Europ. J.clin. Pharmacol. 9, 253-257 (1976) © by Springer-Vexlag 1976

Effect of the Aldosterone Antagonist Canrenone on Plasma Aldosterone Concentration and Plasma Renin Activity, and on the Excretion of Aldosterone and Electrolytes by Man H. C. Erbler, H. Wernze and M. Hilfenhaus I n s t i t u t fHr P h a r m a k o l o g i e der M e d i z i n i s c h e n H o c h s c h u l e H a n n o v e r U n i v e r s i t ~ t s k l i n i k W~rzburg, F e d e r a l R e p u b l i c of G e r m a n y

Received:

December

10,

1974,

accepted:

June

and M e d i z i n i s c h e

4, 1975

summery. C a n r e n o n e was a d m i n i s t e r e d in doses of 2 x 82 mg and 2 x 164 mg per day over a p e r i o d of 10 days to d i a b e t i c p a t i e n t s w i t h o u t c a r d i o v a s c u l a r , liver or kidney involvement. A l d o s t e r o n e e x c r e t i o n and p l a s m a a l d o s t e r o n e i n c r e a s e d only s l i g h t l y d u r i n g both regimes. There was a c l e a r - c u t i n c r e a s e in a l d o s t e r o n e excretion only after d i s c o n t i n u a t i o n of canrenone. E x c r e t i o n of sodium, p o t a s s i u m and fluid was not s i g n i f i c a n t l y c h a n g e d e i t h e r d u r i n g or a f t e r treatment. The lack of e f f e c t of c a n r e n o n e on the k i d n e y was in c o n t r a s t to the s i g n i f i c a n t d e c r e a s e in s e r u m s o d i u m and i n c r e a s e in s e r u m p o t a s s i u m , and the significant, d o s e - d e p e n d e n t rise in p l a s m a r e n i n a c t i v i t y f o l l o w i n g c a n r e n o n e a d m i n i s t r a t i o n . The i n c r e a s e d p l a s m a r e n i n a c t i v i t y p e r s i s t e d for some days after d i s c o n t i n u a t i o n of canrenone. It is s u g g e s t e d that c a n r e n o n e p r i m a r i l y e x e r t e d its effect in the d i s t a l p a r t of the large i n t e s t i n e w h e r e ionic m o v e m e n t s are m o s t a f f e c t e d by a l d o s t e r o n e . The d i s p r o p o r t i o n a t e l y slight i n c r e a s e in p l a s m a a l d o s t e r o n e c o n c e n t r a t i o n and aldos t e r o n e excretion, in spite of the g r e a t l y e l e v a t e d p l a s m a r e n i n a c t i v i t y and serum p o t a s s i u m level, is c o n s i d e r e d to be due to a d i r e c t i n h i b i t o r y e f f e c t of c a n r e n o n e on a l d o s t e r o n e p r o d u c t i o n in the adrenals.

Key words:

Aldosterone,

renin,

aldosterone

S p i r o n o l a c t o n e and c a n r e n o a t e - p o t a s s i u m have been w i d e l y used as a l d o s t e r o n e a n t a g o n i s t s in d i f f e r e n t states of h y p e r a l d o s t e r o n i s m . C a n r e n o n e is cons i d e r e d to be the actual active agent of both a l d o s t e r o n e a n t a g o n i s t s . The oral e f f e c t i v e n e s s of c a n r e n o n e has been d e m o n s t r a t e d in rats, but its m i n e r a l o c o r t i c o i d - a n t a g o n i s m was less p r o n o u n c e d than that of s p i r o n o l a c t o n e or c a n r e n o a t e - p o t a s s i u m (for r e v i e w see Herken, 1969). A d m i n i s t r a t i o n of a single dose of c a n r e n o n e to m a n c a u s e d an i n c r e a s e in s o d i u m and fluid e x c r e t i o n and, in addition, a m a r k e d and s u s t a i n e d s u p p r e s sion of p l a s m a a l d o s t e r o n e c o n c e n t r a tion, and a fall in a l d o s t e r o n e excretion in h y p e r a l d o s t e r o n i s m i n d u c e d by a s a l u r e t i c (Erbler, 1974a).

antagonist,

canrenone,

man.

