K.), High Wycombe, Bucks, England Division of Scientific Affairs, G. D. Searle & Co. Ltd.
Spironolactone (Aldactone) and potassium canrenoate (Soldactone)* are steroid compounds (Fig. I) that have properties consistent with specific competitive antagonism of aldosterone and other mineralocorticoids. 3, 11, 14 Both drugs are used clinically in conditions such as congestive heart failure, hepatic ascites, primary aldosteronism, and essential hypertension. 17 There are certain points of similarity in the pharmacokinetics of these compounds. When spironolactone is administered to man it is rapidly metabolized and the parent drug does not appear in plasma or urine in measurable quantities. The 7 a-acetylthio substituent is Received for publication March 4, 1976. Accepted for publication March 22, 1976. Reprint requests to: Lawrence Ramsay, Department of Medicine, Gardiner Institute, Western Infirmary. Glasgow Gil 6NT, United Kingdom. 'Other names, canrenoate-K, SC-14266.
eliminated from 80% of the administered dose24 to yield canrenone* (Fig. I) as the principal unconjugated metabolite. When potassium canreno ate is given to man the major metabolites in plasma are canrenoate (Fig. 1) and canrenone, with the former predominant. 16 Spironolactone and potassium canrenoate are currently thought to be equipotent as mineralocorticoid antagonists in man. 18 , 23 Sactee, Dagcioglu, and Schroder23 showed that the two drugs gave similar levels of canrenone in plasma when administered to man in equimolar dosage. Since canrenone is active as a mineralocorticoid antagonist in animals,13 they suggested that it was the principal active agent of both drugs and, since plasma levels were similar, that the drugs were equipotent. They have in fact been shown to be equipotent in rats.14 Sactee and 'Other names, aldadiene. SC-9376.
167
168
Ramsay et al.
Clinical Pharmacology and Therapeutics
_,m
,COOK OH
O~cx;r-
POTASSIUM CANRENOATE
n
.0\ I..
r
coo-
0.CJ5D ' o.COXS CANRENONE
OH
CANRENOATE
Fig. 1. Structures of compounds referred to in text. co-workers have pointed out more recently that equipotency of these drugs in man was an assumption,25 and there is unequivocal evidence that canrenone is not the major active metabolite of spironolactone in either rats or dogs. 13 We have recently described methods for accurate quantitative comparison of the pharmacologic activity of single doses of aldosterone antagonists in healthy subjects,19, 20 and in the studies described here have used these methods to re-evaluate spironolactone and potassium canrenoate. The precise objectives were to determine whether the drugs are equipotent in man as related to renal antimineralocorticoid activity, and whether the pharmacologic activity of each drug can be attributed to canrenone. Subjects and methods
Outline. The methods used in the two studies have been described previouslyl9, 20 and are based on the observations of ROSS21 that a single dose of the synthetic mineralocorticoid fludrocortisone administered to healthy subjects in the evening causes reproducible changes in the electrolyte excretion in overnight urine, and that aldosterone antagonists reverse the fludrocortisone-induced urinary electrolyte changes. Activity of aldosterone antagonists is manifest as increased sodium excretion, elevation of the urine Na/K ratio, and, usually, decreased potassium excretion. In the present studies, a further dose of fludrocortisone was given on the
following morning and a second urine collection was obtained, a modification introduced to allow examination of the later phases of activity of the two antagonists. Subjects. Twenty-four healthy male subjects (12 in each study) were admitted to the studies after medical history, physical examination, and laboratory screening. All medication, and specifically aspirin, which may block the renal activity of spironolactone ,26 was prohibited from one week before the study until its completion, and alcohol was not allowed during each phase of the studies. Design of studies. Study 1 was a 3-phase crossover study, balanced for order of medication, involving 12 subjects. The three treatments compared were spironolactone, potassium canrenoate (each at a single dose level), and placebo. Study 2 was a 6-phase crossover study with a balanced design, consisting of two William's squares,2 using 12 subjects. The six treatments tested were spironolactone, at 3 dose levels, and potassium canrenoate at 3 dose levels. This design aimed to yield a 2-drug, 3-dose parallel line bioassay. The phases of each study were separated by intervals of one week. Treatments. Study I. Single oral doses of spironolactone 100 mg (200 ml aqueous suspension) and potassium canrenoate 95 mg (200 ml aqueous solution) were compared to placebo (200 ml water). These doses of the two aldosterone antagonists are equimolar. Study 2. The six oral single-dose treatments were spironolactone 25 mg, 50 mg, and 100 mg (all as 300 ml aqueous suspension) and potassium canrenoate 100 mg, 150 mg, and 200 mg (all as 300 ml aqueous solution). These doses of potassium canrenoate were chosen in light of the results of Study 1, and the dose range was deliberately restricted to improve the chance of obtaining a log dose-response on the steep portion of the curve. 4 All treatments were dispensed open-label. Spironolactone was given as a suspension because of the difficulty of dissolving these amounts of the drug in any suitable inert and nontoxic vehicle. It was expected that the difference in dosage forms would benefit potassium canrenoate slightly as
