| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
a address correspondence to this author at: Abteilung Klinische Chemie, Zentrum Innere Medizin, Georg-August-Universität, Robert Koch Strasse 40, D-37075 Göttingen, Germany
Mycophenolic acid (MPA), the active moiety of mycophenolate mofetil (MMF), is an antiproliferative agent that acts by inhibition of inosine monophosphate dehydrogenase type II (IMPDH-II), a key enzyme in the de novo purine biosynthetic pathway (1)(2). Several studies have documented that MMF is effective in the treatment of refractory rejection in renal, heart, and liver transplant recipients (2). The major pathway for elimination of MPA involves glucuronidation (3) at the phenolic hydroxyl group to form mycophenolate 7-O-glucuronide (7-O-MPAG). Modification of this phenolic hydroxy residue leads to a loss of pharmacological activity toward IMPDH-II (4)(5).
Most studies on the pharmacokinetics of MPA have utilized HPLC procedures (6)(7) to measure both MPA and MPAG. Recently, the first immunoassay became available for the quantification of MPA (Emit-MPA, Dade Behring). 7-O- MPAG does not cross-react with this assay. Comparison of plasma MPA concentrations from clinical samples determined with HPLC showed an overestimation in relation to those obtained with the Emit of up to 100%, with an average of 35% in a group of 37 kidney recipients, which accounts for a mean overestimation of 20% for the calculated areas under the concentration-time curve (8). Through a modification of our HPLC procedure (7), we were able to identify two putative MPA metabolites, M-1 and M-2, from the plasma of transplant recipients, of which M-2 was found to cross-react in the immunoassay (8). Recently, we showed that in clinical samples from heart, kidney, and liver recipients, the relative amounts of M-2 correlate with the bias between MPA values determined with HPLC and the immunoassay (9). In pharmacokinetic studies, it was shown that the areas under the concentration-time curve for both M-1 and M-2 can account for~10% of those of MPA, whereas M-2 can be present in predose trough samples at concentrations up to those of MPA (9). Mass spectrometric analyses are consistent with M-1 being the 7-O-glucoside conjugate and M-2 being an acyl glucuronide of MPA (10). In addition, both metabolites could be produced by incubation of MPA with human liver microsomes and the respective cosubstrates (10). In a recent review Bullingham et al. (11) pointed out that an acyl glucuronide conjugate of MPA was observed in the urine of patients in amounts similar to those of MPA. Knowledge of the pharmacological potential of drug metabolites is essential for judging their role in drug therapy. We, therefore, investigated the ability of the newly discovered metabolites to inhibit human IMPDH-II
MPA, 7-O-MPAG, M-1, and M-2 were isolated from the plasma of transplant recipients under treatment with MMF, using HPLC as described elsewhere (10). After collection, the fractions containing metabolites were desalted by column chromatography, concentrated by evaporation, and reconstituted in water. The purity of the isolated metabolites was confirmed by rechromatography on HPLC.
Human IMPDH-II was expressed in Escherichia coli (DH10B;
Life Technologies), using the pProEX-HTb plasmid (Life Technologies),
which was modified to express the unmodified recombinant human
IMPDH-II. Briefly, an 85-bp fragment of pProEX-HTb containing the
ribosome binding site was enzymatically amplified with a restriction
site for RsaI at the 5' end. The 3' end was constructed to
include the translation initiation codon and a 22-bp fragment of the
human IMPDH-II mRNA sequence. ssDNA of this construct was used as the
forward primer with a human lymphocyte cDNA library, whereas the
specific reverse primer was equipped with a NotI restriction
site. After enzymatic amplification, the resulting construct was
ligated into the plasmid after RsaI and NotI
digestion of both insert and plasmid with a T4 DNA ligase (Life
Technologies). The IMPDH-II insert was confirmed by complete sequencing
using a LI-COR Model 4200 infrared dye sequencer (MWG-Biotec). Cells
were harvested 10–20 h after induction of expression in transformed
E. coli with isopropyl-ß-D-thiogalactoside,
and recombinant human IMPDH-II (rh-IMPDH-II) was extracted with 4 mol/L
urea and purified by chromatography over phosphocellulose followed by
blue-Sepharose as described elsewhere (12). The purity after
these steps was >99% as estimated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (Fig. 1
). The integrity of the protein was confirmed by N-terminal
amino acid sequencing. The specific activity of the rh-IMPDH-II was
determined to be 800 U/g protein. The conditions for determination of
IMPDH-II activity were as follows (final concentrations in reaction):
100 mmol/L Tris-HCl, 200 µmol/L IMP, 300 µmol/L NAD+, 3
mmol/L EDTA, 1 mmol/L dithiothreitol, and 1.5 U/L rh-IMPDH-II.
