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Therapeutic Drug Monitoring of Mycophenolic Acid: Comparison of HPLC and Immunoassay Reveals New MPA Metabolites
Therapeutic Drug Monitoring of Mycophenolic Acid: Comparison of HPLC and Immunoassay Reveals New MPA Metabolites E. Schu¨tz, M. Shipkova, V.W. Armstrong, P.D. Niedmann, L. Weber, B. To¨nshoff, K. Pethig, T. Wahlers, F. Braun, B. Ringe, and M. Oellerich
T
HE PRODRUG mycophenolate mofetil (MMF) is a new antiproliferative agent that is being administered to an increasing extent after solid organ transplantation as an alternative to azathioprine in triple immunosuppressive regimens. MMF is rapidly converted in vivo to the active metabolite mycophenolic acid (MPA), which inhibits inosine monophosphate dehydrogenase 2 (IMPHD2), resulting in suppression of purine de novo synthesis, particularly in lymphocytes. MPA is metabolized in the liver to MPA-b-glucuronide (MPAG), an inactive metabolite which is normally present in plasma at about 40-fold higher concentrations than the parent drug. MPAG is primarily cleared through the kidney. The results of clinical studies comparing a fixed dose of MMF in combination with cyclosporine (CyA) and steroids to either azathioprine or placebo have been encouraging concerning the incidence of rejection.1–3 However, adverse effects such as leukopenia and gastrointestinal disturbances have been described. The role of therapeutic drug monitoring for MPA is currently under investigation.4 Preliminary studies suggest that there is an inverse correlation between the area under the concentration time curve (AUC) and the incidence of acute rejection.5 Recently an immunoassay for MPA has been developed6 based on the EMIT™ technique (Behring-Syva). We have now compared the results from this assay, performed on a Cobas Mira Plus analyzer (Roche, Grenzach, Germany), with those obtained using a reversed-phase (RP) HPLC method.7 A bias between the two methods led us to look for putative metabolites of MPA in plasma of patients under immunosuppression with MMF.
PATIENTS AND METHODS Four patient populations were studied: 25 pediatric kidney recipients (140 samples from AUC monitoring), 12 adult kidney recipients (95 trough samples, ,3 months after transplantation), 10 adult liver recipients (202 trough samples, ,3 months after transplantation), and 20 stable heart recipients (182 samples from AUC monitoring, .2 months after transplantation). Kidney recipients received a CyA-based immunosuppression, whereas liver recipients were under tacrolimus therapy. Fifteen heart recipients were receiving tacrolimus, whereas five were being administered CyA. Plasma concentrations of MPA were quantified by RP-HPLC with UV detection as described elsewhere.7 For detection and isolation of the metabolites, a Hypersil C18, 250 3 4 mm column (MZ Analysentechnik, Mainz, Germany) was used. The gradient used in Andreeva et al7 was modified as follows: mobile phase A was as in Andreeva et al7 and mobile phase B consisted of 450 mL acetonitrile and 550 mL 40 mmol/L phosphate buffer, pH 6.5. The EMIT immunoassay (Behring, Marburg, Germany) was performed on a Cobas Mira plus analyzer (Roche) according to the manufacturer’s instructions. Calibration was performed with the material provided with the test kit. Results from the two methods were compared by plotting the relative difference in the MPA concentrations obtained with the two methods ([MPAEMIT 2 MPAHPLC]/meanMPA) against the mean MPA concentration.8 Pearson’s r and Kendall’s t
From the Abteilung fu¨r Klinische Chemie and Abteilung fu¨r Transplantationschirurgie, Georg-August Universita¨t, Goettingen; Klinik fu¨r Thorax- and Gefa˙bchirurgie der Medizinischen Hochschule, Hannover; and Kinderklinik, Ruprecht-Karls-Universita¨t, Heidelberg, Germany. Address reprint requests to E. Schu¨tz MD, Abteilung fu¨r Klinische Chemie, Georg-August Universita¨t Goettingen, Robert-Koch-Strasse 40, D-37070 Goettingen, Germany.
