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Clinical Chemistry 45: 1047-1050, 1999;
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(Clinical Chemistry. 1999;45:1047-1050.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

The Apparent Inhibition of Inosine Monophosphate Dehydrogenase by Mycophenolic Acid Glucuronide Is Attributable to the Presence of Trace Quantities of Mycophenolic Acid

Magdalena Korecka1, Dejan Nikolic2, Richard B. van Breemen2 and Leslie M. Shaw1,a

1 Departments of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104.

2 Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL 60612.
a Author for correspondence. Fax 215-662-7529; e-mail shawlmj{at}mail.med.upenn.edu


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Mycophenolic acid glucuronide, the primary metabolite of the immunosuppressive agent mycophenolic acid, affords weak inhibition of proliferating and resting lymphocytes and recombinant human inosine monophosphate dehydrogenase in comparison to the active drug. We evaluated the hypothesis that mycophenolic acid is a trace contaminant of the glucuronide metabolite preparation and that this accounts for the observed effects of mycophenolic acid glucuronide on human inosine monophosphate dehydrogenase catalytic activity both in lymphocytes and the pure enzyme.

Methods: We used negative ion electrospray HPLC-mass spectrometry (HPLC-MS) and HPLC-tandem MS (HPLC-MS-MS) to identify mycophenolic acid as a contaminant of mycophenolic acid glucuronide. Quantification of the mycophenolic acid contaminant was achieved using a negative ion electrospray HPLC-MS method in the selected-ion monitoring mode.

Results: Trace amounts of mycophenolic acid were detected and definitively identified in the mycophenolic acid glucuronide preparation by the HPLC-MS-MS analysis. In addition to having identical HPLC retention times, pure mycophenolic acid and the contaminant produced the following major fragments upon HPLC-MS-MS analysis: deprotonated molecular ion, m/z 319; and fragment ions, m/z 275, 243, 205, and 191 (the most abundant fragment ion). Using the negative ion electrospray HPLC-MS procedure in the selected-ion monitoring mode, the quantity of the contaminant mycophenolic acid was determined to be 0.312% ± 0.0184% on a molar basis.

Conclusion: These data provide strong support for the proposal that the apparent inhibition of the target enzyme inosine monophosphate dehydrogenase by mycophenolic acid glucuronide is attributable to the presence of trace amounts of contaminant mycophenolic acid.© 1999 American Association for Clinical Chemistry


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mycophenolic acid (MPA),1 the active metabolite of the pro-drug mycophenolate mofetil (MMF), provides effective immunosuppression by inhibiting inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo pathway of DNA synthesis that is responsible for guanine production. In vitro studies have shown that incubation of proliferating lymphocytes with MPA causes reduction of the guanine nucleotide pool by 40% to 60% (1)(2) and at the same time arrests the cycling of these cells at the G1/S interface. The phenolic glucuronide of mycophenolic acid, (MPAG), the primary metabolite of MPA, is produced by UDP-glucuronosyltransferase(s) in liver and possibly other tissues such as kidney and small intestine. Plasma concentrations of MPAG exceed MPA concentrations in all transplant patients who receive MMF, and reach values >100-fold higher in renally impaired patients (3). Three recent studies have reported that compared with MPA, MPAG weakly inhibits IMPDH in resting (4) and activated lymphocytes (5), and also weakly inhibits recombinant human IMPDH (6). These findings have prompted the question: Does MPAG provide immunosuppression in addition to that afforded by MPA in transplant patients treated with MMF? If the answer is yes, it would be important to measure MPAG along with MPA in pharmacokinetic-clinical response studies and in therapeutic drug monitoring programs.

In an earlier study, we proposed that the weak inhibition provided by MPAG could be explained by the possible presence of trace amounts of MPA in the MPAG preparation. This proposal was based on the appearance of a small HPLC peak with a retention time equal to that of MPA calibrator (6) upon analysis of the MPAG preparation. Support for this explanation was obtained using the technique of tight binding enzyme inhibitor kinetics (7), which indicated that the presence of a trace quantity of MPA in the MPAG preparation could account for the observed IMPDH inhibition (6). To rigorously evaluate the possibility that MPA is a trace contaminant present in the MPAG preparation, we have used the techniques of HPLC-tandem mass spectrometry (MS-MS) and a recently developed negative ion electrospray HPLC-MS method for MPA detection (8).


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
MPA was obtained from Fluka Chemical, and the sodium salt of MPAG was a gift from Roche Bioscience, Palo Alto, CA. The aqueous solutions of MPA and MPAG used for the analyses in this study were prepared just before use.

