Mineralocorticoid Receptor Ligands: Biochemical, Pharmacological, and Clinical Aspects W. Sutanto* and E.R. de Kloet Center for Bio-Pharmaceutical Sciences, Sylvius Laboratoria, University of Leiden, P.O. Box 9503, 2300 RA Leiden, The Netherlandst and Rudolf MQgnUS Institute, Medical Faculty, University of Utrecht, 3521 GD Utrecht, The Netherlands Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Mineralocorticoid Receptor Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . A. Definition ... ... . ..... ........_........ ......... ...... .... ........... ... B. MR Ligands: Agonists.. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 1. Endogenous Corticosteroids. . . .................................... 2. Synthetic Compounds.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. MR Ligands: Antagonists.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Spironolactone, Potassium Canrenoate, and Derivatives . . . . . . . . . . . . . . . . . 2. Epoxyspirolactones and Other Spirolactone Derivatives . . . . . . . . . . . . . . . . . . 3. 15,lGMethylene Derivatives of Spirolactones . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Canrenone, Canrenone Derivatives, and Other Spirolactone Metabolites . . 5. 7a-Propyl Spirolactones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. llp,l8-Epoxypregnane Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. MR and MR Ligands in the CNS Distribution and Putative Functions . . . . . . . . . . A. Distribution of MR in the CNS . . . . . . . . . ... .... . . . . . . .. . . .. ...... .. . .. . . .. B. Possible Function(s) of MR and MR Ligands in the CNS.. . .. ... ... . . . .. . . .. 111. Clinical Aspects of Mineralocorticoid Receptor Ligands . . . . . . . . . . . . . .. . . . . . . . . . A. Mineralocorticoid Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mineralocorticoid Antagonists . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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INTRODUCTION Decades have passed since the importance of adrenocorticosteroids in the regulation of bodily functions [includingthe functioning of the central nervous system (CNS)] was first noted. Mineralocorticoidscontrol sodium homeostasis while glucocorticoids, in coordination with the peptide and amine components of the brain-pituitary-adrenocorticalaxis, regulate circadian events, terminate the stress response, and control subsequent adaptive behavior of the organism (for reviews, see Refs. 1 and 2). Only recently, however, have we come to understand the mechanism whereby corticosteroids exert their action@)in the target tissue or cell. In the CNS, corticosteroid action is mediated by two distinct intracellular (i.e., cytoplasmic or cytosolic)receptors, currently referred to as the mineralocorticoid receptor (Type I), which is physicochem*Author to whom correspondence should be addressed. tCorrespondence address. Medicinal Research Reviews, Vol. 11, No. 6, 617-639 (1991) CCC 0198-6325/91/060617-23$04.00 0 1991 John Wiley & Sons, Inc.

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ically similar to the renal mineralocorticoid receptor (MR), and the glucocorticoid receptor (GR), which is identical to the classic liver GR.'jS5 Both receptor types have been cloned, their primary structures resolved, and their genes found expressed in the CNS.&12 The discovery of the MR and GR primary structure and the fact that the chemical structure of these receptors is identical in each corticosteroid target tissue has led to a redefinition of the nomenclature. MR and GR were previously termed Type I and Type 11, re~pectively.~ The Type I receptor has two functional expressions in the brain; for this reason, Type I was further defined as subtypes MR (aldosterone specific) and CR (corticosterone referr ring).^,^^ There has been some controversy and uncertainty as to the precise nomenclature for hippocampal MR, Type I receptor. In a recent review by Canny,I4 it was argued, based on a series of pharmacological and physiological observations, that the hippocampal MR, Type I is in fact a glucocorticoid sensing system, and as such it cannot be referred to as an MR. On the other hand, not only does the hippocampal MR, Type I bind aldosterone with the same range of affinity as corticosterone, it has also been shown that the renal MR and the hippocampal corticosterone-binding species have identical intrinsic steroid-binding specificity since they share the same intrinsic hierarchy of affinity for a range of natural and synthetic steroid^.'^-" More importantly, data from molecular studies on this receptor has shown unequivocally that the nucleotide and deduced amino acid sequence for the rat hippocampal MR display an extensive homology to an MR cDNA isolated from the human kidney, suggesting that they are orthologous genes. Furthermore, Southern Blot analysis suggests that there is only one gene for MR and in vitro expression of the receptor generates a high-affinity corticosterone-binding protein.l1 It is likely that a single gene, or perhaps slightly different ones, encode for an identical MR in the kidney and the Type I in the brain (although certainly, differential gene splicing cannot be excluded). At present, in order to avoid further confusion, we will refer to the Type I and Type I1 receptor systems simply as MR and GR, respectively, with the former showing aldosterone selectivity (kidney, heart, parotid glands) or corticosterone selectivity (hippocampus). It has also been shown recently that certain classes of both the natural and synthetic analogs of corticosteroids (including especially antimineralocorticoids) and of sex steroids may interact with specific binding sites on the membrane-bound y-aminobutyric acid-A (GABAA)receptor complex. Steroid metabolites such as 3a,5a-tetrahydrodeoxycorticosterone(THDOC) and 3ahydroxy-5a-dihydroprogesterone(3a-DHP) lack affinity for the intracellulai MR and GR in the hippocampus but bind with low nh4 affinities to the GABAA Dr. Win Sutanto was born on May 13, 1957, in Bandung, Indonesia and educated in England where he obtained a PhD. degree in physiologyhiochemistry under the supervision of the late Professor Mortyn Jones. Since 1986, he has been a postdoctoral research fellow of Professor E.R. de Kloet at the Rudolf Magnus Institute, University of Utrecht, and, since October 1, 1990, at the Sylvius Laboratoria, Center for BioPharmaceutical Sciences, University of Leiden, The Netherlands. Professor Dr. E. Ronald de Kloet was born on August 19, 1944, in Maarssen, The Netherlands and obtained his PhD. degree in 1972. He has been a senior member of academic staff of the University of Utrecht at the Rudolf Magnus Institute, and is currently the Professor of Medical Pharmacology at the Center for Bio-Pharmaceutical Sciences, University of Leiden, The Netherlands.

