1521-009X/44/4/570–575$25.00 http://dx.doi.org/10.1124/dmd.115.067397 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:570–575, April 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Identification of Human Sulfotransferases Involved in Lorcaserin N-Sulfamate Formation

Abu J. M. Sadeque, Safet Palamar,1 Khawja A. Usmani, Chuan Chen, Matthew A. Cerny,2 and Weichao G. Chen3

Department of Drug Metabolism and Pharmacokinetics, Arena Pharmaceuticals, Inc., San Diego, California

Received September 30, 2015; accepted January 7, 2016

ABSTRACT Lorcaserin [(R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benza- and among the SULT isoforms SULT1A1 was the most efficient. The zepine] hydrochloride hemihydrate, a selective serotonin 5-hydroxy- order of intrinsic clearance for lorcaserin N-sulfamate is SULT1A1 > Downloaded from tryptamine (5-HT) 5-HT2C receptor agonist, is approved by the U.S. SULT2A1 > SULT1A2 > SULT1E1. Inhibitory effects of lorcaserin Food and Drug Administration for chronic weight management. N-sulfamate on major human cytochrome P450 (P450) enzymes Lorcaserin is primarily cleared by metabolism, which involves were not observed or minimal. Lorcaserin N-sulfamate binds to multiple enzyme systems with various metabolic pathways in human plasma protein with high affinity (i.e., >99%). Thus, despite humans. The major circulating metabolite is lorcaserin N-sulfamate. being the major circulating metabolite, the level of free lorcaserin Both human liver and renal cytosols catalyze the formation of N-sulfamate would be minimal at a lorcaserin therapeutic dose and lorcaserin N-sulfamate, where the liver cytosol showed a higher unlikely be sufficient to cause drug-drug interactions. Considering its dmd.aspetjournals.org catalytic efficiency than renal cytosol. Human sulfotransferases formation kinetic parameters, high plasma protein binding affinity, (SULTs) SULT1A1, SULT1A2, SULT1E1, and SULT2A1 are involved minimal P450 inhibition or induction potential, and stability, the in the formation of lorcaserin N-sulfamate. The catalytic efficiency of potential for metabolic drug-drug interaction or toxicological effects these SULTs for lorcaserin N-sulfamate formation is widely variable, of lorcaserin N-sulfamate is remote in a normal patient population. at ASPET Journals on September 25, 2021

Introduction of cytosolic enzymes, catalyze this reaction by transferring a sulfuryl 9 9 Lorcaserin hydrochloride hemihydrate*, a selective serotonin group from its cosubstrate 3 -phosphoadenosine-5 -phosphosulfate 5-hydroxytryptamine (5-HT) 5-HT receptor agonist, is approved (PAPS) to a nucleophilic acceptor molecule (substrate), producing a 2C 9 9 by the U.S. Food and Drug Administration for chronic weight sulfate conjugate and desulfated 3 -phosphodenosine 5 -phosphate management. It is a secondary amine-containing benzazepine [(R)-8- (Schwartz, 2005). In general, sulfate conjugation leads to an increase chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine] (Fig. 1). in hydrophilicity, and thus facilitates the excretion of the conjugated Lorcaserin is primarily cleared by metabolism. Multiple meta- molecule. Therefore, sulfate conjugation is often considered to be an bolic pathways such as oxidation, glucuronide conjugation, and inactivation and detoxification process for xenobiotics and endobiotics sulfo-conjugation have been identified both in vitro and in vivo. Mul- (Pacifici and Coughtrie, 2005). Chemically stable sulfo-conjugates, such tiple cytochrome P450 (P450) (Usmani et al., 2012) and UDP- as N-sulfo-conjugates of aromatic amines, are known to be readily glucuronosyltransferase (Sadeque et al., 2012) enzymes associated excreted without toxicological consequences (Stillwell et al., 1994; Glatt, with major metabolic pathways of lorcaserin have been reported; 2005). However, sulfo-conjugates of certain chemicals, such as benzylic however, the sulfotransferases (SULTs) involved in its sulfo-conjugation and allylic alcohols and aromatic hydroxylamines, are short-lived and have not yet been reported. Lorcaserin N-sulfamate is the major circulating electrophilic (DeBaun et al., 1970; Miller, 1970, 1994; Glatt, 2005). metabolite of lorcaserin. These may react with DNA and other cellular nucleophiles. Such sulfo- Sulfo-conjugation is an important metabolic reaction in the de- conjugations occur primarily through O-sulfation rather than N-sulfation. toxification, biosynthesis, and homeostasis of essential endogenous While sulfo-conjugates are generally biologically inactive, some can compounds (Coughtrie, 2002; Strott, 2002). SULTs, a supergene family retain their biologic activity—such as cholesterol sulfate, which stimu- lates the differentiation of skin epidermal cells (Elias et al., 1984), and pregnenolone sulfate, which binds and suppresses the GABA receptor 1 Current affiliation: Drug Metabolism & Pharmacokinetics, Novartis Institutes (Corpéchot et al., 1981). Lorcaserin N-sulfamate shows no binding for BioMedical Research, East Hanover, New Jersey. affinity to serotonin 5-HT receptors 5-HT2A,5-HT2B, and 5-HT2C. 2Current affiliation: Pharmacokinetics, Dynamics and Metabolism Department, Among other metabolic transformations reported earlier (Sadeque Pfizer Global Research and Development, Groton, Connecticut. 3Current affiliation: Drug Metabolism & Pharmacokinetics, Vertex Pharmaceu- et al., 2012; Usmani et al., 2012), lorcaserin undergoes sulfo-conjugation ticals, San Diego, California. through the secondary amine nitrogen, forming an N-sulfamate (Fig. 1). *Trade Name of Lorcaserin hydrochloride hemihydrate is BELVIQ. Therefore, the lorcaserin N-sulfo-conjugate does not fit the characteristics dx.doi.org/10.1124/dmd.115.067397. of chemically unstable sulfo-conjugates reported previously (Glatt,

