Facile fabrication of a novel disposable pencil graphite electrode for simultaneous determination of promising immunosuppressant drugs mycophenolate mofetil and tacrolimus in human biological fluids.

An innovative electrochemical sensor was proposed for simultaneous determination of mycophenolate mofetil (Mph) and tacrolimus (TAC) for the first time. A novel sensor based on electro-polymerization of multi-walled carbon nanotubes (MWCNTs) and a novel Cu-1N-allyl-2-(2,5-dimethoxyphenyl)-4,5-diphenyl-1H-imidazole metal organic framework (Cu-ADPPI MOF) on disposable pencil graphite electrode (dPGE). Many techniques were used to characterize the electrochemical activity and surface structure of the fabricated sensor. The proposed sensor exhibited good catalytic performance towards Mph and TAC oxidation due to the synergistic effect. Under optimal conditions, the proposed sensor has achieved a linear range of 0.85-155 × 10-8 M and 1.1-170.0 × 10-8 M with LODs of 0.28 × 10-8 M and 0.36 × 10-8 M for Mph and TAC, respectively. The designated sensor showed good reproducibility, repeatability, stability, and selectivity for the determination of Mph and TAC. Moreover, the simultaneous determination of Mph and TAC in different human biological fluids was carried out with acceptable results. As a result, the proposed sensor opens a new venue for the use of electro-polymerized MOFs in combination with other conductive materials such as MWCNTs for electrochemical sensing of different analytes with the desired sensitivity and selectivity. Graphical abstract Construction of disposable graphite electrode, based on electro-deposition of multilayer films of multi-walled carbon nanotubes and a new generation of Cu-MOFs, for simultaneous analysis of tacrolimus and mycophenolate mofetil for the first time.

Adsorptive anodic stripping differential pulse voltammetric determination of CellCept at Fe3O4 nanoparticles decorated multi-walled carbon nanotubes modified glassy carbon electrode.

A simple and sensitive method based on adsorptive anodic stripping differential pulse voltammetry (AASDPV) for the determination of cellcept, using a magnetic Fe3O4 nanoparticles and functionalized (carboxylated) multi-walled carbon nanotubes modified glassy carbon electrode (f-MWCNs/Fe3O4/GCE) was developed. In phosphate buffer solution (pH = 5), the voltammogram of cellcept exhibited tow anodic peaks and the well-defined peak at about 0.611 V vs SCE was used for its monitoring. The modified electrode was characterized by different methods such as electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and cyclic voltammetry (CV). The experimental parameters, such as pH, deposition potential and time, as well as scan rate were optimized. Under the optimized conditions, Ip (µA) was proportional to the cellcept concentration in the range of 0.05-200 µM (R2 = 0.9989) with a detection limit of 9.0 nM and limit of quantification of 30.2 nM. The recovery was >98%. The practical analytical utilities of the modified electrode were demonstrated by the determination of cellcept in human urine and blood serum samples. Modified electrode showed an adequate sensitivity and stability for evaluated samples.

Simultaneous determination of mycophenolate mofetil and its active metabolite, mycophenolic acid, by differential pulse voltammetry using multi-walled carbon nanotubes modified glassy carbon electrode.

A highly sensitive electrochemical sensor for the simultaneous determination of mycophenolate mofetil (MPM) and mycophenolic acid (MPA) was fabricated by multi-walled carbon nanotubes modified glassy carbon electrode (MWCNTs/GCE). The electrochemical behavior of these two drugs was studied at the modified electrode using cyclic voltammetry and adsorptive differential pulse voltammetry. MPM and MPA were oxidized at the GCE during an irreversible process. DPV analysis showed two oxidation peaks at 0.87V and 1.1V vs. Ag/AgCl for MPM and an oxidation peak at 0.87V vs. Ag/AgCl for MPA in phosphate buffer solution of pH5.0. The MWCNTs/GCE displayed excellent electrochemical activities toward oxidation of MPM and MPA relative to the bare GCE. The experimental design algorithm was used for optimization of DPV parameters. The electrode represents linear responses in the range 5.0×10(-6) to 1.6×10(-4)molL(-1) and 2.5×10(-6)molL(-1) to 6.0×10(-5)molL(-1) for MPM and MPA, respectively. The detection limit was found to be 9.0×10(-7)molL(-1) and 4.0×10(-7)molL(-1) for MPM and MPA, respectively. The modified electrode showed a good sensitivity and stability. It was successfully applied to the simultaneous determination of MPM and MPA in plasma and urine samples.

