Population pharmacokinetic modeling for mycophenolic acid and its main glucuronide metabolite in Chinese kidney transplant recipients

OBJECTIVE To develop an integrated model of MPA and MPAG in kidney recipients, and to evaluate the effect of clinical covariates and genotypes on mycophenolic acid(MPA) and 7-O-mycophenolic acid glucuronide(MPAG) disposition. METHODS Data were collected from 191 adult Chinese mycophenolic acid,including 24 patients with full concentration-time profiles and 167 with trough concentrations. They contained 962 MPA measurements and 746 MPAG measurements.Population pharmacokinetic analysis was performed using NONMEM~?. RESULTS The PK of MPA and MPAG were best described by a three-chain compartment model. Significant correlations were found between the clearance of MPA(CLMPA) and albumin levels(ALB), and between the clearance of MPAG(CLMPAG) and the creatinine clearance(CCR).CLMPA was 13.7 L·h-1 and the CLMPAG was 1.3 L · h~(-1) for the Chinese kidney transplant recipients with ALB 42 g · L~(-1) and CCR 72 m L · min~(-1).CONCLUSION The MPA data was described adequately by a 2-compartment model with linear elimination, while MPAG was described using a 1-compartment model.ALB, CCR affected CLMPA and CLMPAG respectively.The impact of gene polymorphisms of enzymes and transporters did not affect pharmacokinetic of MMF in kidney transplant recipients in our study, including UGT1 A9,UGT1 A8, UGT2 B7, OATP1 B3, MRP2.     

Pharmacokinetics of mycophenolic acid and its phenolic-glucuronide and ACYl glucuronide metabolites in stable thoracic transplant recipients

Mycophenolate mofetil is an immunosuppressant commonly used in solid organ transplantation. Its active metabolite, mycophenolic acid (MPA), is metabolized to the inactive 7-O-mycophenolic acid glucuronide (MPAG) and the active acyl glucuronide (AcMPAG). Most pharmacokinetic (PK) studies have been focused on MPA, but not its metabolites, in kidney transplant recipients. Pharmacokinetic studies of MPA and its metabolites in thoracic transplant recipients are scarce. Because neither the heart nor lung is involved in MPA metabolism or excretion, the thoracic transplant population may exhibit unique PKs. This open-label study aimed to characterize and compare PKs of MPA and its metabolites in stable lung or heart transplant recipients. Fifty thoracic (27 lung, 23 heart) transplant recipients were recruited. Subjects were also taking cyclosporine (11 lung, 14 heart) or tacrolimus (16 lung, nine heart), and prednisone (27 lung, one heart). Blood samples were obtained at 0, 20, 40, 60, and 90 minutes and 2, 4, 6, 8, 10, and 12 hours postdose. Plasma was used for drug level analysis (MPA, MPAG, and AcMPAG) by a high-performance liquid chromatography-ultraviolet detection method; in a subset of subjects, free MPA concentrations were also determined. Conventional PK parameters (dose-normalized) were determined by noncompartmental methods. There was wide interpatient variability of MPA, MPAG, and AcMPAG PKs with coefficients of variation exceeding 70% for most PK parameters measured. Other findings (P < 0.05) included: lower MPA area under the curve, maximum concentration, and minimum concentration; higher apparent clearance and MPAG/MPA metabolic ratio in the lung versus heart transplant group; lower MPA area under the curve and minimum concentration, and higher apparent clearance and MPAG/MPA metabolic ratio in lung transplant recipients concurrently taking cyclosporine versus tacrolimus; and lower minimum concentration in heart transplant recipients taking cyclosporine versus tacrolimus. Despite large interpatient variability in the PKs of MPA, MPAG, and AcMPAG among thoracic transplant recipients, there appear to be significant differences between lung and heart patients, which warrant further study.     

