Relative potency of prorenoate potassium and spironolactone in

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1 Br. J. clin. Pharmac. (1984), 18, Relative potency of prorenoate potassium and spironolactone in attenuating diuretic induced hypokalaemia G. T. McINNES*, I. R. HARRISON, J. R. SHELTON, R. M. PERKINS & JOAN M. CLARKE European Clinical Research, G. D. Searle & Co Ltd, High Wycombe, Bucks HP12 4HL 1 The plasma potassium responses to the aldosterone antagonists prorenoate K (10 mg/day and 40 mg/day) and spironolactone (25 mg/day and 100 mg/day) were compared following treatment for 11 days in combination with the diuretic metolazone (2.5 mg/day) in a double-blind crossover study in twelve healthy men. 2 The best estimate of the potency of prorenoate K relative to spironolactone in attenuating metolazone induced hypokalaemia was 5.6 with 95% confidence limits The method employed allowed a statistically valid quantitative comparison of the potassium sparing properties of the mineralocorticoid antagonists after repeated doses and may be useful in the preclinical evaluation of these drugs. Keywords aldosterone antagonists diuretic potassium prorenoate K spironolactone Introduction The major clinical role of the aldosterone antagonist, spironolactone (Figure 1), is attenuation of hypokalaemia induced by diuretics such as the thiazides. Prorenoate potassium (Figure 1) also possesses properties compatible with competitive antagonism of both endogenous and exogenous mineralocorticoids (Hofmann et al., 1975; Levine et al., 1976; Ramsay et al., 1975, 1976). On a weight basis, this drug is more potent than spironolactone in altering urine electrolyte excretion following fludrocortisone challenge in man (Levine et al., 1976; McInnes etal., 1981, 1982c; Ramsay etal., 1975, 1976) but repeated dose comparisons using this method have been unable to provide a satisfactory estimate of its potency relative to spironolactone as a potassium sparing agent (McInnes et al., 1981, 1982c). Depression of plasma potassium concentration is maximal after about 1 week's treatment *Present address: University Department of Medicine, Western Infirmary, Glasgow GIl 6NT 169 0P Prorenoats potassium COOK %> OH SCOCH3 Spironolactone Figure 1 Structures of prorenoate potassium and spironolactone.

2 170 G. T. Mclnnes et al. with diuretics alone or in combination with spironolactone and thereafter there is little recovery despite continued treatment (Leemhuis & Struyvenberg, 1973; McInnes et al., 1982b; Maronde et al., 1969). In normal subjects given spironolactone and metolazone, a diuretic with properties similar to those of a thiazide (Michelis et al., 1970; Pilewski et al., 1971; Smiley et al., 1972; Suki et al., 1972), linear log spironolactone dose-plasma potassium response relationships have been demonstrated during the period of steady state (McInnes et al., 1983). These observations suggest that plasma potassium responses to relatively short periods of treatment in combination with diuretics may provide a convenient and appropriate method for the quantitative evaluation of the potassium sparing properties of competitive mineralocorticoid antagonists in healthy men. The objectives of the present study were to deter-, mine the potency of prorenoate K relative to spironolactone in attenuating metolazone induced hypokalaemia and to evaluate the bioassay employed as a method for the study of aldosterone antagonists. Methods Subjects Twelve males aged 26 to 39 years, considered healthy after full medical history, physical examination and laboratory screening participated in the study. Written consent was obtained after full explanation of the procedure and its risks. The protocol was approved by an independent ethical committee. Design In a double-blind, crossover study, the subjects received each of four treatment regimens of 11 days' duratiop, separated by at least 11 day intervals. Metolazone 2.5 mg was given in combination with prorenoate K and spironolactone, each at two dose levels, the intention being to yield a parallel line bioassay of aldosterone antagonists in the presence of a fixed dose of diuretic. The order of medication was according to a balanced treatment schedule and the subjects were allocated randomly to the treatment sequences. Plasma