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receptor picrotoxin (barbiturate-like)binding domain on an identified subunit of the cloned receptor.'*-z4The result of such an interaction, which has direct effects on the GABA-ergic synaptic transmission, is normally rapid in its onset (secondsto minutes) but of short duration, and is associated with anaesthetic, anxiolytic, and sedative properties of the steroid. Despite this recently acquired wealth of knowledge on the steroid-GABA, interaction, the physicochemical properties of the steroid binding sites (or probably "receptor") on the GABAAreceptor complex and the resultant physiological significance of this type of interaction are presently not clearly (if at all) defined. In contrast, the study of intracellular corticosteroid systems has advanced to a much greater degree. Dexamethasone (DEX)and triamcinolone acetonide (TA) have been known as high-affinity GR binders, and in just under a decade other synthetic corticosteroids that bind specifically with high affinity to GR (RU26988, RU28362, and RU38486), or to MR have made the distinction between the two receptor systems much clearer. Some steroids have been considered as potent mineralocorticoidagonists or antagonists (i.e., antialdosterone) by binding competitively to MR. These steroids include 901fluorocortisol (9a-F) , 19nor-progesterone, and spirolactone steroids such as and spironolactone (Aldactone), ZK91587 (15p,l6f3-rnethylene-mexrenone), RU26752.25-29 It is important to bear in mind that these antimineralocorticoids and mineralocorticoids are not tissue specific. In other words, they bind equally well to both the hippocampal MR and kidney (renal) MR. The renal MR (but not the hippocampal MR) is described as aldosterone-specific receptor, since under normal physiological conditions the naturally and predominantly occurring glucocorticoid (corticosterone in the rat; cortisol in the hamster and the human) binds with high affinity to the hippocampal MR but not to the renal MR. The kidney possesses an enzyme, 11p-hydroxysteroid dehydrogenase (11p-OHSD), which converts corticosterone (or cortisol, but not mineralocorticoids) to its ll-oxometabolite.3~36 These metabolites do not have affinity for the MR, which may therefore explain why the renal MR is selective for aldosterone. Note that 11P-OHSD is also present in the CNS including the h i p p o ~ a m p u s ,although ~ ~ , ~ its mode of action and physiological significance are not yet clearly understood. The purpose of this review is to highlight selected ligands that bind specifically to MR and/or show (anti)mineralocorticoid activity, both in corticosterone-secreting species such as the rat and in cortisol-secreting species such as the hamster, dog, and human. Since MR is present in the CNS (including especially the hippocampus) and the kidney, we will specify from which tissue the findings from the in nitro (and in v i m ) studies presented herein have been obtained. The biochemical and pharmacological properties, together with the physiological and clinical implications that these (anti)mineralocorticoidsmay have, will be desuribed. I. MINERALOCORTICOID RECEPTOR LIGANDS A. Definition

The characteristic responses of steroidal hormones require their binding to specific receptor proteins in target tissues; the pharmacological, or physiological, response(s)thus triggered (or induced) depends very much upon the

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Table I Relative Binding Affinities of Selected Endogenous and Synthetic Corticosteroids for MR and GR Sites in the Hippocampal Cytosol Steroids

MR ICM(nM)

GR ICM(nM)

Corticosterone Cortisol Aldosterone Deoxycorticosterone THDOC Progesterone 19-Nor progesterone 3a-DHP

1.2 2.1 1.5 3.9 460.0 1.0 0.01 >1000.0

5.2 19.7 26.0 14.0 >lOOo.O 29.0 13.7

Dexamethasone RU28362 RU28318 RU26752 ZK91587 Spironolactone K+-Canrenoate 9a-F Epoxyprorenone Epoxyspironolactone Epoxvmexrenone

10.0 >1000.0 800.0 4.0 1.0 4.9 >lOOo.O 2.5 100.0 100.0

6.0 2.1 >lOOo.O 77.5 550.0 88.0 >1000.0 70.0 620.0 23.0 930.0

>lOOo.O

>lOOo.O

interaction of the steroid-receptor complex with the nuclear chromatin. The precise details of such an interaction, and the role played by the steroid in the process, which have been a puzzle for many years, have begun to become unravelled recently with the advent of advanced and much more refined techniques in molecular and cellular endocrinology. By definition, mineralocorticoid receptor (MR)ligands are those compounds which bind specifically to MR. In reality, not all of these compounds are classified as mineralocorticoids or antimineralocorticoids. Furthermore, one particular MR ligand that binds with high affinity to the brain MR may not necessarily have appreciable affinity for the renal MR. Thus, corticosterone in the rat (cortisol in the dog, human and hamster) binds with higher affinity to hippocampal MR than they do to GR.'6,39 In the kidney cytosol in vitro (obtained from adrenalectomized rats), corticosterone binds to MR equally well, as do the mineralocorticoids aldosterone and deoxycorticosterone (see Table I). However, under normal physiological conditions, corticosterone can not compete with aldosterone for MR, due to the preferential binding of corticosterone by plasma corticosterone-binding globulin (CBG, transcortin) as initially t h o ~ g h t , ~or' as more recently proposed, due to the presence of the enzyme llp-OHSD.31,32The latter thesis has been partly substantiated by the fact that the level of circulating transcortin is extremely low in 10-day-old rats and yet, in these animals, the renal MR shows an almost absolute preference to bind aldosterone than cortic~sterone.~~ In addition, transcortin is present in the brain and yet it does not induce aldosterone selectivity in this organ. What makes a ligand such as aldosterone an MR binder? It is beyond doubt that structural details within a steroid molecule will influence receptor affinity,

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activation, translocation, nuclear interaction, and the resultant physiological response. In their elegant study using x-ray crystallography, Duax and coworkers"~~~ have provided information concerning preferred conformations (i.e., three-dimensional shapes), relative stabilities, and substituent influence on the interactive potential of steroid hormones. They established that highaffinity binding to MR appears to be correlated to a complementary fit between amino acid sequences on the receptor (or binding site) and a flat 4en-3-one A ring of the steroid^.^,^^ Note that the measurement of binding affinity of a steroid for its (specific) receptor is not always a good index for the steroids b i ~ a c t i v i t yNevertheless, .~~ data on receptor binding have been a useful tool in the development of new bioactive steroid^.^' In the study on structureactivity relationship on 18 steroidal aldosterone antagonists, the affinity for mineralocorticoid receptors in vitro and the antialdosterone activity in vivo were compared.48This study shows in general that such a comparison is a valuable procedure in the search for new antimineralocorticoid substances.