The p u r p o s e of the p r e s e n t study was to a s c e r t a i n the e f f e c t of c a n r e n o n e on e l e c t r o l y t e and fluid e x c r e t i o n in the urine, and on the a c t i v i t y of the r e n i n a n g i o t e n s i n - a l d o s t e r o n e - s y s t e m in man, d u r i n g and after c a n r e n o n e t r e a t m e n t for a p e r i o d of 10 days.

M A T E R I A L AND M E T H O D S The studies were p e r f o r m e d in h o s p i t a l ized female d i a b e t i c s (aged 47 to 61 years) w h o s e c a r b o h y d r a t e m e t a b o l i s m was well c o n t r o l l e d by a s t a n d a r d diet, i n s u l i n or oral a n t i d i a b e t i c s . On a c l i n i c a l basis and a c c o r d i n g to functional and b i o c h e m i c a l c r i t e r i a none had any c a r d i o v a s c u l a r , liver or k i d n e y damage. The diet c o n s i s t e d of 150 g

254 carbohydrate, 80 g protein and 70 g fat. The sodium content of the diet ranged between 90 and 110 mEq and the potassium content between 50 and 75 mEq. No drugs known to influence electrolyte balance or the endocrine systems studied were given. Diabetic patients were selected for the study since they are familiar with daily collection of urine and the necessity of total food consumption. The investigation was divided into a control period of three days before treatment, a canrenone period of 10 days, and a second control period of 7 days after discontinuation of the therapy. Canrenone was administered in micronized form in capsules in two doses - 2 x 82 mg and 2 x 164 mg per day after meals in the morning and evening. Blood for the determination of plasma aldosterone and renin activity and serum electrolytes was taken between 07:30 and 08:30. The patients were advised not to leave their beds before the blood samples had been taken. Clotting was prevented by the addition of EDTA and the blood was immediately cooled to OOC. The plasma was divided into several portions to prevent untoward effects due to repeated thawing and freezing of the samples and was stored at -2OOC until assayed. 24-h urine samples were collected daily from 07:00 to 07:00. Aliquots were stored at -20oc until the determination of sodium, potassium and aldosterone was carried out. Plasma aldosterone concentration (normal range O.024-O.105 ng/ml) and urinary aldosterone (normal range 1.2 - 9.6 ~g/24 h) were measured by radio-immunoassay. Determination of Plasma Aldosterone

Some 12 hours prior to extraction, 10 pg of 1,2,6,7-3H-aldosterone (specific activity 103 Ci/mmol, Amersham-Buchler GmbH) was mixed thoroughly with the samples for calculation of losses during the work-up procedure. The samples were then stored at 4oc until extracted in separating funnels at 4°C with methylene chloride. The organic phase was filtered off using filters previously cleaned with methanol and moistened with methylene chloride. The methylene chloride extracts were evaporated under nitrogen in a rotary vacuum evaporator, dissolved in acetone and quantitatively applied with modified Desaga autoliners on to DC-aluminium foil silica gel 60 F254 (Merck). The precoated thin-layer plates had previously been run 3 times in a chloroform-ethanol mixture (1:1; v/v) to remove impurities. Chromato-

graphy was carried out in the benzene: acetone: H20 (3:2:0.02 v/v) solvent system which separates aldosterone from cross-reacting corticosteroids, canrenone and polar metabolites of canrenone. After separation, the aldosterone band was located on the thinlayer foil under UV light by means of a standard and was cut out. The aldosterone was extracted from the silica gel layer in a simple piece of apparatus in which the cut-out section of aluminium foil is inserted at an angle and acetone containing I% H20 is dropped on at the upper end of the silica gel layer. Almost complete desorption of aldosterone from the silica gel layer was achieved by this simple and rapid continuous elution method. The solvent was then evaporated and the residue dissolved in borate buffer pH 8.0. One aliquot was used to estimate the recovery and the other for the radio-immunoassay. Between 75 and 85 per cent recovery was achieved by a single extraction of plasma samples with methylene chloride. The radio-immunoassay determination of aldosterone was done according to the main details of the method of Vecsei and Gless (1975). Determination of Urinary Aldosterone

