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Research ArticleArticle

Involvement of Carboxylesterase 1 and 2 in the Hydrolysis of Mycophenolate Mofetil

Nobuhiro Fujiyama, Masatomo Miura, Shoutaro Kato, Tomomichi Sone, Masakazu Isobe and Shigeru Satoh
Drug Metabolism and Disposition December 2010, 38 (12) 2210-2217; DOI: https://doi.org/10.1124/dmd.110.034249

Abstract

Mycophenolate mofetil (MMF) is the ester prodrug of the immunosuppressant agent mycophenolic acid (MPA) and is rapidly activated by esterases after oral administration. However, the role of isoenzymes in MMF hydrolysis remains unclear. Although human plasma, erythrocytes, and whole blood contain MMF hydrolytic activities, the mean half-lives of MMF in vitro were 15.1, 1.58, and 3.20 h, respectively. Thus, blood esterases seemed to contribute little to the rapid MMF disappearance in vivo. In vitro analyses showed that human intestinal microsomes exposed to 5 and 10 μM MMF exhibited hydrolytic activities of 2.38 and 4.62 nmol/(min · mg protein), respectively. Human liver microsomes exhibited hydrolytic activities of 14.0 and 26.1 nmol/(min · mg protein), respectively, approximately 6-fold higher than those observed for intestinal microsomes. MMF hydrolytic activities in human liver cytosols were 1.40 and 3.04 nmol/(min · mg protein), respectively. Because hepatic cytosols generally contain 5-fold more protein than microsomes, MMF hydrolysis in human liver cytosols corresponded to approximately 50% of that observed in microsomes. Fractions obtained by 9000g centrifugation of supernatants from COS-1 cells expressing human carboxylesterase (CES) 1 or 2 exhibited MMF hydrolytic activity, with CES1-containing fractions showing higher catalytic efficiency than CES2-containing fractions. The CES inhibitor bis-p-nitrophenylphosphate inhibited MMF hydrolysis in human liver microsomes and cytosols with IC50 values of 0.51 and 0.36 μM, respectively. In conclusion, both intestinal and hepatic CESs and in particular CES1 may be involved in MMF hydrolysis and play important roles in MMF bioactivation. Hepatic CES1 activity levels may help explain the between-subject variability observed for MMF usage.

Introduction

Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF) (Fig. 1), is a noncompetitive and reversible inhibitor of inosine monophosphate dehydrogenase and therefore inhibits the de novo pathway of guanine nucleotide synthesis. Because T- and B-lymphocytes are dependent on de novo purine synthesis for proliferation, MPA has a cytostatic effect on lymphocytes (Allison and Eugui, 1993).

Fig. 1.
Fig. 1.

Chemical structures of MMF and MPA.

MMF was designed to improve MPA bioavailability as an ester-type oral prodrug. The oral bioavailability of MPA after MMF administration is reported to be 80.7 to 94% (Bullingham et al., 1998; Pescovitz et al., 2000; Staatz and Tett 2007), such that MMF is essentially completely hydrolyzed to MPA by esterases (Bullingham et al., 1998). Because MMF was almost completely absent in plasma from patients with renal transplants (Bullingham et al., 1996; Armstrong et al., 2005), the conversion from MMF to the active form MPA is considered to occur rapidly in the blood, intestine, and liver, after gastrointestinal absorption (Lee et al., 1990).

Esterases in the blood, intestine, and liver play important roles in drug metabolism and detoxication (Satoh et al., 2002). Butylcholinesterase in plasma, arylesterase, and carboxylesterase (CES) 1 in the erythrocyte cytosol and acetylcholinesterase in the erythrocyte membrane have been described previously (La Du and Snady, 1971; Ott et al., 1975; Oertel et al., 1983). Human intestine and liver tissues express mainly CES2 and CES1/CES2, respectively, and CES expression in both intestine and liver is extremely high compared with that in other organs (Satoh et al., 2002). CESs play central roles in the hydrolysis and bioactivation of various drugs, including cocaine, heroin and irinotecan (Pindel et al., 1997; Slatter et al., 1997). In addition, CES1 and CES2 expressed in the liver cytosol are reported to be required for the bioactivation of irinotecan and capecitabine (Xu et al., 2002; Tabata et al., 2004a,b).