Pharmacokinetic role of protein binding of mycophenolic acid and its glucuronide metabolite in renal transplant recipients

Mycophenolic acid (MPA), the active compound of mycophenolate mofetil (MMF), is used to prevent graft rejection in renal transplant recipients. MPA is glucuronidated to the metabolite MPAG, which exhibits enterohepatic recirculation (EHC). MPA binds for 97% and MPAG binds for 82% to plasma proteins. Low plasma albumin concentrations, impaired renal function and coadministration of cyclosporine have been reported to be associated with increased clearance of MPA. The aim of the study was to develop a population pharmacokinetic model describing the relationship between MMF dose and total MPA (tMPA), unbound MPA (fMPA), total MPAG (tMPAG) and unbound MPAG (fMPAG). In this model the correlation between pharmacokinetic parameters and renal function, plasma albumin concentrations and cotreatment with cyclosporine was quantified. tMPA, fMPA, tMPAG and fMPAG concentration–time profiles of renal transplant recipients cotreated with cyclosporine (n = 48) and tacrolimus (n = 45) were analyzed using NONMEM. A 2- and 1-compartment model were used to describe the pharmacokinetics of fMPA and fMPAG. The central compartments of fMPA and fMPAG were connected with an albumin compartment allowing competitive binding (bMPA and bMPAG). tMPA and tMPAG were modeled as the sum of the bound and unbound concentrations. EHC was modeled by transport of fMPAG to a separate gallbladder compartment. This transport was decreased in case of cyclosporine cotreatment (P < 0.001). In the model, clearance of fMPAG decreased when creatinine clearance (CrCL) was reduced (P < 0.001), and albumin concentration was correlated with the maximum number of binding sites available for MPA and MPAG (P < 0.001). In patients with impaired renal function cotreated with cyclosporine the model adequately described that increasing fMPAG concentrations decreased tMPA AUC due to displacement of MPA from its binding sites. The accumulated MPAG could also be reconverted to MPA by the EHC, which caused increased tMPA AUC in patients cotreated with tacrolimus. Changes in CrCL had hardly any effect on fMPA exposure. A decrease in plasma albumin concentration from 0.6 to 0.4 mmol/l resulted in ca. 38% reduction of tMPA AUC, whereas no reduction in fMPA AUC was seen. In conclusion, a pharmacokinetic model has been developed which describes the relationship between dose and both total and free MPA exposure. The model adequately describes the influence of renal function, plasma albumin and cyclosporine co-medication on MPA exposure. Changes in protein binding due to altered renal function or plasma albumin concentrations influence tMPA exposure, whereas fMPA exposure is hardly affected.     

Automated determination of free mycophenolic acid and its glucuronide in plasma from renal allograft recipients

Mycophenolic acid, the active moiety of mycophenolate mofetil, inhibits the enzyme inosine monophosphate dehydrogenase. The main metabolite, mycophenolic acid glucuronide, has no immunosuppressive effect. Reported protein bindings are 97% for mycophenolic acid and 82% for mycophenolic acid glucuronide. Considerable intraindividual and interindividual variability in mycophenolic acid pharmacokinetics has been observed. Data on the variability of mycophenolic acid free fraction in plasma are sparse but may be relevant when discussing whether therapeutic drug monitoring of this drug is warranted. The authors describe a fully automated method for the determination of free concentrations by dialysis across a membrane followed by concentration of the dialysate on a trace enrichment column and liquid chromatography. Total concentrations are measured by protein precipitation and direct injection on the trace enrichment column. Plasma concentrations as low as 6 ng/mL free mycophenolic acid and 1 microg/mL free mycophenolic acid glucuronide can be measured with between-day coefficient of variation less than 15% and 6%, respectively. Stability testing confirmed that plasma samples could be stored for 14 days at 4 degrees C or -20 degrees C and at room temperature for approximately 12 hours without significant changes in free concentrations. Predose total and free concentrations of mycophenolic acid and mycophenolic acid glucuronide were determined in 27 samples from stable renal allograft recipients treated with mycophenolate mofetil, cyclosporin, and steroids. Total concentrations ranged from 0.57 to 16.2 microg/mL mycophenolic acid and 36 to 199 microg/mL mycophenolic acid glucuronide. Free concentrations ranged from 13 to 210 ng/mL mycophenolic acid and 8 to 58 microg/mL mycophenolic acid glucuronide. The method presented here has been successfully applied to measure free mycophenolic acid and free mycophenolic acid glucuronide in clinical samples. Further investigations may provide important data to support the identification of principles and target ranges for the monitoring of mycophenolic acid in the immunosuppressive therapy of organ transplant recipients.     

