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Research ArticleArticle

Identification of Human Sulfotransferases Involved in Lorcaserin N-Sulfamate Formation

Abu J. M. Sadeque, Safet Palamar, Khawja A. Usmani, Chuan Chen, Matthew A. Cerny and Weichao G. Chen
Drug Metabolism and Disposition April 2016, 44 (4) 570-575; DOI: https://doi.org/10.1124/dmd.115.067397

Abstract

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

Introduction

Lorcaserin hydrochloride hemihydrate*, a selective serotonin 5-hydroxytryptamine (5-HT) 5-HT2C receptor agonist, is approved by the U.S. Food and Drug Administration for chronic weight management. It is a secondary amine-containing benzazepine [(R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine] (Fig. 1). Lorcaserin is primarily cleared by metabolism. Multiple metabolic pathways such as oxidation, glucuronide conjugation, and sulfo-conjugation have been identified both in vitro and in vivo. Multiple cytochrome P450 (P450) (Usmani et al., 2012) and UDP-glucuronosyltransferase (Sadeque et al., 2012) enzymes associated with major metabolic pathways of lorcaserin have been reported; however, the sulfotransferases (SULTs) involved in its sulfo-conjugation have not yet been reported. Lorcaserin N-sulfamate is the major circulating metabolite of lorcaserin.

Fig. 1.
Fig. 1.

In vitro formation of lorcaserin N-sulfamate from lorcaserin catalyzed by cytosol from human liver, kidney and by recombinant SULTs in the presence of PAPS.

Sulfo-conjugation is an important metabolic reaction in the detoxification, biosynthesis, and homeostasis of essential endogenous compounds (Coughtrie, 2002; Strott, 2002). SULTs, a supergene family of cytosolic enzymes, catalyze this reaction by transferring a sulfuryl group from its cosubstrate 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to a nucleophilic acceptor molecule (substrate), producing a sulfate conjugate and desulfated 3′-phosphodenosine 5′-phosphate (Schwartz, 2005). In general, sulfate conjugation leads to an increase in hydrophilicity, and thus facilitates the excretion of the conjugated molecule. Therefore, sulfate conjugation is often considered to be an inactivation and detoxification process for xenobiotics and endobiotics (Pacifici and Coughtrie, 2005). Chemically stable sulfo-conjugates, such as N-sulfo-conjugates of aromatic amines, are known to be readily excreted without toxicological consequences (Stillwell et al., 1994; Glatt, 2005). However, sulfo-conjugates of certain chemicals, such as benzylic and allylic alcohols and aromatic hydroxylamines, are short-lived and electrophilic (DeBaun et al., 1970; Miller, 1970, 1994; Glatt, 2005). These may react with DNA and other cellular nucleophiles. Such sulfo-conjugations occur primarily through O-sulfation rather than N-sulfation. While sulfo-conjugates are generally biologically inactive, some can retain their biologic activity—such as cholesterol sulfate, which stimulates the differentiation of skin epidermal cells (Elias et al., 1984), and pregnenolone sulfate, which binds and suppresses the GABA receptor (Corpéchot et al., 1981). Lorcaserin N-sulfamate shows no binding affinity to serotonin 5-HT receptors 5-HT2A, 5-HT2B, and 5-HT2C.

Among other metabolic transformations reported earlier (Sadeque et al., 2012; Usmani et al., 2012), lorcaserin undergoes sulfo-conjugation through the secondary amine nitrogen, forming an N-sulfamate (Fig. 1). Therefore, the lorcaserin N-sulfo-conjugate does not fit the characteristics of chemically unstable sulfo-conjugates reported previously (Glatt, 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 interactions for lorcaserin.

