Acyl Glucuronide Metabolites Research Articles

Plasma Stability-Dependent Circulation of Acyl Glucuronide Metabolites in Humans: How Circulating Metabolite Profiles of Muraglitazar and Peliglitazar Can Lead to Misleading Risk Assessment

Muraglitazar and peliglitazar, two structural analogs differing by a methyl group, are dual peroxisome proliferator-activated receptor-α/γ activators. Both compounds were extensively metabolized in humans through acyl glucuronidation to form 1-O-β-acyl glucuronide (AG) metabolites as the major drug-related components in bile, representing at least 15 to 16% of the dose after oral administration. Peliglitazar AG was the major circulating metabolite, whereas muraglitazar AG was a very minor circulating metabolite in humans. Peliglitazar AG circulated at lower concentrations in animal species than in humans. Both compounds had a similar glucuronidation rate in UDP-glucuronic acid-fortified human liver microsomal incubations and a similar metabolism rate in human hepatocytes. Muraglitazar AG and peliglitazar AG were chemically synthesized and found to be similarly oxidized through hydroxylation and O-demethylation in NADPH-fortified human liver microsomal incubations. Peliglitazar AG had a greater stability than muraglitazar AG in incubations in buffer, rat, or human plasma (pH 7.4). Incubations of muraglitazar AG or peliglitazar AG in plasma produced more aglycon than acyl migration products compared with incubations in the buffer. These data suggested that the difference in plasma stability, not differences in intrinsic formation, direct excretion, or further oxidation of muraglitazar AG or peliglitazar AG, contributed to the observed difference in the circulation of these AG metabolites in humans. The study demonstrated the difficulty in doing risk assessment based on metabolite exposure in plasma because the more reactive muraglitazar AG would not have triggered a threshold of concern based on the recent U.S. Food and Drug Administration guidance on Metabolites in Safety Testing, whereas the more stable peliglitazar AG would have.

UPLC Analysis of Mycophenolic Acid and Its Phenol and Acyl Glucuronide Metabolites in Human Plasma

A simple, rapid, and reliable UPLC method has been validated for simultaneous analysis of mycophenolic acid (MPA) and its phenol glucuronide (MPAG) and acyl glucuronide (AcMPAG) metabolites in human plasma. Separation was performed on a UPLC BEH C18 column. The mobile phase was a gradient prepared from 10 mM ammonium formate and acetonitrile; the flow rate was 0.5 mL min−1, the total run time 5.5 min, and the analytes were quantified at 254 nm. Sample preparation was by liquid–liquid extraction. The method is specific, sensitive, and linear in the concentration ranges 0.062–126 μg mL−1 for MPA, 0.080–188 μg mL−1 for MPAG, and 0.031–15.9 μg mL−1 for AcMPAG.

Determination of Mycophenolic Acid and its Phenyl Glucuronide in Human Plasma, Ultrafiltrate, Blood, DBS and Dried Plasma Spots

Analysis of mycophenolic acid (MPA), the active form of the immunosuppressive drug mycophenolate mofetil, and its glucuronide metabolite MPAG is required for therapeutic monitoring and postmarketing clinical studies. Dried blood spots (DBS) and dried plasma spots (DPS) could be alternatives to conventional assays for small-volume sampling and easy shipment. A LC-MS/MS method with online SPE was established using stable isotope labeled analytes as internal standards. The quantitation limits were set at 0.1 and 1 µg/ml, for total MPA and MPAG, respectively, in plasma, blood, DBS and DPS, but 100-fold lower for free MPA in ultrafiltrate. Ahlstrom 226 or Whatman FTA(®) DMPK-B cards were well suited for DBS and DPS analyses. MPA and MPAG were analyzed in human plasma and blood either as liquid or dried on cards with similar assay quality. Care should be taken to avoid back-conversion of an instable acyl glucuronide metabolite to MPA.

