DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.110.032870 DMD FastThis article Forward. has not beenPublished copyedited on and March formatted. 19, The 2010 final version as doi:10.1124/dmd.110.032870 may differ from this version. DMD #32870

TITLE PAGE

SHORT COMMUNICATION

Spironolactone and canrenone inhibit UGT2B7-catalyzed human and Downloaded from microsomal 18β-glucuronidation: A potential interaction.

Kathleen M Knights, Kushari Bowalgaha and John O Miners dmd.aspetjournals.org

Department of Clinical Pharmacology, Flinders University School of Medicine,

Adelaide, Australia at ASPET Journals on September 28, 2021

1

Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics. DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.110.032870 This article has not been copyedited and formatted. The final version may differ from this version.

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RUNNING TITLE PAGE

Running title: and canrenone inhibit aldosterone glucuronidation

Corresponding author: Professor Kathleen M Knights, Department of Clinical

Pharmacology, Flinders University School of Medicine, Flinders Medical Centre,

Bedford Park, SA 5042, Australia Downloaded from Telephone: 61 8 8204 4331

Fax: 61 8 82045114;

E-mail: [email protected] dmd.aspetjournals.org

Word count: Abstract - 248, Introduction - 382, Results and Discussion - 829

Text pages: 18 at ASPET Journals on September 28, 2021

Number of tables: 1

Number of figures: 2

Number of references: 23

Abbreviations: ALDO, aldosterone; HLM, human liver microsomes; HKCM, human kidney cortical microsomes; Ki, inhibitor constant; 4MU, 4-methylumbelliferone;

UGT, UDP-.

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ABSTRACT

Elevated plasma concentrations of aldosterone (ALDO) are observed in patients treated with spironolactone. Since ALDO is eliminated via UGT2B7-catalyzed 18β- glucuronidation, this study aimed to determine whether spironolactone and its primary metabolites canrenone and inhibit ALDO 18β-glucuronidation by recombinant UGT2B7 and by human liver- (HLM) and human kidney cortical-

(HKCM) microsomes. Initial experiments characterized the effects of all three Downloaded from compounds on 4-methylumbelliferone (4MU) and ALDO glucuronidation by recombinant human UGT2B7. IC50 values for spironolactone and canrenone were in

the range 26-50 µM whereas canrenoic acid was a weak inhibitor. Ki values for dmd.aspetjournals.org spironolactone and canrenone inhibition of ALDO 18β-glucuronidation were subsequently determined with HLM, HKCM and UGT2B7 as the enzyme sources.

β

Spironolactone and canrenone were competitive inhibitors of ALDO 18 - at ASPET Journals on September 28, 2021 glucuronidation by HLM, HKCM and UGT2B7. Mean (±) Ki values for spironolactone were 52±22 µM (HLM) and 34±4 µM (HKCM), and for canrenone 41

±19 µM (HLM) and 23±2 µM (HKCM). Ki values for spironolactone and canrenone inhibition of ALDO 18β-glucuronidation by recombinant UGT2B7 were 23µM and

11 µM, respectively. ‘Actual’ Ki values for spironolactone and canrenone inhibition of ALDO 18β-glucuronidation, which take into account the role of endogenous microsomal inhibitors, are predicted to be 3-5 µM and 2-4 µM, respectively. The data indicate that the elevated ALDO concentrations observed in patients treated with spironolactone may be due, at least in part, to a pharmacokinetic interaction and spironolactone and canrenone should be considered potential inhibitors of the

UGT2B7 mediated metabolic clearance of in both liver and kidney.

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INTRODUCTION

Aldosterone (ALDO) promotes sodium and water retention thereby contributing to the development and maintenance of hypertension. Additionally, ALDO induces left ventricular hypertrophy, augments myocardial, vascular and renal fibrosis, and plays a pivotal role in the progression of congestive . Recognition of the clinical benefits of aldosterone antagonism in hyperaldosteronism, oedematous states, ascites and heart failure led to the introduction of spironolactone (3-(3-oxo-7α acetylthio- Downloaded from 17β-hydroxy-4-androsten-17α-yl) propionic acid γ-lactone), a receptor antagonist, into clinical practice in 1960. Although an effective drug, lack of

selectivity manifests as progestogenic and antiandrogenic adverse effects and this led dmd.aspetjournals.org to a decline in use. However, in 1999 the Randomized Aldactone Evaluation Study reported a reduction in mortality of 30% in patients with advanced heart failure

treated with low dose spironolactone (Pitt et al., 1999). This led to a resurgence in at ASPET Journals on September 28, 2021 both the use of spironolactone to treat heart failure and the incidence of hyperkalemia, a well documented adverse effect of spironolactone (Juurlink et al., 2004). Since drives ALDO secretion, a predicted pharmacodynamic response to spironolactone is an increase in plasma renin and ALDO concentrations (Garthwaite et al., 2004).