In this paper, we describe the characterization of lorcaserin N-sulfamate through identification of SULTs that catalyze its formation, 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-chloro-1-methyl-4,5-dihydro-1H-benzo[d]azepine-3(2H)-sulfonic acid, and lorcaserin N-sulfamate-d6 (internal standard) were synthesized at Arena Pharmaceuticals, Inc. PAPS tetralithium salt, tris-HCl, and trizma-base were purchased from Sigma (St. Louis, MO). High-purity, high-performance liquid chromatography (HPLC)–grade acetonitrile and high-purity HPLC-grade deionized water were purchased from Burdick and Jackson (Muskegon, MI). Ultrapure distilled water was purchased from Invitrogen Corporation (Carlsbad, CA). Formic acid was purchased from EMD Chemicals Inc. (Gibbstown, NJ). Human liver microsomes (pooled mixed gender, n = 50), liver and renal cytosol (pooled mixed gender, n = 20) were purchased from Xenotech (Lenexa, KS). Human recombinant SULTs, SULT1A1, SULT1A2, SULT1A3, SULT1E1, and SULT2A1, produced from insect cells transfected with a baculovirus containing human SULT cDNA, were purchased from Invitrogen Corporation. Male and female human plasma pooled from different donors with sodium heparin as an anticoagulant were obtained from Bioreclamation, Inc. (East Meadow, NY).

Metabolic Assays.

Initially, the liver cytosol preparation was used to optimize the reaction conditions using para-nitrophenol, a probe substrate commonly used to measure SULT activity, according to the method described previously (Gamage et al., 2003). Following initial optimization, the incubation conducted for lorcaserin N-sulfamate formation for each enzyme system used is described subsequently.

Enzyme kinetics assays for lorcaserin N-sulfamate formation with cytosol.

The incubation mixtures (500 μl final volume) consisted of human liver or renal cytosol (1 mg/ml), tris-HCl buffer (100 mM, pH 7.4), and an appropriate concentration of lorcaserin (0–10 mM). The reaction mixture was preincubated at 37°C in a shaking water bath for 3 minutes, and then the reaction was initiated by the addition of PAPS (100 μM). Incubations were continued for 25 minutes, and then terminated by the addition of 500 μl acetonitrile. All incubations were performed in triplicate. Incubation with para-nitrophenol was conducted as a positive substrate control. Incubations without the addition of PAPS were also conducted as a negative control under identical conditions to rule out the possible involvement of a nonenzymatic process.

Assay for lorcaserin N-sulfamate formation with human recombinant SULTs.

Incubations were carried out as described subsequently with the final reagent concentrations given in parentheses. The incubation mixtures consisted of tris-HCl buffer (100 mM, pH 7.5), PAPS (100 μM), and lorcaserin (0–10 mM as appropriate depending on the SULT used). The incubation mixture was preincubated at 37°C in a shaking water bath for 3 minutes, and then the reaction was initiated by the addition of human recombinant SULT enzymes (15–20 μg protein/ml), bringing the final volume to 500 μl. The incubations were continued for 20 minutes, and then terminated by the addition of 500 μl acetonitrile and mixed thoroughly. Incubations without the addition of PAPS were conducted as the negative control (blank) under identical conditions. Incubations were performed in triplicate.

Assay for P450 enzyme inhibition potential by lorcaserin N-sulfamate.

The metabolic incubations in human liver microsomes with specific P450 probe substrates and lorcaserin N-sulfamate were conducted using a 96-well plate format. The incubation mixture contained 0.25 mg/ml microsomal protein, 0–200 μM lorcaserin N-sulfamate (test compounds as an inhibitor), P450-specific substrates (at respective Km values), 1 mM β-NADPH, and 100 mM potassium phosphate buffer containing 3 mM MgCl2 and 1 mM EDTA (pH 7.4) in a final volume of 100 μl. The following P450-specific substrates were added to the incubation mixture at a concentration close to their respective Km values: phenacetin (36 μM), bupropion (100 μM), paclitaxel (5 μM), tolbutamide (150 μM), (S)-mephenytoin (50 μM), dextromethorphan (5 μM), and midazolam (8 μM) for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. The reaction mixture was incubated at 37°C for 20 minutes for CYP1A2, 30 minutes for CYP2C8, and 10 minutes for CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Parallel control incubations conducted with P450 substrates without the addition of lorcaserin N-sulfamate (as an inhibitor) were used as the positive control. Parallel incubations were also performed without the addition of β-NADPH in the reaction mixture as a blank control (negative control). At the end of the incubation period, the incubations were terminated with the addition of an equal volume of acetonitrile containing the internal standard. The mixtures were then centrifuged at 4000 rpm for 20 minutes and the supernatant was transferred to a clean 96-well microtiter plate. The samples were subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis.