Identification of glucoside and carboxyl-linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil

1. Mycophenolic acid (MPA), is primarily metabolized in the liver to 7-O-MPA-beta-glucuronide (MPAG). Using RP-h.p.l.c. we observed three further MPA metabolites, M-1, M-2, M-3, in plasma of transplant recipients on MMF therapy. To obtain information on the structure and source of these metabolites: (A) h.p.l.c. fractions containing either metabolite or MPA were collected and analysed by tandem mass spectrometry; (B) the metabolism of MPA was studied in human liver microsomes in the presence of UDP-glucuronic acid, UDP-glucose or NADPH; (C) hydrolysis of metabolites was investigated using beta-glucosidase, beta-glucuronidase or NaOH; (D) cross-reactivity of each metabolite was tested in an immunoassay for MPA (EMIT). 2. Mass spectrometry of M-1, M-2, MPA and MPAG in the negative ion mode revealed molecular ions of m/z 481, m/z 495, m/z 319 and m/z 495 respectively. 3. Incubation of microsomes with MPA and UDP-glucose produced M-1, with MPA and UDP-glucuronic acid MPAG and M-2 were formed, while with MPA and NADPH, M-3 was observed. 4. Beta-Glucosidase hydrolysed M-1 completely. Beta-Glucuronidase treatment led to a complete disappearance of MPAG whereas the amount of M-2 was reduced by approximately 30%. Only M-2 was labile to alkaline treatment. 5. M-2 and MPA but not M-1 and MPAG cross-reacted in the EMIT assay. 6. These results suggest that: (i) M-1 is the 7-OH glucose conjugate of MPA; (ii) M-2 is the acyl glucuronide conjugate of MPA; (iii) M-3 is derived from the hepatic CYP450 system.     

Population pharmacokinetics of mycophenolic acid and its glucuronide metabolite in lung transplant recipients with and without cystic fibrosis

1.?Cystic fibrosis (CF) is a disease affecting multiple organs that may reduce the systemic exposure of some drugs. The objective of this work was to characterize and compare the population pharmacokinetics (PK) of the immunosuppressant mycophenolic acid (MPA), and its glucuronide metabolite (MPAG) in adult lung transplant recipients with and without CF (NCF) following repeated oral administration of the prodrug mycophenolate mofetil (MMF).

2.?A population PK model was developed, with simultaneous modeling of MPA and MPAG, using nonlinear mixed effects modeling. MPA and MPAG serum concentration-time data were adequately described by a compartmental model including enterohepatic recirculation (EHR). Both MPA and MPAG apparent clearance values were significantly elevated (>65%) in patients with CF (24.1 and 1.95?L/h, respectively) compared to the values in the NCF patients (14.5 and 1.12?L/h, respectively), suggesting a notable influence of CF on MPA absorption and disposition.

3.?The population PK model developed from our study successfully characterized the absorption, distribution, elimination and EHR of MPA and the metabolite MPAG in lung transplant recipients with or without CF. This model may help to further understand the impact of CF to the overall clinical effects of MPA therapy including immunosuppression and gastrointestinal side effects.     

Pharmacokinetics of mycophenolic acid and metabolites in diabetic kidney transplant recipients

Mycophenolate mofetil (MMF), the prodrug of mycophenolic acid (MPA), is an immunosuppressive agent commonly used after organ transplantation. Because diabetes mellitus may affect disposition of pharmacologic agents, we investigated the influence of diabetes on the pharmacokinetics of MPA, unbound MPA (fMPA) and its phenyl and acyl glucuronide metabolites (MPAG and AcMPAG respectively). The study included 13 diabetic and 11 nondiabetic, stable, kidney-transplant recipients who were receiving a triple maintenance immunosuppressive regimen. Serial plasma samples were obtained predose and at regular intervals for 12 hours. Gastric emptying was assessed using an acetaminophen absorption test and glomerular filtration rate was estimated using iohexol clearance. Treatment groups were well matched. The time to maximum concentration (Tmax) of MPA was 86.4 +/- 41.4 minutes versus 52.8 +/- 31.8 minutes in D and ND patients respectively (P = 0.04) indicating a delay in MMF absorption. Neither the maximum MPA concentration nor the 0- to 12-hour area under the concentration-time curve were different. All parameters derived for fMPA and the MPA metabolites were comparable between the 2 groups, except for the metabolite ratio of MPAG and AcMPAG, which was higher for diabetic patients (P = 0.03). Delayed gastric emptying seemed to have reduced the initial rate but not the extent of MPA absorption in diabetic patients. The profiles of fMPA were similar in both patient groups. With the exception of metabolite concentration ratio, none of the other parameters associated with MPA metabolism were different between the 2 groups.     