ALDO is metabolised in the liver to dihydro and tetrahydro derivatives that are subsequently glucuronidated. In contrast, direct glucuronidation of ALDO by the kidney accounts for ~80% of the formation of ALDO 18β-glucuronide (ALDO 18β-

G), which is excreted in urine (Bledsoe et al., 1966). Human kidney cortical (HKCM) and human liver (HLM) microsomal ALDO 18β-G formation is catalyzed primarily by UGT2B7 (Knights et al., 2009), which is expressed in both liver and kidney. In humans spironolactone is metabolised to canrenone (~79%), which exists in

4 DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.110.032870 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #32870 equilibrium with its hydrolysis product canrenoic acid (Sadee et al., 1973; Karim

1987). Spironolactone reduces the urinary excretion of ALDO 18β-G in humans when the plasma ALDO concentration is elevated (Vetter et al., 1977), and inhibition of

ALDO glucuronidation by spironolactone has been demonstrated in the rat (Tsai et al., 1980). Thus, the question arises whether the elevated plasma ALDO concentration observed with spironolactone treatment (Rossing et al., 2005; Schjoedt et al., 2006) arises solely via a pharmacodynamic mechanism or is due, at least in part, to Downloaded from inhibition of ALDO 18β-glucuronidation. The aim of this study was to determine whether spironolactone, canrenone and canrenoic acid inhibit ALDO 18β-G formation

by recombinant UGT2B7, HLM and HKCM. dmd.aspetjournals.org at ASPET Journals on September 28, 2021

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MATERIALS AND METHODS

Materials

Alamethicin (from Trichoderma viride), ALDO, canrenoic acid, 4- methylumbelliferone (4-MU), 4-methylumbelliferone-β-D-glucuronide (4-MUG), spironolactone and UDP- (UDPGA; sodium salt) were purchased from

Sigma-Aldrich (Sydney, Australia). Canrenone was purchased from Steraloids Inc

(Newport, RI). Solvents and other reagents used were of analytical reagent grade. Downloaded from

Methods

Enzyme sources dmd.aspetjournals.org

HKCM and HLM were prepared from human kidney and liver tissue by differential centrifugation, as described previously (Tsoutsikos et al., 2004; Bowalgaha et al.,

2005). The four human (H7, H12, H29 and H40) and kidneys (K5, K9, K 10 at ASPET Journals on September 28, 2021 and K11) were obtained from either the human liver ‘bank’ of the Department of

Clinical Pharmacology, Flinders Medical Centre or the joint Flinders Medical

Centre/Repatriation General Hospital Tissue Bank. HKCM and HLM were activated by pre-incubation with alamethicin, 50 µg/mg microsomal protein (Boase and Miners,

2002). UGT2B7 was stably expressed in a human embryonic kidney cell line

(HEK293) according to Stone et al. (2003). Cells expressing UGT2B7 were lysed by sonication. The lysate was centrifuged at 12,000 g for 1 min at 4°C and the supernatant fraction was separated and stored in phosphate buffer (0.1 M, pH 7.4) at –

80°C until use. Expression was demonstrated by immunoblotting with an antibody raised against purified mouse UGT (Uchaipichat et al., 2004) and activity measurement (see Results).

Spironolactone, canrenone and canrenoic acid inhibition studies

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Spironolactone, canrenone and canrenoic acid (10, 100, and 250 μM in phosphate buffer 0.1M, pH 7.4) were screened initially as inhibitors of recombinant UGT2B7 using 4MU and ALDO as the probe substrates. The concentrations of 4MU and

ALDO present in incubations were 400 µM and 300 µM respectively, which correspond to the approximate S50/Km values for each compound (Uchaipichat et al.,

2004; Knights et al., 2009). Subsequently, Ki values were determined for spironolactone and canrenone inhibition of ALDO 18β-glucuronidation with HLM, Downloaded from HKCM and UGT2B7 as the enzyme sources. Inhibition experiments included five inhibitor concentrations (5, 10, 20, 30 and 50 μM) at each of three ALDO

concentrations (150, 300 and 600 µM). Control activities were measured in the dmd.aspetjournals.org absence of inhibitor. All experiments were performed in duplicate. Equations for competitive, mixed and non-competitive inhibition were fit to untransformed data

using EnzFitter (version 2.0.18.0: Biosoft, Cambridge). Goodness of fit was assessed at ASPET Journals on September 28, 2021 by comparison of the F-statistic, parameter standard error of fit, and 95% confidence interval. The statistical significance of differences between Ki values were assessed by student’s t-tests for paired and unpaired samples.