B. MR Ligands: Agonists 1. Endogenous Corticosteroids

The naturally occurring endogenous MR Iigands are therefore predominantly aldosterone and deoxycorticosterone, although steroids such as progesterone also show a high-affinity binding to MR. Aldosterone (llp,21-dihydroxy-3,20-dioxo-4-pregnen-18-a1) is the most potent regulator of electrolyte excretion and is therefore absolutely essential for life. This steroid, which is isolated, elucidated structurally, and was synthesized in the is believed to exist in at least five isomers. It is also believed that the isomerism of aldosterone may have a direct bearing upon its biological activity, since these isomers will have different degrees of affinity for the various steroid receptors or metabolizing enzymes present in ~ i v oSubstituted .~~ aldosterone and their derivatives have been synthesized since the early 1960s. An example of this is 17a-hydroxy-18,21-anhydroaldosterone.51 An aldosterone dimer, as well as 18,21-anhydroaldosterone,are acid-catalyzed products of aldoster ~ n ewith ,~~ the former being a highly lipophilic form of aldosterone. Another form of substituted aldosterone is 19-noraldosterone, a potent mineralocort i ~ o i dHarnik . ~ ~ and co-workers have also described the preparation of three forms of tetrahydro derivatives of 19-noraldosterone by chemical synthesis and microbial biocon~ersion.~~ Some fundamental questions (that presently remain unanswered) arise from these studies. Do these substituted aldosterone derivatives bind to MR in the CNS and/or kidney? If so, are they of any physiological significance? 19-Noraldosteroneis present in the urine of normal and hypertensive humans, albeit in small amounts.% Its high mineralocorticoid potency was determined in the adrenalectomized rat bioassay, by shortcurrent measurements in the toad bladder,55and by a radioreceptor assay in human mononuclear leukocytes,% Indeed, 19-noraldosterone binds to MR in the CNS and/or kidney. Deoxycorticosterone (deoxycortone, DOC; A4-pregnene-21-01-3,2O-dione) is a naturally occurring mineralocorticoid that binds to both MR in hippocampal cytosol with higher affinity (in the same affinity range as aldosterone) than it does to GR (see Table I and Ref. 21). The steroid also shows a similar

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binding pattern to the renal MR (Schut, Sutanto, and de Kloet, unpublished observations). The naturally occurring metabolite of DOC, namely THDOC, does not show affinity for hippocampal MR (see Table I and Ref. 21). DOC, like aldosterone, is chiefly concerned with the regulation of water and salt metabolism. It is normally used in acetate form (Deoxycortoneacetate, DOCA) or propionic ester. Progesterone and 19-nor progesterone are by definition progestagens. Surprisingly, however, they are potent MR binders in vitro (see Table I and Ref. 57). Moreover, progesterone antagonizes the action of aldosterone and induces sodium excretion both in humans and animals,%whereas 19-nor progesterone behaves like a partial agonist and causes sodium retention, polydipsia, and hypertension in the rat.59 Chemical modifications of 19-nor progesterone (for example, 17a-hydroxy-19-norprogesterone) resuIt in a dramatic decrease in the binding affinity to renal MR in vitro (and the accompanying in vivo effect on fluid and electrolyte homeostasis) and a proportional increase in progestin activity.60One metabolite of progesterone, 3a-hydroxy5a-dihydroprogesterone 3a-DHP, displays no affinity for the hippocampal MR (see Table I and Ref. 21).

2. Synthetic Compounds

The synthetic analog 9a-fluorocortisol(9a-fluorohydrocortisone, 9a-F, Fludrocortisone), a potent mineralocorticoid agonist, is structurally related to aldosterone (with the same 4-en-3-one A-ring). The attachment of a fluorine atom at the 9a position increases the binding affinity of the steroid for the renal MR.15,47,61 In the hippocampal cytosol (Table I), the steroid binds to MR with much higher affinity than to GR. A synthetic analog of DOC has recently been elucidated and described.62 The compound, All-DOC (21-hydroxypregna-4,ll-diene-3,20-dione) binds to the renal MR in vitro with three times greater affinity than DOC, although the in vivo mineralocorticoid activity of the two compounds is the same. Dexamethasone (DEX) is not generally regarded as an MR ligand. In fact, until recently when a more specific GR ligand (e.g./ RU28362) was available, DEX has always been regarded as a classical GR binder. This potent synthetic glucocorticoid analog exerts its action by interacting with GR. However, DEX binds to MR with appreciable affinity.63These findings have recently been substantiated by the evidence from Luttge and co-workers,64who show that in mouse brain cytosol pretreated with dextran-coated charcoal (DCC) combined with 300 mM KC1 (procedures that will interfere greatly with binding to GR but apparently has little effect on MR binding) DEX still binds substantially and to the degree observed for [3H]aldosterone-detectedMR complexes under the same conditions. In a further study,65it is suggested that the high-affinity binding sites for DEX and aldosterone may be equivalent (or at least, overlapping) as evidenced from the fact that these steroids have similar affinity for MR as determined in the Scatchard analyses and in the binding competition studies. Carbenoxolone [a derivative of glycyrrhetinic acid (GA)] is not, strictly

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speaking, a mineralocorticoid receptor ligand. However, carbenoxolone, together with GA and glycyrrhizic acid (GI), have considerable mineralocorticoid activity.& GA and GI are collectively known as glycyrrhizin, the active mineralocorticoid-mimetic constituent of liquorice. These compounds have been actively used in the treatment of peptic ulcers, as anti-inflammatory and antiallergic agent. Some of these effects have been attributed to their mineralocorticoid-like action. However, the precise mechanism of their action is not fully understood. Early findings indicated that they show considerable affinity for the renal cytosolic MR.67In contrast, other laboratories show that they do not have affinity for renal or hippocampal MR in vitro (see Table I and Ref. 68), despite their structural similarities to aldosterone. It is more likely therefore that their mineralocorticoid activity is due to the inhibition of As a consequence, the cortisol-cortisone shutthe activity of 11p-OHSD.31,32*66 tle is impaired resulting in a free access of the glucocorticoid to act on MR and to act as a MR ligand. It has also been reported that GI and GA directly inhibit the Na+/K+-ATPase activities of renal homogenate and basolateral membrane, due to the decrease in the affinity of the enzyme for ATP.69The resulting increases in sodium retention may explain the symptom of pseudoaldosteronism caused by long-term use of glycyrrhizin. Recent evidence indicate that GA is also a potent inhibitor of cytosolic 5Preductase, one of the major hepatic steroid-metabolizing enzymes.70Equally important, GA specifically inhibits microsomal 3P-hydroxysteroid dehydrogenase enzyme activity in a competitive manner, resulting in a buildup of the intermediate, 5a-dihydro-aldosterone. GA thus appears to have a profound effect on hepatic ring A-reduction of aldosterone. The result of the inhibition of 5P-reductase and 3P-hydroxysteroid dehydrogenase is a decreased synthesis of both 3a,5P-tetrahydroaldosterone and 3P,5au-tetrahydroaldosterone and hence, accumulation of aldosterone and 5a-dihydroaldosterone,both potent mineralocorticoids. Thus more than one enzyme may be involved in the mechanism by which GA (or glycyrrhizin in general) causes glucocorticoid such as cortisol or corticosterone to display mineralocorticoidmimetic action.70

C. MR Ligands: Antagonists While it is the A ring (or the moieties contained therein) that actually determines the affinity of a ligand for MR, it is the specificinteractions between the B, C, and D ring of the steroid with the receptor that determine agonist or antagonistic behavior of the steroid subsequent to binding.44,45,71 A substantial number of steroids that are aldosterone antagonists (i.e., antialdosterone) have been developed in the past three decades and used clinically in the treatment of disorders with primary and secondary aldosterone excess or hyperaldosteronism, as described in the Sec. I.C.l. In the following sections, some known antimineralocorticoids and their derivatives are described. Some of these steroids which are MR ligands in vitro show little (if any) antimineralocorticoid activity in vivo (and vice versa). Furthermore, most of the derivatives of known antimineralocorticoids have been designed and synthesized