Influence of drug transporters and UGT polymorphisms on pharmacokinetics of phenolic glucuronide metabolite of mycophenolic acid in Japanese renal transplant recipients

Mycophenolic acid (MPA) is mainly glucuronized by uridine diphosphate-glucuronosyltransferases (UGTs) into the phenolic MPA glucuronide (MPAG). MPAG is excreted by transporters such as organic anion-transporting polypeptide (gene SLCO), multidrug resistance protein 2 (gene ABCC2), breast cancer resistance protein (BCRP, gene ABCG2) or P-glycoprotein (gene ABCB1). This study investigated the association of UGTs, SLCOs, ABCB1, ABCC2, and ABCG2 polymorphisms with MPAG pharmacokinetics in 80 Japanese renal transplant recipients. Eighty recipients were given repeated doses of combination immunosuppressive therapy consisting of mycophenolate mofetil and tacrolimus every 12 hours at a designated time (0900 and 2100). On day 28, after renal transplantation, plasma concentrations of MPA and MPAG were measured by high-performance liquid chromatography. There were no significant differences in the area under the plasma concentration-time curve (AUC) ratio of MPAG/MPA between UGT1A1, UGT1A6, UGT1A7, UGT1A8, and UGT1A9 I399C/T genotypes. On the other hand, the median dose-adjusted AUC0-12 of MPAG in SLCO1B1 1a/1a+1a/1b+1b+1b (n = 53) and 1a/*15 + 1b/*15+*15/*15 (n = 27) were 1549 and 1134 mg.h L g, respectively (P = 0.03004 in multivariate analysis). The median dose-adjusted AUC0-12 of MPAG in SLCO1B3 334T/T+T/G (699G/G+G/A, n = 46) and 334G/G (699A/A, n = 34) was 1191 and 1580 mg.h L g, respectively (P = 0.02792 in multivariate analysis). There were no significant differences in the dose-adjusted AUC0-12 of MPAG between the ABCB1 C3435T and ABCC2 C-24T genotypes. However, the dose-adjusted AUC0-12 of MPAG was significantly lower in recipients with ABCG2 421C/A+A/A (n = 44) than in those with C/C (n = 36) (P = 0.0295). In conclusion, our findings showed that MPAG pharmacokinetics were significantly influenced by SLCO1B1 and SLCO1B3 polymorphisms and not by UGT polymorphisms. BCRP rather than multidrug resistance protein 2 seems to be the transporter associated with biliary excretion of MPAG.     

Mycophenolic acid and mycophenolic acid glucuronide pharmacokinetics in pediatric liver transplant recipients: effect of cyclosporine and tacrolimus comedication

Determinants of the wide interindividual variability of the pharmacokinetics of mycophenolic acid (MPA) in 21 stable pediatric liver transplant recipients were investigated in relation to the kinetics of the drug's major phenolic glucuronide metabolite (MPAG), cyclosporin (CsA), or tacrolimus (Tac) co-medication and liver and renal function. Trough concentrations (C(0) ) most reliably predicted the area under the curve (AUC) of 0-7 hours MPA plasma concentrations (r (2) = 0.650). Co-medication with CsA demanded higher MPA mofetil (MMF) doses to achieve equivalent trough levels than Tac (362 vs. 178 mg per mg/L, P= 0.004). Median MPA C(0) (range) was significantly lower during CsA co-therapy when corrected for MMF dose (2.8 vs. 5.6 mg MPA/L for Tac, P= 0.006). The AUC of MPAG was correspondingly higher during CsA co-medication (229 vs. 94 mg/L/h for Tac, P = 0.012) with the MPA-to-MPAG ratio at C(0) correspondingly lower (0.10 vs. 0.14, respectively, P = 0.04). This suggested contrasting effects of CsA and Tac on MPA glucuronidation or its excretion and enterohepatic recirculation. MPAG AUC was correlated to body weight and creatinine clearance. Children with elevated aspartate transaminase (AST; but with no evidence of rejection on liver biopsy, n = 7) had significantly lower MPA trough levels compared with those in whom AST was normal (0. 77 vs. 1.76 mg/L, P = 0.05), but there was no difference in the MMF dose per body weight. Examination of the MPA profiles in these subjects showed significantly lower MPA concentrations from 120 minutes after dose until the end of the 7-hour profile and suggest an accelerated clearance or decreased enterohepatic recirculation.)     