Change in pharmacokinetics of mycophenolic acid as a function of age in rats and effect of coadministered amoxicillin/clavulanate.

Changes of mycophenolic acid (MPA) pharmacokinetics with aging were investigated in rats. We also compared the effect of concomitant amoxicillin/clavulanate combination (CVA/AMPC) on the pharmacokinetics of MPA in 4-week-old and 12-week-old rats (the package insert of CVA/AMPC warns of possible interaction with MPA). Four-week-old rats showed a 1.4-fold higher total body clearance of MPA and a lower volume of distribution of MPA (65%), compared to the values in 12-week-old rats. However, the difference in MPA pharmacokinetics disappeared when enterohepatic circulation was eliminated by bile duct cannulation (BDC). Concomitant CVA/AMPC significantly reduced plasma MPA concentration in intact rats of both age groups, and the age-dependent difference of MPA pharmacokinetics was no longer apparent. The effect of CVA/AMPC was not seen in rats that had undergone BDC, suggesting that the drug-drug interaction can be attributed to inhibition of enterohepatic circulation by CVA/AMPC. These results indicate that the aging-related alteration of MPA pharmacokinetics is a consequence of immature enterohepatic circulation in 4-week-old rats. Higher doses of MPA may be necessary in juveniles.

A high-throughput U-HPLC-MS/MS assay for the quantification of mycophenolic acid and its major metabolites mycophenolic acid glucuronide and mycophenolic acid acyl-glucuronide in human plasma and urine.

Mycophenolic acid (MPA) is used as an immunosuppressant after organ transplantation and for the treatment of immune diseases. There is increasing evidence that therapeutic drug monitoring and plasma concentration-guided dose adjustments are beneficial for patients to maintain immunosuppressive efficacy and to avoid toxicity. The major MPA metabolite that can be found in high concentrations in plasma is MPA glucuronide (MPAG). A metabolite usually present at lower concentrations, MPA acyl-glucuronide (AcMPAG), has been implicated in some of the adverse effects of MPA. We developed and validated an automated high-throughput ultra-high performance chromatography-tandem mass spectrometry (U-HPLC-MS/MS) assay using liquid-handling robotic extraction for the quantification of MPA, MPAG, and AcMPAG in human EDTA plasma and urine. The ranges of reliable response were 0.097 (lower limit of quantitation) to 200 µg/mL for MPA and MPAG and 0.156-10 µg/mL for AcMPAG in human urine and plasma. The inter-day accuracies were 94.3-104.4%, 93.8-105.0% and 94.4-104.7% for MPA, MPAG and AcMPAG, respectively. Inter-day precisions were 0.7-7.8%, 0.9-6.9% and 1.6-8.6% for MPA, MPAG and AcMPAG. No matrix interferences, ion suppression/enhancement and carry-over were detected. The total assay run time was 2.3 min. The assay met all predefined acceptance criteria and the quantification of MPA was successfully cross-validated with an LC-MS/MS assay routinely used for clinical therapeutic drug monitoring. The assay has proven to be robust and reliable during the measurement of samples from several pharmacokinetics trials.

High-performance liquid chromatography-mass spectroscopy/mass spectroscopy method for simultaneous quantification of total or free fraction of mycophenolic acid and its glucuronide metabolites.