ABBREVIATIONS: CLint, intrinsic clearance; HPLC, high-performance liquid chromatography; 5-HT, 5-hydroxytryptamine; LC-MS/MS, liquid chromatography–tandem mass spectrometry; PAPS, 39-phosphoadenosine-59-phosphosulfate; P450, cytochrome P450; SULT, sulfotransferase.

570 Human Sulfotransferases Involved in Lorcaserin Metabolism 571

2005), and instead is stable and can be readily excreted. Its P450 inhibition, formation kinetic parameters, and plasma protein binding characteristics may help to further rule out potential drug-drug interac- tions for lorcaserin. In this paper, we describe the characterization of lorcaserin N-sulfamate through identification of SULTs that catalyze its forma- tion, inhibition, and induction potential on P450 enzymes and its affinity for plasma proteins. In light of these characteristics, we discuss the potential of lorcaserin N-sulfamate for toxicological consequences and metabolic drug-drug interactions.

Materials and Methods Chemicals and Enzyme Source Lorcaserin hydrochloride hemihydrate, (R)-8-chloro-1-methyl-2,3,4,5- tetrahydro-1H-benzo[d]azepine hydrochloride hemihydrate, was synthesized by Cilag AG (Schaffhausen, Switzerland). Lorcaserin N-sulfamate, (R)-8- Fig. 2. Michaelis-Menten kinetic plot of lorcaserin N-sulfamate formation by chloro-1-methyl-4,5-dihydro-1H-benzo[d]azepine-3(2H)-sulfonic acid, and human liver cytosol. Downloaded from lorcaserin N-sulfamate-d6 (internal standard) were synthesized at Arena Pharmaceuticals, Inc. PAPS tetralithium salt, tris-HCl, and trizma-base were protein/ml), bringing the final volume to 500 ml. The incubations were continued purchased from Sigma (St. Louis, MO). High-purity, high-performance liquid for 20 minutes, and then terminated by the addition of 500 ml acetonitrile and chromatography (HPLC)–grade acetonitrile and high-purity HPLC-grade mixed thoroughly. Incubations without the addition of PAPS were conducted as deionized water were purchased from Burdick and Jackson (Muskegon, MI). the negative control (blank) under identical conditions. Incubations were Ultrapure distilled water was purchased from Invitrogen Corporation (Carlsbad, performed in triplicate. dmd.aspetjournals.org CA). Formic acid was purchased from EMD Chemicals Inc. (Gibbstown, NJ). Assay for P450 enzyme inhibition potential by lorcaserin N-sulfamate. The Human liver microsomes (pooled mixed gender, n = 50), liver and renal cytosol metabolic incubations in human liver microsomes with specific P450 probe (pooled mixed gender, n = 20) were purchased from Xenotech (Lenexa, KS). substrates and lorcaserin N-sulfamate were conducted using a 96-well plate Human recombinant SULTs, SULT1A1, SULT1A2, SULT1A3, SULT1E1, and format. The incubation mixture contained 0.25 mg/ml microsomal protein, SULT2A1, produced from insect cells transfected with a baculovirus 0–200 mMlorcaserinN-sulfamate (test compounds as an inhibitor), P450- containing human SULT cDNA, were purchased from Invitrogen Corporation. specific substrates (at respective Km values), 1 mM b-NADPH, and 100 mM Male and female human plasma pooled from different donors with sodium potassium phosphate buffer containing 3 mM MgCl2 and 1 mM EDTA (pH 7.4) heparin as an anticoagulant were obtained from Bioreclamation, Inc. (East m in a final volume of 100 l. The following P450-specific substrates were added at ASPET Journals on September 25, 2021 Meadow, NY). to the incubation mixture at a concentration close to their respective Km values: Metabolic Assays. Initially, the liver cytosol preparation was used to phenacetin (36 mM), bupropion (100 mM), paclitaxel (5 mM), tolbutamide optimize the reaction conditions using para-nitrophenol, a probe substrate (150 mM), (S)-mephenytoin (50 mM), dextromethorphan (5 mM), and commonly used to measure SULT activity, according to the method described midazolam (8 mM) for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, previously (Gamage et al., 2003). Following initial optimization, the incubation CYP2D6, and CYP3A4, respectively. The reaction mixture was incubated at conducted for lorcaserin N-sulfamate formation for each enzyme system used is 37C for 20 minutes for CYP1A2, 30 minutes for CYP2C8, and 10 minutes for described subsequently. CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Parallel control Enzyme kinetics assays for lorcaserin N-sulfamate formation with cytosol. incubations conducted with P450 substrates without the