Hepatic Clearance of Reactive Glucuronide Metabolites of Diclofenac in the Mouse Is Dependent on Multiple ATP-Binding Cassette Efflux Transporters

Diclofenac is an important analgesic and anti-inflammatory drug that is widely used for the treatment of postoperative pain, rheumatoid arthritis, and chronic pain associated with cancer. Diclofenac is extensively metabolized in the liver, and the main metabolites are hydroxylated and/or glucuronidated conjugates. We show here that loss of multidrug resistance protein 2 (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2) in mice results in highly increased plasma levels of diclofenac acyl glucuronide, after both oral and intravenous administration. The absence of Mrp2 and Bcrp1, localized at the canalicular membrane of hepatocytes, leads to impaired biliary excretion of acyl glucuronides and consequently to elevated liver and plasma levels. Mrp2 also mediates the biliary excretion of two hydroxylated diclofenac metabolites, 4'-hydroxydiclofenac and 5-hydroxydiclofenac. We further show that the sinusoidal efflux of diclofenac acyl glucuronide, from liver to blood, is largely dependent on multidrug resistance protein 3 (MRP3/ABCC3). Diclofenac acyl glucuronides are chemically instable and reactive, and in patients, these metabolites are associated with rare but serious idiosyncratic liver toxicity. This might explain why Mrp2/Mrp3/Bcrp1(-/-) mice, which have markedly elevated levels of diclofenac acyl glucuronides in their liver, display acute, albeit very mild, hepatotoxicity. We believe that the handling of diclofenac acyl glucuronides by ATP binding cassette transporters may be representative for the handling of acyl glucuronide metabolites of many other clinically relevant drugs.

Challenges in the indirect quantitation of acyl‐glucuronide metabolites of a cardiovascular drug from complex biological mixtures in the absence of reference standards

This paper describes the quantitation of acyl-glucuronide metabolites (M26 and M5) of a cardiovascular-drug (torcetrapib) from monkey urine, in the absence of their reference standards. LC/MS/MS assays for M1 and M4 (aglycones of M26 and M5, respectively) were characterized from normal and base-treated urine, as their respective reference standards were available. The in vivo study samples containing M26 and M5 were treated with 1 n sodium hydroxide to hydrolyze them to their respective aglycones. The study samples were assayed for M1 and M4 before and after alkaline hydrolysis and the difference in the concentrations provided an estimate of the urinary levels of M26 and M5. Prior to the main sample analysis, conditions for alkaline hydrolysis of the glucuronides were optimized by incubating pooled study samples. During incubations, a prolonged increase in M4 levels over time was observed, which is inconsistent with the base-hydrolysis of an acyl-glucuronide (expected to hydrolyze rapidly). Possible interference of the metabolite M9 (an ether-glucuronide metabolite isobaric to M4) was investigated to explain this observation using chromatographic and wet-chemistry approaches. The strategies adopted herein established that the LC/MS/MS assay and our approach were reliable. The metabolite exposure was then correlated to toxicological observations to gain initial insights into the physiological role of these metabolites.

The acyl glucuronide metabolite of mycophenolic acid induces tubulin polymerization in vitro

Objectives The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) forms covalent protein adducts and possesses antiproliferative properties independent of IMPDH inhibition. The underlying mechanism is unknown. Disorganized tubulin polymerization prevents cell cycle progression. We investigated whether AcMPAG interacts with tubulin polymerization. Design and methods AcMPAG (1.0–100 μM) was incubated with bovine tubulin in the presence of GTP. Polymerization was followed at 340 nm. The time until onset and the extent of polymerization were determined. MPA (100 μM), phenolic glucuronide MPAG (100 μM), and paclitaxel (10 μM) served as controls. Results MPAG was without effect. The AcMPAG effect on tubulin polymerization was dose dependent and significantly stronger (about 2.5-fold) than that of MPA ( n = 4; p < 0.05), but weaker than paclitaxel. Conclusions MPA and AcMPAG can induce tubulin polymerization in the presence of GTP with AcMPAG being significantly stronger. This property of AcMPAG may contribute to its IMPDH independent antiproliferative effect.