Measurement of unbound fractions of mycophenolic acid and its metabolites may prove useful in explaining the complicated pharmacokinetic and pharmacodynamic behavior of this drug as well as in therapeutic drug monitoring. We developed a reliable, accurate, and sensitive liquid chromatography-tandem mass spectrometric method for the simultaneous quantification of mycophenolic acid (MPA), MPA glucuronide (MPAG), and MPA acyl-glucuronide (AcMPAG), total or unbound, in plasma, urine, and tissue extract. This method uses a single internal standard, carboxy-butoxy ether of mycophenolic acid (MPAC), and involves a simple sample preparation step. Aliquots of plasma, urine, or dissolved tissue extract (100 microL) or plasma ultrafiltrate for free analytes (50 microL) are treated with acetonitrile/formic acid mixture (99.5/0.5 v/v) followed by centrifugation and dilution with water. The prepared samples are then injected onto an extraction column (Eclipse XDB-C18 12.5 x 4.1 mm; Agilent Technologies, Palo Alto, CA) and washed with mobile phase composed of acetonitrile/water/formic acid (10/89.5/0.5 v/v/v) at a flow rate of 2.8 mL/min. A switching valve is activated 1 minute after sample injection. The analytes are eluted onto the analytical column (Eclipse XDB-C18 150 x 4.1 mm; Agilent Technologies) with a gradient of 0.5% aqueous formic acid, methanol, acetonitrile, and water. We used a tandem mass spectrometer with electrospray ion source, in which the tandem mass spectroscopy transitions were (m/z): 338-->207 for MPA, 438-->303 for MPAC, and 514-->303 for MPAG and AcMPAG. The dynamic ranges (lower limit of quantitation and upper limit of quantitation) were as follows: 0.05 to 30 mg/L for total MPA and 1 to 300 microg/L for free MPA; 0.5 to 300 mg/L of total MPAG and 0.2 to 60 mg/L for free MPAG; and 0.025 to 15 mg/L of total AcMPAG and 1 to 60 microg/L for free AcMPAG. The precision at lower limit of quantitation was in the range of 8.0% to 11.9% for all three total analytes and 13.8 to 18.7% for the free analytes. Accuracy at lower limit of quantitation was in the range of 100% to 105% for total and 97% to 99% for free analytes. Between-day precision of quality control samples was 4.0% to 6.3% for human plasma spiked with total analytes and 4.5% to 14.4% for spiked plasma ultrafiltrate for free analytes. Mean absolute recovery ranged from 98.5% to 101.7% for MPA (both total and free), from 78.1% to 103.4% for MPAG and from 91.5% to 110.4% for AcMPAG. No significant ion suppression was found under these conditions for any of the analytes. Carryover effect was found to be at a maximum level of 0.02%. This method was successfully applied to analyze over 11,000 samples for total analytes, and over 8000 samples for free analytes in plasma, and has been in operation for nearly 3 years without loss of performance.

Impact of calcineurin inhibitors on urinary excretion of mycophenolic acid and its glucuronide in kidney transplant recipients.

Concomitant cyclosporine interacts with mycophenolic acid (MPA) through inhibition of the biliary excretion of its glucuronide (MPAG). The aim of this study was to evaluate the influence of calcineurin inhibitors on the plasma disposition and urinary excretion of MPA and MPAG in kidney transplant recipients. Twelve recipients treated with tacrolimus and 18 treated with cyclosporine at 30 days after transplantation were enrolled. AUC from 0 to 12 hours (AUC(0-12)) of MPA was significantly higher in tacrolimus-treated than in cyclosporine-treated recipients. In contrast, there was no significant difference in MPAG AUC(0-12) between calcineurin inhibitor medications. Unbound fractions of MPA and MPAG did not change significantly in a comparison between the tacrolimus and cyclosporine treatments (0.90% vs 1.27% in MPA; 20.0% vs 19.3% in MPAG). The ratio of renal clearance to creatinine clearance (CL(R)/CL(Cr)) of MPA was significantly lower in tacrolimusthan in cyclosporine-treated recipients (0.054 vs 0.100). In contrast, no significant difference was observed in the CL(R)/CL(Cr) of MPAG between the tacrolimus and cyclosporine treatments (0.19 vs 0.18). In conclusion, concomitant calcineurin inhibitors influenced the urinary excretion of MPA but not MPAG in kidney transplant recipients. The results suggest the presence of renal tubular secretion in the urinary excretion process of MPA.