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for the purpose of further elucidating the steroid-receptor interaction, and thus data on their biological and pharmacological properties are not available. 1. Spironolactone, Potassium Canrenoate, and Derivatives

The most widely used, and certainly the best documented, antialdosterone agents are spirolactones, steroidal drugs that contain at C-17 position a ylactone [e.g., spironolactone (Aldactonem)]or a y-hydroxy acid moiety [e.g., potassium canrenoate (Soldactone@')]. Spironolactone (7a-acetylthi0-3-0~0-17apregn-4-ene-21, 17-carbolactone)was the first competitive aldosterone antagonist used in clinical m e d i ~ i n eand ~ ~ has , ~ ~remained the standard for over 30 years. Spironolactone has been shown to bind with high affinity to both the renal MRz8 and hippocampal MR (see Table I and Refs. 21 and 26). Theoretically then, with a few possible exceptions, aldosterone antagonists will bind to the receptor site normally occupied by aldosterone, but since no translocation is likely to take place, an antagonist-receptor interaction does not produce the effect of the agonist. On the other hand, antagonists may exert their effects by interfering with the synthesis or metabolism of a specific steroid (or any proteins) that are essential to the expression of the physiological response of that particular steroid.& However, since most binding data are obtained from in vitro studies, it is not a dogma that a good binder in vitro should be a reflection of its efficacy in vivo.46Potassium canrenoate is a typical example. Structurally, it is similar to spironolactone; spironolactone has a 7athioacetyl group at the C-7 position while potassium canrenoate has a C-6,7 double bond. Despite the fact that in vitro potassium canrenoate does not have appreciable binding affinity for hippocampal MR (see Table I) or the renal MR (Schut, Sutanto, and de Kloet, unpublished, and Ref. 28), the steroid is an antimineralocorticoid and has been used in the clinics as an antialdost e r ~ n eIn . ~the ~ study of structure-activity relationship of spironolactone derivative~,~~ compounds which do not show any affinity for the renal cytosolic MR (e.g., 17a-hydroxypropylspironolactoneand 17~-hydroxyspironolactone) have an equal or greater relative antialdosterone potency. Spirorenone (A1-6P,7P; 15P,16P-dimethylene spirolactone) and dihydrospirorenone are a relatively new class of potent aldosterone antagonist^^^,^^,^ Their mean relative potency as antimineralocorticoidsis approximately 8.6fold that of spironolactone, which, however, is not reflected by an increase in their receptor binding; the relative binding affinity of spirorenones is only 73% that of spiron~lactone.~~ Note that these steroids show an increase in their relative binding affinity to androgen receptor compared to spironolactone.77 There is therefore a tremendous dissociation between the relative affinity for MR in vitro and the in vivo pharmacological activity (which may require metabolism of the parent drug) for certain groups of antimineralocorticoid compounds.48Prorenone (6P,7P-methylene spironolactone), having a spironolactone structure but with the 7a-thioacetylreplaced by 6P,7P-methyl group, is more potent than spironolactone both in vivo and in vitro.75*78,79 However, potassium prorenoate (6P-7P-methylene-17a-propionate,potassium) is almost without affinity for the renal MR in vitro and yet showing an in vivo activity superior to spironolactone.75~80~8*

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2. Epoxyspirolactonesand Other Spirolactone Derivatives

Three new epoxy-spirolactone derivatives have been synthesized by CibaGeigy Ltd., Switzerland and first described by de Gasparo and co-workers.82 These steroids are the 9a,lla-epoxy-derivatives of known aldosterone antagonists spironolactone, prorenone, and mexrenone (hence epoxyspironolactone, epoxyprorenone, and epoxymexrenone).The attachment of the epoxy group results in a slight decrease in the in vitro binding to renal MR (epoxyspironolactone and epoxyprorenone) compared to their parent compounds, or in a dramatic decrease by 15-20-fold (epoxymexrenone) compared to mexrenone or spironolactone. However, epoxymexrenone displaces 50% of the in vivo binding of [3H]aldosteroneat a lower dose [0.8 mg/kg body weight (bw)] than does spironolactone (1.7 mg/kg bw). All three epoxy derivatives, which do not show high-affinity binding to the rat hippocampal cytosol (Table I), have markedly less (between 10- and 500-fold) affinity for the androgen and progesterone receptors. In vivo, all three epoxy derivatives were equipotent as (or more potent than) spironolactone as an antimineralocorticoid. Further, parallel to the decreased affinity for the androgen and progesterone in vitro, there was a 3-10-fold decrease in the antiandrogenic (in the rat) and progestagenic (in the rabbit) effect, compared to spironolactone. Fluorescent spirolactone derivatives are a new class of spirolactones described recently by Auzou and co-workers.83 However, these compounds were developed as a new approach to study the hormone-receptor interaction. The principle behind this method is the use of fluorescent-labelledderivatives (which are synthesized by attaching a coumarin-type fluorescent label at position C7a), which undergo changes in fluorescence polarization as they interact with the nonfluorescent receptor. When tested for their binding affinity to the renal MR, these steroids, designated RU46742 (17P-hydroxy-3-oxo-7acarbonyl oxyl] propyll-17a-pregn-4[3-[[[(2-oxo-2H-1-benzopyran-3-yl)amino] ene-21,17-carbolactone)and RU46744 (17~-hydroxy-3-oxo-7~~-[3[[[(7-methoxy2-oxo-2H-l-benzopyran-3-yl)amino] carbonyl] oxyl] propyl1-17a-pregn-4-ene21,17-carbolactone),do not show any affinity to MR and little affinity for GR.= These authors concluded that to maintain good affinity for aldosterone receptors (i.e., renal MR and GR), the fluorescent derivativeshave to be attached to positions other than C-3, C-7, and C-17. 3. 15,16-Methylene Derivatives of Spirolactones

The biochemical, pharmacological, and antimineralocorticoid properties of 15p,16P-methylene derivatives (7a-acetylthio-, 7a-methylthio-, 7a-methoxycarbonyl-, and A*) of spirolactoneshave been described elsewhere.25-27*29,84-86 More recently, the properties of 7a-alkoxycarbonyl-l5~,16~-methylene spirolactone have also been described.87From this series of compounds, only those showing potentials as an MR ligand and/or an antimineralocorticoid are described below. ZK91587 (15(3,16p-methylene mexrenone: 7a-methoxycarbonyl-l5~,16Pmethylene-3-oxo-17a-pregn-4-ene-21,17-carbolactone) is the 15f3,lbP-methylene derivative of mexrenone (ZK32055). The steroid was first shown to be a potent antimineralocorticoid that binds with high affinity to the renal MRMjB5 and subsequently was shown to bind with high affinity to the hippocampal