High-Field Asymmetric Waveform Ion Mobility Spectrometry for Determining the Location of In-Source Collision-Induced Dissociation in Electrospray Ionization Mass Spectrometry

The understanding and control of the in-source collision-induced dissociation (CID) of analytes is important for the accurate LC-MS/MS quantitation of drugs and metabolites in biological samples. Accordingly, it was of interest to us to establish whether such in-source CID takes place after and/or before the orifice of an electrospray ionization (ESI) mass spectrometer. A high-field asymmetric waveform ion mobility spectrometry (FAIMS) system that is physically located between the sprayer and the orifice of a mass spectrometer can serve as an ion filter to control ions entering the orifice of the mass spectrometer. In such a configuration, FAIMS could conceivably be used to determine if the in-source CID of an analyte occurs after and/or before the mass spectrometer orifice. We demonstrated this capability of FAIMS using ifetroban acylglucuronide metabolite as a model compound. Under the conditions used, the results showed that the in-source CID conversion of the acylglucuronide metabolite to its parent drug ifetroban occurred almost entirely after the orifice of the mass spectrometer, with the conversion upstream of the orifice accounting for only 5.6% of the conversion. Under the circumstance, the term "post-orifice CID" rather than "in-source CID" may be more appropriate in describing such a dissociation occurring in the front end of a mass spectrometer.

Population Pharmacokinetics of Mycophenolic Acid and Its 2 Glucuronidated Metabolites in Kidney Transplant Recipients

The population pharmacokinetics of mycophenolic acid (MPA) and its phenolic (MPAG) and acyl (AcMPAG) glucuronide metabolites were studied in patients taking enteric-coated mycophenolate sodium. Plasma samples (n = 232), obtained from 18 renal transplant recipients, were analyzed for MPA, MPAG, and AcMPAG using a validated high-performance liquid chromatography/ultraviolet assay. Population pharmacokinetic analysis was performed using NONMEM. The pharmacokinetics of MPA were best described by a 2-compartment model, with MPAG and AcMPAG produced from the central compartment and with enterohepatic recirculation of MPA via these 2 metabolites. Population mean estimates for MPA were apparent clearance (CL/F) of 10.6 L/h (interindividual variability [IIV] = 21.4%) and apparent volume of distribution of the central compartment (V(1)/F) of 25.9 L (IIV = 87.8%). Mean elimination rate constants of MPAG and AcMPAG were 0.323 h(-1) (IIV = 29.1%) and 0.206 h(-1) (IIV = 48.8%), respectively. The mean fraction of MPA converted to MPAG and AcMPAG, normalized by their volumes of distribution (FM(AG) and FM(AC), respectively), was also estimated. The elimination rate constant for MPAG and FM(AC) was influenced by glomerular filtration rate in patients with renal impairment. The visual predictive check, based on 100 simulated data sets each for MPA, MPAG, and AcMPAG, found that the final pharmacokinetic model adequately predicts the observed concentrations of all 3 species.

AcylMPAG Plasma Concentrations and Mycophenolic Acid-Related Side Effects in Patients Undergoing Renal Transplantation Are Not Related to the UGT2B7-840G&gt;A Gene Polymorphism