Negative ion electrospray HPLC-MS analyses of MPAG were performed using a recently described method for MPA analysis (8). We used a Hewlett-Packard 5989B mass spectrometer, an ion guide and electrospray ionization source from Analytica, and a Hewlett-Packard 1050 HPLC system to achieve HPLC-MS detection, using full-scan analysis from m/z 140 to m/z 550 at 2 s/scan. The HPLC column used for these studies was a Hewlett-Packard C18 Hypersil column (2 x 250 mm; 5 µm particle size). The mobile phase was an aqueous solution of 430 mL/L acetonitrile and 0.5 mL/L formic acid, and the flow rate was 0.15 mL/min.

Negative ion electrospray tandem mass spectra were obtained using a Micromass Quattro II triple quadrupole mass spectrometer that was tuned to unit resolution using water clusters. To enhance the formation of deprotonated molecules [M-H]-, during electrospray ionization, a solution of 50 mL/L ammonia in methanol was added postcolumn at 20 µL/min. There was no splitting of the mobile phase between the HPLC and the electrospray mass spectrometer. The same model HPLC system and conditions was used as described above for the HPLC-MS system.

Initially, HPLC-MS analysis was used to identify HPLC peaks according to their retention times and molecular weights. The range of m/z 150 to m/z 500 was scanned in ~2 s. The structures of the compounds eluting from the HPLC were then confirmed using HPLC-MS-MS, and the quantity of the MPA contaminating the MPAG sample was determined using HPLC-MS with selected ion monitoring (SIM), as described previously (8).

During HPLC-MS-MS analysis, the deprotonated molecule of MPA at m/z 319 was selected in the first quadrupole, fragmented in the second quadrupole using collision-induced dissociation (CID), and the product ions were analyzed using the third quadrupole by scanning the range m/z 50–325 in ~2 s/scan. Argon at 1.8 x 10-4 kPa was used as the collision gas for CID. The collision energy was 25 eV, and the capillary, counter electrode, and cone voltages were 3.04 kV, 0.5 kV, and 30 V, respectively.

For negative ion electrospray-MS-MS analysis of the MPA calibrator, a solution of pure MPA (3120 mg/L in 500 mL/L methanol, 10 mL/L ammonia, 490 mL/L water) was infused at a flow rate of 10 µL/min into the ion source of the mass spectrometer. The flow rate of the carrier solution (500 mL/L methanol, 10 mL/L ammonia, 490 mL/L water) was 10 µL/min. All other instrumental conditions used for the MS-MS analysis of the MPA calibrator were the same as described above for the HPLC-MS-MS analyses.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HPLC-MS was used to analyze the MPAG calibrator for impurities. A single contaminant peak was detected at a retention time of 5.7 min, which is later than the more polar MPAG peak at 2.4 min (Fig. 1A ). The molecular weight of the contaminant was determined to be 320, based on the observation (Fig. 1B ) of a base peak at m/z 319, which corresponded to the deprotonated molecule, a less abundant adduct ion at m/z 341, [M-2H+Na]-, and no ions corresponding to the deprotonated molecule of MPAG at m/z 495 (Fig. 1 , B and C ). HPLC-MS analysis of authentic MPA showed an identical retention time and negative ion electrospray mass spectrum. For additional, and more rigorous structural confirmation and identification of the contaminant, HPLC-MS-MS was used to compare its fragmentation pattern with that of the MPA calibrator.



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  		Figure 1. Total ion chromatogram of MPAG (A), and mass spectra for the trace MPA contaminant peak (B) and the MPAG peak (C).

(A), 10 µL of an aqueous solution of 25 mg/L MPAG was injected onto a Hewlett-Packard C18 Hypersil column (2 x 250 mm, 5 µm particle size). The mobile phase was an aqueous solution of 430 mL/L acetonitrile and 0.5 mL/L formic acid, and the flow rate was 0.15 mL/min. A Hewlett-Packard 5989B mass spectrometer, an ion guide, and electrospray ionization were used for detection, with full-scan analysis from m/z 140 to m/z 550 achieved in 2 s. The retention time of the MPA contaminant was 5.75 min, and the retention time of the MPAG peak was 2.40 min.

Because the ion at m/z 319, corresponding to the deprotonated molecule of MPA, was selected by the first quadrupole of the triple quadrupole mass spectrometer, no signals were recorded for the sodium adduct of MPA at m/z 341. The ion at m/z 319 was then fragmented using CID in the second quadrupole, and the product ion spectrum was recorded by scanning the third quadrupole mass spectrometer. The negative ion electrospray-MS-MS product ion spectrum of the MPA impurity is shown in Fig. 2 A. For comparison, the electrospray-MS-MS product ion spectrum of the MPA calibrator is shown in Fig. 2B .