addition of lorcaserin The incubation mixtures (500 ml final volume) consisted of human liver or renal N-sulfamate (as an inhibitor) were used as the positive control. Parallel cytosol (1 mg/ml), tris-HCl buffer (100 mM, pH 7.4), and an appropriate incubations were also performed without the addition of b-NADPH in the concentration of lorcaserin (0–10 mM). The reaction mixture was preincubated reaction mixture as a blank control (negative control). At the end of the at 37C in a shaking water bath for 3 minutes, and then the reaction was initiated incubation period, the incubations were terminated with the addition of an by the addition of PAPS (100 mM). Incubations were continued for 25 minutes, equal volume of acetonitrile containing the internal standard. The mixtures and then terminated by the addition of 500 ml acetonitrile. All incubations were were then centrifuged at 4000 rpm for 20 minutes and the supernatant was performed in triplicate. Incubation with para-nitrophenol was conducted as a transferred to a clean 96-well microtiter plate. The samples were subjected to positive substrate control. Incubations without the addition of PAPS were also liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. conducted as a negative control under identical conditions to rule out the possible Enzymatic assay sample processing. After termination of the incubation with involvement of a nonenzymatic process. an equal volume of acetonitrile, samples were vortexed and centrifuged for Assay for lorcaserin N-sulfamate formation with human recombinant 10 minutes at 13,000 rpm in a microcentrifuge. The supernatant was transferred SULTs. Incubations were carried out as described subsequently with the final to a clean 96-well microtiter plate and subjected to LC-MS/MS analysis. reagent concentrations given in parentheses. The incubation mixtures consisted Assay for P450 enzyme induction by lorcaserin N-sulfamate in human of tris-HCl buffer (100 mM, pH 7.5), PAPS (100 mM), and lorcaserin (0–10 mM hepatocytes. Lorcaserin N-sulfamate (0.2, 2, and 20 mM) and the known P450 as appropriate depending on the SULT used). The incubation mixture was inducers 3-methylcholanthrene, phenobarbital, and rifampicin were incubated in preincubated at 37C in a shaking water bath for 3 minutes, and then the reaction cultures of human hepatocytes from three separate donors (two male and one was initiated by the addition of human recombinant SULT enzymes (15–20 mg female) for three consecutive days. Cells were harvested and microsomes were prepared (LeCluyse et al, 2000). The activities of CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 were determined using P450-selective marker substrates (described in the previous section). Messenger RNA levels for each of these P450 isozymes were analyzed by quantitative reverse-transcription polymerase chain reaction (TaqMan). Plasma protein binding assay for lorcaserin N-sulfamate. Plasma protein Fig. 1. In vitro formation of lorcaserin N-sulfamate from lorcaserin catalyzed by binding of lorcaserin N-sulfamate was determined using equilibrium dialysis. cytosol from human liver, kidney and by recombinant SULTs in the presence of Three concentrations of lorcaserin N-sulfamate (0.1, 1, and 10 mM) and human PAPS. plasma proteins were incubated for 24 hours in a 37C humidified incubator. 572 Sadeque et al.

Results Lorcaserin N-Sulfamate Formation Catalyzed by Liver and Renal Cytosol Formation of lorcaserin N-sulfamate is catalyzed by human cytosolic SULTs in the presence of PAPS (Fig. 1). In this study, the formation rate of lorcaserin N-sulfamate in vitro in liver and renal cytosol of humans was examined. Incubations were conducted with liver and renal cytosol fractions in the presence of PAPS. The rate constants Km and Vmax for lorcaserin N-sulfamate formation for liver and renal cytosol were calculated from a nonlinear least-square fit to the Michaelis- Menten equation (Figs. 2 and 3). The kinetic parameters Km and Vmax and the intrinsic clearance (CLint = Vmax/Km) are presented in Table 1. The formation of lorcaserin N-sulfamate in human liver cytosol was inhibited by approximately 80% in the presence of 1 mM of 2,6- Fig. 3. Michaelis-Menten kinetic plot of lorcaserin N-sulfamate formation by dichloro-4-nitrophenol (data not shown), a known inhibitor generally human renal cytosol. used for the inhibition of SULT activity (Seah and Wong, 1994). Downloaded from

