The Biotransformation of Prasugrel , a New Thienopyridine Prodrug , by

3 DMD #20248 Prasugrel is a novel thienopyridine prodrug with demonstrated inhibition of platelet aggregation and activation. The biotransformation of prasugrel to its active metabolite, R-138727, requires ester bond hydrolysis, forming the thiolactone R-95913, followed by cytochrome P450-mediated metabolism to the active metabolite. The presumed role of the human liverand intestinaldominant carboxylesterases, hCE1 and hCE2, respectively, in the conversion of prasugrel to R95913 was determined using expressed and purified enzymes. The hydrolysis of prasugrel is at least 25-times greater with hCE2 than hCE1. Hydrolysis of prasugrel by hCE1 demonstrated Michaelis-Menten kinetics yielding an apparent Km of 9.25 μM and an apparent Vmax of 0.725 nmol of product/min/μg of protein. Hydrolysis of prasugrel by hCE2 showed a mixture of Hill kinetics at low substrate concentrations and substrate inhibition at high concentrations. At low concentrations, prasugrel hydrolysis by hCE2 yielded an apparent Ks of 11.1 μM, an apparent Vmax of 19.0 nmol/min/μg, and an apparent Hill coefficient of 1.42; while at high concentrations, an apparent IC50 of 76.5 μM was obtained. In humans, no in vivo evidence of inhibition exists. In vitro transport studies using the intestinal Caco-2 epithelial cell model demonstrated a high in vivo absorption potential for prasugrel and rapid conversion to R-95913. In conclusion, the human carboxylesterases efficiently mediate the conversion of prasugrel to R95913. These data help explain the rapid appearance of R-138727 in human plasma, where maximum concentrations are observed 0.5 hour after a prasugrel oral dose, and the rapid onset of action of prasugrel. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from 4 DMD #20248 Platelet aggregation and activation is a serious concern for patients who undergo percutaneous coronary intervention and stent placement. The underlying mechanism of platelet aggregation is mediated through two G-protein coupled P2 receptors, P2Y1 and P2Y12 (Gachet, 2001). P2Y1 activation leads to a transient aggregation, while P2Y12 activation maintains a sustained aggregation. To reduce platelet aggregation, the development of P2Y12 selective inhibitors has yielded the thienopyridine prodrugs, which include ticlopidine, clopidogrel (structures available in Farid et al., 2008), and prasugrel (Figure 1), a novel thienopyridine currently in clinical development. While the active metabolites for prasugrel and clopidogrel have equipotency at the P2Y12 receptor in vitro (Sugidachi et al., 2007), orally administered prasugrel is 10 and 100 times more effective on an equal dose basis in inhibiting platelet aggregation than clopidogrel and ticlopidine, respectively (Niitsu et al., 2005). Clopidogrel and prasugrel differ markedly in the biotransformation pathways leading to their activation. Prasugrel (Figure 1) has a single dominant metabolic pathway leading to the active metabolite (Farid et al., 2007a). However, clopidogrel has two competing metabolic pathways for the parent compound, with the major pathway leading to the formation of an inactive metabolite, clopidogrel carboxylic acid derivative (Caplain et al., 1999). The clopidogrel carboxylic acid derivative is formed through ester hydrolysis by the human carboxylesterase (hCE) 1 (Tang et al., 2006). The minor pathway in clopidogrel metabolism yielding the active metabolite requires two sequential steps of cytochrome P450 (CYP) biotransformation (Kurihara et al., 2005). Whereas, prasugrel bioactivation requires the hydrolysis of the ester then oxidation of the formed thiolactone, R95913 (Farid et al., 2007a) (Figure 1) to form the active metabolite of prasugrel. The oxidation This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from 5 DMD #20248 of R-95913 has been shown to be mediated by several CYP enzymes, but primarily by CYP3A and CYP2B6 (Rehmel et al., 2006). The carboxylesterases are a multigene family that hydrolyze compounds containing an ester, amide, or