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cytosol in the rat (Table I), but not in the hamster.25,26 In the dog hippocampal cytosol, ZK91587 has twice the binding affinity for MR as that shown by spironolactone but approximately eight-fold less potent as an MR binder than ald~sterone.~~ These observations bring out one fundamental criterion in the selection of a ligand for one particular receptor type; steroids that have been shown to be specific, high-affinity ligand in one species (in this case, spironolactone and ZK91587 in the rat) may not necessarily display the same (or similar) specificity in another species. Results from our laboratory in the in vitro autoradiography study using guinea pig (which secretes cortisol and corticosterone, as the predominantly circulating glucocorticoids) and human brain sections also show that the distribution pattern of [3H]ZK91587binding is similar to that of GR bindingz6In these studies, both the guinea pig and human were adrenally intact. Under these conditions, MR sites are supposed to be almost fully occupied by the endogenous corticosteroids.'3~88 In another series of studies using in vivo autoradiography, adrenalectomized hamsters were injected intravenously (i.v.) with a tracer dose (50 pCi/animal) of [3H]ZK91587.The autoradiogram thus obtained revealed a high-density binding to the hippocampus, in the same pattern of binding to MR following i.v. injection of [3H]cortisol.5It appears therefore that in vivo ZK91587 binds with high affinity to MR in this species. It remains to be seen if the binding of [3H]ZK91587in vivo is due to the steroid itself or its metabolite(s). In the rat brain, the binding of (3H]ZK91587is not confined to just the MR in the hippocampus. Specific binding sites for ZK91587 are distributed throughout the brain (similar to that of [3H]aldosteronebinding to MR) with CA1 and CA3 regions of the hippocampus, some amygdaloid nuclei, and lateral septum containing most of the binding sites.29Furthermore, the binding of [3H]ZK91587in vivo was readily displaced by aldosterone in physiological amounts, in brain regions such as the hippocampus, amygdala, and the preoptic area.29 Mespirenone is an example of a 15@,16P-methylenethioacetyl spirolactone that is structurally similar to spironolactone except for the presence of the 15,16 methylene bridge and the double bond at C-1 position. Mespirenone as well as ZK91587, are of (ZK94697; A1-15~,16~-methylenespironolactone), interest since they have been found to have two (ZK91587) to three times (mespirenone)the antimineralocorticoidpotency (in a diuresis test) compared with s p i r o n ~ l a c t o n eEqually . ~ ~ ~ ~ important, these steroids have less affinity for gestagen and androgen receptors than does spironolactone. The binding profile of mespirenone (and of mexrenone) to the renal (or hippocampal) MR is presently not known. 4. Canrenone, Canrenone Derivatives, and Other Spirolactone Metabolites

It is now recognized that many, if not all, of the effects of spironolactone are mediated by metabolites of the steroid. It has been regarded for a long time that canrenone is the major metabolite of both spironolactone and potassium c a n r e n ~ a t e(for ~ ~review, ,~ see Ref. 74). Since canrenone also shows some antimineralocorticoid activity, the biological effects of the parent compounds were attributed to this metabolite. However, pharmacokinetic studies

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indicate clearly that canrenone can only account for a small part of the antimineralocorticoid activity of its precursor^.^' Since then, however, it has been shown that the principal metabolites are in fact sulphur-containing steroids such as 7a-thio-spironolactone and, to a greater extent, 7a-thiomethyl-spiron~lactone.~ These ~ - ~ ~two sulphur-containing metabolites, but not canrenone, have been shown to have an appreciable affinity for the renal MR.28*95 The conversion of spironolactone to 7a-thio- and 7a-thiomethyl-metabolites have been characterized in the hepatic and renal% as well as adrenocortical and testicular microsomal and cytosolic fractions.%In the adrenal gland and testes, spironolactone inhibits steroidogenesis, due partly to the destruction of cytochrome(s) P-450by the steroid. It is believed that this phenomenon is one of the reasons for the side effects of spironolactone. However, since metabolism of spironolactone is so rapid and very little of the parent compound is detectable in the urine or spironolactone-treatedhumans or animals, it is likely that the sulphur-containing metabolites are the factors responsible for the cytochrome destruction. In the testes, such a destruction has been observed in all species studies,97while adrenal destruction seems to be confined to species that secrete cortisol as the major adrenal glucocorticoid. It is likely that such species differences are due to the differential metabolism of spironolactone.98 In spite of the substantial evidence for the role(s) of these sulphur-containing metabolites (and the fact that they bind to the renal MR with high affinity), is the major meas well as the findings that 7a-thiomethylspironolactone92 tabolite in both the rat and the human, it is interesting to note that when these metabolites were given to healthy subjects, their antimineralocorticoid potency is only 12.0-62.0% of that for spiron~lactone,~~ which lead these authors to conclude that these sulphur-containing intermediate metabolites are unlikely to contribute to the renal antimineralocorticoid activity of spironolactone. It has been reported that other metabolites of spironolactone, apart from the ones mentioned above, are also important in mediating the biological effects of the parent drug.* Three biologically active dihydroxylated canrenone derivatives have been reported to exist naturally in urine and plasma of man and animal.loo Recently, these isomers (6P,7a-dihydroxy-6,7-dihydrocanrenone, 6p,7p- and 6a,7a-dihydroxy-6,7-dihydroca~enone, together with 6a,7a-epoxycanrenone) have been synthesized. Their structures were elucidated and confirmed by proton nuclear magnetic resonance spectra (NMR).'O' Their binding profiles for the corticosteroid receptors, however, have not yet been studied. 5 . 7a-Propyl Spirolactones Steroids belonging to this series are structurally similar to spironolactone or potassium canrenoate except for the substitution of the thioacetyl by a propyl moiety at position C-7a. RU26752 (7a-propylspirolactone: [3-(3-oxo7a-n-propyl-l7~-hydroxy-4-androsten-17a-yl)propionic acid y-lactone) and RU28318 (potassium salt of 7a-propyl-spirolactone)are two examples of 7apropyl spirolactone.