Lorcaserin N-Sulfamate Formation Catalyzed by Recombinant After incubation for 24 hours, 200-ml aliquots of the solutions in the donor and SULTs receiver chambers for each condition were transferred to 1.2-ml 8-well tube Initial metabolic screening was performed with five recombinant strips. A 200-ml aliquot of the dosing solution was also sampled for analysis. The human SULTs, SULT1A1, SULT1A2, SULT1A3, SULT1E1, and proteins were precipitated with the addition of three volumes of ice-cold SULT2A1 (Fig. 4). Of these, SULT1A3 did not catalyze lorcaserin acetonitrile containing internal standard (100 ng/ml lorcaserin sulfamate-d6) dmd.aspetjournals.org followed by centrifugation at 3000g for 30 minutes. The resulting supernatant N-sulfamate formation and was therefore excluded from further (70 ml) was mixed with an equal volume of HPLC-grade water on a 96-well kinetic analyses. The remaining SULTs showed differential activity, sample plate. The samples were then analyzed by LC-MS/MS. The analyte with SULT1A1 exhibiting the highest activity and SULT1E1 the lowest concentration was determined by comparing with a standard curve of lorcaserin activity. SULT1A2 and SULT2A1 showed modest activity. The kinetic N-sulfamate generated under identical conditions of protein binding sample parameters are reported in Table 1 and a representative Michaelis-Menten preparation. kinetic plot with SULT1A1 is shown in Fig. 5. The order of catalytic Samples analysis by LC-MS/MS. Analysis of lorcaserin N-sulfamate was efficiency of human SULTs for lorcaserin N-sulfamate formation is as carried out with an Aria LX-2 HPLC system coupled with an MDS Sciex API- at ASPET Journals on September 25, 2021 follows: SULT1A1 (CLint = 255 ml/min/mg) . SULT2A1 (CLint = 4000 triple quadruple mass spectrometer (Applied Biosystems/MDS Sciex, 19.31 ml/min/mg) . SULT1A2 (CL = 2.86 ml/min/mg) . SULT1E1 Foster City, CA). A HALO C18, 3 Â 30 mm and 2.7 mm (Mac-Mod Analytical int (CL = 1.85 ml/min/mg). Inc., Chadds Ford, PA) column was used for separating lorcaserin N-sulfamate int and the internal standard at room temperature. The mobile phase consisted of Human Liver P450 Enzyme Inhibition and Induction. To solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in evaluate the inhibitory effect of lorcaserin N-sulfamate on major acetonitrile). The analysis was performed with a stepwise gradient from 10% to P450 enzymes, the IC50 values for the inhibition of the activity of 95% of solvent B over a time period of approximately 2 minutes, followed by seven major P450 enzymes was evaluated. The IC50 value for the re-equilibration with initial solvent conditions (10% solvent B). The flow rate inhibition of CYP1A2-mediated phenacetin O-deethylase, CYP2B6- of the mobile phase was 0.6 ml/min throughout the run. The detection of mediated bupropion hydroxylase, CYP2C8-mediated paclitaxel lorcaserin N-sulfamate was achieved using electrospray ionization in negative- 6a-hydroxylase, CYP2C19-mediated (S)-mephenytoin 49-hydroxylase, ion mode. The source temperature was set at 500Cwithanion-sprayvoltage CYP2D6-mediated dextromethorphan O-demethylase, and CYP3A4- 2 of 4200 V. Multiple-reaction monitoring was used to acquire data. The Q1/Q3 mediated midazolam 19-hydroxylase was .100 mM (Fig. 6). However, (precursor/product) ions monitored for lorcaserin N-sulfamate and the internal the IC50 value for CYP2C9-mediated tolbutamide 49-hydroxylase was standard were 274.0/79.8 and 280/79.8 atomic mass units, respectively. The m quantification of lorcaserin N-sulfamate was achieved using a reference 10.3 M (2840 ng/ml) (Table 2). As shown in Table 2, in the presence standard curve generated with authentic lorcaserin N-sulfamate (0.001– of increasing concentrations of human serum albumin in microsomal 10 mM concentration range). The samples for the determination of inhibitory incubations, the IC50 value for CYP2C9 inhibition was increased such effect of lorcaserin N-sulfamate on the activity of human P450–probe that at a physiologically relevant albumin concentration of 500 mM, the substrates were analyzed according to the method described previously IC50 value observed for CYP2C9 inhibition was .200 mM (Fig. 7). (Usmanietal.,2012). There was no induction observed for major P450 enzymes when Enzyme kinetic analysis. The data obtained were analyzed for enzyme kinetic parameters Km and Vmax, using Sigma Plot software (Systat Software Inc., Richmond, CA), which generated a nonlinear least-square fit to the Michaelis- TABLE 1

Menten equation. The apparent Km value (Michaelis-Menten constant for Kinetic parameters of lorcaserin N-sulfamate formation catalyzed by cytosol from substrate affinity) was calculated as the substrate concentration at half of the human liver, (coma) kidney and by human recombinant SULTs

Vmax value (maximum velocity of the reaction). The intrinsic clearance (CLint) SULT Enzyme Source Km Vmax CLint was calculated as CLint = Vmax/Km (Segel, 1976). Calculation for percentage of plasma protein bound. The percentage of mM nmol/min/mg protein ml/min/mg protein the free lorcaserin N-sulfamate [fraction unbound (%fu)] was calculated Human liver cytosol 1700 0.220 0.130 according to the formula described previously (Wright et al., 1996), i.e., %fu = Human renal cytosol 1470 0.0125 0.00850 [(concentration in receiver chamber) 4 (concentration in donor chamber)] Â SULT1A1 742.07 189.26 255 100%. The percentage of the lorcaserin N-sulfamate bound to plasma proteins SULT1A2 5419.56 15.52 2.86 SULT1E1 368.30 0.68 1.85 [plasma protein bound (%PB)] was determined according to the following SULT2A1 3210.06 61.98 19.31 formula: %PB = 100 2 %fu. Human Sulfotransferases Involved in Lorcaserin Metabolism 573