thioester linkage. Carboxylesterases are broadly expressed throughout the body with two major forms in humans, hCE1 and hCE2. Although both forms have high mRNA expression in the liver, hCE1 levels exceed those of hCE2 (Satoh et al., 2002). Importantly, the extrahepatic expression differs between hCE1 and hCE2 (Satoh et al., 2002). For hCE1, the liver-dominant form, extrahepatic mRNA expression observed in decreasing order are the stomach, testis, kidney, spleen, and colon. The intestinal-dominant form, hCE2, has extrahepatic mRNA expression in decreasing order in the colon, small intestine, and heart. In addition to the studies examining mRNA expression, the relative activity of the two enzymes has been recently demonstrated in the liver and small intestine (Imai et al., 2006). Using tissue preparations, hCE1 and hCE2 were separated in non-denaturing gels, and the hydrolysis of 1naphthylbutyrate was utilized to determine the relative activity of the two enzymes. Both enzymes were detected in the liver, but hCE1 was clearly the dominant form. Unlike the liver, the small intestine demonstrated that hCE2 was the dominant form with a minor contribution by hCE1. This pattern of tissue distribution has been further demonstrated (Taketani et al., 2007). Although hCE1 and hCE2 have overlapping substrate recognition, clear evidence of ester-based substrate specificity has been observed (Satoh et al., 2002). Two products result from ester hydrolysis, an alcohol and an acyl moiety. In general, hCE1 prefers substrates with a large acyl moiety, while hCE2 prefers substrates with a large alcohol substituent. Based upon this substrate-activity relationship, prasugrel would be predicted to be a preferred substrate for hCE2. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from 6 DMD #20248 This study aims to investigate the role of hCE1 and hCE2, the dominant forms in the liver and intestinal tract, respectively, in the bioactivation of prasugrel. To accomplish this endeavor, hCE1 and hCE2 enzymes were expressed and purified and used to determine the formation kinetics of R-95913 from prasugrel. Additional experiments were conducted with Caco-2 monolayers to assess the conversion of prasugrel to R-95913 and their relative transit across this intestinal model. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from Materials and Methods 7 DMD #20248 In Vitro Hydrolysis Assays For the hydrolysis assays, each reaction tube contained buffer, enzyme, prasugrel, and acetonitrile, in a total volume of 600 μL. The buffer used was Dulbecco's Phosphate Buffered Saline (D-PBS; 14040-117; Invitrogen, Corp.; Carlsbad, CA). A 5 mM stock of prasugrel was prepared in acetonitrile. The final reaction volume contained a total of 2% acetonitrile. Prasugrel concentrations used with hCE1 were 0.855, 1.71, 3.42, 6.84, 13.7, 27.4, 54.7, and 109 μM. In addition to the concentrations used with hCE1, the hCE2 studies also used 21.9, 40.5, 71.2, and 87.6 μM. The expression and purification of hCE1 and hCE2 were previously described (Williams et al., 2008). All protein preparations are homogenous based upon analysis of an SDS-PAGE gel stained with SimplyBlue SafeStain (Invitrogen, Corp.; data not shown). The tubes containing the various prasugrel concentrations were pre-incubated in a water bath at 37°C for approximately 1 minute. The enzyme, either 1 μg/mL of hCE1 or 0.25 μg/mL of hCE2, was pre-incubated with buffer for approximately 5 minutes at 37°C and added to the tubes with prasugrel to start the reaction. A 100 μL aliquot was removed from the reaction tube at 1, 2, 3, and 6 minutes after reaction initiation then a 100 μL of acetonitrile containing 2 μg/mL of a d4-labeled R-95913 as the internal standard was added to each aliquot to terminate the reaction. All studies were conducted in triplicate. For the standards, a stock solution of R-95913 was prepared in acetonitrile at 2 mg/mL. The concentrations ranged from 4.88 ng/mL to 40 μg/mL, in 2% acetonitrile in D-PBS. Each standard (100 μL) was added to 100 μL of acetonitrile with internal standard. In addition to