Trough levels of mycophenolic acid and its glucuronidated metabolite in renal transplant recipients

OBJECTIVE: The prophylactic use of the immunosuppressant prodrug, mycophenolate mofetil (MMF) to prevent graft rejection in renal transplant patients is continuing to increase. We measured trough levels of the active metabolite, mycophenolic acid (MPA) and its inactive glucuronide (MPAG) in renal recipients with the aim of characterizing individual variability and of ascertaining factors influencing trough levels, in particular the effect of differences in renal function and the effect of drugs given concurrently. METHODS: Laboratory and clinical data obtained in 35 renal recipients treated with triple therapy (MMF, cyclosporin A (CsA), steroids) were included in this retrospective study. Trough levels of MPA and MPAG were obtained after transplantation and up to 16 months post transplantation where the mean observation period was 5.7 months. Plasma levels were measured using a validated HPLC assay. RESULTS: A total of 212 plasma concentrations of MPA and 209 of MPAG were measured. There was considerable intra- and interindividual variability in MPA and MPAG trough levels especially in the early post-transplantation phase. At a fixed dose of 2 g/d MMF, the mean MPA level during the first 30 days averaged 1.46 +/- 1.31 microg/ml vs. 1.87 +/- 0.89 microg/ml after 30 days and later (p = 0.130) and the mean MPAG concentration averaged 188.1 = 142.8 [microg/ml vs. 98.09 +/- 52.4 microlg/ml (p 0.003). The MPAG levels were positively correlated with the serum creatinine concentrations (r = 0.815, p < 0.001), and in the case of MPA there was a correlation with the serum protein concentrations (r = 0.258, p = 0.001). Concomitant drug treatment using CsA, steroids and furosemide were without effect of the measured plasma concentrations, but in the case of xipamide (+) and diltiazem (-) an effect on MPA and MPAG levels and a co-effect depending on the serum creatinine could not be excluded. Neither CsA trough levels nor hemoglobin levels were related to MPA and MPAG trough levels. CONCLUSIONS: The data of this study demonstrate that there is substantial individual variability in the trough levels of MPA and MPAG after renal transplantation which may be associated with the functional status of the graft and the serum protein level. Whether comedication with xipamide and diltiazem affects the plasma levels of MPA and MPAG remains to be clarified in further investigations.     

Relationship between mycophenolate mofetil side effects and mycophenolic acid plasma trough levels in renal transplant patients

There has been limited experience with routine therapeutic monitoring of mycophenolic acid (MPA; CAS 24280-93-1), which is the active metabolite of the new immunosuppressive prodrug mycophenolate mofetil (MMF; CAS 115007-34-6). MMF was introduced with recommendation for fixed oral dosing (1 g twice daily) in combination with cyclosporin A (CSA) and a glucocorticoid for the prevention of renal allograft rejection. In the course of routine CSA monitoring a MPA monitoring was performed in adult renal transplant patients receiving MMF in combination with CSA and methylprednisolone (MEP). For 30 consecutive patients with 234 plasma samples the relationship of MMF doses used and MPA plasma through levels estimated at steady state (C88 min) to clinical outcome was evaluated retrospectively. The MPA concentrations were determined with the enzyme-multiplied immunoassay technique (EMIT mycophenolic acid assay) on a Cobas Mira Plus analyzer. The within-run (n = 10) and between-run (n = 10) coefficients of variation were 3.6%, 3.5%, 3.1% and 3.6%, 5.1%, 6.7% analysing three MPA level plasma controls (1.25 mg/l, 7.5 mg/l, 12.5 mg/l), respectively. The data analysis of the MPA plasma trough levels resulted in a high variability between patients (0.3 to 3.4 mg/l) received the recommended fixed MMF dose (2 g/day). There was a higher incidence of adverse reactions with increasing MPA plasma trough levels (2.13 +/- 1.35 mg/l in 13 patients with side effects versus 1.53 +/- 0.67 mg/l in 17 patients without side effects; p < 0.001), regardless of reduction of MMF dose (1.77 +/- 0.3 g/day versus 1.89 +/- 0.2 g/day; NS), respectively. No acute rejection episodes occured under MMF administration in combination with CSA and MEP. The study shows that the to date recommended MMF dose resulted in individual, quite different MPA plasma trough levels, which were associated with incidence of side effects rather than the MMF doses. Therefore, monitoring of plasma MPA trough levels and individual dose adjustment could be helpful to reduce the incidence of adverse reactions and to increase the safety of MMF therapy.     