Multicriteria optimization methodology in development of HPLC separation of mycophenolic acid and mycophenolic acid glucuronide in human urine and plasma.

Multicriteria optimization methodology was applied for development of isocratic reversed-phased HPLC method for simultaneous determination of mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) in human urine and plasma. In the first stage of method development, pH value of the water phase, percentage of acetonitrile, temperature of the column and flow rate of the mobile phase were investigated using fractional factorial design. Afterwards, the optimal conditions were found employing central composite design and Derringer's desirability function. Two goals were considered, the retention factor of the MPAG to be in the range, between 0.8 and 1.118 which allowed well separation of MPAG from the urine and plasma peaks, and the shortest possible total analysis run time. Then, the obtained sigmoid functions were used to transform the optimization criteria into the desirability values. The satisfactory chromatographic conditions were obtained with mobile phase consisted of acetonitrile-phosphate buffer (pH 2.4; 0.04 M KH(2)PO(4)) (28:72, v/v). The separation was performed on C(18) Chromolith column (100 mm x 4.6 mm) with flow rate of 5 mL/min, the temperature of the column was 25 degrees C and the chosen wavelength for simultaneous determination of MPA and MPAG was 215 nm. The MPAG eluted at 0.552 min and the duration of run was 3.092 min. Afterwards, the method was subjected to validation. Linearity was observed over the concentration range of 1-50 microg/mL for MPA and 1-500 microg/mL for MPAG in urine and 1-60 microg/mL for MPA and 1-70 microg/mL for MPAG in plasma matrix. The method showed intra-day and inter-day precision with relative standard deviation lower then 5% and accuracy as recovery (%) between 100+/-5%.

Pharmacokinetics of mycophenolate mofetil and its glucuronide metabolites in healthy volunteers.

UNLABELLED: We previously reported that polymorphisms in the UGT2B7 and UGT1A9 genes are associated with significant alteration in the disposition of mycophenolic acid (MPA) in healthy volunteers. AIM: This study further evaluates the impact of genetic polymorphisms at the UGT1A1, UGT1A7 and ABCC2 loci. METHODS: Genetic analyses of five UGT candidate genes and ABCC2 were completed on 47 healthy subjects who received a single dose of 1.5 g mycophenolate mofetil and completed a 12-h pharmacokinetic profile. RESULTS: Multivariate analyses indicate that the ABCC2 -24T promoter polymorphism is associated with a 25% increase in acyl mycophenolic acid phenolic glucuronide level. Subjects with combined ABCC2 -24T and UGT1A9*3 genotypes present a 169% increased exposure to AcMPAG. Homozygosity for UGT1A7 387G/391A (129Lys/131Lys) is associated with a modest but significant 7% reduction in MPA level. When these additional genetic factors are considered in the model, the effects of previously described UGT1A9 and UGT2B7 variations remain significant. No significant effect is observed for UGT1A1*28, UGT1A7 622T/C (Trp208Arg), UGT1A9 -440TC/-331CT, UGT1A9 -118 TA(9/10) and seven other ABCC2 SNPs. CONCLUSION: We demonstrate that MPA disposition is a multigenic process, and that additional studies are required to ascertain the relationship between UGT, ABCC2 genotypes and MPA pharmacokinetics in transplant recipients.

Application of experimental design in optimization of solid phase extraction of mycophenolic acid and mycophenolic acid glucuronide from human urine and plasma and SPE-RP-HPLC method validation.

The aim of this study was to develop and optimize a solid phase extraction (SPE) procedure for purification of mycophenolic acid (MPA) and its metabolite mycophenolic acid glucuronide (MPAG) in biological samples. During optimization process chemometric approach was applied. First, in screening experiments fractional factorial design (FFD) was used for selecting the variables which affected the extraction procedure. The ionic strength of the phosphate buffer in the washing step and the percentage of acetonitrile in the elution step were statistically significant for the recovery of MPAG while the percentage of acetonitrile and pH of the washing solution were statistically significant for that of MPA. Afterwards, the significant variables were optimized using central composite design (CCD). The developed SPE method included phosphate buffer (pH 2.4; 0.056 M) in the washing step, and the mixture of acetonitrile and phosphate buffer of which pH was adjusted to 2.4 (70:30, v/v) in the elution step. The investigation was applied to both urine and plasma and the nature of biological matrix appeared to be of no importance. The extraction from both matrixes showed good repeatability with relative standard deviations up to 6% for MPAG and 8% for MPA, and recovery around 100% for both substances. Furthermore, new SPE-RP-HPLC method for determination of MPA and MPAG in both humane urine and plasma has been validated. The great advantage of this method is the chromatographic run of only 3 min.