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RU26752 (soluble in 9:l benzene ethanol but not in 100%ethanol) was found to bind with high affinity to the renal MR (Refs. 85, 102, 103, and Schut, Sutanto, and de Kloet, unpublished observation). In the brain, RU26752 also binds with high affinity to MR. Thus in the rat hippocampal cytosol, RU26752 binds with 1.5-2.5 times the potency of aldosterone (see Table I and Refs. 25, 26, 104), or approximately five times the affinity shown by spironolactone.26 RU26752 was first described as an antimineralocorticoid by Torelli and cow o r k e r ~and ~ ~later ~ confirmed when long-term administration of RU26752 was shown to prevent aldosterone-induced hypertension.lo6 In this study, RU26752 on its own was shown to be devoid of any pressure effect and of any agonistic activities as indicated by the unaltered saline consumption, diuresis pattern, or sodium excretion. The steroid, given alone or in combination with aldosterone, does not show any damaging effect on the kidney or heart as observed by microscopic examination. The mineralocorticoid specificity of RU26752 in the kidney is beyond doubt; it does not bind to transcortin (Ref. 107and Sutanto and deKloet, unpublished) or to non-mineralocorticoid target tissues such as the liver and lung.lo7It has been suggested that in the kidney, RU26752-receptor complex (and ZK91587receptor complex) is physicochemically and kinetically distinct from that of aldosterone-receptor complex.85-102,103 It appears that in this tissue, the antagonist (RU26752) may occupy the classically defined high-affinity, low-capacity aldosterone sites, as well as separate binding sites that specifically accept RU26752 but not aldosterone. This phenomenon may explain the need for a very high amount of RU26752 to antagonize the in vivu aldosteroneinduced hypertension.lo6In other words, regardless of the fact that RU26752 displays a very high affinity for renal MR in vitru, it is likely that in vivo a larger amount of the steroid is required to exert its effect on high-affinity aldosterone binding sites. Other possible explanations for the need of high doses of the steroid (for example, solubility, transport, ease of penetration into target cell), however, cannot be excluded but presently remained unanswered. RU28318, the ethanol-soluble potassium salt analog of RU26752, shows very little binding affinity in vitro for hippocampal MR (see Table I and Refs. 26, 104), renal MR (Schut, Sutanto, and de Kloet, unpublished observations), or progesterone and androgen receptors.lo5In vivo, however, the uptake of [3H]aldosteronein the forebrain is suppressed approximately 95%by infusion of RU28318.lo' Intracerebroventricular (icv) fusion of this steroid blocks the hypertension produced by subcutaneous (sc) administration of aldosterone to saline-drinking uninephrectomized rats. This study provides evidence of the importance of the CNS in the pathogenesis of hypertension produced by systemic mineralocorticoid excess (see also Sec. I1 of this review). Data of the efficacy of RU28318 in vitru can only be obtained from one report."O In this study, using perifused frog adrenal glands, RU28318 was shown to block ACTH- or angiotensin 11-induced aldosterone biosynthesis. 6 . 12/3,28-Epuxypregnune Derivatives

In the search for "better" aldosterone antagonists, Schmidlin and Wettstein (1961)and Kondo, Mitsugi, and Tori (1967) synthesized 18-deoxyaldosterone

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(21-hydroxy-ll~-18-epoxypregn-4-ene-3,20-dione), which is structurally similar to aldosterone, with the aldehyde hemiacetal group replaced by a more stable llP,l&epoxy ring. This steroid binds to the renal MR in vitro with an affinity approximately one-third that of aldosterone, and shows 2:l antagonist:agonist ratio in both toad bladder and adrenalectomized rat bioassays.'l* The steroid, 1Sdeoxyaldosterone, was later modified structurally by %-fluorination to increase its activity and by replacing the 17p-hydroxyacetylchain with 17P-hydroxy-17-propionicacid y-lactone to improve its antagonistic nature.62,112The resulting steroid, one of the llp-Wepoxypregnane derivatives (3-(9a-fluoro-l7~-hydroxy-3-oxoandrost-4-en-l7a-yl)propionic acid y-lactone), possesses a fairly strong affinity for the renal MR in vitro as well as exhibiting a stronger antimineralocorticoid activity in vivo compared to spironolactone. Unfortunately, it also shows a slight, but not negligible (especially at higher doses) agonistic activity. On the other hand, the steroid shows no affinity for glucocorticoid, progestin, and androgen receptors. These same authors also modified the structure of the already known MR ligand A"-DOC (see Sec. I.B.2) by replacing the 17P-hydroxylacetyl group with 17y-spirolactone. The rationale behind this approach is the introduction of a double bond at C-11 (All) to certain pregnane derivatives, which will increase the affinity of the molecule for MR. However, the substitution of C-17 with 17y-lactone does not always produce the desired or expected antimineralocorticoid activity. In fact, most of the steroids from this series of 11,12-dehydropregnane derivatives show agonistic, rather than antagonistic activity (although generally they bind to the renal cytosolic MR with greater affinity than DOC or spironolactone). Further modifications on these molecular structures are therefore necessary to find a ligand from this series that shows high affinity to MR in vitro and have a potent antimineralocorticoid in vivo.62Another form of 1Sdeoxyaldosterone derivative is 18-deoxy-19-noraldosterone,which has been found to be an antimineralocorticoid as potent as spiron~lactone."~ 11. MR AND MR LIGANDS IN THE CNS: DISTRIBUTION AND PUTATIVE FUNCTIONS The evidence for the presence of MR in the CNS has been accumulated from different experimental approaches: biochemical studies, topography, and molecular biology. In the CNS, corticosteroid interaction with MR and GR is believed to mediate the differential role($ of the steroids. A. Distribution of MR in the CNS The localization of MR (and GR) is best illustrated in an in vivo autoradiography as shown in Fig. 1. Adrenalectomized rats were injected intravenously with a tracer dose of either [3H]corticosterone or [3H]aldosterone to label the MR, or [3H]RU28362to label the GR. The administered corticosterone or aldosterone was equivalent to 1.0 pg/lOO g bw, the dose that has been shown previously to occupy most of MR and very little of GR.4,'6 The autoradiograms thus obtained from the brain sections (Fig. 1)and the corresponding optical density quantification by an image analysis system reveal the highest density of MR in the septo-hippocampal region. MR as labelled by [3H]aldosteronehas a similar, but not identical, pattern of distribution. Thus,

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Figure 1. In om0 autoradiography of ~Hlcorticosterone,[%iJaldoeterone, and [3H]RU28362in the rat brain. Adrenaleaomized rats were given intravenous tram doeee of [3I]corticosterone or [%iJaldosterone to identify the mineralocorlicoid recepton, (MR), or of [3I]RU28362 to identify the g l u e receptors (GR). F" = paraventrirular nudeus; SON = supraoptic nudeus.

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a substantial amount of [3H]aldosterone,but not [3H]corticosterone,is bound to MR in anterior hypothalamic nuclei, amygdaloid nuclei, circumventricular organs, and layer I1 of the cortex. Recently, in situ hybridization techniques using specific probes for MR (and GR) in the brain show that high density of MR-mRNA was found in the neurons of the hippocampal formation, lateral septum, medial and central amygdala, olfactory nucleus, layer I1 of the cortex, and in brain-stem sensory and motor neurone~.*,~ These results essentially confirm the findings from the biochemical" and aut~radiographical~ studies.

