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 liver and kidney Downloaded from microsomal aldosterone 18β-glucuronidation: A potential drug 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. DMD #32870 RUNNING TITLE PAGE Running title: Spironolactone 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-glucuronosyltransferase. 2 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 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 canrenoic acid 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 drugs in both liver and kidney. 3 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 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 heart failure. 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 mineralocorticoid 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 potassium 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 5 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 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-glucuronic acid (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 livers (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 6 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 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