Sensitive high-performance liquid chromatography-tandem mass spectrometry method for quantitative analysis of mycophenolic acid and its glucuronide metabolites in human plasma and urine.

A method to determine total and free mycophenolic acid (MPA) and its metabolites, the phenolic (MPAG) and acyl (AcMPAG) glucuronides, using HPLC and mass spectrometry was developed. Mean recoveries in plasma and urine samples were >85%, and the lower limits of quantification for MPA, MPAG and AcMPAG were 0.05, 0.05 and 0.01 mg/L, respectively. For plasma, the assay was linear over 0.05-50 mg/L for MPA and MPAG, and from 0.01 to 10mg/L for AcMPAG. A validation study demonstrated good inter- and intra-day precision (CV

Rifampin induces alterations in mycophenolic acid glucuronidation and elimination: implications for drug exposure in renal allograft recipients.

BACKGROUND: Exposure to mycophenolic acid (MPA) and its main metabolites (MPA 7-O-glucuronide [MPAG] and MPA acyl-glucuronide [AcMPAG]) is characterized by a large interindividual and intraindividual variability, resulting in part from variability in glucuronidation (via uridine diphosphate-glucuronosyltransferase isoforms) and excretion via multidrug resistance-associated protein 2 (MRP2). It can be hypothesized that drugs interfering with glucuronidation and excretion will alter (Ac)MPA(G) exposure. METHODS: This prospective, open-label, nonrandomized, controlled pharmacokinetic interaction study included 8 stable renal allograft recipients, all treated with mycophenolate mofetil. Rifampin (INN, rifampicin), administered once daily (600 mg/d) for 8 days, was used as the probe drug because of its known effects on both uridine diphosphate-glucuronosyltransferase activity and MRP2 transport capacity. A 12-hour pharmacokinetic time-concentration profile was assessed before rifampin administration was started, and this was repeated on the last day of rifampin administration. Total and free MPA, MPAG, and AcMPAG concentrations in plasma and urine were measured by use of HPLC with tandem mass spectrometry detection. RESULTS: Total MPA area under the plasma concentration-time curve (AUC) from 0 to 12 hours decreased significantly after rifampin coadministration (17.5% decrease [95% confidence interval (CI), 5.18%-29.9%]; P=.0234). This was mainly a result of a decrease in total MPA AUC from 6 to 12 hours (32.9% decrease [95% CI, 15.4%-50.4%]; P=.0078), representing decreased enterohepatic recirculation. Free MPA AUC from 6 to 12 hours decreased significantly, by 22.4% (95% CI, 4.71%-49.5%; P=.0391). Total MPAG and AcMPAG AUC from 0 to 12 hours increased by 34.4% (95% CI, 13.5%-55.4%; P=.0156) and 193% (95% CI, 30.3%-355%; P=.0078) respectively. Urinary recovery of MPAG and AcMPAG increased significantly (P=.0078), but renal clearance of these glucuronides did not change after rifampin coadministration. CONCLUSION: This study demonstrates an interaction between mycophenolate mofetil and rifampin, which is a result of induction of MPA glucuronidation and possibly also rifampin-associated alterations in MRP2-mediated transport of MPAG and AcMPAG. This interaction should be taken into account when rifampin or other drugs influencing pregnane X receptor activity are coadministered with mycophenolate mofetil.

Characterization of a phase 1 metabolite of mycophenolic acid produced by CYP3A4/5.