Effect of cyclosporine on mycophenolic acid area under the concentration-time curve in pediatric kidney transplant recipients

Mycophenolic acid (MPA), the active compound of mycophenolate mofetil (MMF), shows substantial interindividual and intraindividual variability. It was recently shown that in vitro calcineurin inhibitors alter the bioavailability of MPA by dose-dependent inhibition of MPA glucuronidation. The authors retrospectively analyzed full 10-point profiles for both MPA and cyclosporine (CyA) in 23 pediatric patients receiving MMF and cyclosporine microemulsion (Neoral; Novartis Pharmaceuticals Canada; Dorval, Quebec, Canada). Mycophenolic acid was measured using a commercially available EMIT (Novartis Pharmaceuticals, Canada) assay. As the majority of patients were treated with low doses of cyclosporine after adding MMF, the area under the concentration-time curve (AUC) for cyclosporine showed a wide scatter ranging from 296 to 6400 ng x h/mL. The mean cyclosporine dose was 100 +/- 76 mg/m2 per day (range: 28 to 331). There was no correlation between MPA AUC and MPA dose, and there was substantial interindividual variation. However, there was a significant negative correlation between dose-normalized MPA AUC and cyclosporine AUC ( r2 = 0.23, p < 0.0220). When dividing the MPA profiles into two groups (11 and 12 patients) with a CyA AUC less than or greater than 1600 ng x h/mL, there was a significantly higher 8-hour concentration in the patients with the lower CyA AUC, secondary to a higher second peak. The data demonstrate that the cyclosporine AUC is a determining factor for the MPA AUC and that MPA dose should be reduced when cyclosporine dose is reduced to achieve the same MPA AUC. The significantly higher peak in the group with a lower CyA profile supports the concept of a dose-dependent cyclosporine-induced inhibition of MPA glucuronidation.     

Pharmacokinetics and protein adduct formation of the pharmacologically active acyl glucuronide metabolite of mycophenolic acid in pediatric renal transplant recipients

The acyl glucuronide metabolite (AcMPAG) of mycophenolic acid (MPA) has been found to possess both immunosuppressive and pro-inflammatory activity in vitro. In this study its pharmacokinetics were determined in pediatric renal transplant recipients receiving cyclosporine, steroids, and mycophenolate mofetil. Twelve-hour concentration-time profiles for AcMPAG, MPA, and the phenolic glucuronide (MPAG) were determined by high-performance liquid chromatography (HPLC) in the initial (1-3 wk; n = 16) and stable (3-12 mo; n = 22) phases after transplantation. In addition, the formation of covalent adducts between AcMPAG and plasma albumin (AcMPAG-Alb) was investigated using Western Blot analysis. AcMPAG-AUC(12h) showed significant (p < 0.05) correlations with MPA-AUC(12h) (r = 0.78) and MPAG-AUC(12h) (r = 0.78). In molar equivalents the median AcMPAG-AUC(12h) was 10.3% (range, 4.6%-45.5%) of MPA-AUC(12h). Values (median [range]) of AcMPAG-AUC(12h) (10.1 [3.30-30.1] mg.h/L), AcMPAG-C(0) (0.48 [0.08-1.43] mg/L), and AcMPAG-C(max) (1.95 [0.88-5.35] mg/L) were significantly (p < 0.05) higher in the stable phase than in the initial phase: 3.54 [2.07-20.0] mg.h/L for AUC(12h); 0.25 [<0.04-0.97] mg/L for C(0), and 1.12 [0.32-2.44] mg/L for C(max). The increases in the AcMPAG pharmacokinetic variables were paralleled by significant increases in the corresponding MPA variables. In addition, a strong negative correlation (r = -0.69; p < 0.05) was found between AcMPAG concentrations and the creatinine clearance. AcMPAG-Alb adducts were detected in all patient samples. They showed considerable interindividual variation and increased significantly with time from the initial phase to the stable phase. AcMPAG-Alb correlated significantly (p < 0.05) with AcMPAG-AUC(12h) (r = 0.70) and plasma albumin (r = 0.40). AcMPAG plasma concentrations are dependent on renal function, MPA disposition, and glucuronidation. The pharmacokinetics of AcMPAG is characterized by broad interindividual variation. In some patients AcMPAG may significantly contribute to the immunosuppression during mycophenolate mofetil therapy. AcMPAG-Alb adduct formation may serve as a marker for extended AcMPAG exposure. The association of AcMPAG with adverse effects must be further investigated.     