Fig. 4. Formation of lorcaserin N-sulfamate catalyzed by human recombinant SULTs. A wide range of lorcaserin concentrations was used (0–1000 mM) for initial screening of recombinant SULT activity toward lorcaserin N-sulfamate formation prior to kinetic analysis. Downloaded from cultured human hepatocytes were treated with lorcaserin N-sulfamate (Elias et al., 1984), and pregnenolone sulfate, which binds and (up to 20 mM) for 72 hours, suggesting that lorcaserin N-sulfamate is suppresses the GABA receptor (Corpéchot et al., 1981). However, not a human P450 enzyme inducer (data not shown). sulfo-conjugation via N-sulfation, such as N-sulfonation of aromatic amines, yields stable sulfo-conjugates that are readily excreted without Plasma Protein Binding toxicological consequences (Stillwell et al., 1994; Glatt, 2005). In this paper, we describe the formation and systematic characterization of m N dmd.aspetjournals.org Three concentrations, 0.1, 1.0, and 10 M, of lorcaserin -sulfamate lorcaserin N-sulfamate, formed by human liver or renal cytosol and were used for its plasma protein binding affinity determination. There recombinant SULTs. The lorcaserin sulfo-conjugate is not the product , N , was 1% of lorcaserin -sulfamate unbound ( 1%fu) to human of O-sulfonation, but rather N-sulfonation, and is known to be . m plasma proteins ( 99% bound) at 10 M, whereas with low concen- chemically stable (Stillwell et al., 1994; Glatt, 2005). Thus, toxicolog- m trations (0.1 and 1 M) the unbound fraction was below the limit of ical consequences of lorcaserin N-sulfamate are considered to be quantification. The data are presented in Table 3. unlikely. Prior to the characterization of lorcaserin N-sulfamate formation kinetics, the SULT activity of the cytosolic preparations was verified at ASPET Journals on September 25, 2021 Discussion using para-nitrophenol, a known substrate for SULTs. Typical biphasic Sulfo-conjugates of xenobiotics and endobiotics are generally kinetics with initial product formation (para-nitrophenol-sulfate) at low biologically inactive and hydrophilic; therefore, this represents a para-nitrophenol concentration followed by inhibition of its own product biologically important excretion pathway (Stillwell et al., 1994; formation at higher concentration was observed (data not shown) as Coughtrie, 2002; Strott, 2002; Glatt, 2005). However, the structural reported previously (Gamage et al., 2003). The kinetic parameters for features of certain sulfo-conjugated molecules may elicit various lorcaserin N-sulfamate formation showed that the Vmax and CLint values biological effects. For example, the O-sulfo-conjugates of aromatic for liver cytosol are about 17- and 15-fold higher, respectively, than that hydroxylamines, benzylic and allylic alcohols are known to be short- for renal cytosol, suggesting that lorcaserin N-sulfamate is predominantly lived and electrophilic, and may therefore bind to cellular nucleophiles, formed by liver SULTs. An initial screening with recombinant SULTs including DNA (DeBaun et al., 1970; Glatt, 2005). In addition, some indicated that SULT1A1, SULT1A2, SULT1E1, and SULT2A1 were sulfo-conjugates show biological activity, such as cholesterol sulfate, involved in lorcaserin N-sulfamate formation, whereas SULT1A3 which is known to stimulate differentiation of skin epidermal cells showed minimal or no activity (Fig. 4). The CLint values of SULT1A1

Fig. 6. Inhibitory effect of lorcaserin N-sulfamate on human liver microsomal P450 enzymes. The inhibitory effects of lorcaserin N-sulfamate on the catalytic activities Fig. 5. Michaelis-Menten kinetic plot for lorcaserin N-sulfamate formation by of P450 selective probe substrates in human liver microsomes were measured in the human recombinant SULT1A1. presence of various concentrations of lorcaserin N-sulfamate (0–200 mM). 574 Sadeque et al.