Table 1. Correlation Analysis of MPA Concentrations or AUC Determined With Either RP-HPLC or EMIT
n r t
Adult Kidney*
Adult Liver†
Adult Heart*
Adult Heart†
Pediatric Kidney*‡
Pediatric Kidney*§
95 0.97 0.81
202 0.98 0.78
51 0.99 0.96
131 0.98 0.90
140 0.96 0.87
19 0.94 0.78
*Concomitant CyA immunosuppression. † Concomitant tacrolimus immunosuppression. ‡ Correlation of MPA plasma concentrations. § Correlation of AUC.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)00201-2
Transplantation Proceedings, 30, 1185–1187 (1998)
1185
SCHU¨TZ, ANDREEVA, ARMSTRONG ET AL
1186
bias of only 7% was seen in those heart transplant patients on tacrolimus therapy (Fig 1F). Inspection of the plots in Fig 1 revealed that there was a tendency to a higher relative bias at low MPA levels. To gain further insight into the cause of this methodologic bias, which was not correlated to the MPAG concentrations (data not shown), the HPLC method was modified to obtain a better resolution of the peaks eluting from the column. At least two peaks could be identified that showed a UV spectrum almost identical to either that of MPAG (metabolite M-1) or that of MPA (metabolite M-2). The first metabolite (M-1) eluted shortly after MPAG, while the second (M-2) had a longer retention time but eluted prior to MPA (Fig 2). The fractions containing these metabolites as well as those containing either MPA or MPAG were collected, desalted, and reconcentrated. Only MPA (0.93 mg/L) as well as the metabolite M-2 (0.64 mg/L) yielded a measurable result with the EMIT assay. DISCUSSION
Fig 1. Relative differences of either MPA concentrations (A, B, C, E, F) or AUC (D), determined with EMIT or HPLC, plotted against the mean of the results obtained by the two methods. (A) Adult liver recipients (tacrolimus), (B) adult kidney recipients (CyA), (C) pediatric kidney (CyA) recipients (MPA concentrations), (D) pediatric kidney (CyA) recipients (AUC), (E) stable heart transplant recipients on concomitant CyA immunosuppression, (F) stable heart transplant recipients on concomitant tacrolimus immunosuppression. The solid line reflects the median difference between EMIT and HPLC.
were calculated for correlation estimation. The AUC was calculated from the time points 0, 20, 40, and 75 minutes, and 2, 4, 6, 8, and 12 hours according to the linear trapezoidal rule.
RESULTS
A good correlation was observed between the two methods with regard to both the individual MPA concentrations, irrespective of transplant type or patient age, and the AUC in pediatric kidney recipients (Table 1). However, analysis of the data according to the procedure recommended by Bland and Altman8 revealed a substantial overestimation of 30% to 35% by the EMIT assay compared to HPLC in adult liver (Fig 1A) and both adult and pediatric kidney (Figs 1B, C) transplant recipients. In the case of the AUC for the pediatric kidney recipients (Fig 1D), the values derived from the EMIT measurements were around 20% higher than those obtained with HPLC. The bias between EMIT and HPLC was associated with the concomitant immunosuppressant in stable heart recipients. Thus, the EMIT values were on the average around 24% higher than the HPLC values in CyA-treated patients (Fig 1E), whereas a
A critical issue in drug monitoring of immunosuppressants is the analytical specificity of the methods available. Most studies into MPA plasma concentrations have been based on RP-HPLC procedures, which are specific for the parent drug. The observed positive bias between the MPA EMIT immunoassay and HPLC led us to presume that crossreactive metabolites of MPA may be present in the plasma of organ transplant recipients under immunosuppression with MMF. The major metabolite of MPA, MPAG could be excluded as the source of this crossreactivity.6 Through HPLC (Fig 2), we were able to identify two putative metabolites that showed an almost identical UV spectrum to either MPA (M-2) or MPAG (M-1). One of these metabolites (M-2) was also immunoreactive in the EMIT assay, suggesting that it may be a major cause for the discrepancies observed between EMIT and HPLC. The greatest bias (30% to 35%) was seen in liver patients on tacrolimus immunosuppression early after transplantation, as well as in kidney recipients on CyA, also early after transplantation. A bias of around 24% was seen in stable heart transplant recipients on CyA, whereas in those heart recipients on tacrolimus the positive bias was much lower (7%) and the metabolite M-2 was barely detectable. In general, the relative bias was most pronounced in those samples with low MPA concentrations representing trough as well as late AUC time points (Fig 1). The generation of the metabolite by the liver could be confirmed in isolated human liver microsomes (Shipkova M, unpublished observation) by HPLC analysis of the incubation medium that had been supplemented with MPA before incubation. The putative presence of the metabolite M-2 at relatively high concentrations in liver recipients early after transplantation, as reflected by the bias between EMIT and HPLC, is consistent with the accumulation of hepatic metabolites of other immunosuppressants such as CyA. Since in stable heart recipients the bias between EMIT and HPLC was associated with CyA, but not
THERAPEUTIC DRUG MONITORING
1187
Fig 2. HPLC chromatogram of a plasma sample obtained from a liver transplant recipient on MMF therapy (upper panel). MPA, mycophenolic acid; MPAG, mycophenolic acid b-glucuronide; MPC, carboxy butoxy ether of MPA (internal standard); M-1, putative metabolite of MPA that does not crossreact with the EMIT MPA assay; M-2, putative metabolite of MPA that crossreacts with the EMIT MPA assay. UV-spectrum overlay (lower panel) of MPA and M-2 reveals almost complete identity.