Mycophenolic acid (MPA) is metabolized primarily by glucuronidation to form the biologically inactive 7-O-glucuronide conjugate (MPAG), which is the major urinary excretion product. MPA is also converted to acyl-glucuronide metabolite (AcylMPAG), which has been suggested to be involved in the generation of MPA-related adverse events such as diarrhea or leucopenia. This conversion of MPA to AcylMPAG is catalyzed by UDP-glucuronosyltransferase 2B7 (UGT2B7). We studied the impact of the -840G>A polymorphisms in the UGT2B7 gene on the pharmacokinetics of AcylMPAG. We also investigated whether the plasma concentrations of AcylMPAG are correlated with MPA-related toxicity to further evaluate its potential clinical significance. In a randomized, controlled trial, comparing fixed-dose mycophenolate mofetil (MMF) with concentration-controlled MMF therapy, patients undergoing renal transplantation were treated with a calcineurin inhibitor, MMF, and corticosteroids. Informed consent was obtained from 332 patients for genotyping. In all patients, blood samples were drawn (three samples within the first 2 hours after administration) on Day 3, Day 10, Week 4, and Months 3, 6, and 12 to measure MPA and AcylMPAG plasma concentrations. The pharmacokinetics of AcylMPAG were correlated with the -840G>A single nucleotide polymorphism (SNP) in the UGT2B7 gene. Heterozygosity for the -840G>A SNP in the UGT2B7 gene was found in 145 patients (145 of 332 [44%]) and 93 (93 of 332 [28%]) patients were homozygous for the -840G>A allele. No difference was found in the dose-normalized AcylMPAG trough (C0) levels and dose-normalized AcylMPAG areas under the concentration-time curve (AUCs) at each visit between carriers and noncarriers of the -840G>A SNP. Also, metabolic ratios, expressed as AcylMPAG/MPA and AcylMPAG/MPAG, were not related to UGT2B7 genotype. The dose-normalized AcylMPAG-C0 and AcylMPAG AUC were higher in the cyclosporine-treated group compared with the tacrolimus-treated patients at each visit. There was no difference in AcylMPAG concentrations (trough or AUC) or AcylMPAG/MPAG ratio between patients with compared with patients without diarrhea. None of the -840G>A UGT2B7 SNPs was disproportionately present among the patients with diarrhea. There was a higher incidence of diarrhea in tacrolimus-treated patients [26 of 163 (16.0%)] compared with cyclosporine-treated individuals [five of 51 (9.8%)], although AcylMPAG concentrations were lower in tacrolimus-treated patients. In this study, we have found no influence of the -840G>A UGT2B7 SNP on AcylMPAG exposure in patients undergoing renal transplantation. There also was no association between this variant genotype and the incidence of diarrhea or leucopenia, two adverse events for which a role for AcylMPAG has been suggested.

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.

Pharmacokinetics and metabolism of KW-4490, a selective phosphodiesterase 4 inhibitor: Difference in excretion of KW-4490 and acylglucuronide metabolites between rats and cynomolgus monkeys

1. The pharmacokinetics and metabolism of KW-4490, a selective phosphodiesterase 4 inhibitor, were investigated in rats and monkeys. After oral administration, KW-4490 was rapidly absorbed, and then its plasma concentrations apparently declined with half-lives of approximately 5 h in rats and 3.5 h in cynomolgus monkeys; however, a number of secondary peaks were apparent in the profiles for both species. The plasma pharmacokinetics of KW-4490 were comparable between rats and monkeys.2. After oral administration, KW-4490 was mainly eliminated by metabolism to acylglucuronides and renal excretion in the unchanged form. KW-4490 acylglucuronides were found in monkey but not rat urine. In rats, KW-4490 acylglucuronides were excreted only in bile. Although the pathway of excretion of acylglucuronides differed between rats and monkeys, cumulative excretion in the two animals was very similar, as expected from comparable hepatic clearance for glucuronidation in rat and monkey liver microsomes.3. The glomerular filtration rate of unbound KW-4490 indicated that renal tubular secretion was significant in monkeys, whereas reabsorption was significant in rats. These species differences in urinary excretion of KW-4490 and its acylglucuronide metabolites are most likely due to substrate specificity of active transporters in rat and monkey kidney.

Determination of a novel gamma-secretase inhibitor in human plasma and cerebrospinal fluid using automated 96 well solid phase extraction and liquid chromatography/tandem mass spectrometry

A method for determination of a gamma-secretase inhibitor, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]propanoic acid ( A), in human plasma and cerebrospinal fluid (CSF) has been developed to support the clinical investigation of compound A for its potential treatment of Alzheimer's disease. The method is based on HPLC with atmospheric pressure chemical ionization tandem mass spectrometric detection (APCI-MS/MS) in the negative ionization mode using a heated nebulizer interface. The addition of phosphoric acid at the ratio of 10–30 μL per milliliter of human plasma or CSF was required during clinical sample collection to stabilize an acylglucuronide metabolite ( C), which was potentially present in human plasma and CSF. Tween 20 (10% solution) was added at the ratio of 20 μL per milliliter of CSF during CSF sample collection to prevent the loss of compound A during the storage of clinical samples. The compound A and its analog internal standard ( B) in treated plasma or CSF were isolated from human plasma or CSF using solid phase extraction (SPE) in the 96 well format. The isolated analyte and internal standard were chromatographed on a Phenomenex Synergi ® Polar RP analytical column (50 mm × 3.0 mm, 4 μm), using a mobile phase consisting of 60/40 (v/v, %) acetonitrile/water at a flow-rate of 0.5 mL/min. Tandem mass spectrometric detection was performed using a Sciex API 3000 tandem mass spectrometer operated in the multiple reaction monitoring (MRM) mode using precursor to product ion transitions of 441 → 175 for A and 469 → 175 for B, respectively. The assays were validated over the concentration range of 0.5–200 ng/mL for human plasma and CSF. Replicate analyses ( n = 5) of spiked standards for both assays yielded a linear response with coefficients of variation less than 7% and accuracy within 5% of the nominal concentrations. In addition, the assays were automated to improve sample throughput by utilizing a Packard Multi PROBEII automated liquid handling system and a Tom-Tec Quadra 96 system. Numerous clinical studies have been supported using these assays.