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  		Figure 2. Negative ion electrospray MS-MS product ion mass spectra of the deprotonated molecule, m/z 319, of the MPA impurity obtained by HPLC-MS-MS analysis of MPAG (A) and the authentic MPA calibrator (B).

(A), 20 µL of a 1.92 g/L aqueous solution of the sodium salt of MPAG was analyzed by the negative ion electrospray HPLC-MS-MS method as described in Materials and Methods. (B), the procedure for analysis of the MPA calibrator by direct infusion into the ion source of the mass spectrometer is described in Materials and Methods.

The major fragmentation patterns of MPA obtained during MS-MS analysis with CID are summarized in Fig. 2AUp . Upon deprotonation, the negative charge is probably localized either on the carboxylic acid group (as a carboxylate anion) or on the phenolic group (to form a phenolate anion). Following formation of the carboxylate anion, decarboxylation would generate the fragment ion detected at m/z 275. Decarboxylation combined with elimination of methanol from the aromatic ring would form the fragment ion at m/z 243 (see fragmentation pattern in Fig. 2A). Localization of the negative charge to the phenolate anion would lead to different fragmentation pathways, including cleavage of the hexanoyl chain accompanied by a hydrogen transfer to form the product ions at m/z 191 (the most abundant fragment ion) and m/z 205.

The quantity of MPA contaminating the MPAG sample was determined using the negative ion electrospray SIM HPLC-MS method (8). We used the electrospray HPLC-MS method in the SIM mode to perform three replicate analyses of the MPAG preparation for the purpose of determining the relative amount of the contaminant MPA. Deprotonated MPA, m/z 319, and MPAG, m/z 495, were recorded. The percentage of MPA on a molar basis was calculated from the ratio of the respective areas under the deprotonated MPA and MPAG peaks corrected for the 2.04-fold greater response for the deprotonated MPAG peak. On the basis of this analysis, the MPA content was 0.312% ± 0.0184%.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This investigation provides unequivocal identification and quantification of trace quantities of MPA in the MPAG preparation, using the techniques of negative ion electrospray HPLC-MS-MS and negative ion electrospray SIM HPLC-MS. These study data provide substantiation for the proposal that the apparent weak inhibition afforded by MPAG, and reported independently by three different laboratories (4)(5)(6), is attributable to the presence of MPA itself and not the glucuronide metabolite. The presence of trace quantities of contaminant MPA is sufficient to explain the observed weak inhibition of IMPDH catalytic activity according to a recent investigation in our laboratory, which used the principle of tight-binding enzyme inhibitor kinetics (6). According to tight-binding inhibitor kinetics, IC50 values for tight-binding inhibitors increase, whereas IC50 values for weak-binding inhibitors do not change, as the target enzyme concentration is increased (6)(7). The reason for this characteristic behavior for IC50 values can be appreciated from an analysis of the tight-binding enzyme inhibitor kinetic equation. According to this equation: IC50 = 1/2 [E0] + Ki(1 + Km/S), where E0 is the molar concentration of the target enzyme and Ki(1 + Km/S) is the apparent inhibition constant (7). The concentration of enzyme typically is in the nanomolar range, and the concentrations of tight-binding inhibitors are in a similar concentration range; however, the concentrations of weak-binding inhibitors are in the micromolar range. Thus, for tight-binding inhibitors, as enzyme concentration increases, so will the IC50 values, whereas for weak-binding inhibitors, the IC50 value will be unaffected by the increase in enzyme concentration (7). In the previously reported study from our laboratory, the IC50 for pure MPA increased from 13.7 to 132 nmol/L, whereas as expected, the IC50 for xanthine monophosphate, a well-known, weaker binding competitive inhibitor of IMPDH, remained unchanged (mean ± SD, 713 ± 116 µmol/L) as the concentration of human IMPDH was increased from 26 to 260 nmol/L. On the other hand, the IC50 for MPAG, an apparent weak-binding inhibitor, increased from 14 to 79.4 µmol/L over the same 10-fold concentration range for the target enzyme, IMPDH (6). This degree of increase in IC50 could be explained by the presence of trace quantities of MPA (as little as 0.2% on a molar basis) in the MPAG preparation (6).