Enzymatic assay sample processing.

After termination of the incubation with an equal volume of acetonitrile, samples were vortexed and centrifuged for 10 minutes at 13,000 rpm in a microcentrifuge. The supernatant was transferred to a clean 96-well microtiter plate and subjected to LC-MS/MS analysis.

Assay for P450 enzyme induction by lorcaserin N-sulfamate in human hepatocytes.

Lorcaserin N-sulfamate (0.2, 2, and 20 μM) and the known P450 inducers 3-methylcholanthrene, phenobarbital, and rifampicin were incubated in cultures of human hepatocytes from three separate donors (two male and one 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 binding of lorcaserin N-sulfamate was determined using equilibrium dialysis. Three concentrations of lorcaserin N-sulfamate (0.1, 1, and 10 μM) and human plasma proteins were incubated for 24 hours in a 37°C humidified incubator. After incubation for 24 hours, 200-μl aliquots of the solutions in the donor and receiver chambers for each condition were transferred to 1.2-ml 8-well tube strips. A 200-μl aliquot of the dosing solution was also sampled for analysis. The proteins were precipitated with the addition of three volumes of ice-cold acetonitrile containing internal standard (100 ng/ml lorcaserin sulfamate-d6) followed by centrifugation at 3000g for 30 minutes. The resulting supernatant (70 μl) was mixed with an equal volume of HPLC-grade water on a 96-well sample plate. The samples were then analyzed by LC-MS/MS. The analyte concentration was determined by comparing with a standard curve of lorcaserin N-sulfamate generated under identical conditions of protein binding sample preparation.

Samples analysis by LC-MS/MS.

Analysis of lorcaserin N-sulfamate was carried out with an Aria LX-2 HPLC system coupled with an MDS Sciex API-4000 triple quadruple mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA). A HALO C18, 3 × 30 mm and 2.7 μm (Mac-Mod Analytical Inc., Chadds Ford, PA) column was used for separating lorcaserin N-sulfamate and the internal standard at room temperature. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The analysis was performed with a stepwise gradient from 10% to 95% of solvent B over a time period of approximately 2 minutes, followed by re-equilibration with initial solvent conditions (10% solvent B). The flow rate of the mobile phase was 0.6 ml/min throughout the run. The detection of lorcaserin N-sulfamate was achieved using electrospray ionization in negative-ion mode. The source temperature was set at 500°C with an ion-spray voltage of −4200 V. Multiple-reaction monitoring was used to acquire data. The Q1/Q3 (precursor/product) ions monitored for lorcaserin N-sulfamate and the internal standard were 274.0/79.8 and 280/79.8 atomic mass units, respectively. The quantification of lorcaserin N-sulfamate was achieved using a reference standard curve generated with authentic lorcaserin N-sulfamate (0.001–10 μM concentration range). The samples for the determination of inhibitory effect of lorcaserin N-sulfamate on the activity of human P450–probe substrates were analyzed according to the method described previously (Usmani et al., 2012).

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-Menten equation. The apparent Km value (Michaelis-Menten constant for substrate affinity) was calculated as the substrate concentration at half of the Vmax value (maximum velocity of the reaction). The intrinsic clearance (CLint) was calculated as CLint = Vmax/Km (Segel, 1976).

Calculation for percentage of plasma protein bound.