To characterize a phase 1 metabolite of mycophenolic acid (MPA) and the human cytochrome P450 isoform(s) (CYP) involved in its formation. MPA metabolites were investigated in blood and urine samples from transplant patients under mycophenolate mofetil therapy (n = 5) as well as with in vitro incubation of MPA with human liver microsomes. The CYP isoforms involved in the oxidative metabolism were investigated in vitro on human liver microsomes with isoform-specific inhibitors as well as in human embryonic kidney cell lines expressing recombinant human CYPs. The analytic methods used were based on LC-MS/MS. A 6-O-desmethyl-MPA (DM-MPA) metabolite and 2 related glucuronides were identified in patients' blood and urine. Human liver microsomes produced DM-MPA with an apparent Km = 0.83 +/- 0.06 mmol/L and Vmax = 5.57 +/- 0.29 pmol/mg/min. The CYP3A inhibitor ketoconazole was found to inhibit DM-MPA formation by 50.3% with respect to the control, and trimethoprim (CYP2C8 inhibitor) reduced it by 30.1%. However, DM-MPA was produced only by the transfected cell lines expressing CYP3A4 and, to a lesser extent, CYP3A5. In vitro, MPA at concentrations above the plasma therapeutic range was found to decrease the metabolism of tacrolimus, suggesting a possible competition for CYP3A. No effect of MPAat therapeutic or higher level was found on cyclosporin metabolism. The phase 1 metabolite of MPA previously known as M-3 was identified as 6-O-desmethyl-MPA and is produced by CYP3A4/5 and probably CYP2C8. MPA might compete with other drugs on CYP3A because of its high therapeutic concentrations, although this was not the case for cyclosporin and to only a small extent for tacrolimus.

Simple reversed-phase ion-pair liquid chromatography assay for the simultaneous determination of mycophenolic acid and its glucuronide metabolite in human plasma and urine.

A simple and reproducible reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method using isocratic elution with UV absorbance detection is presented for the simultaneous quantitation of mycophenolic acid (MPA) and MPA-glucuronide (MPAG) in human plasma and urine. The sample preparation procedures involved simple protein precipitation for plasma and 10-fold dilution for urine. Each analytical run was completed within 15min, with MPAG and MPA being eluted at 3.8 and 11.4min, respectively. The optimized method showed good performance in terms of specificity, linearity, detection and quantitation limits, precision and accuracy. This assay was demonstrated to be applicable for clinical pharmacokinetic studies.

High-performance liquid chromatographic method for mycophenolic acid and its glucuronide in serum and urine.

OBJECTIVE: To develop a simple analytical method for monitoring serum and urine concentrations of mycophenolic acid (MPA), an active metabolic constituent of the immunosuppressive pro-drug mycophenolate mofetil, and its glucuronide. METHODS: Serum samples were prepared by solid-phase extraction (SPE), while urine samples were simply diluted with water. Serum was added to an SPE cartridge, then washed twice with 5% methanol solution. The analytes were eluted with methanol containing benzoic acid as internal standard for mycophenolic acid glucuronide (MPAG). The resultant eluate was directly injected into a high-performance liquid chromatograph (HPLC) to determine MPAG. For the assay of MPA, the remaining eluate was dried under nitrogen and resolved in a mixture of acetonitrile and 20 mM phosphate buffer (pH 3.0). RESULTS: The present methods were reproducible and accurate based on the intra- and inter-assay, and had detection limits of 0.225 microg/mL for MPA and 9.0 microg/mL for MPAG. The present methods enabled us to monitor the time course of changes in the concentrations of MPA and MPAG in serum and urine in a patient with a renal transplant during 12 h after ingestion of mycophenolate mofetil. CONCLUSION: The HPLC method described should be useful for the routine monitoring of serum and urine concentrations of MPA and MPAG during immunosuppressive medication for renal transplantation.

Determination of mycophenolic acid and its phenol glucuronide metabolite in human plasma and urine by high-performance liquid chromatography.