B. Possible Function(s) of MR and MR Ligands in the CNS There is a substantialbody of evidence that MR ligands, by interacting with MR in the CNS, are involved in the centrally regulated processes. On the one hand, aldosterone action via MR is involved in sodium retention in the kidney, and the interaction with the MR in the CNS is responsible for selected central functions of the steroid. On the other hand, in corticosterone-selective tissue such as the hippocampus, MR exerts tonic influence on brain function. This subject has been reviewed elsewhere.'t2 These centrally regulated processes include the negative feedback regulation by corti~osteroids,~'~ the effect of aldosterone and deoxycorticosterone on salt appetite,108*115 and the regulation of an autonomic system such as blood pressure.61,109,116'18 In the CNS, the influence of corticosteroidson neuronal activities, a subject of uncertainty for many years, has been given fresh evidence in a series of elegant electrophysiological studies by Dr. M. Joels,llp.'zl which show that the coordinate antagonistic MR- and GR-mediated corticosteroid effects are a physiological feature of living cells (in this case, hippocampal CA1 neurones) that express both MR and GR. In these studies, the MR- and GR-mediated effects on the excitability of hippocampal neurones are evaluated in the context of the innervating aminergic and serotoninergic neurones. Corticosteroids, including MR ligands such as aldosterone, spironolactone, and ZK91587, may also influence neuronal activity in another manner, i.e., by interacting with the GABAA receptor complex (see Introduction). The brain MR has also been shown to be involved in the mediation of the memory-enhancing effects of piracetam.'= In another series of studies, spironolactone was shown to inhibit the release of ACTH from primary culture of the rat anterior pituitary cells in a similar way to that of DEX and aldosterone.'= This effect of aldosterone is also inhibited by spironolactone at the pituitary level. It appears that spironolactone antagonizes aldosterone in the kidney and pituitary. Recently, Dr. A. Ratka from our laboratory has shown that RU28318, a spirolactone analog, antagonizes corticosterone action in the maintenance of basal activity of the HPA axis."" 111. CLINICAL ASPECTS OF MINERALOCORTICOID RECEPTOR LIGANDS

MR ligands, both mineralocorticoid agonists and antagonists, have been used successfully in clinical medicine. For a review on steroid therapy see Azarnoff.'" In the following subsections, their potential usage, as well as the limitation of that usage, will be described, together with some recent findings

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on the improvement of structurally modified analogs of antimineralocorticoids for therapeutic purposes.

A. Mineralocorticoid Agonists Aldosterone has sodium-retaining properties when administered to patients with Addison’s disease. The steroid is suspended in oil and injected intramuscularly. The effect is seen after 2 h and lasts for up to 8 h. It is generally used as a substitution therapy in Addison’s disease as regards metabolism of electrolytessince, unlike cortisone, it does not correct the abnormality in water excretion and it has only 1%activity in carbohydrate effects, compared to cortisone. DOC, like aldosterone, is administered (in oil suspension) intramuscularly to patients with Addison’s disease. Unlike aldosterone, however, it has only 10% potency as an electrolyte regulator compared to aldosterone. However, DOC can be prepared synthetically and is available in acetate form (DOCA), pivalate, or trimethylacetate. Deoxycortone pivalate (21-Pivaloyl-oxypregn-4ene-3,20-dione) and deoxycortone trimethylacetate have a long-lasting action of 2-3 weeks (pivalate) or 3-4 weeks (trimethylacetate). Fludrocortisone (9a-F) has the same actions of hydrocortisone but is much more potent, causing salt retention. Normally it is used in acetate form (Fludrocortisone acetate) to suppress inflammation in the treatment of certain allergic dermatoses. It is also given orally in patients with Addison’s disease.

B. Mineralocorticoid Antagonists Antimineralocorticoids have been used for therapeutic purposes for well over 30 years. Spirolactones [spironolactone (Aldactonem)and potassium canrenoate (Soldactone@)]have been successfully used, alone or in combination with other d r ~ g ~in ,a variety ~ ~ ~ of, diseases,lZ5 ~ ~ ~ including primary hyperaldosteronism, better known as Conn’s ~ y n d r o m e . ’ ~ Spironolactone ~,~~* corrects the electrolyte imbalance (or abnormalities, i.e., hypokalaemia resulting from increased sodium retention accompanies by potassium depletion) due to the excess production of aldosterone. Spironolactone has also been used in conditions of secondary hyperaldosteronism such as hepatic cirrhosis,129,130 and essential hypertension, where as an congestive cardiac antihypertensive drug spironolactone is normally used alone or in combination with other hypertensive drug such as hydrochlorothiazide.126,132-134 Other clinical uses for spironolactone are in Apparent Mineralocorticoid Excess Syndrome (AMS).135AMS is a juvenile disease characterized by low or undetectable levels of renin and plasma aldosterone accompanied by hypokalaemia and severe hypertension. This syndrome, due to either congenital deficiency or acquired inhibition of the enzyme 11P-OHSD,30results in cortisol acting as a potent mineralocorticoid and is associated with a prolonged plasma cortisol half-life despite normal plasma levels, raised urinary-free cortisol, and a reduced daily cortisol-production rate. Spironolactone is also used in the treatment of acne and h i r ~ u t i s mwhich ,~~~ emphasizes once again the antiandrogenic property of these steroids, and in combination with testolactone in the treatment of male precocious p~berty.’~’ Although spirolactone drugs (especially spironolactone) have been used