potassium was determined immediately before and 24 h after each treatment period. Baseline plasma concentrations of prorenone and canrenone, quantitatively the major active metabolites of prorenoate K and spironolactone respectively Karim et al., 1976; McInnes et al., 1982c) were estimated prior to therapy. Drugs Metolazone 2.5 mg/day was administered as 2.5 mg tablets (Zaroxolyn, Pennwalt). Prorenoate K 10 mg/day and 40 mg/day was dispensed as 10 mg tablets and spironolactone 25 mg/day and 100 mg/day as 25 mg tablets with matching placebo prorenoate K and placebo spironolactone tablets as necessary. Procedure On days 1 to 11 of each treatment period, medications were taken once daily at h with 300 ml water. Venous blood was sampled on day 1 at h immediately prior to therapy for determination of plasma potassium concentration, and plasma concentrations of prorenone and canrenone. A further venous blood sample was taken at h on day 12, 24 h after the last dose in each treatment period, for estimation of plasma potassium concentration. Blood was taken with the subjects seated and relaxed. Minimal venous stasis was employed and forearm exercise avoided. Plasma was separated within 1 h and stored at -20 C until assay. Laboratory screening including urinalysis, haematological analysis, plasma electrolytes, urea and liver function tests was carried out 2 weeks prior to the study, immediately before and after each treatment period, and 1 week after the completion of the study. The subjects were ambulant but avoided unusually violent exercise for 24 h before each blood sample. Other medications were forbidden and alcohol was restricted on the day prior to blood sampling. Laboratory Plasma potassium concentration was assayed by flame photometry using autoanalyser methods, plasma canrenone concentration by a fluorimetric method (Gochman & Gantt, 1962) and plasma prorenone concentration by radioimmunoassay (McInnes et al., 1982c). Samples were analysed in batches each corresponding to one treatment period. Statistical analysis The mean plasma potassium concentrations before and following the four treatment regimens were compared by the analysis of variance appropriate to the Latin square design, allowing for subject and phase effects. To validate the relative potency estimates, the between treat-

3 Aldosterone antagonists and plasma potassium ment variation was subdivided to test the statistical significance of the slope of the doseresponse relationship for each aldosterone antagonist, the slope of the average doseresponse and the deviation from parallelism of the dose response trend for each drug. The C conditions for validity (statistically significant,.o zx P<0.05, dose response trend for each compound or for the average trend, and non-significant.4 deviation from parallelism) were satisfied for the 2 < post-treatment results allowing a relative potency estimation with 95% confidence limits One subject failed to complete the final 0 treatment phase while taking metolazone 2.5 mg plus prorenoate K 10 mg/day. The missing post- o treatment value was estimated separately by ez- Yates' method (minimising the residual sum of X squares and reducing the corresponding degrees < E e E of freedom by one) to allow analysis as for a. o 2 complete design. The mean result following 2 O. - prorenoate K 10 mg/day includes the estimated missing value, in the interest of an unbiased treatment comparison. The statistical methods employed are described by Armitage (1973).,u _ 26 Io Results Plasma potassium D o a) 0~~ E 0 c Mean + s.e. mean results for plasma potassium a r C 0 concentration before and after treatment, and the results of the statistical tests are shown in Table 1. After 11 days, there were significant differences between the four treatment regi- 2 mens, but the pre-treatment means did not differ 2s +:+i significantly. Nor were there differences between the phases of the study with regard to mean concentration before treatment (P<0.1). Although the phase means after treatment S2 4 2 varied (P 0.019), = (A ra n there was no trend suggestive of treatment carry-over effects. None of the Ce regimens abolished completely the tendency XCu towards hypokalaemia, the mean post-treatment concentrations of plasma potassium after pro- + renoate K 40 mg and spironolactone 100 mg Ci being 0.28 and 0.44 mmol/l respectively less than the overall pre-treatment mean. 2 *, 4;. S +1 2 E Relative potency prorenoate K: spironolactone +1 E V 2 +1 t+ =~~~~~~~~~~~a I's_ll After trea;ment, prorenoate K exhibited a o marked log dose-plasma potassium response = A relationship while that for spironolactone approached statistical significance. The slope of.. o the average dose-response was highly significant m < Z statistically and there was no contraindication to parallelism between the trends for each drug (Figure 2), allowing a valid estimate of the 171