Without prior extraction, 250 ~i of urine were pipetted with 10 pg 1,2,6,73H-aldosterone (specific activity 103 Ci/mmol) into 5 ml of p H I TitrisolR buffer (glycine-HCl buffer, Merck) and left to stand in the dark for 20 hours at 22oc. Extraction and thin-layer chromatographic separation of the samples were carried out by the methods used for the determination of plasma aldosterone. It was not necessary to wash the methylene chloride with NaOH after extraction of the urine. Indeed, it is not to be recommended as aldosterone is unstable in an alkaline milieu, and if it were done, recovery decreased substantially. When very small volumes of urine were analysed the recovery rate was between 60 and 70 per cent. The higher losses compared with those found in the determination of plasma aldosterone can be explained by increased adsorption of aldosterone onto active sites of the silica gel in the thin-layer chromatography. On chromatography of plasma extracts or of larger volumes of urine, they are saturated by substances migrating in front of the aldosterone. The radio-immunoassay part of the method followed the technique of Vecsei and Gless (1975). The results for urinary aldosterone

255

e x c r e t i o n o b t a i n e d by r a d i o - i m m u n o a s s a y w e r e c o n f i r m e d by gas c h r o m a t o g r a p h y . The gas c h r o m a t o g r a p h i c m e t h o d has b e e n p u b l i s h e d in d e t a i l e l s e w h e r e (Erbler, 1974b). P l a s m a r e n i n a c t i v i t y was m e a s u r e d by i n c u b a t i n g p l a s m a at pH 5.9 for 15, 45 and 90 min. The a n g i o t e n s i n I g e n e r ated in 50 pl of the i n c u b a t e was m e a s u r e d by r a d i o - i m m u n o a s s a y u s i n g a c o m m e r c i a l l y a v a i l a b l e kit (Sorin, Isot o p e n d i e n s t West). The r e s u l t s w e r e exp r e s s e d as ng a n g i o t e n s i n I/ml x h. The c o e f f i c i e n t of v a r i a t i o n for low r e n i n levels (4.0 n g / m l x h) b e t w e e n 7 and 11 per cent. The s o d i u m and p o t a s s i u m c o n t e n t of s e r u m and u r i n e w e r e m e a s u r e d by f l a m e photometry. S t a t i s t i c a l a n a l y s i s was d o n e by S t u d e n t ' s t-tes£; all v a l u e s h a v e b e e n r e p o r t e d as the m e a n ± S.E. D r u g s used in the s t u d y were: canrenone (3'-(3-oxo-178-hydroxy-4,6androstadiene-17~-yl) p r o p i o n i c acid - y - l a c t o n e ) , B o e h r i n g e r M a n n h e i m GmbH, in m i c r o n i z e d form and a d m i n i s t e r e d in c a p s u l e s c o n t a i n i n g 82 mg. This dose of c a n r e n o n e is e q u i m o l a r to 100 mg of s p i r o n o l a c t o n e . The c a n r e n o n e was chrom a t o g r a p h i c a l l y pure.

RESULTS

s e r u m s o d i u m and p o t a s s i u m c o n c e n t r a tions r e t u r n e d to n o r m a l w i t h i n a few days. A l d o s t e r o n e e x c r e t i o n (Fig. IC) s h o w e d a t e n d e n c y to i n c r e a s e d u r i n g t r e a t m e n t from the 6th day onwards, but there was no s i g n i f i c a n t d i f f e r e n c e from the c o n t r o l v a l u e s t h r o u g h o u t the e n t i r e p e r i o d of t r e a t m e n t . O n l y a f t e r c a n r e n o n e had b e e n d i s c o n t i n u e d was a c l e a r - c u t and s i g n i f i c a n t i n c r e a s e in a l d o s t e r o n e e x c r e t i o n o b s e r v e d - over 4 days (p

Effect of aldosterone antagonist canrenone on plasma aldosterone concentration and plasma renin activity, and on the excretion of aldosterone and electrolytes by man.

Europ. J.clin. Pharmacol. 9, 253-257 (1976) © by Springer-Vexlag 1976 Effect of the Aldosterone Antagonist Canrenone on Plasma Aldosterone Concentrat...
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