the standards, two blanks were also included. One blank was lacking R-95913, but included the internal standard. The second blank was lacking both R-95913 and internal standard. Samples and standards had two dilution schemes for analysis, 1:25 and 1:275 dilution, with the diluent This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from Materials and Methods 8 DMD #20248 being methanol:water (1:1, v:v). The appropriate dilution was utilized for analysis to ensure samples did not saturate the detector response of the mass spectrometer. Study samples were analyzed by LC-MS/MS using a Sciex API 4000 triple quadrupole mass spectrometer (Applied Biosystems/MDS; Foster City, CA) equipped with a TurboIonSpray interface, and operated in positive ion mode. The analytes were chromatographically separated using a Betasil C18 2.1x20 mm, 5 μm, Javelin HPLC column (Thermo Fisher Scientific, Inc.; Waltham, MA), with a gradient LC system composed of water:1 M ammonium bicarbonate, (200:1, v:v) (Mobile Phase A), and methanol:1 M ammonium bicarbonate, (200:1, v:v) (Mobile Phase B). The pumps were a Shimadzu LC-10AD with a SCL-10A controller (Kyoto, Japan). Also, a Gilson 215 liquid handler (Middleton, WI) was used. The gradient profile changed from 30% B at 0 min, 42% B at 0.01 min to 0.10 min, 75% B at 0.20 to 0.30 min, and 98% at 0.31 to 0.76 min, at a flow rate of 1.5 mL/min. Chromatography was performed at ambient temperature, with 1 mL/min directed to the mass spectrometer between 0.18 and 0.5 min (0.5 mL/min split to waste). Selected reaction monitoring (M+H) transitions m/z 332.2 > 177.1 and 336.2 > 149.2 were monitored for R-95913, and the internal standard, respectively. The TurboIonSpray temperature was maintained at 725oC, with collision, curtain, nebulizing, and desolvation gas (nitrogen) settings of 8, 10, 50, and 70, respectively. The ionspray voltage was set to 4000 V, while the respective declustering, entrance, collision, and exit potentials were 55, 10, 27, and 4 for R-95913, and 55, 10, 33, and 8 for the internal standard. The mass spectrometer quadrupoles were tuned to achieve unit resolution (0.7 DA at 50% FWHM). Data were acquired and processed with Analyst 1.4.1 (Applied Biosystems). For the assays with hCE1, the standards for the 1:25 dilution ranged from 19.5 to 2500 ng/mL. A correlation coefficient of 0.999 was achieved with less than a 7% deviation from the This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from Materials and Methods 9 DMD #20248 relative mean using a quadratic curve with a weighting of 1/X2. The standards for the 1:275 dilution ranged from 156 to 20000 ng/mL. A correlation coefficient of 0.998 was achieved with less than a 12% deviation from the relative mean using a quadratic curve with a weighting of 1/X2. For the assays with hCE2, the standards for the 1:25 dilution ranged from 78.1 to 2500 ng/mL. A correlation coefficient of 0.998 was achieved with less than a 9% deviation from the relative mean using a quadratic curve with a weighting of 1/X2. The standards for the 1:275 dilution ranged from 625 to 10000 ng/mL. A correlation coefficient of 0.999 was achieved with less than a 6% deviation from the relative mean using a quadratic curve with a weighting of 1/X2. Enzyme Kinetic Modeling Hydrolysis reaction rates (nmol of product/min/μg of protein) were calculated from the linear portion of the product concentration versus time curve. Fitting the rate data to standard kinetic models (Copeland, 1996) was accomplished using WinNonlin (Pharsight Corp.; Mountain View, CA). Michaelis-Menten, substrate inhibition, and Hill kinetic models with differing weighting were used. For the results with hCE1, Michaelis-Menten kinetics using a weighting of 1/Y2 provided the best fit. However, the unusual kinetics of the formation of R95913 by hCE2 could not be fit to the models described above as demonstrated by the standard errors of the fit being large as compared to the kinetic values. Therefore, the product formation rate data obtained with incubation with hCE2 between 0.855 and 40.5 μM of prasugrel were found to best fit the Hill model using a weighting of 1/Ŷ2. The rate data with hCE2 and prasugrel concentrations between 27.4 and 109 μM were fit to an IC50 model using Prism (GraphPad Software, Inc.; San Diego, CA). The various estimated parameters are reported as the value ± the standard error of the estimate. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from Materials and Methods 10 DMD #20248 Intestinal Transport and Metabolism Assays The Caco-2 cell line was obtained from the American Type Culture Collection (ATCC; Rockville, MD) and grown under standard culture conditions of 37oC in a humidified atmosphere containing 5% CO2. Caco-2 cell monolayers were cultured in 175 cm 2 flasks in Dulbecco's Modified Eagle Medium (DMEM; 12430-054; Invitrogen, Corp.) supplemented with 10% fetal bovine serum (FB-01; Omega Scientific, Inc.; Tarzana, CA), 1 mM sodium pyruvate (25-000-CI; Mediatech, Inc.; Herndon, VA), 100 mM non-essential amino acids (25-025-CI), 2 mM L-glutamine, 100 U/mL penicillin (30-002-CI), and 100 μg/mL streptomycin. Cells were seeded at a density of 60,000 cell/cm onto collagen-coated 12-well Costar Transwell polycarbonate membranes (0.4 μm pore size, 1.13 cm surface area) and used between 21 to 28 days post-seeding. The culture medium was changed every other day for 10 days after seeding onto Transwell filters, and daily afterwards. Cells of passage number 61 were used for these studies. Studies were performed with Hank's Balanced Salt Solution (14065-056; Invitrogen, Inc.) containing 10 mM HEPES (15630-080) and 15 mM glucose (G-5400; Sigma-Aldrich, Corp.; St. Louis, MO), pH 7.4 (HBSS+) at 37oC with 5% CO2 in a humidified incubator. At the start of the experiment, monolayers were rinsed twice with HBSS+ and the transepithelial electrical resistance (TEER) of each cell monolayer was measured using an Endohm-12 resistence meter. Monolayers having TEER values outside the range of 450-650 Ω·cm were discarded. Volumes in the apical and basolateral chambers were 0.5 mL and 1.5 mL, respectively. All studies were conducted in triplicate. The apical-to-basolateral studies used an apical dosing solution of 5 μM prasugrel along with reference compounds, 100 μM atenolol (A-7655; Sigma-Aldrich, Corp.) and 10 μM This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from Materials and Methods 11 DMD #20248 pindolol (P-0778; Sigma-Aldrich, Corp.). At each sampling time (0, 15, 30, 60, 90, and 120 minutes), a 200 μL aliquot of drug solution was removed from the basolateral receiver chamber and immediately replaced with an equal volume of drug-free buffer. Similarly, a 20 μL aliquot was removed from the apical donor chamber without replenishing the donor solution. Monolayers were dosed with 0.5 mM Lucifer Yellow (L-453; Invitrogen, Inc.) to determine post-experimental monolayer integrity. Lucifer yellow was measured with a BMG Fluostar 403 microplate reader using an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Each determination was performed in triplicate and was not significantly greater than baseline. The basolateral-to-apical studies used a basolateral dosing solution of 5 μM prasugrel along with reference compounds, 100 μM atenolol and 10 μM pindolol. At each sampling time (0, 15, 30, 60, 90, and 120 minutes), a 200 μL aliquot of drug solution was removed from the apical receiver chamber and immediately replaced with an equal volume of drug-free buffer. Similarly, a 20 μL aliquot was removed from the basolateral donor chamber without replenishing the donor solution. A post-experimental monolayer integrity check was conducted with Lucifer Yellow, as described above. A 10 mM stock solution of prasugrel and R-95913 in DMSO was prepared. Further dilution in 1:1 ACN:water gave a 100 μM stock. Atenolol stocks (10 mM in water and 100 μM in 1:1 ACN:water) and pindolol stocks (10 mM in DMSO and 10 μM in 1:1 ACN:water) were prepared previously. Standard curves were obtained by serial dilution in 1% formic acid in ACN:Hanks buffer, pH 7.4 (1:1). Caco-2 study samples were diluted in 1% formic acid in ACN:Hanks buffer, pH 