Glucuronidation is an important phase II metabolic pathway for MPA, and the molecular mechanisms underlying this process have been demonstrated by various researchers (Bernard and Guillemette, 2004; Picard et al., 2005). However, although we recently reported that MMF underwent hydrolysis as a phase I pathway via CES located in the hepatic microsome fraction in humans (Fujiyama et al., 2009), the systemic mechanisms responsible for MPA formation have not yet been fully elucidated. In addition, it is possible that interindividual variations in MPA oral bioavailability might be due to phase I metabolism by CESs. Therefore, to clarify the mechanisms of systemic MMF hydrolysis, we characterized the MMF hydrolytic pathway using human whole blood, erythrocytes, plasma, tissue microsomes, and cytosolic fractions. In addition, we investigated the involvement of CES family enzymes in MMF hydrolysis using tissue fractions from CES1- and CES2-expressing COS-1 cells.

Materials and Methods

Reagents and Chemicals.

MMF and MPA were supplied by Roche Palo Alto (Palo Alto, CA). 2-(6-Methoxy-2-naphthyl)propenoic acid (naproxen), diisopropyl fluorophosphate (DFP), bis-p-nitrophenylphosphate (BNPP), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco's modified Eagle's medium, fetal calf serum, and Lipofectamine Plus reagent were obtained from Life Technologies Japan (Tokyo, Japan). All other chemicals and solvents were of the highest grade commercially available.

Enzyme Sources.

Blood samples were collected into tubes containing EDTA from healthy Japanese volunteers (two men and three women aged 23–59 years) (samples HB-1 to HB-5). Informed consent was obtained from all subjects. After an aliquot of whole blood was taken, erythrocytes and plasma were separated by centrifugation at 3000 rpm for 5 min. Erythrocytes were washed using an equal volume of saline. All samples were assayed on the same day as collection, and no hemolysis was observed.

Pooled human liver cytosols (HLC), pooled human intestinal microsomes (HIM), and pooled human liver microsomes (HLM) were purchased from BD Gentest (Woburn, MA). Pooled human jejunal cytosols (HJC) and human ileal cytosols (HIC) were purchased from KAC (Kyoto, Japan).

Expression of each human carboxylesterase isoform in COS-1 cells was performed as described previously (Yamaori et al., 2006). In brief, COS-1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-incubated fetal calf serum. cDNA fragments encoding human CES1 or CES2 were ligated into linearized mammalian expression vectors and transfected into COS-1 cells using Lipofectamine Plus reagent according to the manufacturer's instructions. After 48 h, the transfected cells were collected and sonicated to prepare cell lysates. The lysates were centrifuged at 9000g for 20 min, and the resultant supernatants (S9 fractions) were homogenized and kept at −80°C until assay. An S9 fraction from COS-1 cells transfected with a plasmid carrying CES2 cDNA ligated in the reverse orientation was used as a negative control. Protein concentrations were estimated using a BCA Protein Assay Kit (Takarabio, Shiga, Japan) with bovine serum albumin used as a standard.

Characterization of MMF Hydrolysis In Vitro.