Inhibition of the enzymatic activity of rh-IMPDH-II was used to assess
the pharmacological activity of the metabolites as follows: 2.0–12
µL of sample containing either metabolite, MPA, or H2O as
control was mixed with 250 µL of reaction mixture without
NAD+, and after incubation for 5 min, the reaction was
started by the addition of 30 µL of NAD+ solution; the
enzymatic activity was followed by measurement of absorbance at 340 nm
for 5 min. The enzyme reactions were carried out at 37 °C on a Cobas
Mira Plus analyzer (Hoffmann-La Roche).
|
As shown in Table 1
, M-2 isolated from the plasma of a liver transplant recipient
under treatment with MMF displayed a concentration-dependent inhibition
of rh-IMPDH-II similar to that obtained with MPA. The ability of M-2 to
inhibit rh-IMPDH-II was further investigated by use of a total of 21
separate HPLC preparations of this metabolite obtained from the plasma
of six kidney transplant recipients, one heart transplant recipient,
and one liver transplant recipient at different time points after
transplantation. The concentration of M-2 in each preparation was
estimated using the MPA Emit assay and compared with the MPA
concentration that caused the same inhibition of rh-IMPDH-II within the
same analytical run (inhibition/concentration ratio). The inhibitory
effect of M-2 from different preparations was found to be somewhat
variable compared with that of MPA: the median inhibition/concentration
ratio of M-2 to MPA, as a measure of the relative effectiveness
compared to MPA, was 0.91 (range, 0.48–1.23; n = 21).
|
In contrast, 7-O-MPAG, also fractionated by HPLC, showed no measurable enzyme inhibition. When five fresh preparations of 7-O-MPAG from the plasma of kidney recipients with final concentrations of 8–23 mg/L were tested immediately, the enzyme activity in the presence of 7-O-MPAG was not significantly different from that of the respective control assay using H20 (ratio of enzyme activity with 7-O-MPAG to the control activity:median, 0.998; range, 0.94–1.03; P = 0.61). The purity of these preparations was confirmed by HPLC analysis.
The 7-O-glucoside metabolite M-1, which like 7-O-MPAG does not cross-react in the immunoassay, also did not inhibit rh-IMPDH-II at concentrations up 500 µg/L estimated by HPLC; this concentration estimation was based on the assumption that both 7-O-MPAG and M-1 have a similar ultraviolet absorption at 215 nm.
This is the first study to demonstrate the presence of a metabolite of MPA that possesses a pharmacological potency almost as high as that of the parent drug. Like MPA, this metabolite (M-2) cross-reacts with the antibody used in the Emit immunoassay. M-2 has been observed regularly in the plasma of liver, kidney, and heart transplant recipients undergoing treatment with MMF (8) and has been identified as the acyl glucuronide of MPA (10). This is in agreement with the observation that modifications at the acyl residue do not lead to a substantial loss of IMPDH-II inhibitory efficacy, in contrast to modifications at the phenolic hydroxyl group (13). Results from a recent study (3) into the glucuronidation of MPA with human UDP-glucuronosyl transferase (UGT1A10) showed that in addition to the 7-OH glucuronide, a second product was formed, which was proposed to be the acyl glucuronide of MPA. Some variability was observed in the ability of different preparations of M-2 to inhibit IMPDH-II. This may be related to the fact that acyl glucuronides undergo intramolecular rearrangement at physiological pH from position 1 to positions 2, 3, and 4 in the glucuronic acid moiety (14). The metabolite M-2 isolated from the plasma of patients is, therefore, likely to be a mixture of these different isomers. Whether these isomers differ in their ability to inhibit rh-IMPDH-II must await their separation and isolation.