4 172 G. T. Mclnnes et al. potency of prorenoate K relative to spironolactone (5.6 with 95% confidence limits 2.4 to 35.2). Plasma drug metabolites In all cases plasma prorenone concentrations immediately before treatment were < 1.0 ng/ml, the lower limit of sensitivity of the assay. Pretreatment mean canrenone concentrations were 0.97,ug/100 ml, 0.83,ug/100 ml, 0.83,ug/ml and 0.92 plg/100 ml for treatment periods 1-4 respectively. These results indicate no important pharmacokinetic carryover effects between successive treatments. Side effects and laboratory screens A number of minor adverse effects, notably headaches and muscle cramps, were reported particularly after the high dose of each aldosterone antagonist but there were no obvious differences between prorenoate K and spironolactone in this respect. No clinically significant abnormalities in laboratory screens were observed. Discussion In attenuating the reduction in plasma potassium concentration induced by metolazone, pro renoate K was superior to spironolactone on a weight basis, with a relative potency 5.6 and 95% confidence limits 2.4 to This estimate is consistent with data from steady state comparisons of the drugs in antagonising the potassium response following fludrocortisone challenge (McInnes et al., 1981, 1982c), but, in the absence of a spironolactone dose-urinary potassium response relationship (McInnes et al., 1982a), little confidence could be placed on the findings in these studies. The present study provided a quantitative comparison of the aldosterone antagonists after repeated doses in their usual clinical role as potassium sparing agents in diuretic treated subjects. Although extrapolation of results in healthy men should be cautious, this information was achieved conveniently without the need to involve patients in prolonged investigation. The reproducibility of the spironolactone dose-plasma potassium response relationship with that noted previously (McInnes et al., 1983), and its parallelism with that for prorenoate K support the validity of this method for the assessment of the potassium sparing properties of mineralocorticoid antagonists. Nevertheless the relative potency estimate lacked precision (wide confidence limits). We attempted to reduce or eliminate sampling error by analysing potassium in plasma rather than in serum (Bergstrom, 1973), and by avoiding inconsistency in the collecting and processing of 0 E E E E 3.25 r- 10 <. I Dose (mg) log scale Figure 2 Log dose-plasma potassium response relationships for prorenoate K (0) and spironolactone (0) after repeated doses in combination with metolazone 2.5 mg/day. Mean (vertical bars, s.e. mean) results in 12 subjects after 11 days' continuous treatment. 100

5 samples. The precision of the assay might be improved by replicating samples during the period of steady state to compensate for day to day and diurnal fluctuations, thus reducing intrasubject variability. A sensitive within subject comparison depends on minimising the variance in each individual's response. As only a proportion of subjects exhibit marked hypokalaemia following diuretics (Schwartz & Swartz, 1974), pre-selection of such individuals might result in further reduced intra-subject variability and also steeper dose-response relationships (Ramsay et al., 1980). These subjects may form an appropriate study group, representing those most likely to benefit from potassium sparing therapy. In keeping with our previous findings (McInnes et al., 1982b, 1983), even at high ratios of spironolactone to metolazone by weight Aldosterone antagonists and plasma potassium 173 the attenuation of metolazone induced hypokalaemia was incomplete. Although prorenoate K was more potent