The percentage of the free lorcaserin N-sulfamate [fraction unbound (%fu)] was calculated according to the formula described previously (Wright et al., 1996), i.e., %fu = [(concentration in receiver chamber) ÷ (concentration in donor chamber)] × 100%. The percentage of the lorcaserin N-sulfamate bound to plasma proteins [plasma protein bound (%PB)] was determined according to the following formula: %PB = 100 − %fu.

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 μM of 2,6-dichloro-4-nitrophenol (data not shown), a known inhibitor generally used for the inhibition of SULT activity (Seah and Wong, 1994).

Fig. 2.
Fig. 2.

Michaelis-Menten kinetic plot of lorcaserin N-sulfamate formation by human liver cytosol.

Fig. 3.
Fig. 3.

Michaelis-Menten kinetic plot of lorcaserin N-sulfamate formation by human renal cytosol.

View this table:
TABLE 1

Kinetic parameters of lorcaserin N-sulfamate formation catalyzed by cytosol from human liver, (coma) kidney and by human recombinant SULTs

Lorcaserin N-Sulfamate Formation Catalyzed by Recombinant SULTs

Initial metabolic screening was performed with five recombinant human SULTs, SULT1A1, SULT1A2, SULT1A3, SULT1E1, and SULT2A1 (Fig. 4). Of these, SULT1A3 did not catalyze lorcaserin N-sulfamate formation and was therefore excluded from further kinetic analyses. The remaining SULTs showed differential activity, with SULT1A1 exhibiting the highest activity and SULT1E1 the lowest activity. SULT1A2 and SULT2A1 showed modest activity. The kinetic parameters are reported in Table 1 and a representative Michaelis-Menten kinetic plot with SULT1A1 is shown in Fig. 5. The order of catalytic efficiency of human SULTs for lorcaserin N-sulfamate formation is as follows: SULT1A1 (CLint = 255 μl/min/mg) > SULT2A1 (CLint = 19.31 μl/min/mg) > SULT1A2 (CLint = 2.86 μl/min/mg) > SULT1E1 (CLint = 1.85 μl/min/mg).

Fig. 4.
Fig. 4.

Formation of lorcaserin N-sulfamate catalyzed by human recombinant SULTs. A wide range of lorcaserin concentrations was used (0–1000 μM) for initial screening of recombinant SULT activity toward lorcaserin N-sulfamate formation prior to kinetic analysis.

Fig. 5.
Fig. 5.

Michaelis-Menten kinetic plot for lorcaserin N-sulfamate formation by human recombinant SULT1A1.

Human Liver P450 Enzyme Inhibition and Induction.

To evaluate the inhibitory effect of lorcaserin N-sulfamate on major P450 enzymes, the IC50 values for the inhibition of the activity of seven major P450 enzymes was evaluated. The IC50 value for the inhibition of CYP1A2-mediated phenacetin O-deethylase, CYP2B6-mediated bupropion hydroxylase, CYP2C8-mediated paclitaxel 6α-hydroxylase, CYP2C19-mediated (S)-mephenytoin 4′-hydroxylase, CYP2D6-mediated dextromethorphan O-demethylase, and CYP3A4-mediated midazolam 1′-hydroxylase was >100 μM (Fig. 6). However, the IC50 value for CYP2C9-mediated tolbutamide 4′-hydroxylase was 10.3 μM (2840 ng/ml) (Table 2). As shown in Table 2, in the presence of increasing concentrations of human serum albumin in microsomal incubations, the IC50 value for CYP2C9 inhibition was increased such that at a physiologically relevant albumin concentration of 500 μM, the IC50 value observed for CYP2C9 inhibition was >200 μM (Fig. 7). There was no induction observed for major P450 enzymes when cultured human hepatocytes were treated with lorcaserin N-sulfamate (up to 20 μM) for 72 hours, suggesting that lorcaserin N-sulfamate is not a human P450 enzyme inducer (data not shown).

Fig. 6.
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 of P450 selective probe substrates in human liver microsomes were measured in the presence of various concentrations of lorcaserin N-sulfamate (0–200 μM).