In accordance with the results of previous investigations (5), purified 7-O-MPAG did not inhibit rh-IMPDH-II. The higher IMPDH-II inhibition of 7-O-MPAG seen in lymphoblasts (15) could not be confirmed with the recombinant enzyme. According to our experience, during 7-O-MPAG isolation and storage, hydrolysis to MPA can occur in trace amounts, which then inhibit IMPDH activity, when 7-O-MPAG is tested at high concentrations (data not shown). This phenomenon may also explain the apparent IMPDH inhibition by 7-O-MPAG obtained in a test system that used a cell lysate as the source of IMPDH-II (15). The second metabolite, M-1, has been shown to be the 7-O-glucoside of MPA (10). In accordance with the modification at the phenolic hydroxyl group, this metabolite, like the 7-O-MPAG, did not inhibit rh-IMPDH-II when tested at concentrations up to 40-fold higher than those that achieved a 50% inhibition with MPA or M-2. The 7-O-glucoside metabolite, like 7-O-MPAG, did not cross-react in the immunoassay. In conclusion, a pharmacologically active metabolite of MPA has been identified, which is present in the plasma of patients undergoing immunosuppressive therapy with MMF. The magnitude of the observed rh-IMPDH-II inhibition and the presence of M-2 in the plasma of transplant recipients (9) in concentrations up to those of MPA in predosetrough samples is of interest with regard to its contribu-tion to the immunosuppressive and toxic effects of MPA.
Acknowledgments
We thank Reiner Andag, Tanja Schneider, and Hamza Sinanoglu for their excellent and skillful technical assistance. M. Shipkova was supported by a grant from the Volkswagen Stiftung. We give special thanks to H. Eiffert from the Abteilung Medizinische Mikrobiologie for performing N-terminal amino acid sequencing of the expressed rh-IMPDH-II.
Footnotes
Abteilung Klinische Chemie, Georg-August-University, D-37075 Göttingen, Germany
fax 49 (0)551-398551, e-mail eschuetz{at}med.uni-goettingen.de
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  O. Gensburger, N. Picard, and P. Marquet Effect of Mycophenolate Acyl-Glucuronide on Human Recombinant Type 2 Inosine Monophosphate Dehydrogenase Clin. Chem., May 1, 2009; 55(5): 986 - 993. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. M. Davies, J. Grinyo, R. Heading, B. Maes, H.-U. Meier-Kriesche, and M. Oellerich Gastrointestinal side effects of mycophenolic acid in renal transplant patients: a reappraisal Nephrol. Dial. Transplant., September 1, 2007; 22(9): 2440 - 2448. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Jacobson, K. Green, J. Rogosheske, C. Brunstein, B. Ebeling, T. DeFor, P. McGlave, and D. Weisdorf Highly Variable Mycophenolate Mofetil Bioavailability Following Nonmyeloablative Hematopoietic Cell Transplantation J. Clin. Pharmacol., January 1, 2007; 47(1): 6 - 12. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Bernard, J. Tojcic, K. Journault, L. Perusse, and C. Guillemette Influence of Nonsynonymous Polymorphisms of UGT1A8 and UGT2B7 Metabolizing Enzymes on the Formation of Phenolic and Acyl Glucuronides of Mycophenolic Acid Drug Metab. Dispos., September 1, 2006; 34(9): 1539 - 1545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. S. Ting, N. Partovi, R. D Levy, K W. Riggs, and M. H. Ensom Pharmacokinetics of Mycophenolic Acid and Its Glucuronidated Metabolites in Stable Lung Transplant Recipients Ann. Pharmacother., September 1, 2006; 40(9): 1509 - 1515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. S. Westley, L. R. Brogan, R. G. Morris, A. M. Evans, and B. C. Sallustio ROLE OF MRP2 IN THE HEPATIC DISPOSITION OF MYCOPHENOLIC ACID AND ITS GLUCURONIDE METABOLITES: EFFECT OF CYCLOSPORINE Drug Metab. Dispos., February 1, 2006; 34(2): 261 - 266. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. R. Ingle and T. Shah Enteric-coated mycophenolate sodium for transplant immunosuppression Am. J. Health Syst. Pharm., November 1, 2005; 62(21): 2252 - 2259. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Picard, D. Ratanasavanh, A. Premaud, Y. Le Meur, and P. Marquet IDENTIFICATION OF THE UDP-GLUCURONOSYLTRANSFERASE ISOFORMS INVOLVED IN MYCOPHENOLIC ACID PHASE II METABOLISM Drug Metab. Dispos., January 1, 2005; 33(1): 139 - 146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. G Morris Immunosuppressant Drug Monitoring: Is the Laboratory Meeting Clinical Expectations? Ann. Pharmacother., January 1, 2005; 39(1): 119 - 127. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. K. Basu, L. Kole, S. Kubota, and I. S. Owens HUMAN UDP-GLUCURONOSYLTRANSFERASES SHOW ATYPICAL METABOLISM OF MYCOPHENOLIC ACID AND INHIBITION BY CURCUMIN Drug Metab. Dispos., July 1, 2004; 32(7): 768 - 773. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Neumann, M. Haidinger, H. Jager, H. Grutzmacher, A. Griesmacher, M. M. Muller, P. M. Bayer, and F. Thomas Meisl Pharmacokinetics of Mycophenolate Mofetil in Patients with Autoimmune Diseases Compared Renal Transplant Recipients J. Am. Soc. Nephrol., March 1, 2003; 14(3): 721 - 727. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. T. Weber, M. Shipkova, V. W. Armstrong, N. Wagner, E. Schutz, O. Mehls, L. B. Zimmerhackl, M. Oellerich, and B. Tonshoff Comparison of the Emit Immunoassay with HPLC for Therapeutic Drug Monitoring of Mycophenolic Acid in Pediatric Renal-Transplant Recipients on Mycophenolate Mofetil Therapy Clin. Chem., March 1, 2002; 48(3): 517 - 525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. T. Weber, M. Shipkova, V. W. Armstrong, N. Wagner, E. Schuutz, O. Mehls, L. B. Zimmerhackl, M. Oellerich, and B. Tonshoff The Pharmacokinetic-Pharmacodynamic Relationship for Total and Free Mycophenolic Acid in Pediatric Renal Transplant Recipients: A Report of the German Study Group on Mycophenolate Mofetil Therapy J. Am. Soc. Nephrol., March 1, 2002; 13(3): 759 - 768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Mourad, J. Malaise, D. Chaib Eddour, M. De Meyer, J. Konig, R. Schepers, J. P. Squifflet, and P. Wallemacq Pharmacokinetic Basis for the Efficient and Safe Use of Low-Dose Mycophenolate Mofetil in Combination with Tacrolimus in Kidney Transplantation Clin. Chem., July 1, 2001; 47(7): 1241 - 1248. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Oellerich and V. W. Armstrong Prodrug Metabolites: Implications for Therapeutic Drug Monitoring Clin. Chem., May 1, 2001; 47(5): 805 - 806. [Full Text] [PDF]
|
|
|
|
|
|
T. M. Annesley, S. Kurzyniec, G. D. Nordblom, N. Buchanan, W. Pool, M. Reily, R. Talaat, and W. L. Roberts Glucuronidation of Prodrug Reactive Site: Isolation and Characterization of Oxymethylglucuronide Metabolite of Fosphenytoin Clin. Chem., May 1, 2001; 47(5): 910 - 918. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Mourad, J. Malaise, D. Chaib Eddour, M. De Meyer, J. Konig, R. Schepers, J. P. Squifflet, and P. Wallemacq Correlation of Mycophenolic Acid Pharmacokinetic Parameters with Side Effects in Kidney Transplant Patients Treated with Mycophenolate Mofetil Clin. Chem., January 1, 2001; 47(1): 88 - 94. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Millan, F. Oppenheimer, M. Brunet, J. Vilardell, I. Rojo, J. Vives, and J. Martorell Assessment of Mycophenolic Acid-induced Immunosuppression: A New Approach Clin. Chem., September 1, 2000; 46(9): 1376 - 1383. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Morissette, M. Beaulieu, and B. Vinet A Specific Artificial Antibody toward Mycophenolic Acid Prepared by Molecular Imprinting Clin. Chem., September 1, 2000; 46(9): 1516 - 1518. [Full Text] [PDF]
|
|
|
|
|
|
M. Shipkova, E. Schutz, V. W. Armstrong, P. D. Niedmann, M. Oellerich, and E. Wieland Determination of the Acyl Glucuronide Metabolite of Mycophenolic Acid in Human Plasma by HPLC and Emit Clin. Chem., March 1, 2000; 46(3): 365 - 372. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