Influence of carboxylesterase 2 genetic polymorphisms on mycophenolic acid pharmacokinetics in Japanese renal transplant recipients

1. Mycophenolic acid (MPA), converted from the prodrug mycophenolate mofetil (MMF), is generated by intestinal and hepatic esterases. The role of carboxylesterase (CES) in MMF hydrolysis was examined in vitro using human liver microsomes. V_max and K_m values of MMF hydrolysis in pooled human liver microsomes were 1368?±?44 nmol min^?1 mg^?1 protein and 1030?±?65?μM, respectively.

2. Hydrolytic activity was inhibited by the CES inhibitors phenylmethylsulfonylfluoride, bis-p-nitorophenylphosphate and diisopropylfluorophosphate, with IC₅₀ values of 77.1, 3.59 and 0.0312?μM, respectively.

3. Eighty Japanese renal transplant recipients that received repeated-doses of MMF, tacrolimus and prednisolone, were evaluated for MPA pharmacokinetics 28 days after transplantation to investigate the relationship between MPA pharmacokinetics and CES2 genetic polymorphisms.

4. No significant differences in MPA pharmacokinetics were observed between CES2 A4595G, C8721T or A-1548G genotype groups. CES2 allelic variants also did not appear to affect plasma MPA concentrations between individuals.

5. In conclusion, the study demonstrated that while CES1 and/or CES2 are involved in the hydrolysis of MMF to MPA, CES2 allelic variants appeared to make only a minor contribution to inter-personal differences in MPA pharmacokinetics.

     

HPLC测定肾移植患者血浆中的霉酚酸

目的 采用HPLC法测定肾移植患者血浆中的霉酚酸酯活性代谢物霉酚酸.方法 采用Symmetry C18柱(150 mm× 3.9 mm,5μm),流动相为乙腈-32 mmol· L-1甘氨酸缓冲液(pH9.2)(17∶83),柱温30℃,流速1.0 mL· min-1,用荧光检测法,激发波长342 nm,发射波长425 nm,进样量为20 μL.结果 霉酚酸0.01 ～40.00 μg·mL-1与峰高的线性关系良好(r=0.999),最低定量限为0.01μg·mL-1;提取回收率、加样回收率分别为92.29％ ～ 95.01％、100.69％ ～ 110.57％,日内、日间RSD均＜8％.结论 所用方法灵敏、快速、准确,符合生物样品分析方法的基本要求,适用于霉酚酸血药浓度的测定.     

HPLC determination of mycophenolic acid and mycophenolic acid glucuronide in human plasma with hybrid material

Mycophenolic acid (MPA), the active metabolite of the prodrug mycophenolate mofetil is an immunosuppressive agent which inhibits inosine monophosphate dehydrogenase. MPA is metabolised to phenolic glucuronide (MPAG) that may be hydrolysed in vivo to form free MPA. Drug monitoring is required in patients with multi-organ failure. Here, we report a HPLC method with organic/inorganic hybrid material for the simultaneous analysis of MPA and MPAG in human plasma. MPA and MPAG and carboxy butoxy ether mycophenolic acid (MPAC) used as internal standard were analysed on a bonded X-Terra column with a linear gradient elution mode using orthophosphoric acid and acetonitrile as eluents. Sample treatment procedure consists of deproteinisation with acetonitrile. Analytical recoveries were higher than 98 and 89% at concentrations ranging from 1 to 25 and 20 to 200mg/L for MPA and MPAG, respectively. Calibration curves fitted by plotting the peak area ratio (compound of interest/internal standard) versus concentration were linear in the range 0.2-50mg/L for MPA and in the range 1-500mg/L for MPAG. The quantification limit was 0.2mg/L for MPA and 1mg/L for MPAG with a coefficient of variation less than 20% for a 500microL sample volume. Intra- and inter-assay coefficient of variation was lower than 7% for all compounds. Detection was performed at 215nm. Peak identity was confirmed through library matching by comparison with reference spectra. The X-Terra column provides good peak shape and may be used at low pH with a long life-time column. This HPLC method using a simple sample treatment procedure appears suitable for therapeutic drug monitoring in organ-transplant patients. The method is sensitive enough for monitoring MPA and MPAG during pharmacokinetic studies.     