Simultaneous determination of mycophenolic acid (MPA) and mycophenolate phenol glucuronide (MPAG) in plasma and urine was accomplished by isocratic HPLC with UV detection. Plasma was simply deproteinated with acetonitrile and concentrated, whereas urine was diluted prior to analysis. Linearity was observed from 0.2 to 50 microg/ml for both MPA and MPAG in plasma and from 1 to 50 microg/ml of MPA and 5 to 2000 microg/ml MPAG in urine with extraction recovery from plasma greater than 70%. Detection limits using 0.25 ml plasma were 0.080 and 0.20 microg/ml for MPA and MPAG, respectively. The method is more rapid and simple than previous assays for MPA and MPAG in biological fluids from patients.

Pharmacokinetics of mycophenolic acid after mycophenolate mofetil administration in liver transplant patients treated with tacrolimus.

The pharmacokinetics of mycophenolic acid (MPA) was studied after oral administration of mycophenolate mofetil (MMF) in 8 liver transplant patients. The mean (+/- SD) maximum MPA plasma concentration of 10.6 (+/- 7.5) mg/ml was achieved within 0.5 to 5 hours. The mean (+/- SD) steady-state area under the plasma concentration versus time curve (AUC(0-12)) was 40 (+/- 30.9) mg/ml/h. The mean (+/- SD) half-life was 5.8 (+/- 3.8) hours. There was poor correlation between trough blood concentrations of tacrolimus (r = -0.004) or serum creatinine (r = 0.689) with MPA AUC, while the serum bilirubin concentrations correlated (r = 0.743) well with MPA AUC, suggesting impairment in MPA conjugation in patients with liver dysfunction. The mean (+/- SD) ratio of the AUC of mycophenolic acid glucuronide (MPAG) to MPA was 64 (+/- 84), which correlated significantly with serum creatinine (r = 0.72) but not with serum bilirubin concentrations (r = 0.309), indicating accumulation of MPAG in patients with renal dysfunction. In 7 primary liver transplant patients on the same dose of MMF, the trough plasma concentrations of MPA during the first week of therapy ranged from < 0.3 to 1.5 microg/ml. The MPA concentrations increased by several folds during the next few weeks, which correlates well with increases in serum albumin concentrations. Changes in albumin appear to partially contribute to the variations in the pharmacokinetics of MPA in liver transplant patients.

Mycophenolic acid glucuronidation and its inhibition by non-steroidal anti-inflammatory drugs in human liver and kidney.

OBJECTIVE: The aims of this investigation were to study the glucuronidation of mycophenolic acid (MPA) in human liver and kidney and to search for a compound that inhibits MPA glucuronidation among the non-steroidal anti-inflammatory drugs (NSAIDs). METHODS: A sensitive and reproducible radiometric assay was developed to measure the rate of MPA glucuronidation in human liver and kidney microsomes. The assay employed uridine 5'-diphosphate-[U-14C]-glucuronic acid (UDPGA) and MPA-glucuronide was isolated by TLC. The final concentrations of UDPGA and MPA necessary were 1 mM (liver), and MPA concentration was 0.5 mM (kidney). The inhibition of MPA glucuronidation was studied with 18 NSAIDs and tacrolimus. RESULTS: Glucuronosyl transferase activity followed Michaelis-Menten kinetics and the Km (mean +/- SD; mM) was 0.31+/-0.06 (liver; n = 5) and 0.28+/-0.07 (kidney; n = 5; P = 0.555); the Vmax (mean SD; nmol/mg per minute) was 5.2+/-1.4 (liver; n = 5) and 10.5+/-1.2 (kidney; n = 5; P = 0.0005). The MPA glucuronidation rates (mean +/- SD; nmol/min/mg) were 3.3+/-0.9 (liver; n = 10) and 7.8+/-1.5 (kidney; n = 10; P = 0.0002). The rate of MPA glucuronidation ranged between 2.0 and 5.1 nmol/ mg per minute with a 2.5-fold variation (liver) and between 5.7 and 9.8 nmol/mg per minute with a 1.7-fold variation (kidney). The inhibition study was performed in liver and revealed that the percentage of control ranged from 8%+/-3% (niflumic acid) to 119%+/-16% (Ketoralac). The inhibition curves for MPA glucuronidation rate were determined with the four most effective inhibitors: niflumic acid, flufenamic acid, mefenamic acid and diflunisal. Their IC50 estimates (microM) were 8+/-1, 19+/-9, 63+/-8 and 109+/-15, respectively (liver), and 8+/-2, 13+/-2, 49+/-4 and 122+/-18, respectively (kidney). The IC50 estimate for niflumic acid was eightfold lower than the peak plasma levels after a single oral dose of 250 mg of this drug. CONCLUSION: The human liver and kidney are important sites of MPA glucuronidation. MPA glucuronidation was inhibited to various extents by different NSAIDs and the four most effective inhibitors were niflumic acid, flufenamic acid, mefenamic acid and diflunisal. These drugs have similar molecular structures consisting of two aromatic rings bearing a carboxylic group.