View this table:
TABLE 2

IC50 values for the inhibition of CYP2C9-mediated tolbutamide 4′-hydroxylase activity by lorcaserin N-sulfamate in the presence of human serum albumin

Fig. 7.
Fig. 7.

Inhibitory effect of lorcaserin N-sulfamate on human liver microsomal CYP2C9 in the presence 10, 100, and 500 μM of human serum albumin. The inhibitory effect of lorcaserin N-sulfamate on CYP2C9 decreased with increasing concentration of human serum albumin in the incubation mixture.

Plasma Protein Binding

Three concentrations, 0.1, 1.0, and 10 μM, of lorcaserin N-sulfamate were used for its plasma protein binding affinity determination. There was <1% of lorcaserin N-sulfamate unbound (<1%fu) to human plasma proteins (>99% bound) at 10 μM, whereas with low concentrations (0.1 and 1 μM) the unbound fraction was below the limit of quantification. The data are presented in Table 3.

View this table:
TABLE 3

Percentage of lorcaserin N-sulfamate bound to male and female human plasma proteins

Discussion

Sulfo-conjugates of xenobiotics and endobiotics are generally biologically inactive and hydrophilic; therefore, this represents a biologically important excretion pathway (Stillwell et al., 1994; Coughtrie, 2002; Strott, 2002; Glatt, 2005). However, the structural features of certain sulfo-conjugated molecules may elicit various biological effects. For example, the O-sulfo-conjugates of aromatic hydroxylamines, benzylic and allylic alcohols are known to be short-lived and electrophilic, and may therefore bind to cellular nucleophiles, including DNA (DeBaun et al., 1970; Glatt, 2005). In addition, some sulfo-conjugates show biological activity, such as cholesterol sulfate, which is known to stimulate differentiation of skin epidermal cells (Elias et al., 1984), and pregnenolone sulfate, which binds and suppresses the GABA receptor (Corpéchot et al., 1981). However, sulfo-conjugation via N-sulfation, such as N-sulfonation of aromatic amines, yields stable sulfo-conjugates that are readily excreted without toxicological consequences (Stillwell et al., 1994; Glatt, 2005). In this paper, we describe the formation and systematic characterization of lorcaserin N-sulfamate, formed by human liver or renal cytosol and recombinant SULTs. The lorcaserin sulfo-conjugate is not the product of O-sulfonation, but rather N-sulfonation, and is known to be chemically stable (Stillwell et al., 1994; Glatt, 2005). Thus, toxicological consequences of lorcaserin N-sulfamate are considered to be unlikely.