Assay for 4MU glucuronidation

Incubations, in a total volume of 0.2 ml, contained, MgCl2 (4 mM), UDPGA (5 mM),

HEK293 cell lysate expressing UGT2B7 (1 mg/ml), 4MU (400 µM), and spironolactone, canrenone or canrenoic acid (10, 100, and 250 μM) in phosphate buffer (0.1 M, pH 7.4). Reactions were performed in air at 37°C for 60 min in a shaking water bath. Following termination of the reaction (Uchaipichat et al., 2004),

4MUG formation was quantified by HPLC as described previously (Lewis et al.,

2007).

Assay for ALDO 18β- glucuronide formation

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Incubations (0.2 ml) contained MgCl2 (4 mM), UDPGA (5 mM), ALDO (see Figures

1 and 2 for concentrations), the enzyme source (HLM, 0.5 mg/ml; HKCM, 0.2 mg/ml; or HEK293 cell lysate expressing UGT2B7, 1 mg/ml), with and without inhibitor (see

Figures 1 and 2 for spironolactone, canrenone and canrenoic acid concentrations) in phosphate buffer (0.1 M, pH 7.4). Reactions were carried out in air at 37°C (shaking water bath) for 45 min and then terminated by the addition of 200 μl of ice cold 4% v/v acetic acid in methanol. Mixtures were vortex mixed then centrifuged (4000 g for Downloaded from

10 min). A 30 μl aliquot of the supernatant fraction was analyzed by HPLC for quantitation of ALDO 18β-glucuronide formation (Knights et al., 2009).

dmd.aspetjournals.org at ASPET Journals on September 28, 2021

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RESULTS AND DISCUSSION

Initial experiments characterized the inhibitory effects of spironolactone, canrenone and carenoic acid on the glucuronidation of the prototypic glucuronidated substrate

4MU with recombinant UGT2B7 as the enzyme source. Following demonstration of inhibition (Figure 1A), effects of the three compounds on UGT2B7 catalyzed ALDO glucuronidation were investigated (Figure 1B). Inhibition data were broadly consistent between the two substrates. IC50 values for spironolactone and canrenone Downloaded from were in the range 26 to 50 µM whereas canrenoic acid was a weak inhibitor. Thus, only Ki values for spironolactone and canrenone inhibition of ALDO glucuronidation

were determined in subsequent kinetic experiments. dmd.aspetjournals.org

Ki values for spironolactone and canrenone inhibition of ALDO glucuronidation by

HLM, HKCM and UGT2B7 are given in Table 1, while representative Dixon plots

are shown in Figure 2. Spironolactone and canrenone inhibition of ALDO at ASPET Journals on September 28, 2021 glucuronidation by all three enzyme sources was well described by the equation for competitive inhibition. The mean Ki values for canrenone inhibition of ALDO glucuronidation were approximately 20 to 30% lower than those for spironolactone with both HLM (p = 0.039) and HKCM (p = 0.05) as the enzyme sources. The difference (ca. 50%) was somewhat larger for experiments conducted with recombinant UGT2B7. Although the mean Ki values for spironolactone and canrenone inhibition of ALDO glucuronidation by HKCM were 35 to 45% lower than those of HLM, the difference was not statistically significant (p > 0.05). The lack of statistical significance may reflect the small sample size typically associated with in vitro kinetic studies of this type.

Ki values for UGT2B7 substrates determined using recombinant enzyme, HLM and

HKCM as the enzyme sources are now known to over-estimate the ‘true’ parameter. It

9 DMD Fast Forward. Published on March 19, 2010 as DOI: 10.1124/dmd.110.032870 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #32870 has been demonstrated recently that long chain unsaturated fatty acids released from the microsomal membrane and HEK293 cell lysate during the course of an incubation act as potent inhibitors of UGT2B7, resulting in artificially high estimates of Km and

Ki (Rowland et al., 2007). Accurate values of these parameters may be obtained by adding bovine serum albumin or fatty acid free serum albumin to incubations to sequester long chain unsaturated fatty acids. This approach was not feasible in the current study due to the high binding of spironolactone (and canrenone) to albumin Downloaded from

(fuplasma < 0.1). ‘Actual’ Km and Ki values are approximately 10-fold lower than those generated with microsomes as the enzyme source and 5-fold lower than those

obtained with recombinant UGT2B7 expressed in HEK293 cells (Rowland et al., dmd.aspetjournals.org

2006, 2007, 2008; Uchaipichat et al., 2006). On this basis we predict Ki values of 3 to

5 µM for spironolactone inhibition of ALDO 18β-glucuronidation, and 2 to 4 µM

with carenone as the inhibitor. It should be noted that the somewhat higher Ki values at ASPET Journals on September 28, 2021 for spironolactone and canrenone inhibition of ALDO glucuronidation by HLM presumably reflects a contribution of an enzyme other than UGT2B7 to hepatic

ALDO glucuronide formation (Knights et al., 2009).