The pharmacokinetics of a single oral dose of mycophenolate mofetil in patients with varying degrees of renal function.

BACKGROUND: The purpose of this study was to determine the effect of renal function on the elimination and disposition of mycophenolic acid and its glucuronide metabolite (MPAG) after oral administration of the pro-drug mycophenolate mofetil. In addition, this study sought to examine hemodialysis removal of mycophenolic acid and its MPAG. METHODS: Subjects were stratified into five groups on the basis of iohexol clearance. After an overnight fast, all subjects received a single 1 gm dose of mycophenolate mofetil. Plasma concentrations of mycophenolic acid and MPAG were measured from 0 to 96 hours after administration. Mycophenolic acid and MPAG maximum plasma concentration (Cmax) and the time to reach Cmax (tmax) for each group were determined from the mean plasma concentration-time profiles. Area under the plasma concentration-time curve values for mycophenolic acid and MPAG were calculated by the trapezoidal rule. The half-lives of mycophenolic acid and MPAG were calculated from the terminal portions of the concentration-time profiles. RESULTS: Mycophenolic acid clearance was not associated with changes in glomerular filtration rate (GFR). Cmax tended to increase as GFR declined. MPAG clearance correlated well with GFR (r2 = 0.905). Clearance of mycophenolic acid and MPAG were unaffected by hemodialysis. CONCLUSIONS: Clearance of mycophenolic acid after a single 1 gm oral dose of mycophenolate mofetil is unaffected by renal function. Clearance of mycophenolic acid is unaffected by hemodialysis. Diminished renal function should not require preemptive adjustment of 1 gm doses of mycophenolate mofetil; however dosage adjustment may be warranted on the basis of adverse effects or toxicity in individual patients. Mycophenolate mofetil can be administered irrespective of hemodialysis session without effect on mycophenolic acid exposure.

Pharmacokinetics of oral mycophenolate mofetil in volunteer subjects with varying degrees of hepatic oxidative impairment.

Eighteen patients with compensated alcoholic cirrhosis participated in a single-dose pharmacokinetic study of oral mycophenolate mofetil (MMF). Participants were divided into groups of 6 patients each with mild, moderate, or severe hepatic oxidative impairment as defined by the aminopyrine breath test (APBT). Clinically, hepatic disease was of mild or moderate severity. Six healthy volunteers were included as control subjects. Plasma and urine samples were collected over 96 hours and assayed for the active metabolite mycophenolic aced (MPA) and the glucuronide conjugate, MPAG. Plasma protein binding of MPA also was determined in 6 unrelated patients with cirrhosis. Cirrhosis did not grossly affect plasma pharmacokinetics or plasma binding of MPA. Maximum plasma concentrations (C(max)) and area under the curve (AUC) of MPA and MPAG consistently decreased, increased, and then decreased as oxidative impairment declined from normal to severe. Patients with cirrhosis had comparable or greater recovery of administered drug substance in urine than controls, showing that cirrhosis did not affect the extent of MMF absorption. Urine clearance of MPAG was two times higher in the group with severe impairment than in the other groups. Creatinine clearance was similar in all groups. These results suggest progressive impairment of hepatic glucuronidation of MPA and induction of renal glucuronidation in patients with severe hepatic oxidative impairment.