TABLE 2 TABLE 3

IC50 values for the inhibition of CYP2C9-mediated tolbutamide 49-hydroxylase Percentage of lorcaserin N-sulfamate bound to male and female human activity by lorcaserin N-sulfamate in the presence of human serum albumin plasma proteins

Human Serum Albumin IC50 Percentage of Plasma Protein Lorcaserin N-Sulfamate Bound (Mean 6 S.D., n =3) mM mM Concentration 0 10.3 (9.27a) Male Female 10 14.6 mM%% 100 85.8 6 6 500 . 200 10.0 99.97 0.00 99.97 0.01 1.0 BLOQa BLOQ aThis value was calculated based on the free fraction of lorcaserin N-sulfamate determined in 0.1 BLOQ BLOQ human liver microsomal protein. BLOQ, below the limit of quantification. aThe concentration of lorcaserin N-sulfamate in the receiver chamber of the equilibrium dialysis device was below the limit of quantification. are about 89-, 13-, and 137-fold higher compared with SULT1A2, SULT2A1, and SULT1E1, respectively, suggesting that SULT1A1 is the predominant SULT that catalyzes lorcaserin N-sulfamate formation in to SUL1A2, SULT2A1 also showed low affinity for lorcaserin humans (Table 1). SULT1A1, SULT2A1, and SULT1E1 account for N-sulfamate formation. The active site of SULT2A1 appears to Downloaded from 53%, 27%, and 6%, respectively, of total SULTs in human liver, whereas accept more hydrophobic substrates such as steroids, steroid derivates, in the human kidney these values are 40%, 1%, and 0%, respectively and bile acids (Comer et al., 1993; Huang et al., 2010). Additionally, (Riches et al., 2009). Therefore, the SULTs that are involved in lorcaserin specific binding orientation in SULT2A1 is required for specific N-sulfamate catalysis constituted approximately 86% of the total SULTs substrates such as dehydroepiandrosterone and androsterone (Lu expressed in human liver compared with about 41% in kidney, suggesting et al., 2008). This may be the reason why lorcaserin is not a high- that the liver is the primary metabolic site for lorcaserin N-sulfamate affinity substrate for SULT2A1. formation. It is noteworthy that three members of the SULT1 family Comparing all of the SULTs used for lorcaserin N-sulfamate for- dmd.aspetjournals.org (SULT1A1, SULT1A3, and SULT1E1) show very different kinetic mation, SULT1A1 appears to be the most catalytically efficient. Based behavior for lorcaserin N-sulfamate formation. This may reflect the on the CLint values, the order of catalytic efficiency is SULT1A1 . differential nature of the substrate binding pocket of these isoforms SULT2A1 . SULT1A2 . SULT1E1. As indicated by the kinetic (Gamage et al., 2003). The SULT1A1 binding pocket accepts a wide parameters, lorcaserin does not appear to be a prototypical high-affinity range of molecules, such as simple planar-substituted phenols (Brix et al., substrate for human SULT enzymes. Thus, drug-drug interactions arising 1999), as well as very large lipophilic substrates (Harris et al., 2000; Li through the SULT-mediated metabolic pathway are not anticipated. et al., 2001) and polycyclic aromatic compounds (Grimm et al., 2013). Additionally, although lorcaserin N-sulfamate is the major metabolite in at ASPET Journals on September 25, 2021 Lorcaserin is a secondary amine-containing benzazepine that is more circulation, it accounts for only approximately 3% of the total lorcaserin lipophilic due to the aryl chloride portion of the molecule and thereby dose in human urine, whereas the major clearance pathways of lorcaserin may be well suited for binding to the hydrophobic pocket of SULT1A1. are oxidation by multiple P450 enzymes (Usmani et al., 2012) and Although lorcaserin is a nonplanar molecule, the strongly hydrophobic glucuronidation by multiple UDP-glucuronosyltransferase enzymes and flexible nature of the active site of SULT1A1 may accommodate (Sadeque et al., 2012). The observed pharmacokinetic half-life of lorcaserin as a better substrate compared with other SULTs reported in lorcaserin N-sulfamate after lorcaserin dosing in humans is much longer ; ; this study. Lorcaserin has a pKa value of 9.53 and the secondary amine in than the parent ( 40 versus 11 hours). One likely explanation for the its cyclic aliphatic moiety may become protonated under physiologic high levels of lorcaserin N-sulfamate in circulation and the prolonged reaction pH (7.4). However, lorcaserin appears not to be a substrate for observed half-life is that the very high plasma protein binding affinity SULT1A3, which may be due to the lack of an aliphatic amine containing (.99%) renders only a small free fraction of the metabolite in circulation an open side chain with at least two carbons, a characteristic of preferred available for tissue distribution and elimination. We also observed that substrates (e.g., dopamine or tyramine) (Brix et al., 1999). SULT1E1 lorcaserin N-sulfamate inhibits human liver microsomal tolbutamide shows high selectivity for endogenous estrogens such as 17b-estradiol hydroxylation with an IC50 value of ;10 mM, a reaction catalyzed by as well as for a number of other important drugs including 17a-ethinylestradiol (Forbes-Bamforth and Coughtrie 1994; Zhang et al., 1998). Poor catalytic efficiency of SULT1E1 for lorcaserin N-sulfonation is consistent with previous literature reports wherein fused heterocyclic compounds with secondary amines underwent very negligible sulfonation by SULT1E1; however, such compounds are efficiently catalyzed by SULT1A1 (Cole et al., 2010), which is consistent with the finding that lorcaserin is a better substrate for SULT1A1 than SULT1E1. Despite SULT1A2 being closely related to SULT1A1 in amino acid sequence homology, their substrate affinities are very different. Unlike SULT1A1, simple phenolic compounds are poor substrates for SULT1A2. In the case of SULT1A2, the phenolic ends of two tyrosine moieties, Tyr149 and Tyr240, extend into the substrate binding pocket, which makes it more hydrophilic (thus, compounds such as aromatic hydroxamic acids are preferred), possibly through interaction of its hydrophilic aliphatic region (Meinl et al., Fig. 7. Inhibitory effect of lorcaserin N-sulfamate on human liver microsomal CYP2C9 in the presence 10, 100, and 500 mM of human serum albumin. The 2002; Lu et al., 2010). The hydrophobic chloro-benzene moiety of inhibitory effect of lorcaserin N-sulfamate on CYP2C9 decreased with increasing lorcaserin likely makes it a poor substrate for SULT1A2. Similar concentration of human serum albumin in the incubation mixture. Human Sulfotransferases Involved in Lorcaserin Metabolism 575