All MMF hydrolysis assays were performed as described previously with some modifications (Yamaori et al., 2006; Fujiyama et al., 2009). In brief, 450-μl aliquots of each human blood fraction (whole blood, erythrocytes, or plasma) were preincubated at 37°C for 5 min. Reactions were initiated by the addition of 50 μl of MMF solution (final concentration 100 μM). Incubations were performed at 37°C for 0, 10, 45, 90, and 135 min and terminated by the addition of 500 μl of acetonitrile. To determine the kinetic parameters of MMF hydrolysis, MMF (5–1600 μM) was incubated with or without each human tissue fraction (25 μg of HIM protein, 20 μg of HIC protein, 20 μg of HJC protein, 25 μg of HLM protein, 50 μg of HLC protein, or 25 or 50 μg of S9 fraction protein obtained from COS-1 cells expressing human CES1 and CES2, respectively) in 100 mM sodium phosphate buffer (pH 7.4) to give a final volume of 0.5 ml in 5-ml test tubes. Mixtures were incubated using a shaking water bath at 37°C for 30 min for intestinal fractions (HIM, HIC, and HJC), 5 min for HLM, 15 min for HLC, or 10 and 45 min for S9 fractions from COS-1 cells expressing human CES1 and CES2, respectively. Reactions were terminated by immersion in an ice bath and the addition of 0.5 ml of acetonitrile. Before extraction, naproxen (1.0 μg) in dimethyl sulfoxide (10 μl) was added to the reaction mixtures as an internal standard, and the solutions were vortexed for 30 s and then centrifuged for 10 min at 3000g. Aliquots of the clear supernatants were filtered through Millipore filters (0.45 μm, Millex-LH; Nihon Millipore, Tokyo, Japan). The final concentration of organic solvent in all reaction mixtures was <1.0%.

Samples were then directly injected into a high-performance liquid chromatography apparatus (AS-2057 plus intelligent sampler, PU-2080 plus chromatography pump, UV-2075 plus ultraviolet detector, and 807-IT integrator; JASCO, Tokyo, Japan) equipped with a CAPCELL PAK C18 MG II column (250 mm × 4.6 mm, 5 μm; Shiseido, Tokyo, Japan). The mobile phase was 0.5% KH2PO4 (pH 3.5)-acetonitrile (1:1). Elution was performed at a flow rate of 0.5 ml/min. MPA was monitored at a wavelength of 254 nm, and the lower limit of MPA detection was 25 ng/ml.

In preliminary experiments, reaction conditions were confirmed to ensure initial rates of MPA formation. MPA enzymatic production rates were calculated by subtracting nonenzymatic production rates obtained after incubation without added enzyme sources.

Inhibition Studies.

Inhibitory effects on the hydrolysis of MMF to MPA were evaluated using the inhibitors DFP, BNPP, and PMSF. Inhibitors were preincubated with human tissue fractions at 37°C for 1 min to give final inhibitor concentrations of 0.001 to 10 μM (DFP), 0.01 to 100 μM (BNPP), and 0.1 to 1000 μM (PMSF). Reactions were started by the addition of MMF solution, and the MMF levels were quantitated in reactions containing 12.5 μM liver fractions, 25 μM intestine fractions, or S9 fractions from COS-1 cells expressing human CES1 or CES2.

Data Analysis.

MMF concentration-time data obtained from MPA production reactions were fitted to the equation [C(t) = Ae−at] using SigmaPlot 9.0 software (HULINKS Inc., Tokyo, Japan). Velocity versus MMF concentration plots were fitted to the Michaelis-Menten equation (one- or two-site saturation models) by nonlinear least-squares regression analysis using SigmaPlot 9.0 software). IC50 values were calculated by nonlinear least-squares regression analysis using SigmaPlot 9.0 software.

Statistical Analysis.

Statistical analyses were performed using an unpaired t test or one-way analysis of variance followed by Dunnett's test using InStat 3 software (GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered statistically significant.

Results

MMF Hydrolysis to MPA in Human Blood Fractions.

MMF hydrolysis in human blood samples was evaluated in vitro. MPA formation from nonenzymatic MMF hydrolysis after 10, 45, 90, and 135 min of incubation was 0.35, 1.11, 2.02, and 3.15%, respectively, using 100 μM MMF in 100 mM phosphate buffer without the addition of enzyme sources. As shown in Fig. 2, whole blood, erythrocytes, and plasma formed MPA from MMF time dependently. The elimination rate constants (kel) of MMF in whole blood, erythrocytes, and plasma were 0.24 ± 0.11, 0.46 ± 0.11, and 0.051 ± 0.015 h−1, respectively, giving corresponding MMF half-lives (t1/2) of 3.2 ± 0.94, 1.6 ± 0.43, and 15 ± 6.1 h, respectively (Table 1). The half-life of MMF in whole blood was approximately 2-fold longer than that in erythrocytes, whereas the plasma MMF half-life was approximately 10-fold longer than that in erythrocytes.