Prior to the characterization of lorcaserin N-sulfamate formation kinetics, the SULT activity of the cytosolic preparations was verified using para-nitrophenol, a known substrate for SULTs. Typical biphasic kinetics with initial product formation (para-nitrophenol-sulfate) at low para-nitrophenol concentration followed by inhibition of its own product formation at higher concentration was observed (data not shown) as reported previously (Gamage et al., 2003). The kinetic parameters for lorcaserin N-sulfamate formation showed that the Vmax and CLint values for liver cytosol are about 17- and 15-fold higher, respectively, than that for renal cytosol, suggesting that lorcaserin N-sulfamate is predominantly formed by liver SULTs. An initial screening with recombinant SULTs indicated that SULT1A1, SULT1A2, SULT1E1, and SULT2A1 were involved in lorcaserin N-sulfamate formation, whereas SULT1A3 showed minimal or no activity (Fig. 4). The CLint values of SULT1A1 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 humans (Table 1). SULT1A1, SULT2A1, and SULT1E1 account for 53%, 27%, and 6%, respectively, of total SULTs in human liver, whereas in the human kidney these values are 40%, 1%, and 0%, respectively (Riches et al., 2009). Therefore, the SULTs that are involved in lorcaserin N-sulfamate catalysis constituted approximately 86% of the total SULTs expressed in human liver compared with about 41% in kidney, suggesting that the liver is the primary metabolic site for lorcaserin N-sulfamate formation. It is noteworthy that three members of the SULT1 family (SULT1A1, SULT1A3, and SULT1E1) show very different kinetic behavior for lorcaserin N-sulfamate formation. This may reflect the differential nature of the substrate binding pocket of these isoforms (Gamage et al., 2003). The SULT1A1 binding pocket accepts a wide range of molecules, such as simple planar-substituted phenols (Brix et al., 1999), as well as very large lipophilic substrates (Harris et al., 2000; Li et al., 2001) and polycyclic aromatic compounds (Grimm et al., 2013). Lorcaserin is a secondary amine-containing benzazepine that is more lipophilic due to the aryl chloride portion of the molecule and thereby may be well suited for binding to the hydrophobic pocket of SULT1A1. Although lorcaserin is a nonplanar molecule, the strongly hydrophobic and flexible nature of the active site of SULT1A1 may accommodate lorcaserin as a better substrate compared with other SULTs reported in this study. Lorcaserin has a pKa value of 9.53 and the secondary amine in its cyclic aliphatic moiety may become protonated under physiologic reaction pH (7.4). However, lorcaserin appears not to be a substrate for SULT1A3, which may be due to the lack of an aliphatic amine containing an open side chain with at least two carbons, a characteristic of preferred substrates (e.g., dopamine or tyramine) (Brix et al., 1999). SULT1E1 shows high selectivity for endogenous estrogens such as 17β-estradiol as well as for a number of other important drugs including 17α-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., 2002; Lu et al., 2010). The hydrophobic chloro-benzene moiety of lorcaserin likely makes it a poor substrate for SULT1A2. Similar to SUL1A2, SULT2A1 also showed low affinity for lorcaserin N-sulfamate formation. The active site of SULT2A1 appears to accept more hydrophobic substrates such as steroids, steroid derivates, and bile acids (Comer et al., 1993; Huang et al., 2010). Additionally, specific binding orientation in SULT2A1 is required for specific substrates such as dehydroepiandrosterone and androsterone (Lu et al., 2008). This may be the reason why lorcaserin is not a high-affinity substrate for SULT2A1.

Comparing all of the SULTs used for lorcaserin N-sulfamate formation, SULT1A1 appears to be the most catalytically efficient. Based on the CLint values, the order of catalytic efficiency is SULT1A1 > SULT2A1 > SULT1A2 > SULT1E1. As indicated by the kinetic parameters, lorcaserin does not appear to be a prototypical high-affinity substrate for human SULT enzymes. Thus, drug-drug interactions arising through the SULT-mediated metabolic pathway are not anticipated. Additionally, although lorcaserin N-sulfamate is the major metabolite in circulation, it accounts for only approximately 3% of the total lorcaserin dose in human urine, whereas the major clearance pathways of lorcaserin are oxidation by multiple P450 enzymes (Usmani et al., 2012) and glucuronidation by multiple UDP-glucuronosyltransferase enzymes (Sadeque et al., 2012). The observed pharmacokinetic half-life of lorcaserin N-sulfamate after lorcaserin dosing in humans is much longer than the parent (∼40 versus ∼11 hours). One likely explanation for the high levels of lorcaserin N-sulfamate in circulation and the prolonged observed half-life is that the very high plasma protein binding affinity (>99%) renders only a small free fraction of the metabolite in circulation available for tissue distribution and elimination. We also observed that lorcaserin N-sulfamate inhibits human liver microsomal tolbutamide hydroxylation with an IC50 value of ∼10 μM, a reaction catalyzed by CYP2C9. However, the IC50 value becomes >200 μM in the presence of physiologic concentration of human serum albumin (Fig. 7). Additionally, lorcaserin N-sulfamate has no or minimal inhibitory effect on other major P450 enzymes (Fig. 6) and also is not a time-dependent inhibitor of CYP2C9 (data not shown). On the other hand, although the parent drug, lorcaserin, inhibits CYP2D6 activity (approximately 2-fold increase in the area under the curve of dextromethorphan, see the prescribing information for reference, http://www.belviq.com/documents/Belviq_Prescribing_Information.pdf), it is also not a time-dependent inhibitor of CYP2D6. In a separate experiment we found that only 10% of lorcaserin N-sulfamate is bound to microsomal protein, in contrast to >99% bound to human plasma protein. Thus, in an in vivo environment, the availability of free lorcaserin N-sulfamate in circulation would be very negligible at a clinical dose of lorcaserin and is highly unlikely to cause metabolic inhibition of the CYP2C9 pathway. Furthermore, at concentrations up to 20 μM, lorcaserin N-sulfamate did not induce any P450 enzymes in cultured human hepatocytes treated for 72 hours. In comparison, the plasma Cmax of lorcaserin N-sulfamate exceeds that of lorcaserin [mean Cmax value = 0.29 μM (56.8 ng/ml), following 10 mg twice daily in humans] by 1- to 5-fold. Taken together, these results suggest that lorcaserin N-sulfamate has a very low potential of affecting P450 metabolic pathways by P450 enzyme inhibition or induction.