Comer KA, Falany JL, and Falany CN (1993) Cloning and expression of human liver CYP2C9. However, the IC50 value becomes .200 mM in the presence of dehydroepiandrosterone sulphotransferase. Biochem J 289:233–240. physiologic concentration of human serum albumin (Fig. 7). Addi- Corpéchot C, Robel P, Axelson M, Sjövall J, and Baulieu EE (1981) Characterization and tionally, lorcaserin N-sulfamate has no or minimal inhibitory effect on measurement of dehydroepiandrosterone sulfate in rat brain. Proc Natl Acad Sci USA 78: 4704–4707. other major P450 enzymes (Fig. 6) and also is not a time-dependent Coughtrie MW (2002) Sulfation through the looking glass—recent advances in sulfotransferase inhibitor of CYP2C9 (data not shown). On the other hand, although research for the curious. Pharmacogenomics J 2:297–308. 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Forbes-Bamforth KJ and Coughtrie MW (1994) Identification of a new adult human liver that only 10% of lorcaserin N-sulfamate is bound to microsomal sulfotransferase with specificity for endogenous and xenobiotic estrogens. Biochem Biophys protein, in contrast to .99% bound to human plasma protein. Thus, in Res Commun 198:707–711. Gamage NU, Duggleby RG, Barnett AC, Tresillian M, Latham CF, Liyou NE, McManus ME, an in vivo environment, the availability of free lorcaserin N-sulfamate and Martin JL (2003) Structure of a human carcinogen-converting enzyme, SULT1A1. incirculationwouldbeverynegligible at a clinical dose of Structural and kinetic implications of substrate inhibition. J Biol Chem 278:7655–7662. Glatt H (2005) Activation and inactivation of carcinogens by human sulfotransferases, in Human lorcaserin and is highly unlikely to cause metabolic inhibition of Cytosolic Sulfotransferases (Pacifici GM and Coughtrie MWH eds) pp 279–304, CRC Press/ the CYP2C9 pathway. Furthermore, at concentrations up to 20 mM, Taylor & Francis, Boca Raton, FL. Grimm FA, Lehmler HJ, He X, Robertson LW, and Duffel MW (2013) Sulfated metabolites of lorcaserin N-sulfamate did not induce any P450 enzymes in cultured polychlorinated biphenyls are high-affinity ligands for the thyroid hormone transport protein Downloaded from human hepatocytes treated for 72 hours. In comparison, the plasma transthyretin. Environ Health Perspect 121:657–662. Harris RM, Waring RH, Kirk CJ, and Hughes PJ (2000) Sulfation of “estrogenic” alkylphenols Cmax of lorcaserin N-sulfamate exceeds that of lorcaserin [mean and 17b-estradiol by human platelet phenol sulfotransferases. J Biol Chem 275:159–166. Cmax value = 0.29 mM (56.8 ng/ml), following 10 mg twice daily in Huang J, Bathena SP, Tong J, Roth M, Hagenbuch B, and Alnouti Y (2010) Kinetic analysis of bile acid sulfation by stably expressed human sulfotransferase 2A1 (SULT2A1). Xenobiotica humans] by 1- to 5-fold. Taken together, these results suggest that 40:184–194. lorcaserin N-sulfamate has a very low potential of affecting P450 LeCluyse E, Madan A, Hamilton G, Carroll K, DeHaan R, and Parkinson A (2000) Expression and regulation of cytochrome P450 enzymes in primary cultures of human hepatocytes. metabolic pathways by P450 enzyme inhibition or induction. J Biochem Mol Toxicol 14:177–188. dmd.aspetjournals.org In this study, we have demonstrated that lorcaserin undergoes Li X, Clemens DL, Cole JR, and Anderson RJ (2001) Characterization of human liver thermo- N-sulfation, thus forming a chemically stable sulfo-conjugate and an stable phenol sulfotransferase (SULT1A1) allozymes with 3,39,5-triiodothyronine as the sub- strate. J Endocrinol 171:525–532. excretory pathway (Glatt, 2005). We also demonstrated that although Lu J, Li H, Zhang J, Li M, Liu MY, An X, Liu MC, and Chang W (2010) Crystal structures of lorcaserin N-sulfo-conjugation is catalyzed by multiple SULTs, SULT1A2 and SULT1A1 *3: insights into the substrate inhibition and the role of Tyr149 in SULT1A2. Biochem Biophys Res Commun 396:429–434. lorcaserin does not appear to be a high-affinity substrate for these Lu LY, Hsieh YC, Liu MY, Lin YH, Chen CJ, and Yang YS (2008) Identification and charac- enzymes. Lorcaserin N-sulfamate is a minor clearance pathway in terization of two amino acids critical for the substrate inhibition of human dehydroepian- drosterone sulfotransferase (SULT2A1). Mol Pharmacol 73:660–668. humans since it accounts for only ;3% of lorcaserin dose in urine. Meinl W, Meerman JH, and Glatt H (2002) Differential activation of promutagens by alloen-