than spironolactone, a similar limitation was noted for that drug. The spironolactone dose-response for reducing diuretic induced hypokalemia may extend beyond 100 mg (Ramsay et al., 1980), the maximal dose used in our series of studies of metolazone and spironolactone in combination, but such doses are more likely to be accompanied by patient intolerance. Prorenoate K may offer an advantage in potency as a potassium sparing agent but its acceptability to patients requires substantiation in a clinical setting. We are grateful for the assistance given by Dr E. Celinska, Miss E. F. Allan and Mrs M. Porteous. References Armitage, P. (1973). Statistical methods in medical research. Oxford: Blackwell Scientific Publications. Bergstrom, J. (1973). Plasma and muscle electrolytes in patients receiving diuretics: methodological and clinical considerations. Acta Cardiol. [Suppl 171, 28, Gochman, N. & Gantt, C. L. (1962). A fluorimetric method for the determination of a major spironolactone (Aldactone) metabolite in human plasma. J. Pharmac. exp. Ther., 135, Hofmann, L. M., Chinn, L. J., Pedrera, J. A., Krupnick, M. 1. & Suleymanov, 0. D. (1975). Potassium prorenoate: a new steroidal aldosterone antagonist. J. Pharmac. exp. Ther., 194, Karim, A., Zagarella, J., Hribar, J. & Dooley, M. (1976). Spironolactone. I. Disposition and metabolism. Clin. Pharmac. Ther., 19, Leemhuis, M. P. & Struyvenberg, A. (1973). Significance of hypokalaemia due to diuretics. Neth. J. Med, 16, Levine, D., Ramsay, L., Auty, R., Branch, R. & Tidd, M. (1976). Antagonism of endogenous mineralocorticoids in normal subjects by prorenoate potassium and spironolactone. Eur. J. clin. Pharmac., 9, McInnes, G. T., Clarke, J. M. & Shelton, J. R. (1983). Dose-response relationships for spironolactone in combination with a potassium wasting diuretic. Clin. Pharmac. Ther., 33, Mclnnes, G. T., Perkins, R. M., Shelton, J. R. & Harrison, I. R. (1982a). Dose-response relationships for spironolactone at steady-state. Clin. Pharmac. Ther., 31, Mclnnes, G. T., Shelton, J. R. & Harrison, I. R. (1981). Steady-state relative potency of aldosterone antagonists: spironolactone and prorenoate. Clin. Pharmac. Ther., 29, Mclnnes, G. T., Shelton, J. R. & Harrison, 1. R. (1982b). Time course of urine electrolyte and plasma potassium responses to treatment with metolazone and spironolactone. Br. J. clin. Pharmac., 14, 149P- 150P. Mclnnes, G. T., Shelton, J. R., Harrison, 1. R., Perkins, R. M. & Palmer, R. F. (1982c). Comparison of prorenoate potassium and spironolactone after repeated doses and steady state plasma levels of active metabolites. Br. J. clin. Pharmac., 13, Maronde, R. F.. Milgrom, M. & Dickey, J. M. (1969). Potassium loss with thiazide therapy. Am. Heart./., 78, Michelis, M. F., De Rubertis. F., Beck, N. P., McDonald, R. H. & Davis, B. B. (1970). Standard oral water load to determine the site of action of diuretics in man. With data on metolazone, a new diuretic. Clin. Pharmac. Ther., 11, Pilewski, R. M.. Scheib, E. I., Misage, J. R., Kessler, E., Kritcher, E. & Shapiro, A. P. (1971). Technique of controlled drug assay in hypertension. V. Comparison of hydrochlorothiazide with a new quinethazone diuretic, metolazone. Clin. Pharmac. Ther., 12, Ramsay, L., Harrison, 1., Shelton, J. & Tidd. M. (1975). Relative potency of prorenoate and spironolactone in normal man. Clin. Pharmac. Ther., 18, Ramsay, L. E., Hettiarachchi, J., Fraser, R. & Morton, J. J. (1980). Amiloride, spironolactone and potassium chloride in thiazide treated hypertensive patients. Clin. Pharmac. Ther., 27, Ramsay, L. E., Shelton, J. R. & Tidd, M. J. (1976). The pharmacodynamics of single doses of prorenoate potassium and spironolactone in fludrocortisone treated normal subjects. Br. J. clin. Pharmac., 3,

6 174 G. T. Mclnnes et al. Schwartz. A. B. & Swartz, C. D. (1974). Dosage of potassium chloride elixir to correct thiazide induced hypokalaemia. J. Am. med. Ass., 230, Smiley. J. W.. Onesti. G. & Swartz, C. (1972). The acute effects of metolazone on electrolyte and acid excretion in man. Clin. Pharmac. Ther., 13, Suki. W. N.. Dawoud. F., Eknoyan, G. & Martinez- Maldonado. M. (1972). Effects of metolazone on renal function in normal man. J. Pharmac. exp. 'rher., 180,6-12. (Received January 30, 1984, accepted March 31, 1984)