The extent of inhibition of in the liver may be predicted from the Ki and the hepatic input concentration of inhibitor (Miners et al., 2010). Although plasma concentrations of spironolactone and its metabolites have been reported, there is no reliable estimate of absorption from the or absorption rate constant. Thus, calculation of hepatic input concentration is not possible. However, this parameter will exceed the combined plasma concentration of spironolactone and canrenone, which has been reported to be approximately 1µM following a single oral dose of 200 mg (Overdiek et al., 1985). At present the inhibitory potential of other metabolites of spironolactone such as 7α-thiomethylspironolactone and 6β-hydroxy-

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7α-thiomethylspironolactone, whose combined concentrations in plasma are almost double those of spironolactone and canrenone (Overdiek et al., 1985), is unknown.

Although the extent of an inhibitory interaction for a drug cleared by the liver in vivo may be predicted from the Ki and hepatic input concentration of inhibitor (Miners et al, 2010), the majority of ALDO 18β-glucuronidation (ca. 80%) occurs in the kidney.

An effect on renal metabolism cannot be predicted from the ratio of the Ki to the plasma concentration of inhibitor since renal tubular cell concentrations of the Downloaded from inhibitor may be elevated due to reabsorption from urine. The relatively low Ki values for spironolactone and canrenone inhibition of ALDO 18β-glucuronidation predicted

here (2 to 5 µM) provide a basis for an intra-renal interaction. In turn, elevation of the dmd.aspetjournals.org intra-renal ALDO concentration would be expected to result in an enhanced and prolonged decrement in the natriuretic excretory function of the kidney.

Spironolactone and canrenone were shown here to inhibit both ALDO and 4-MU at ASPET Journals on September 28, 2021 glucuronidation by UGT2B7. UGT2B7 is arguably the most important enzyme involved in drug glucuronidation. Drugs with a substantial component of UGT2B7 mediated metabolic clearance include anticonvulsants (e.g. valproic acid), antineoplastics (DMXAA, epirubicin), antivirals (zidovudine), non-steroidal anti- inflammatory agents (e.g. , naproxen), and opioids (, )

(Miners et al., 2010). Moreover, UGT2B7 metabolizes numerous endogenous compounds, notably fatty acids and 3-hydroxy . Thus spironolactone and canrenone should be considered potential inhibitors of all reactions catalyzed by

UGT2B7 in both liver and kidney.

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glucuronidation: Investigations with the model substrate zidovudine. Br J Clin Downloaded from Pharmacol 54: 493-503.

Bowalgaha K, Elliot DJ, Mackenzie PI, Knights KM, Swedmark S and Miners JO

(2005) Naproxen and desmethylnaproxen glucuronidation by human liver dmd.aspetjournals.org

microsomes and recombinant human UDP- (UGT): Role

of UGT2B7 in the elimination of naproxen Br J Clin Pharmacol 60: 423-433.

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Cell Endocrinol 217: 27-31.

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albumin enhance the glucuronidation of UGT1A9 substrates but not UGT1A1 and

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FOOTNOTE

The research was funded by the National Health and Medical Research Council of

Australia.

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LEGENDS TO FIGURES

Figure 1. Spironolactone, canrenone and canrenoic acid inhibition of 4MU (panel A) and aldosterone (panel B) 18β-glucuronidation by UGT2B7. Histograms represent the mean of duplicate measurements (<10% variance).

Figure 2. Representative Dixon plots for the inhibition of aldosterone 18β- Downloaded from glucuronidation by spironolactone (panels A-C) and canrenone (panels D-E) with

HLM, HKCM and UGT2B7 as the enzyme sources. Concentrations of aldosterone

were 150, ■300 and ▲600 μM. Points represent the mean of duplicate dmd.aspetjournals.org measurements (<10% variance). at ASPET Journals on September 28, 2021

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Table 1: Ki values for the inhibition of aldosterone 18β-glucuronidation by spironolactone and canrenone with HLM, HKCM and UGT2B7 as the enzyme sources.

Enzyme Spironolactone Canrenone Downloaded from source Ki (µM) Ki (µM)

HL7 36 23

HL12 79 62 dmd.aspetjournals.org

HL29 60 53

HL40 31 27

Mean ± SD 52 ± 22 41 ± 19 at ASPET Journals on September 28, 2021

HKC5 30 24

HKC9 35 22

HKC10 30 26

HKC11 39 21

Mean ± SD 34 ± 4 23 ± 2

UGT2B7 23 ± 3.5a 11 ± 1a

aParameter ± SE of parameter fit

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