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clinically as early as the late 1 9 5 0 ~ , ~ ,serious " side effects of these agents, especially following prolonged use, have been reported repeatedly. Because of the nature of its physiological action, high doses of spironolactone may cause hyperkalaemia,''' which is even more serious in patients with renal insufficiency or in those receiving potassium supplements.131,138 Other doserelated side effects resulting specificallyfrom long-term exposure to the steroid are endocrine-related disturbances such as gynaecomastia, decreased libido, impotence (in men), or menstrual irregularities (in women).*39 Spironolactone and spirolactone analogs may also cause less commonly occurring side effects including gastrointestinal symptoms (anorexia, nausea, vomiting) and skin rashes. The fact that spirolactone administration may cause neurological disturbances such as weakness, drowsiness, and confusion,138 indicates that the compounds have effects on centrally regulated functions. With the knowledge that spirolactones produce side effects (even though no evidence of toxicity as such has been reported), some measures can be taken to ensure that such harmful effects can be minimized. The first step is to actually reduce the d ~ s e ' ~without , ' ~ ~ diminishing the efficacy of the drug during the treatment. This, naturally, may not work in all instances. A better strategy would therefore involve the design of new molecules that have equal (if not greater) potency as antimineralocorticoidsthan spironolactone. Indeed, attempts have been made in the past years to substitute spironolactone with analogs that interact specifically with the renal MR (and thus acting as an antialdosterone in the manner of spironolactone) but not with other steroid receptor systems such as the androgen, oestrogen, or progesterone receptors. Theoretically, and ideally, then, the steroid should be an antialdosterone that does not bind to receptors other than the aldosterone receptor (i.e., MR), does not interfere with the production or the metabolism of sex steroids and corticosteroids, and is as potent (or more potent than) spironolactone. Potassium canrenoate and spironolactone are competitive aldosterone antagonists. For certain clinical conditions, potassium canrenoate has been shown to be a more suitable antimineralocorticoid than spironolactone. In the clinical evaluation on cirrhotic patients, the efficacy of these two antialdosteronic drugs were compared.lm The greater solubility of potassium anr re no ate,^^ in combination with its lower rate of degradation by the liver, result in an enhanced availability of potassium canrenaote compared to spironolactone for the receptor. In these studies, not only is potassium canrenoate required at a dose of 1:2 compared to spironolactone (to achieve the same effect) but also spironolactone-treated patients have a significantly higher incidence/ prevalence of g y n a e c ~ m a s t i aHowever, .~~~ these findings have to be received with caution since a study in the rat has shown that potassium canrenoate may not be suitable as an antimineralocorticoid in a long-term treatment. It is believed that one or more metabolites of potassium canrenoate are mutagenic. These mutagenic metabolites, unique to this steroid (but not spironolactone), are probably responsible for potassium canrenoate dose-related increases in myelogenous leukaemia and the significant increases in malignant tumours of the liver, thyroid, brain, and mammary glands in the rat. Note that these effects were not observed in rats treated with the same or even higher doses of s p i r o n ~ l a c t o n eWhether .~~~ or not these phenomena are reflected in the human study remains to be seen.

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Because of the drawbacks shown by these major antialdosterone agents, the search for "better" antimineralocorticoidshas actively continued. Such a search has lead to the synthesis, for example, of RU26752 (7a-propylspirolactone) and RU28318 (potassium salt of 7a-propylspirolactone) (both from Roussel-Uclaf Pharmaceuticals, France), the three epoxyspirolactones (CibaGeigy, Switzerland), and the ZK steroid series (Schering AG, Germany). Of these steroids, only RU28318 has been used in clinical trial for its antimineralocorticoid effect in healthy patients with induced exogenous and endogeIn fact, the use of RU28318 instead of spinous hypermineralo~orticism.'~~ ronolactone may be of advantage since spironolactone treatment often results in a significant increase in plasma renin activity which, in turn, may stimulate aldosterone production.144 In contrast, with the administration of RU28318 (at least in an in vitro system in the frog perifused adrenals), the stimulatory effects of ACTH and angiotensin I1 on aldosterone synthesis is almost completely blocked. It has been suggested, based on the findings in the rat,lM that RU26752 may be of potential use in humans since the steroid does not show any mineralocorticoid agonistic activity and the dose of RU26752 needed to antagonize aldosterone is lower than spironolactone.145 However, it is important to first establish whether RU26752 has any affinity for other steroid receptor systems. In the rat, at least, both RU26752 and RU28318 show much less binding affinity for progesterone and androgen receptor^.'^^ To our knowledge, data on clinical trials with RU26752 are not yet available. IV. CONCLUDING REMARKS

A substantial number of mineralocorticoid receptor (MR) ligands, some of which are highlighted in this review, have been synthesized and used clinically. The search for potent MR ligands has been actively pursued in the past three decades since the therapeutic value of these compounds, especially those with antimineralocorticoid properties, was acknowledged in clinical medicine. Thus antimineralocorticoids such as spironolactone (Aldactonem) were used in the treatment of diseases (or conditions) with sodiudpotassium imbalance as the main culprit. With the knowledge that an antialdosterone such as Aldactone does not act solely in the kidney (in other words, it does not interact only with the renal MR), the use of such a drug, especially at high doses and/or for long-term purposes, becomes limited. The "ideal" antimineralocorticoids (or for that matter, mineralocorticoid agonists) are those that interact with MR with high affinity but show no affinity for other steroid receptors (progesterone, androgen, oestrogen, glucocorticoid). It is important to bear in mind, however, that MR also exist in the CNS, which accounts for the fact that potent mineralocorticoid or antimineralocorticoid agents may interfere with centrally regulated processes. Thus not only do they interact with MR in the CNS, they also interact with membranebound neurotransmitter receptor, notably the GABAA receptor complex. MR ligands such as spironolactone, ZK91587, RU26752, aldosterone, deoxycorticosterone, corticosterone, and cortisol, at nanomolar concentrations, have been shown to enhance the binding of [35S]t-butylbicyclopho~phorothionate (TBPS, the "cage convulsant") to the GABAA receptor complex. It has also

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been shown that the metabolites (THDOC and 3a-DHP), at low nanomolar concentrations, displace the binding of TBPS.'8,21It is therefore likely that these steroids influence, under physiological and pathological conditions, the GABAAreceptor complex and thus the activity of the CNS. The fact that antimineralocorticoids, which are designed to act on the kidney, do interact with the MR in the CNS has raised the question of the importance of steroid specificity. We have gained a considerable amount of knowledge in this aspect since the identification and isolation of the enzyme llP-OHSD, which determines the specificity of an MR for either aldosterone (i.e., the renal MR) or for corticosterone (i.e., hippocampalMR).An MRligand that is targeted specifically for the CNS must therefore contain the C-llp hydroxyl group. The availability of an 11-OH-containing MR ligand will be of special interest since the steroid will be inactivated in the kidney and yet still retain its ability to act in the CNS. To our knowledge, however, such a steroid is currently not yet available. ACKNOWLEDGMENTS We are grateful to Roussel-Uclaf Pharmaceuticals,Romainville, France, for the generous supply of RU28318, RU28362, and RU26752; New England Nuclear, Boston for [3H]RU2R362; Schering AG, Berlin, Germany, for ZK91587; Organon Internationals, Oss, The Netherlands, for corticosterone, cortisol, aldosterone, deoxycorticosterone,and dexamethasone; Searle Research and Development, St. Louis, for spironolactone (Aldactone);and Ciba-Geigy, Basle, Switzerland, for epoxy-spirolactones.The work depicted in Table I and Figure 1 was carried out entirely at the Rudolf Magnus Institute, Medical Faculty, University of Utrecht, and partly supported by IBRO/UNESCO postdoctoral Fellowship and by de Stichting Farmacologisch Studiefonds, University of Utrecht, The Netherlands (to WS). The contributions of Dr.Anna Ratka, Margreet Bloemers, Astrid Schut, Freddy de Bree, and Alexander Diamandidis in some part of this work is also gratefully acknowledged.

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