Simultaneous determination of mycophenolic acid and its acyl and phenol glucuronide metabolites in human serum by capillary zone electrophoresis

A simple capillary electrophoretic method was developed for simultaneous determination of mycophenolic acid (MPA) and its metabolites, acyl glucuronide (AcMPAG) and phenol glucuronide (MPAG), in human serum. The method utilized only running buffer in the separation, prior acidification of serum, and extraction with ethyl acetate. Separation was performed by capillary zone electrophoresis using 20 mM sodium acetate–acetic acid (pH 4.9) as running buffer, applied voltage of 15 kV, and UV detection at 217 nm. Each electrophoretic run was completed within 14 min. The optimized method demonstrated good performance concerning specificity, linearity ( r > 0.998), sensitivity (limit of detection: for MPA, 0.10 μg/ml; for AcMPAG, 0.10 μg/ml; MPAG, 0.42 μg/ml), accuracy (87–108%) and precision (<9.3%). This method was successfully applied to measurements of MPA, AcMPAG, and MPAG in renal transplant patient samples and could be a useful alternative to HPLC-based methods.

Stability of mycophenolic acid and glucuronide metabolites in human plasma and the impact of deproteinization methodology

In recent years there is growing interest in therapeutic drug monitoring of mycophenolic acid (MPA) and its glucuronide metabolites MPAG and AcMPAG. Like other acyl glucuronide metabolites, AcMPAG has a limited stability, but this aspect has received little attention. Plasma sample deproteinization with perchloric acid 2 M (method A) was compared to metaphosphoric acid 15% (method B). Stability of MPA, MPAG and AcMPAG in acidified and non-acidified plasma stored at room temperature, 4 degrees C, -20 degrees C and -80 degrees C was assessed over short and long time intervals using HPLC-UV methodology. The area ratio of AcMPAG/IS on spiked plasma at pH 2.5 with method A was 63% of the respective ratio in water, in contrast to 102% with method B, suggesting partial deconjugation and/or incomplete release of AcMPAG from proteins with method A. At room temperature, AcMPAG concentrations in both whole blood and non-acidified plasma decreased significantly after 2-5 h. MPA, MPAG and AcMPAG concentrations remained stable in acidified plasma stored at -20 degrees C and -80 degrees C, but not longer than 5 months after collection. It is concluded that adequate sample collection, storage measures and deproteinization methods should be applied in order to avoid deconjugation and hence underestimation of MPA, MPAG and AcMPAG concentrations.

Cyclization of the Acyl Glucuronide Metabolite of a Neutral Endopeptidase Inhibitor to an Electrophilic Glutarimide: Synthesis, Reactivity, and Mechanistic Analysis

The neutral endopeptidase inhibitor (2R)-2-[(1-{[(5-ethyl-1,3,4-thiadiazol-2-yl)amino]carbonyl}cyclopentyl)methyl]pentanoic acid 2 is metabolized to acyl glucuronide 3. Unprecedentedly, at pH 7.4, 3 does not undergo the O-acyl migration characteristic of acyl glucuronides but rapid, eliminative cyclization (t1/2 at 37 degrees C, 10.2 min) to glutarimide 4. Glucuronide 3 was synthesized efficiently via acylation of benzylglucuronate with N-benzyloxymethyl-protected 2. Glucuronide and imide reacted rapidly in aqueous solution, pH 7.4, with amino acids and glutathione to form stable amides and unstable thioesters. Imide 4 acylated eight lysine Nepsilon-amino groups of human serum albumin. Rapid cyclization of 3 was attributed to attack on the ester linkage by an unusually nucleophilic glutaramide NH (pKa in 2 = 9.76). N-propyl 3 was refractory to acyl migration and cyclization. This suggested a synthetic strategy for preparing analogues of 2 that form chemically stable acyl glucuronides.