Furthermore, its high plasma protein binding affinity (.99%) is zymes of human sulfotransferase 1A2 expressed in Salmonella typhimurium. Pharmacoge- at ASPET Journals on September 25, 2021 netics 12:677–689. consistent with its long half-life in circulation. Importantly, it is Miller JA (1970) Carcinogenesis by chemicals: an overview—G. H. A. Clowes Memorial Lec- neither a clinically relevant P450 enzyme inhibitor nor an inducer. In ture. Cancer Res 30:559–576. Miller JA (1994) Sulfonation in chemical carcinogenesis—history and present status. Chem Biol conclusion, lorcaserin N-sulfamate has a very low probability of causing Interact 92:329–341. metabolic drug-drug interactions in a normal patient population. Pacifici GM and Coughtrie MW, editors (2005) Human Cytosolic Sulfotransferases, CRC Press/ Taylor & Francis, Boca Raton, FL. Riches Z, Stanley EL, Bloomer JC, and Coughtrie MW (2009) Quantitative evaluation of the Acknowledgments expression and activity of five major sulfotransferases (SULTs) in human tissues: the SULT “pie”. Drug Metab Dispos 37:2255–2261. The authors thank Laurie Turbeville, Paul Mawson, and Nathan Kozon for Sadeque AJ, Usmani KA, Palamar S, Cerny MA, and Chen WG (2012) Identification of human providing some enzymatic assay support and to Michael Ma and Salma Sarwary UDP-glucuronosyltransferases involved in N-carbamoyl glucuronidation of lorcaserin. Drug Metab Dispos 40:772–778. from the Department of Drug Metabolism and Pharmacokinetics at Arena Schwartz NB (2005) PAPS and sulfoconjugation, in Human Cytosolic Sulfotransferases (Pacifici Pharmaceuticals, Inc., for providing some liquid chromatography mass spec- GM and Coughtrie MWH eds) pp 43–60, CRC Press/Taylor & Francis, Boca Raton, FL. trometry analytical support. Seah VM and Wong KP (1994) 2,6-Dichloro-4-nitrophenol (DCNP), an alternate-substrate in- hibitor of phenolsulfotransferase. Biochem Pharmacol 47:1743–1749. Segel HI (1976) Biochemical Calculations, 2nd ed. Wiley, New York. Authorship Contributions Stillwell WG, Turesky RJ, Gross GA, Skipper PL, and Tannenbaum SR (1994) Human urinary excretion of sulfamate and glucuronide conjugates of 2-amino-3,8-dimethylimidazo[4,5- Participated in research design: Sadeque, Usmani, Palamar, C. Chen, f]quinoxaline (MelQx). Cancer Epidemiol Biomarkers Prev 3:399–405. Cerny, W. G. Chen. Strott CA (2002) Sulfonation and molecular action. Endocr Rev 23:703–732. Usmani KA, Chen WG, and Sadeque AJ (2012) Identification of human cytochrome P450 and Conducted experiments: Palamar, Usmani, C. Chen. flavin-containing monooxygenase enzymes involved in the metabolism of lorcaserin, a novel Performed data analysis: Palamar, Usmani, C. Chen. selective human 5-hydroxytryptamine 2C agonist. Drug Metab Dispos 40:761–771. Wrote or contributed to the writing of the manuscript: Sadeque. Wright JD, Boudinot FD, and Ujhelyi MR (1996) Measurement and analysis of unbound drug concentrations. Clin Pharmacokinet 30:445–462. Zhang H, Varlamova O, Vargas FM, Falany CN, and Leyh TS (1998) Sulfuryl transfer: the References catalytic mechanism of human estrogen sulfotransferase. J Biol Chem 273:10888–10892.

Brix LA, Barnett AC, Duggleby RG, Leggett B, and McManus ME (1999) Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: site-directed mu- Address correspondence to: Abu J. M. Sadeque, Arena Pharmaceuticals, Inc., tagenesis and kinetic studies. Biochemistry 38:10474–10479. 6154 Nancy Ridge Drive, San Diego, CA 92121. E-mail: asadeque@arenapharm. Cole GB, Keum G, Liu J, Small GW, Satyamurthy N, Kepe V, and Barrio JR (2010) Specific estrogen sulfotransferase (SULT1E1) substrates and molecular imaging probe candidates. Proc com Natl Acad Sci USA 107:6222–6227.