with tacrolimus, an interaction between the metabolism of MPA and CyA in the liver can be implied. This is further supported by the large bias seen in kidney recipients on CyA without any sign of liver disorder. The exact structure of both of these metabolites as well as their ability to inhibit IMPDH2 is under investigation. The answer to the question whether these metabolites are pharmacologically active will be essential in judging the utility of the EMIT assay for MPA as well as for the interpretation of the results.
NOTE ADDED IN PROOF In extension of these investigations, we could now demonstrate, that MPA metabolite M-2 inhibits rh-IMPDH2.
REFERENCES 1. Sollinger HW for the U.S. Renal Transplant Mycophenolate Mofetil Study Group: Transplantation 60:225, 1995 2. European Mycophenolate Mofetil Cooperative Study Group: Lancet 345:1321, 1995 3. The Tricontinental Mycophenolate Mofetil Renal Transplantation Study Group: Transplantation 61:1029, 1996 4. Shaw LM, Sollinger HW, Halloran P, et al: Ther Drug Monit 17:690, 1995 5. Takahashi K, Ochiai T, Uchida K, et al: Transplant Proc 27:1421, 1995 6. Haley CJ, Jaklitsch A, McGowan B, et al: Ther Drug Monit 17:431, 1995 7. Andreeva M, Niedmann D, Schu ¨tz E, et al: Ther Drug Monit 19:565, 1997 8. Bland JM, Altmann DG: Lancet i:307, 1986
T
HE PRODRUG mycophenolate mofetil (MMF) is a new antiproliferative agent that is being administered to an increasing extent after solid organ transplantation as an alternative to azathioprine in triple immunosuppressive regimens. MMF is rapidly converted in vivo to the active metabolite mycophenolic acid (MPA), which inhibits inosine monophosphate dehydrogenase 2 (IMPHD2), resulting in suppression of purine de novo synthesis, particularly in lymphocytes. MPA is metabolized in the liver to MPA-b-glucuronide (MPAG), an inactive metabolite which is normally present in plasma at about 40-fold higher concentrations than the parent drug. MPAG is primarily cleared through the kidney. The results of clinical studies comparing a fixed dose of MMF in combination with cyclosporine (CyA) and steroids to either azathioprine or placebo have been encouraging concerning the incidence of rejection.1–3 However, adverse effects such as leukopenia and gastrointestinal disturbances have been described. The role of therapeutic drug monitoring for MPA is currently under investigation.4 Preliminary studies suggest that there is an inverse correlation between the area under the concentration time curve (AUC) and the incidence of acute rejection.5 Recently an immunoassay for MPA has been developed6 based on the EMIT™ technique (Behring-Syva). We have now compared the results from this assay, performed on a Cobas Mira Plus analyzer (Roche, Grenzach, Germany), with those obtained using a reversed-phase (RP) HPLC method.7 A bias between the two methods led us to look for putative metabolites of MPA in plasma of patients under immunosuppression with MMF.