Earlier reports, which showed in several proliferating cell lines in culture that MPAG was ineffective as an inhibitor of cell multiplication in contrast to the very significant activity of MPA in the same cell lines, provide further evidence for the inactivity of MPAG as a suppressor of proliferation of rapidly dividing cells (9)(10). Additional support for the lack of inhibitor activity of MPAG is provided in the recent detailed description of the structure and mechanism of IMPDH (11). In the latter investigation, it was shown that the portion of the inhibitor site that surrounds the phenolic hydroxyl group of MPA is tightly packed and cannot easily accommodate additional atoms without undergoing major configurational distortion.

Thus, increased concentrations of MPAG, which have been shown to occur in renal transplant patients with delayed graft function (3) and in transplant patients with chronic renal failure (12), are not likely to contribute directly to immunosuppression. However, the presence of increased concentrations of MPAG in such patients is an important contributor to increases in the pharmacologically active free fraction and free concentration of MPA because MPAG competes for MPA-binding sites on human serum albumin; therefore, MPAG may indirectly contribute to immunosuppression (3)(13).


   Acknowledgments
 
This work was supported by a grant from Roche Bioscience, Palo Alto, CA.


   Footnotes
 
1 Nonstandard abbreviations: MPA, mycophenolic acid; MMF, mycophenolate mofetil; IMPDH, inosine monophosphate dehydrogenase; MPAG, mycophenolic acid glucuronide; MS, mass spectrometry; SIM, selected ion monitoring; and CID, collision-induced dissociation.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dayton JS, Turka LA, Thompson CB, Mitchell BS. Comparison of the effects of mizoribine with those of azathioprine, 6-mercaptopurine, and mycophenolic acid on T lymphocyte proliferation and purine ribonucleotide metabolism. Mol Pharmacol 1992;41:671-676. [Abstract]
  2. Eugui EM, Mirkovich A, Allison AC. Lymphocyte selective antiproliferative and immunosuppressive effects of mycophenolic acid in mice. Scand J Immunol 1991;33:175-183. [Web of Science][Medline] [Order article via Infotrieve]
  3. Shaw LM, Mick R, Nowak I, Korecka M, Brayman KL. Mycophenolic acid pharmacokinetics in renal transplant patients with delayed graft function. J Clin Pharmacol 1998;38:268-275. [Abstract]
  4. Langman LJ, LeGatt DF, Yatscoff RW. Pharmacodynamic assessment of mycophenolic acid-induced immunosuppression by measuring IMP dehydrogenase activity. Clin Chem 1995;41:295-299. [Abstract/Free Full Text]
  5. Griesmacher A, Weigel G, Seebacher G, Muller MM. IMP-dehydrogenase inhibition in human lymphocytes and lymphoblasts by mycophenolic acid and mycophenolic acid glucuronide. Clin Chem 1997;43:2312-2317. [Abstract/Free Full Text]
  6. Nowak I, Shaw LM. Effect of mycophenolic acid glucuronide on inosine monophosphate dehydrogenase activity. Ther Drug Monit 1997;19:358-360. [Web of Science][Medline] [Order article via Infotrieve]
  7. Cha S. Tight-binding inhibitors. I. Kinetic behavior. Biochem Pharmacol 1975;24:2177-2185. [Web of Science][Medline] [Order article via Infotrieve]
  8. Shaw LM, Korecka M, van Breemen RB, Nowak I, Brayman KL. Analysis, pharmacokinetics and therapeutic drug monitoring of mycophenolic acid. Clin Biochem 1998;31:323-328. [Web of Science][Medline] [Order article via Infotrieve]
  9. Franklin TJ, Jacobs V, Jones G, Ple P, Bruneau P. Glucuronidation associated with intrinsic resistance to mycophenolic acid in human colorectal carcinoma cells. Cancer Res 1996;56:984-987. [Abstract/Free Full Text]
  10. Sweeney MJ, Hoffman DH, Esterman MA. Metabolism and biochemistry of mycophenolic acid. Cancer Res 1972;32:1803-1809. [Abstract/Free Full Text]
  11. Sintchak MD, Fleming MA, Futer O, Raybuck SA, Chambers SP, Caron PR, et al. Structure and mechanism of inosine monophosphate dehydrogenase complex with the immunosuppressant mycophenolic acid. Cell 1996;85:921-930. [Web of Science][Medline] [Order article via Infotrieve]
  12. Kaplan B, Gruber SA, Nallamathou R, Katz SM, Shaw LM. Decreased protein binding of mycophenolic acid associated with leukopenia in a pancreas transplant recipient with renal failure. Transplantation 1998;65:1127-1129. [Web of Science][Medline] [Order article via Infotrieve]
  13. Nowak I, Shaw LM. Mycophenolic acid binding to human serum albumin: characterization and relation to pharmacodynamics. Clin Chem 1995;41:1011-1017. [Abstract/Free Full Text]



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