Volume 20 Number 2
Spironolactone and canrenoate
169
Table I. Mean (SEM) results in 12 subjects (Study l)for urinary electrolyte excretion after administration of placebo, potassium canrenoate 95 mg, and spironolactone 100 mg Placebo
Potassium canrenoate 95 mg
Spironolactone 100 mg
2-10 hr Urine volume (ml) Sodium excretion (mmol) Potassium excretion (mmol) log 10 Na/K
333 10.9 21.2 0.64
(47) (2.3) (2.6) (0.08)
353 16.1 15.9 0.99
(43) (1.9) (1.6) (0.07)*
416 24.4 11.2 1.42
(50) (3.0) *t (2.3) * (0.10)*+
12-16 hr Urine volume (ml) Sodium excretion (mmo1) Potassium excretion (mmol) log 10 Na/K
160 6.3 15.6 0.52
(19) (1.5) (2.5) (0.08)
172 9.6 14.7 0.86
(20) (1.9) (2.4) (0.10)*
221 13.7 16.1 0.97
(30) (1.7) * (2.1) (0.09)*
*p < 0.01 vs placebo. tp < 0.05 vs potassium caorenoate. tp < 0.01 vs potassium canrenoate.
related to rapidity, and perhaps completeness, of absorption. The potassium content of potassium canrenoate (0.25 mmol per 100 mg) was considered too low to seriously bias the results for urinary potassium excretion. Procedure. On each study day the subjects followed normal diet and activities until 7.00 P. M., when fludrocortisone I mg was given orally. A prescribed light meal was taken, and the dose of aldosterone antagonist was given at 9.00 P.M. Water (300 ml) was taken at 11.00 P.M., and all urine passed from then until 7.00 A.M. was collected. At 7.00 A.M. a further dose of fludrocortisone (0.5 mg) was given, followed by a prescribed light breakfast. At 9.00 A.M. venous blood was obtained without stasis or forearm exercise for measurement of plasma potassium (Study 2 only), water (300 ml) was taken, and all urine passed from 9.00 A.M. to 1.00 P.M. was collected. Apart from the meals and water mentioned, the subjects took nothing by mouth. This procedure was repeated at weekly intervals. The drug responses were assessed from electrolyte excretion in the two urine collections, i.e., between 2 to 10 and 12 to 16 hr after treatment with the aldosterone antagonists. Metabolite measurements. In Study 1 venous blood for measurement of plasma canrenone and canrenoate was taken at 2, 4, 12, and 16 hr after treatments, and in Study 2 a single sample for plasma canrenone was obtained at 12 hr. Plasma was stored at -20 0 C
until analyzed. In Study 2 the urinary excretion of canrenone was also measured between 2 to 10 and 12 to 16 hr after treatments. Laboratory procedure. Urinary sodium concentration was measured by atomic absorption spectrophotometry, and plasma and urinary potassium concentration by flame photometry using lithium as the internal standard. Canrenone in plasma and urine was measured by the fluorometric method of Gochman and Gantt1 and plasma canrenoate by the method of Sadee, Dagcioglu, and Riegelman.22 The fluorometric assay in urine after spironolactone is nonspecific, measuring several metabolites,24 an aspect that will be discussed later. To ensure that fludrocortisone did not interfere with the ftuorometric assay, plasma samples after placebo treatment in Study 1 (i.e., fludrocortisone alone) were assayed for canrenone. The levels measured. (mean 0.3 ,ug/dl, range 0 to 0.7 ,ug/dl, n = 8) were similar to blank values in plasma from healthy subjects. Statistical evaluation. In each study the analysis of variance was used to compare direct treatment effects, having first isolated variance related to individual subjects and to the phase of study. In the first study the results of the three treatments were compared and where they differed significantly the Newman-Keuls procedure was used to determine which pairs of treatments differed. In the second study the variance related to treatments was partitioned to test the assumptions required for valid estimates
170
Ramsay et al.
Clinical Pharmacology and Therapeutics
Table II. Mean (SEM) results in 12 subjects (Study 2) for urinary electrolyte excretion after potassium canrenoate and spironolactone, each at 3 dose levels* Potassium canrenoate 100 mg 2 to 10 hr Urine volume (ml) Sodium excretion (mmol) Potassium excretion (mmol) Log 10 Na/K
I
150 mg
I
200 mg
I P valuet
S66 22.4 23.S 0.94
(63) (2.7) (1.4) (0.06)
46S (SS) 24.0 (1.4) 20.9 (I. I) 1.06 (0.04)
S76 32.7 23.7 1.12
(81) (4.8) (2.S) (O.OS)
NS