Fig. 2.
Fig. 2.

Plots of MMF concentration as a function of incubation time with human blood fractions. Residual amounts of MMF were calculated by the formation of MPA in human blood fractions (whole blood, erythrocytes, and plasma) at the indicated times. All points represent the mean ± S.D. of five subjects (HB-1 to HB-5).

View this table:
TABLE 1

Kinetic parameters of MMF hydrolysis in vitro in human blood fraction

MMF Hydrolysis to MPA in Human Tissue Fractions.

To calculate the kinetic parameters of MPA formation from MMF, MMF was incubated with HIM, HJC, HIC, HLM, and HLC (Fig. 3). In preliminary experiments, reaction conditions were confirmed to ensure that the initial rates of MPA formation were optimal for each protein concentration and each reaction time, as described under Materials and Methods. Velocity versus MMF concentration plots for HIM, HJC, and HIC are shown in Fig. 3, A to C, with the three data plots best fitted to a two-site saturation model. Typical Eadie-Hofstee plots of MPA formed from MMF in the presence of HIM, HJC, and HIC were clearly biphasic as shown in the insets to Fig. 3. The kinetic parameters of MPA formation from MMF are shown in Table 2. For HIM, the estimated apparent Km, Vmax, and intrinsic clearance (Clint; Vmax/Km) values were 27.3 ± 4.7 μM, 15.6 ± 1.5 nmol/(min · mg protein), and 571 μl/(min · mg protein), respectively, for the high-affinity enzyme reaction and 782 ± 256 μM, 19.7 ± 1.2 nmol/(min · mg protein), and 252 μl/(min · mg protein), respectively, for the low-affinity enzyme reaction. For HJC, the estimated apparent Km, Vmax, and Clint values for MPA formation were 14.9 ± 5.4 μM, 4.58 ± 0.72 nmol/(min · mg protein), and 307 μl(min · mg protein), respectively, for the high-affinity enzyme reaction and 1091 ± 432 μM, 14.2 ± 1.76 nmol/(min · mg protein), and 13 μl/(min · mg protein), respectively, for the low-affinity enzyme reaction. For HIC, the estimated apparent Km, Vmax, and Clint values for MPA formation were 17.5 ± 6.0 μM, 5.94 ± 0.84 nmol/(min · mg protein), and 339 μl/(min · mg protein), respectively, for the high-affinity enzyme reaction and 1656 ± 1355 μM, 12.0 ± 4.2 nmol/(min · mg protein), and 7 μl/(min · mg protein), respectively, for the low-affinity enzyme reaction.

Fig. 3.
Fig. 3.

Velocity versus concentration plots of MPA formation from MMF by gastrointestinal tissue fractions. MMF (5–1600 μM) was incubated with HIM (A), HJC (B), HIC (C), HLM (D), or HLC (E) as described under Materials and Methods. Insets show Eadie-Hofstee plots. Vertical maximum scales in each graph are adjusted to enhance readability. F, MMF hydrolytic activities at 5 μM (□) and 10 μM (■) for each enzyme source. Columns represent the mean of three determinations. v, velocity; [s], MMF concentration.

View this table:
TABLE 2

Kinetic parameters of MMF hydrolysis in human tissue fractions

Values are represented as mean (S.D.).

Velocity versus MMF concentration plots obtained using HLM and HLC are shown in Fig. 3, D and E, respectively, and the data were best fitted to a one-site saturation model. The estimated apparent Km and Vmax values for MPA formation using HLM were 992 ± 69 μM and 3072 ± 112 nmol/(min · mg protein), respectively (Table 2). The Clint of MMF hydrolysis for HLM [3098 nl/(min · mg protein)] was higher than that reported in our previous study [1308 nl/min · mg protein)] (Fujiyama et al., 2009). The estimated Km, Vmax, and Clint values obtained for HLC were 329 ± 20 μM, 75.4 ± 2.0 nmol/(min · mg protein), and 230 nl/(min · mg protein), respectively.