Carboxylic Acid Drug-Induced DNA Nicking in HEK293 Cells Expressing Human UDP-Glucuronosyltransferases: Role of Acyl Glucuronide Metabolites and Glycation Pathways

Glucuronidation of carboxylic-acid-containing drugs can yield reactive acyl (ester-linked) glucuronide metabolites that are able to modify endogenous macromolecules. Previous research has shown that several carboxylic acid drugs are genotoxic in isolated mouse hepatocytes, and that DNA damage is prevented by the glucuronidation inhibitor, borneol. Whether these species induce comparable genetic damage in human cells is unknown. In this study, we investigated the mechanisms of clofibric acid-induced genotoxicity in HEK293 cells expressing the human UDP-glucuronosyltransferases UGT1A3, UGT1A9, or UGT2B7, and screened three other carboxylic acid drugs for UGT-dependent genotoxicity. DNA damage was detected using the alkaline version of the comet assay. HEK293 cells were incubated for 18 h with vehicle (2.5 mM NaOH), 0.1-2.5 mM clofibric acid or 0.1-1.0 mM benoxaprofen, bezafibrate, or probenecid. To identify mechanisms underlying any observed genotoxicity, we treated UGT2B7 transfectants with 10 mM aminoguanidine, 1 mM borneol, or 2 mM desferrioxamine mesylate prior to co-incubation with 1 mM clofibric acid for 18 h. Compared to vehicle, clofibric acid, benoxaprofen, and probenecid produced significant DNA damage in all three UGT-transfected HEK293 cell lines, detectable from the lowest concentration tested. Bezafibrate caused DNA damage only at higher concentrations (1.0 mM) in UGT2B7- and UGT1A9-, but not UGT1A3-transfected cells. No drug-induced DNA damage was detected in untransfected cells, consistent with the limited glucuronidation capacity of these cells. The glycation/glycoxidation inhibitor aminoguanidine and the glucuronidation inhibitor borneol significantly decreased clofibric-acid-mediated DNA damage in UGT2B7 transfected cells by 73.5 and 94.8%, respectively. The inhibitor of transition-metal-catalyzed oxidation, desferrioxamine mesylate, had no significant effect on DNA damage. This study demonstrates the substrate-dependent role of human UGTs in the bioactivation of carboxylic acid drugs to genotoxic acyl glucuronide metabolites that are able to damage nuclear DNA via glycation and/or glycoxidation mechanisms.

Influence of Renal Insufficiency on Pharmacokinetics of ACYL-Glucuronide Metabolite of Mycophenolic Acid in Renal Transplant Patients

The acyl glucuronide of mycophenolic acid (AcMPAG) is a metabolite with in vitro immunosuppressive activity. The chemical properties of acyl glucuronides have been associated with the toxicity of some drugs. The aim of our study was to analyze the influence of renal insufficiency on the pharmacokinetics of AcMPAG. Areas under the 12-hour curve (AUC 0–12h) of MPA, glucuronide of MPA (MPAG), and AcMPAG were determined by high performance liquid chromatography performed in 20 renal transplantation patients under treatment with mycophenolate mofetil (MMF), cyclosporine, and steroids. They were divided between a group with preserved renal function (group I, mean creatinine clearance [Clcr] of 105 ± 7 mL/min) and one with advanced renal insufficiency (group II, mean Clcr of 27 ± 5 mL/min). There was no difference in MMF dose or MPA-AUC 0–12h values between groups. Mean predose levels of AcMPAG-C 0 and AcMPAG-AUC 0–12h were much higher in group II than in group I (0.5 ± 2 vs 1.6 ± 1 μg/mL and 12 ± 2 vs. 32 ± 19 μg*h/mL respectively, P < .005). The present data suggested that AcMPAG, a metabolite with immunosuppressive activity that may be related to toxic effects of MPA, is renally eliminated. Its levels can significantly rise in patients with renal insufficiency. Although further studies with more patients are required to determine the role of AcMPAG in MPA toxicity, we believe that this accumulation may be of clinical relevance.