7.4 (1:1), then analyzed by LC-MS/MS using a Sciex API 3000 triple quadrupole mass spectrometer This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from Materials and Methods 12 DMD #20248 (Applied Biosystems/MDS) equipped with a TurboIonSpray interface, and operated in positive ion mode. The analytes were chromatographically separated using a BDS Hypersil C18 30x2.1 mm 3 μm HPLC column (Thermo Fisher Scientific, Inc.). The buffer consisted of 25 mM NH4OH, adjusted to pH 3.5 with 88% formic acid. The gradient LC system composed of 10% buffer in water (Mobile Phase A) and 10% buffer in acetonitrile (Mobile Phase B). Rheos 2000 micropumps (Thermo Fisher Scientific, Inc.) and CTC Analytics HTC PAL autosampler (Zwingen, Switzerland) were used. The mobile phase composition changed from 0% B at 0 min, 100% B at 0.02 min to 1.00 min, 100% B at 1.00 to 2.50 min, 0% at 2.50 to 2.60 min, and 0% from 2.60 to 4.00 min, at a flow rate of 0.3 mL/min. Chromatography was performed at ambient temperature. Selected reaction monitoring (M+H) transitions m/z 374.0 > 206.1, 332.2 > 109.3, 267.2 > 145.0, and 249.0 > 116.2 were monitored for prasugrel, R-95913, atenolol, and pindolol, respectively. The TurboIonSpray temperature was maintained at 450oC, with collision, curtain, and nebulizer gas settings of 10, 10, and 8, respectively. The ionspray voltage was set to 5000 V, while the respective declustering, focusing, entrance, collision, and exit potentials were 46, 200, 10, 35, and 12 for prasugrel, 31, 150, 10, 50, and 10 for R-95913, 36, 170, 10, 35, and 8 for atenolol, and 36, 170, 10, 35, and 6 for pindolol. The standards for prasugrel, R-95913, pindolol, and atenolol ranged from 10 to 1000 nM. For prasugrel and R-95913, the correlation coefficients were 1.00 and 1.00, respectively, with a maximum deviation of 3% and 4%, respectively, from the relative mean using a quadratic curve with a weighting of 1/X2. For pindolol and atenolol, the correlation coefficients were 0.998 and 0.999, respectively, with a maximum deviation of 8.8% and 10%, respectively, from the relative mean using a linear curve with a weighting of 1/X2. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from Materials and Methods 13 DMD #20248 The formation and disappearance rates of prasugrel and R-95913 were calculated using Microsoft Excel. The rates (nmol/hr) are the result of using the SLOPE function with data points in the linear range, as described in the results section. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from Results 14 DMD #20248 The human carboxylesterases hCE1 and hCE2 efficiently hydrolyzed prasugrel to R95913, as shown in Figure 2. Figure 2A depicts the fit of standard Michaelis-Menten kinetics to the rate data for hCE1, which yields an apparent Km of 9.25 ± 0.78 μM and an apparent Vmax of 0.725 ± 0.035 nmol of product/min/μg of protein. However, the results obtained using hCE2 (Figure 2B) were determined to fit poorly to all standard enzyme kinetic models, including that for substrate inhibition. Therefore, the results obtained with hCE2 were divided into two data sets for modeling. The first data set consisted of the results between prasugrel concentrations of 0.855 and 40.5 μM to model for standard enzyme kinetics. The best fit with this data set was produced using the Hill equation, which gave an apparent Ks of 11.1 ± 2.8 μM, an apparent Vmax of 19.0 ± 2.8 nmol of product/min/μg of protein, and an apparent Hill coefficient (N) of 1.42 ± 0.12. The second portion of the curve resembles an inhibition plot and as such the data between 27.4 and 109 μM was modeled for inhibition to yield an apparent IC50 of 76.5 ± 2.7 μM. Due to the different models needed to fully describe the diverse kinetics observed, the clearance near the therapeutic concentration range (Williams et al., 2002) was also compared. The clearance by each enzyme was calculated using the substrate concentrations between 0 and 14 μM, which resulted in a linear slope of 32 μL/min/μg and 798 μL/min/μg for hCE1 and hCE2, respectively, yielding R2 values of 0.955 and 