MMF hydrolytic activities for all tissue fractions are shown in Fig. 3F. MPA formation from 5 and 10 μM MMF in the presence of HLM was 14.0 and 26.1 nmol/(min · mg protein), respectively, compared with 1.40 and 3.04 nmol/(min · mg protein), respectively, in the presence of HLC, 2.38 and 4.62 nmol/(min · mg protein), respectively, for HIM, 1.11 and 1.92 nmol/(min · mg protein), respectively, for HJC, and 1.29 and 2.33 nmol/(min · mg protein), respectively, for HIC. HLC corresponded to 10.0 and 11.6% of MMF hydrolytic activities of HLM with 5 and 10 μM MMF, respectively.

MMF Hydrolysis by Recombinant Human CES1 and CES2.

Levels of MPA formation in S9 fractions from COS-1 cells expressing human CES1 or CES2 after MMF incubation are shown in Fig. 4A. Hydrolytic activities on 100 μM MMF in response to the negative control, hCES1, and hCES2 were 0.51 ± 0.16, 83.6 ± 6.2, and 7.98 ± 0.43 nmol/(min · mg protein), respectively. COS-1 CES1 and CES2 S9 fractions had significantly higher MMF hydrolytic activities compared with that for the negative control. To characterize MMF hydrolysis by CES1 and CES2, kinetic parameters were calculated from velocity versus MMF concentration plots, as shown in Fig. 4, B and C. In both cases, data best fitted to a one-site saturation model. The estimated Km, Vmax, and Clint values for MPA formation were 225 ± 12 μM, 313 ± 5 nmol/(min · mg protein), and 1391 μl/(min · mg protein), respectively, for hCES1 and 22.3 ± 2.1 μM, 12.3 ± 0.2 nmol/(min · mg protein), and 552 μl/(min · mg protein), respectively, for hCES2 (Table 3).

Fig. 4.
Fig. 4.

MMF hydrolysis catalyzed by S9 fractions from COS-1 cells expressing human CES1 and CES2. A, hydrolysis of 100 μM MMF in S9 fractions from COS-1 cells expressing human CES1 and CES2 was compared with a negative control. Columns represent the mean ± S.D. of three determinations. B and C, velocity (v) versus MMF concentration ([s]) plots of MPA formation from MMF by gastrointestinal tissue fractions. MMF (5–1600 μM) was incubated with S9 fractions from COS-1 cells expressing human CES1 and CES2 as described under Materials and Methods. Insets show Eadie-Hofstee plots. Vertical maximum scales in each graph are adjusted to enhance readability. *, significantly different from the negative control, p < 0.001.

View this table:
TABLE 3

Kinetic parameters of MMF hydrolysis in S9 from COS-1 cells expressing CES1 and CES2

Values are represented as mean (S.D.).

Effects of CES Inhibitors on MMF Hydrolysis to MPA.

Inhibitory effects of DFP, BNPP, and PMSF on MMF hydrolysis in the presence of HIM, HJC, HIC, HLM, HLC, hCES1, and hCES2 are shown in Fig. 5, A to G. MPA formation from all enzymatic sources was inhibited by DFP, BNPP, and PMSF in concentration-dependent manners, except for DFP and BNPP inhibition of MPA formation by HIM. Residual MMF hydrolytic activities in HIM in the presence of 10 μM DFP and 100 μM BNPP were 14.2 and 12.8% of control values, respectively (Fig. 4A). The estimated IC50 values for inhibitory effects as determined from these residual activities are shown in Table 4. Inhibitory effects on MMF hydrolysis by DFP, BNPP, and PMSF were stronger in HLC than in HLM, and the effects of DFP and BNPP were stronger on hCES2 than on hCES1.

Fig. 5.
Fig. 5.

