pharmaceutics Article In Vitro and In Vivo Assessment of Metabolic Drug Interaction Potential of Dutasteride with Ketoconazole 1, 2, 1 1 3 Seong-Wook Seo y, Jin Woo Park y, Dong-Gyun Han , Ji-Min Kim , Sanghyun Kim , Taeuk Park 3, Kyung-Hwa Kang 4,*, Min Hye Yang 1,* and In-Soo Yoon 1,* 1 College of Pharmacy, Pusan National University, Busan 46241, Korea; [email protected] (S.-W.S.); [email protected] (D.-G.H.); [email protected] (J.-M.K.) 2 Department of Pharmacy, College of Pharmacy, Mokpo National University, Jeonnam 58554, Korea; [email protected] 3 Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Korea; [email protected] (S.K.); [email protected] (T.P.) 4 Department of Physiology, College of Korean Medicine, Dongeui University, Busan 47227, Korea * Correspondence: [email protected] (K.-H.K.); [email protected] (M.H.Y.); [email protected] (I.-S.Y.) These authors contributed equally to this work. y Received: 3 November 2019; Accepted: 9 December 2019; Published: 11 December 2019 Abstract: Dutasteride (DUT) is a selective, potent, competitive, and irreversible inhibitor of both type-1 and type-2 5α-reductase (5AR) commonly used in the treatment of benign prostatic hyperplasia and androgenetic alopecia. In the present study, we developed a simple and sensitive high-performance liquid chromatography with fluorescence detection (HPLC-FL) method for simultaneous determination of DUT and its major active metabolite, 6β-hydroxydutasteride (H-DUT). Next, the pharmacokinetic interactions of DUT with ketoconazole (KET), a potent CYP3A inhibitor, were comprehensively investigated. In vivo rat intravenous and oral studies revealed that the pharmacokinetics of DUT and H-DUT were significantly altered by the co-administration of KET. Furthermore, the in vitro microsomal metabolism, blood distribution, and protein-binding studies suggest that the altered pharmacokinetics of DUT could be attributed primarily to the inhibition of the DUT metabolism by KET. To the best of our knowledge, this is the first study to show the drug interaction potential of DUT with azole antifungal drugs including KET, together with a newly developed HPLC-FL method for the simultaneous quantification of DUT and H-DUT. Keywords: dutasteride; 6β-hydroxy dutasteride; HPLC; fluorescence; ketoconazole; CYP3A 1. Introduction Dutasteride (DUT; Figure1), marketed under the brand name Avodart (GlaxoSmithKline), is a selective, potent, competitive, and irreversible inhibitor of 5α-reductase (5AR) that catalyzes the intracellular conversion of testosterone to dihydrotestosterone [1]. Dihydrotestosterone is known to be closely associated with benign prostatic hyperplasia (BPH) and androgenetic alopecia [2–4]. To date, only two isozymes of 5AR have been identified: type 1, which is primarily found in the liver and skin, and type 2, which is predominantly found in the hair follicles and male genitalia [1,5]. DUT, a dual inhibitor of type-1 and type-2 5AR, was approved by the US Food and Drug Administration (FDA) in 2001 for the treatment of symptomatic benign prostatic hyperplasia [6]. Further, it was approved for androgenic alopecia in Korea and Japan [7]. Previous in vitro studies have shown that compared to finasteride, DUT more potently inhibited type-1 and type-2 5AR by 45- and 2.5-fold, respectively [8,9]. Pharmaceutics 2019, 11, 673; doi:10.3390/pharmaceutics11120673 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2019, 11, x FOR PEER REVIEW 2 of 15 Pharmaceuticscompared2019 to, 11finasteride,, 673 DUT more potently inhibited type-1 and type-2 5AR by 45- and 2.5-fold,2 of 16 respectively [8,9]. Dutasteride 6β-hydroxydutasteride Ketoconazole Celecoxib FigureFigure 1. Chemical1. Chemical structures structures of dutasterideof dutasteride (DUT), (DUT), 6β -hydroxydutasteride6β-hydroxydutasteride (H-DUT), (H-DUT), ketoconazole ketoconazole (KET),(KET), and and celecoxib celecoxib (internal (internal standard). standard). InIn humans, humans, the the oral oral bioavailability bioavailability ofof DUTDUT is approximately approximately 60%, 60%, and and ranges ranges between between 40% 40% and and94%. 94%. DUT DUT has has a relatively a relatively large large volume volume of distribu of distributiontion and and high high plasma plasma protein protein binding. binding. The Themajor majorelimination elimination route route of DUT of DUT is cytochrome is cytochrome P450 P450(CYP) (CYP) 3A4/3A5-mediated 3A4/3A5-mediated hepatic hepatic metabolism. metabolism. Among Amongthree threemajor major and andtwo two minor minor metabolites, metabolites, 6β 6-hydroxydutasterideβ-hydroxydutasteride (H-DUT; (H-DUT; FigureFigure1 )1) exerts exerts 5AR 5AR inhibitioninhibition activity activity similar similar to to that that of of DUT DUT [6 ].