0.938, respectively. This comparison indicates that the rate of hydrolysis of prasugrel at low substrate concentrations is about 25times greater for hCE2 than hCE1. The Caco-2 monolayer transport and metabolism study demonstrated the conversion of prasugrel to R-95913 (Figure 3). The active metabolite of prasugrel, R-138727, was not monitored since CYP3A4, which is the primary CYP involved in its formation from the thiolactone, R-95913, (Rehmel et al., 2006), is not routinely expressed in Caco-2 monolayers This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from Results 15 DMD #20248 (Cummins et al., 2004). Figures 3A and B show the apical to basolateral (representing lumen to blood) conversion and transport of the cumulative amounts of prasugrel and R-95913 in the donor and receiver compartments, respectively. As shown in Figure 3A, the appearance rate of R-95913 in the donor (apical) buffer is 1.65 ± 0.40 nmol/hr between 0 and 30 minutes, and the loss of prasugrel between 0 and 30 minutes occurs at a rate of 2.80 ± 0.34 nmol/hr. For Figure 3B, the appearance rate of R-95913 in the receiver (basolateral) buffer is 0.730 ± 0.028 nmol/hr between 0 and 90 minutes, and prasugrel was not detected. Similarly, Figures 3C and D show the basolateral to apical (representing blood to lumen) prasugrel conversion and transport. Figure 3C demonstrates a loss of prasugrel in the donor (basolateral) buffer at a rate of 1.98 ± 0.10 nmol/hr between 30 and 120 minutes, and the appearance rate of R-95913 between 0 and 90 minutes is 1.41 ± 0.08 nmol/hr. In Figure 3D, the appearance rate for R-95913 in the receiver (apical) buffer is 0.510 ± 0.043 nmol/hr between 0 and 90 minutes, and prasugrel was not detected. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from Discussion 16 DMD #20248 These studies demonstrate that both hCE1 and hCE2 have similar Km and Ks values for the ester hydrolysis of prasugrel, suggesting they bind prasugrel with similar affinity. However, the rate of hydrolysis (Vmax) by hCE2 appears to be 26-times higher than that of hCE1. Since the pattern of kinetics for the hydrolysis of prasugrel by hCE1 and hCE2 were found to be quite different, the hydrolysis rates at low substrate concentrations, as previously described (Williams et al. 2002), were used as a potentially more meaningful comparison. This comparison indicates the hydrolysis of prasugrel by hCE2 at low substrate concentrations is 25-times greater than that for hCE1. This pattern of substrate selectivity by CEs is consistent with published data (Satoh et al., 2002). Upon hydrolysis of the ester, thioester, or amide bond, an alcohol and acyl moiety are released as metabolites. The relative sizes of these two moieties have been shown to predict which enzyme will have preferential recognition of the substrate (Satoh et al., 2002). Substrates yielding a smaller alcohol moiety, such as clopidogrel (Tang et al., 2006), are preferred by hCE1. However, hCE2 has a preference for substrates that yield a smaller acyl moiety upon hydrolysis, like prasugrel. Prasugrel appears to have a higher binding affinity (lower Km value) for the human CEs as compared to other characterized CE substrates. When compared to irinotecan (CPT-11), the binding affinity of prasugrel is about four-times greater for hCE1 but ten-times lower for hCE2 (Sanghani et al., 2004). As compared to heroin, the binding of prasugrel to hCE1 and hCE2 is substantially greater, by about 600-times (Kamendulis et al., 1996). Similar to heroin, the binding affinities of cocaine and 4-methylumbelliferyl acetate (Pindel et al., 1997) are lower by at least ten-times for prasugrel with both enzymes. Also, when compared to 4-nitrophenyl butyrate the binding affinity of prasugrel is about ten-times greater (Williams et al., 2008). This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on March 27, 2008 as DOI: 10.1124/dmd.107.020248 at A PE T Jornals on A uust 5, 2017 dm d.aspurnals.org D ow nladed from Discussion 17 DMD #20248 Therefore, prasugrel appears to have a higher binding affinity for the human CEs than most CE