Effects of CES inhibitors on MPA formation by human gastrointestinal organ fractions and S9 fractions from COS-1 cells expressing CES1 and CES2. Inhibitory effects on MMF hydrolysis for each enzyme source were tested using DFP (●, 0.001–10 μM), BNPP (□, 0.01–100 μM), and PMSF (▴, 0.1–1000 μM). The MMF concentration in each reaction mixture was 25 μM (intestinal fraction), 12.5 μM (liver fraction), and 25 μM (S9 fraction from COS-1 cells). Control activities were 7.42 nmol/(min · mg protein) for HIM (A), 2.66 nmol/(min · mg protein) for HJC (B), 3.02 nmol/(min · mg protein) for HIC (C), 23.3 nmol/(min · mg protein) for HLM (D), 4.36 nmol/(min · mg protein) for HLC (E), 29.3 nmol/(min · mg protein) for hCES1 (F), and 5.50 nmol/(min · mg protein) for hCES2 (G). All points represent the mean of two experiments.

View this table:
TABLE 4

IC50 values of DFP, BNPP, and PMSF for MMF hydrolysis

Discussion

Several reports have found that the pharmacokinetics of MPA in humans is characterized by large inter- and intraindividual variability and that glucuronide activity from MPA may be involved in the between-individual variability (Kuypers et al., 2005; Baldelli et al., 2007). In the present study, we focused on the importance of the MMF bioactivation pathway to MPA as a factor other than the MPA glucuronidation pathway previously researched.

At 10 min after peripheral intravenous infusion with MMF, plasma MMF concentrations fall below detection levels in human test subjects, which led to the conclusion that the half-life of MMF in human plasma in vivo was approximately 2 min (Bullingham et al., 1996). However, in the present in vitro study, the estimated mean half-life of MMF in plasma was 15.1 and 1.58 h in erythrocytes. Because the measured mean hematocrit value was 0.468 ± 0.053, esterases located in erythrocytes seemed to be involved in MMF hydrolysis. However, the half-life of MMF in human blood in vitro was much longer than that reported in a previous in vivo study (Bullingham et al., 1996). This finding suggests that blood esterases contribute little to the rapid disappearance of MMF in the human body and that gastrointestinal organs and the liver may be central to MMF hydrolysis.

Our study found that the order of MMF to MPA hydrolysis rate was liver ≫ intestine, with HLM showing the highest MMF hydrolytic activity [Clint for MPA formation in the presence of HLM being 3098 nl/(min · mg protein)]. However, our previous study gave an intrinsic clearance value for HLM of 1308 nl/(min · mg protein) (Fujiyama et al., 2009). The HLM used in the present and previous studies were supplied by BD and consisted of microsomes pooled from 33 and 27 donors, respectively. Because microsomal carboxylesterase activity can exhibit large interindividual variations, the difference in HLM activities between our studies could be due to chance differences in assay validation. The Clint for MMF hydrolysis in response to enzymes from intestinal tissues was 5-fold lower than that from liver cellular fractions. It is known that the intestine predominantly expresses the CES2 isoform rather than CES1 (Satoh et al., 2002; Taketani et al., 2007). Therefore, it is possible that the MMF hydrolytic activity in hepatic fractions was mainly due to CES1, as the liver highly expresses both CES1 and CES2. To clarify the role of CES isoforms in MMF hydrolysis, we examined recombinant human cells expressing CES1 or CES2. Hydrolytic activity on 100 μM MMF by hCES1 was approximately 10-fold higher than that by hCES2, such that the Clint for MPA formation by hCES1 was higher than that by hCES2 [1391 versus 552 nl/(min · mg protein)]. Thus, it appears that CES1 may be important for MPA formation. However, because orally administrated prodrugs encounter intestinal esterases before liver esterases, it is likely that some hydrolysis would still occur in the intestine (Fig. 6).

Fig. 6.
Fig. 6.

Schematic illustration of MMF bioactivation.