In this study, we have demonstrated that lorcaserin undergoes N-sulfation, thus forming a chemically stable sulfo-conjugate and an excretory pathway (Glatt, 2005). We also demonstrated that although lorcaserin N-sulfo-conjugation is catalyzed by multiple SULTs, lorcaserin does not appear to be a high-affinity substrate for these enzymes. Lorcaserin N-sulfamate is a minor clearance pathway in humans since it accounts for only ∼3% of lorcaserin dose in urine. Furthermore, its high plasma protein binding affinity (>99%) is consistent with its long half-life in circulation. Importantly, it is neither a clinically relevant P450 enzyme inhibitor nor an inducer. In conclusion, lorcaserin N-sulfamate has a very low probability of causing metabolic drug-drug interactions in a normal patient population.

Acknowledgments

The authors thank Laurie Turbeville, Paul Mawson, and Nathan Kozon for providing some enzymatic assay support and to Michael Ma and Salma Sarwary from the Department of Drug Metabolism and Pharmacokinetics at Arena Pharmaceuticals, Inc., for providing some liquid chromatography mass spectrometry analytical support.

Authorship Contributions

Participated in research design: Sadeque, Usmani, Palamar, C. Chen, Cerny, W. G. Chen.

Conducted experiments: Palamar, Usmani, C. Chen.

Performed data analysis: Palamar, Usmani, C. Chen.

Wrote or contributed to the writing of the manuscript: Sadeque.

Footnotes

    • Received September 30, 2015.
    • Accepted January 7, 2016.

  • ↵1 Current affiliation: Drug Metabolism & Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, New Jersey.

  • ↵2 Current affiliation: Pharmacokinetics, Dynamics and Metabolism Department, Pfizer Global Research and Development, Groton, Connecticut.

  • ↵3 Current affiliation: Drug Metabolism & Pharmacokinetics, Vertex Pharmaceuticals, San Diego, California.

  • * Trade Name of Lorcaserin hydrochloride hemihydrate is BELVIQ.

  • dx.doi.org/10.1124/dmd.115.067397.

Abbreviations

CLint
intrinsic clearance
HPLC
high-performance liquid chromatography
5-HT
5-hydroxytryptamine
LC-MS/MS
liquid chromatography–tandem mass spectrometry
PAPS
3′-phosphoadenosine-5′-phosphosulfate
P450
cytochrome P450
SULT
sulfotransferase

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Human Sulfotransferases Involved in Lorcaserin Metabolism

Abu J. M. Sadeque, Safet Palamar, Khawja A. Usmani, Chuan Chen, Matthew A. Cerny and Weichao G. Chen
Drug Metabolism and Disposition April 1, 2016, 44 (4) 570-575; DOI: https://doi.org/10.1124/dmd.115.067397
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