Time-dependent clearance of mycophenolic acid in renal transplant recipients

AIMS: Pharmacokinetic studies of the immunosuppressive compound mycophenolic acid (MPA) have shown a structural decrease in clearance (CL) over time after renal transplantation. The aim of this study was to characterize the time-dependent CL of MPA by means of a population pharmacokinetic meta-analysis, and to test whether it can be described by covariate effects. METHODS: One thousand eight hundred and ninety-four MPA concentration-time profiles from 468 renal transplant patients (range 1-9 profiles per patient) were analyzed retrospectively by nonlinear mixed effect modelling. Sampling occasions ranged from day 1-10 years after transplantation. RESULTS: The pharmacokinetics of MPA were described by a two-compartment model with time-lagged first order absorption, and a first-order term for time-dependent CL. The model predicted the mean CL to decrease from 35 l h(-1) (CV = 44%) in the first week after transplantation to 17 l h(-1) (CV = 38%) after 6 months. In a covariate model without a term for time-dependent CL, changes during the first 6 months after transplantation in creatinine clearance from 19 to 71 ml min(-1), in albumin concentration from 35 to 40 g l(-1), in haemoglobin from 9.7 to 12 g dl(-1) and in cyclosporin predose concentration from 225 to 100 ng ml(-1) corresponded with a decrease of CL from 32 to 19 l h(-1). Creatinine clearance, albumin concentration, haemoglobin and cyclosporin predose concentration explained, respectively, 19%, 12%, 4% and 3% of the within-patient variability in MPA CL. CONCLUSIONS: By monitoring creatinine clearance, albumin concentration, haemoglobin and cyclosporin predose concentration, changes in MPA exposure over time can be predicted. Such information can be used to optimize therapy with mycophenolate mofetil.     

Influence of cyclosporine on the serum concentration and biliary excretion of mycophenolic acid and 7-O-mycophenolic acid glucuronide

The authors have investigated whether cyclosporine decreases the serum concentration of mycophenolic acid, the active principle of the immunosuppressant mycophenolate mofetil, and increases that of the inactive metabolite 7-O-mycophenolic acid glucuronide by reducing their enterohepatic recirculation. Rats were treated daily with methylcellulose (1.66 mL/kg PO) plus 0.9% NaCl (6 mL/kg IP), mycophenolate mofetil (20 mg/kg PO) plus 0.9% NaCl (6 mL/kg IP), methylcellulose (1.66 mL/kg PO) plus cyclosporine (5 mg/kg IP), and mycophenolate mofetil (20 mg/kg PO) plus cyclosporine (5 mg/kg IP). After 14 days a bile fistula was installed to measure the biliary excretion of the immunosuppressants and their metabolites. After 90 minutes blood was taken to determine their concentrations in blood or serum by HPLC. Cyclosporine significantly decreased the serum concentration of mycophenolic acid by 39% and increased, not significantly, that of 7-O-mycophenolic acid glucuronide by 53%. The biliary excretion of 7-O-mycophenolic acid glucuronide was significantly reduced by cyclosporine by 57%, whereas that of mycophenolic acid was not affected. Mycophenolate mofetil did not show a significant effect on either the blood concentration or the biliary excretion of cyclosporine and its metabolites AM1, AM9, AM1c, and AM4N. Cyclosporine significantly decreased the serum concentration of active mycophenolate acid and increased, not significantly, the serum concentration of inactive 7-O-mycophenolic acid glucuronide, presumably by reducing the biliary excretion of this inactive metabolite.