Because CESs were reported to be expressed in the inner lumen of the endoplasmic reticulum (Robbi and Beaufay, 1987; Medda and Proia, 1992), it was thought that only compounds located in the lumen underwent hydrolysis and glucuronidation. However, Tabata et al. (2004a) reported that CES1 is present in both microsomes and cytosols and that cytosolic CES plays an important role in the bioactivation of the prodrug, capecitabine. Because there is approximately 5-fold more total cytosolic protein than microsomal protein, when we extrapolated this to the results obtained in the present study, we estimated that cytosolic MMF hydrolysis was equivalent to approximately 37 or 88% of the microsomal MMF hydrolysis. This result is consistent with our finding that HLC showed 46 to 50% lower MMF hydrolytic activity than HLM over the concentration range of 5 to 10 μM MMF. On the basis of these results, our study indicated that the order of MMF to MPA hydrolysis rates was HLM > HLC > HIM > HJC = HIC. Moreover, although MMF has high membrane permeability, MMF hydrolysis in the cytosol may be decreased by formation of MPA glucuronide metabolites by uridine diphosphate glucuronosyltransferases located in the inner lumen of the endoplasmic reticulum because of the low membrane permeability of MPA. Therefore, cytosolic CESs may be an important determinant of MMF pharmacokinetics in vivo.

We further investigated the esterases involved in MMF hydrolysis in human samples using a series of inhibitors. In erythrocytes, the CES inhibitor BNPP (10 μM) inhibited MMF hydrolysis by only 5.6% relative to control values, whereas the arylesterase inhibitor 5,5′-dithiobis(2-nitrobenzoic acid) showed no inhibition at all (Heymann and Krisch, 1967; Costello and Green, 1983) (supplemental data). Therefore, an erythrocyte esterase other than CES and arylesterase, namely acetylcholinesterase, seemed to be responsible for MMF hydrolysis in whole blood. In intestinal fractions, addition of DFP and BNPP to HIM inhibited MMF hydrolysis by 87% relative to control values. In theory, 92% of the hydrolysis of 25 μM MMF by HIM was due to the high-affinity enzyme present in HIM. The kinetic parameters of MPA formation by the high-affinity enzyme in HIM were similar to the kinetics displayed by S9 fractions from COS-1 cells expressing CES2. Therefore, our results indicated that the HIM esterase involved in MMF hydrolysis is CES2. Although intestinal cytosolic fractions HJC and HIC also exhibited the biphasic curves, MMF hydrolysis in HJC and HIC was completely inhibited by BNPP. This result suggested that multiple CESs might be involved in this pathway. Moreover, to our knowledge, the existence of CESs in the intestinal cytosol has not been reported previously. Because both microsomal and cytosolic CESs appear to play key roles in first-pass effects on the esterified prodrug, further study on intestinal cytosolic CESs is required.

In conclusion, MMF was hydrolyzed by human intestinal and hepatic fractions rather than by human blood fractions. Although orally dosed MMF rapidly disappears from the blood, our results suggested that the disappearance of MMF was due mainly to hepatic CESs, in particular CES1.

Footnotes

  • This work was supported by the Japan Research Foundation for Clinical Pharmacology, Tokyo, Japan; and the Research Foundation for Pharmaceutical Sciences, Tokyo, Japan.

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    doi:10.1124/dmd.110.034249.

  • ↵Embedded Image The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    MPA
    mycophenolic acid
    MMF
    mycophenolate mofetil
    CES
    carboxylesterase
    DFP
    diisopropyl fluorophosphate
    BNPP
    bis-p-nitrophenylphosphate
    PMSF
    phenylmethylsulfonyl fluoride
    HLC
    pooled human liver cytosols
    HIM
    pooled human intestinal microsomes
    HLM
    pooled human liver microsomes
    HJC
    pooled human jejunal cytosols
    HIC
    pooled human ileal cytosols
    h
    human.

  • Received May 4, 2010.
  • Accepted September 7, 2010.

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Involvement of Carboxylesterase 1 and 2 in the Hydrolysis of Mycophenolate Mofetil

Nobuhiro Fujiyama, Masatomo Miura, Shoutaro Kato, Tomomichi Sone, Masakazu Isobe and Shigeru Satoh
Drug Metabolism and Disposition December 1, 2010, 38 (12) 2210-2217; DOI: https://doi.org/10.1124/dmd.110.034249
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