PATIENTS AND METHODS Four patient populations were studied: 25 pediatric kidney recipients (140 samples from AUC monitoring), 12 adult kidney recipients (95 trough samples, ,3 months after transplantation), 10 adult liver recipients (202 trough samples, ,3 months after transplantation), and 20 stable heart recipients (182 samples from AUC monitoring, .2 months after transplantation). Kidney recipients received a CyA-based immunosuppression, whereas liver recipients were under tacrolimus therapy. Fifteen heart recipients were receiving tacrolimus, whereas five were being administered CyA. Plasma concentrations of MPA were quantified by RP-HPLC with UV detection as described elsewhere.7 For detection and isolation of the metabolites, a Hypersil C18, 250 3 4 mm column (MZ Analysentechnik, Mainz, Germany) was used. The gradient used in Andreeva et al7 was modified as follows: mobile phase A was as in Andreeva et al7 and mobile phase B consisted of 450 mL acetonitrile and 550 mL 40 mmol/L phosphate buffer, pH 6.5. The EMIT immunoassay (Behring, Marburg, Germany) was performed on a Cobas Mira plus analyzer (Roche) according to the manufacturer’s instructions. Calibration was performed with the material provided with the test kit. Results from the two methods were compared by plotting the relative difference in the MPA concentrations obtained with the two methods ([MPAEMIT 2 MPAHPLC]/meanMPA) against the mean MPA concentration.8 Pearson’s r and Kendall’s t
From the Abteilung fu¨r Klinische Chemie and Abteilung fu¨r Transplantationschirurgie, Georg-August Universita¨t, Goettingen; Klinik fu¨r Thorax- and Gefa˙bchirurgie der Medizinischen Hochschule, Hannover; and Kinderklinik, Ruprecht-Karls-Universita¨t, Heidelberg, Germany. Address reprint requests to E. Schu¨tz MD, Abteilung fu¨r Klinische Chemie, Georg-August Universita¨t Goettingen, Robert-Koch-Strasse 40, D-37070 Goettingen, Germany.
Table 1. Correlation Analysis of MPA Concentrations or AUC Determined With Either RP-HPLC or EMIT
n r t
Adult Kidney*
Adult Liver†
Adult Heart*
Adult Heart†
Pediatric Kidney*‡
Pediatric Kidney*§
95 0.97 0.81
202 0.98 0.78
51 0.99 0.96
131 0.98 0.90
140 0.96 0.87
19 0.94 0.78
*Concomitant CyA immunosuppression. † Concomitant tacrolimus immunosuppression. ‡ Correlation of MPA plasma concentrations. § Correlation of AUC.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)00201-2
Transplantation Proceedings, 30, 1185–1187 (1998)
1185
SCHU¨TZ, ANDREEVA, ARMSTRONG ET AL
1186
bias of only 7% was seen in those heart transplant patients on tacrolimus therapy (Fig 1F). Inspection of the plots in Fig 1 revealed that there was a tendency to a higher relative bias at low MPA levels. To gain further insight into the cause of this methodologic bias, which was not correlated to the MPAG concentrations (data not shown), the HPLC method was modified to obtain a better resolution of the peaks eluting from the column. At least two peaks could be identified that showed a UV spectrum almost identical to either that of MPAG (metabolite M-1) or that of MPA (metabolite M-2). The first metabolite (M-1) eluted shortly after MPAG, while the second (M-2) had a longer retention time but eluted prior to MPA (Fig 2). The fractions containing these metabolites as well as those containing either MPA or MPAG were collected, desalted, and reconcentrated. Only MPA (0.93 mg/L) as well as the metabolite M-2 (0.64 mg/L) yielded a measurable result with the EMIT assay. DISCUSSION
Fig 1. Relative differences of either MPA concentrations (A, B, C, E, F) or AUC (D), determined with EMIT or HPLC, plotted against the mean of the results obtained by the two methods. (A) Adult liver recipients (tacrolimus), (B) adult kidney recipients (CyA), (C) pediatric kidney (CyA) recipients (MPA concentrations), (D) pediatric kidney (CyA) recipients (AUC), (E) stable heart transplant recipients on concomitant CyA immunosuppression, (F) stable heart transplant recipients on concomitant tacrolimus immunosuppression. The solid line reflects the median difference between EMIT and HPLC.
were calculated for correlation estimation. The AUC was calculated from the time points 0, 20, 40, and 75 minutes, and 2, 4, 6, 8, and 12 hours according to the linear trapezoidal rule.