[6]. Only Only trace trace amounts amounts of of unchanged unchanged DUT DUT are are excreted excreted in in thethe urine urine [7 ].[7]. The The terminal terminal half-life half-lif ofe of DUT DUT after after reaching reaching a steadya steady state state is is approximately approximately 5 weeks5 weeks [6 ].[6]. AlthoughAlthough the the toxicodynamics toxicodynamics (relationship (relationship between between drug drug concentration concentration and and drug-related drug-related toxicity) toxicity) of of DUTDUT is currentlyis currently unknown, unknown, the the FDA FDA prescription prescription drug drug label label of of DUT DUT indicates indicates that that metabolic metabolic drug drug interactionsinteractions may may occur occur when when DUT DUT is administeredis administered in in combination combination with with potent potent CYP3A CYP3A inhibitors inhibitors such such asas ketoconazole ketoconazole (KET), (KET), ritonavir, ritonavir, and and troleandomycin troleandomycin [6 ].[6]. To To date, date, however, however, this this issue issue has has not not been been adequatelyadequately addressed addressed by by any any systematic systematic preclinical preclinical and and clinical clinical studies. studies. AsAs shown shown in in Table Table S1, S1, several several methods methods using using liquid liquid chromatography chromatography coupled coupled with with tandem tandem mass mass detectordetector (LC-MS (LC-MS/MS)/MS) and and high-performance high-performance liquid liquid chromatography chromatography coupled coupled with with UV UV/Vis/Vis detector detector (HPLC-UV)(HPLC-UV) were were developed developed for for the the quantitation quantitation of DUT of DUT in rat in andrat and human human plasma. plasma. However, However, some some of theseof these methods methods were notwere fully not validated, fully validated, used a large used sample a large volume, sample and volume,/or had drawbacksand/or had associated drawbacks withassociated sample preparationwith sample procedurespreparation such procedures as liquid–liquid such as liquid–liquid extraction and extraction solid phase and extraction,solid phase i.e.,extraction, high cost, i.e., irregular high cost, recovery, irregular and recovery,/or potential and/or hazards potential associated hazards with associated highly with volatile highly solvents. volatile Moreover,solvents. LC-MS Moreover,/MS systemsLC-MS/MS require systems high instrumentationrequire high instrumentation and maintenance and expenses,maintenance which expenses, may notwhich be aff ordablemay not for be manyaffordable institutions for many in resource-limited institutions in resource-limited conditions [10,11 conditions]. In this regard, [10,11]. HPLC In this withregard, fluorescence HPLC with detection fluorescence (HPLC-FL) detection methods (HPLC- canFL) be amethods good alternative, can be a good because alternative, they are because more accessiblethey are andmore cost accessible effective and than cost LC-MS effective/MS systems,than LC-MS/MS and they systems, generally and off theyer better generally sensitivity offer andbetter selectivitysensitivity than and HPLC-UV selectivity systems than HPLC-UV [12,13]. Additionally, systems [12,13]. the bloodAdditionally, levels of the H-DUT, blood the levels major of activeH-DUT, metabolite,the major need active to metabolite, be monitored need together to be monitored with those to ofgether DUT [with6]. To those the best of DUT of our [6]. knowledge, To the best there of our haveknowledge, been no studies there have showing been a validatedno studies HPLC-FL showing method a validated for the HPLC-FL quantitative method determination for the quantitative of DUT anddetermination H-DUT in biological of DUT and matrices. H-DUT in biological matrices. Pharmaceutics 2019, 11, 673 3 of 16 In this study, a simple bioanalytical method using the HPLC-FL system was developed for the simultaneous quantitation of DUT and H-DUT in rat plasma and was validated for its original application to in vivo rat pharmacokinetic study on DUT. The potential of the pharmacokinetic drug interaction of DUT with KET (Figure1), an azole antifungal drug that is a potent CYP3A inhibitor [ 11,14], was investigated in terms of in vitro rat and human microsomal metabolism, in vitro protein binding, and in vivo rat pharmacokinetics. Approximately 50% of men aged over 50 years have pathological evidence of BPH and this number increases to >80% of men aged over 80 years [15]. Moreover, almost a billion people are estimated to have skin, nail and hair fungal infections, several tens of millions of people have mucosal candidiasis, and more than 150 million people have serious fungal diseases, which are fatal or significantly affect quality of life [16].