RESULTS
A good correlation was observed between the two methods with regard to both the individual MPA concentrations, irrespective of transplant type or patient age, and the AUC in pediatric kidney recipients (Table 1). However, analysis of the data according to the procedure recommended by Bland and Altman8 revealed a substantial overestimation of 30% to 35% by the EMIT assay compared to HPLC in adult liver (Fig 1A) and both adult and pediatric kidney (Figs 1B, C) transplant recipients. In the case of the AUC for the pediatric kidney recipients (Fig 1D), the values derived from the EMIT measurements were around 20% higher than those obtained with HPLC. The bias between EMIT and HPLC was associated with the concomitant immunosuppressant in stable heart recipients. Thus, the EMIT values were on the average around 24% higher than the HPLC values in CyA-treated patients (Fig 1E), whereas a
A critical issue in drug monitoring of immunosuppressants is the analytical specificity of the methods available. Most studies into MPA plasma concentrations have been based on RP-HPLC procedures, which are specific for the parent drug. The observed positive bias between the MPA EMIT immunoassay and HPLC led us to presume that crossreactive metabolites of MPA may be present in the plasma of organ transplant recipients under immunosuppression with MMF. The major metabolite of MPA, MPAG could be excluded as the source of this crossreactivity.6 Through HPLC (Fig 2), we were able to identify two putative metabolites that showed an almost identical UV spectrum to either MPA (M-2) or MPAG (M-1). One of these metabolites (M-2) was also immunoreactive in the EMIT assay, suggesting that it may be a major cause for the discrepancies observed between EMIT and HPLC. The greatest bias (30% to 35%) was seen in liver patients on tacrolimus immunosuppression early after transplantation, as well as in kidney recipients on CyA, also early after transplantation. A bias of around 24% was seen in stable heart transplant recipients on CyA, whereas in those heart recipients on tacrolimus the positive bias was much lower (7%) and the metabolite M-2 was barely detectable. In general, the relative bias was most pronounced in those samples with low MPA concentrations representing trough as well as late AUC time points (Fig 1). The generation of the metabolite by the liver could be confirmed in isolated human liver microsomes (Shipkova M, unpublished observation) by HPLC analysis of the incubation medium that had been supplemented with MPA before incubation. The putative presence of the metabolite M-2 at relatively high concentrations in liver recipients early after transplantation, as reflected by the bias between EMIT and HPLC, is consistent with the accumulation of hepatic metabolites of other immunosuppressants such as CyA. Since in stable heart recipients the bias between EMIT and HPLC was associated with CyA, but not
THERAPEUTIC DRUG MONITORING
1187
Fig 2. HPLC chromatogram of a plasma sample obtained from a liver transplant recipient on MMF therapy (upper panel). MPA, mycophenolic acid; MPAG, mycophenolic acid b-glucuronide; MPC, carboxy butoxy ether of MPA (internal standard); M-1, putative metabolite of MPA that does not crossreact with the EMIT MPA assay; M-2, putative metabolite of MPA that crossreacts with the EMIT MPA assay. UV-spectrum overlay (lower panel) of MPA and M-2 reveals almost complete identity.
with tacrolimus, an interaction between the metabolism of MPA and CyA in the liver can be implied. This is further supported by the large bias seen in kidney recipients on CyA without any sign of liver disorder. The exact structure of both of these metabolites as well as their ability to inhibit IMPDH2 is under investigation. The answer to the question whether these metabolites are pharmacologically active will be essential in judging the utility of the EMIT assay for MPA as well as for the interpretation of the results.
NOTE ADDED IN PROOF In extension of these investigations, we could now demonstrate, that MPA metabolite M-2 inhibits rh-IMPDH2.
REFERENCES 1. Sollinger HW for the U.S. Renal Transplant Mycophenolate Mofetil Study Group: Transplantation 60:225, 1995 2. European Mycophenolate Mofetil Cooperative Study Group: Lancet 345:1321, 1995 3. The Tricontinental Mycophenolate Mofetil Renal Transplantation Study Group: Transplantation 61:1029, 1996 4. Shaw LM, Sollinger HW, Halloran P, et al: Ther Drug Monit 17:690, 1995 5. Takahashi K, Ochiai T, Uchida K, et al: Transplant Proc 27:1421, 1995 6. Haley CJ, Jaklitsch A, McGowan B, et al: Ther Drug Monit 17:431, 1995 7. Andreeva M, Niedmann D, Schu ¨tz E, et al: Ther Drug Monit 19:565, 1997 8. Bland JM, Altmann DG: Lancet i:307, 1986