Plasma Concentrations of Mycophenolic Acid Acyl Glucuronide Are Not Associated with Diarrhea in Renal Transplant Recipients

The aim of this study was to determine whether plasma concentrations of the acyl (AcMPAG) and phenolic (MPAG) glucuronide metabolites of mycophenolic acid (MPA) were related to diarrhoea in renal transplant patients on mycophenolate mofetil (MMF) with cyclosporine (CsA) or tacrolimus (TCL). Blood samples (0, 30, 120 min) were taken at days 3, 10, week 4, months 3, 6 and 12 for determination of MPA, MPAG and AcMPAG. MPA-AUC was estimated using validated algorithms. Two hour AUCs were calculated for MPAG and AcMPAG. Immunosuppressive therapy consisted of CsA/MMF (n= 110) and of TCL/MMF (n= 180). In 70/290 (24%) patients 86 episodes of diarrhoea were recorded during 12 months. Significantly more patients on TCL (31.1%) suffered from diarrhea compared to CsA (12.7%). MMF dose, MPA-AUC and the 2 h AUCs of MPAG and AcMPAG did not differ between patients with and without diarrhoea. Plasma AcMPAG and MPAG concentrations were substantially higher in patients on CsA compared with TCL, while MPA-AUC was lower in the former group. These data support the concept that CsA inhibits the biliary excretion of MPAG and AcMPAG, thereby potentially reducing the risk of intestinal injury through enterohepatic recycling of MPA and its metabolites.

Concentrations of Mycophenolic Acid and Glucuronide Metabolites Under Concomitant Therapy With Cyclosporine or Tacrolimus

Mycophenolate mofetil [MMF, the prodrug of mycophenolic acid (MPA)] is usually administered at double doses with cyclosporine than with tacrolimus because it is believed that MPA exposure is lower during cyclosporine therapy. This study aimed to compare 12 hour, steady-state concentration-time profiles of MPA and its phenol- and acyl-glucuronide metabolites (MPAG and AcMPAG, respectively) in stable kidney transplant recipients maintained either on cyclosporine (n = 12) or tacrolimus (n = 12). During the absorption phase in the cyclosporine group, dose-normalized concentrations of total and free MPA were significantly higher but the overall area under the concentration-time curve (AUC0-12) was not significantly different. Additionally, exposure to AcMPAG was higher in the cyclosporine group (P < 0.05). Ten of 12 patients in the cyclosporine group were on ketoconazole therapy; however, the exposure to MPA or MPAG was not different when MMF was given orally to Sprague-Dawley rats with or without ketoconazole. In conclusion, cyclosporine modulates the disposition of MPA and metabolites differently from tacrolimus; however, patients on cyclosporine may not require double doses of MMF to achieve the same exposure.

Proteins identified as targets of the acyl glucuronide metabolite of mycophenolic acid in kidney tissue from mycophenolate mofetil treated rats

Covalent binding of the acyl glucuronide (AcMPAG) metabolite of the immunosuppressant mycophenolic acid (MPA) to proteins is considered a possible initiating event for organ toxicity. Since the kidney is involved in the formation and excretion of AcMPAG, it can be hypothesized that this tissue may be exposed to relatively high concentrations of this metabolite and would, therefore, be a particularly suitable organ to investigate AcMPAG protein targets. In the present study we identified potential AcMPAG target proteins in kidney tissues from Wistar rats treated with mycophenolate mofetil (40 mg/kg/day over 21 days). Proteins were separated by 2-DE and covalent protein adducts were detected by Western blotting with an antibody specific for MPA/AcMPAG. The corresponding coomassie blue stained proteins from parallel gels were subjected to in-gel tryptic digestion and peptides were characterized on a Q-TOF Ultima Global. The protein targets were further verified by immunoprecipitation with anti-MPA/AcMPAG antibody to purify the modified proteins followed by 1-DE and MS analysis. Database searches revealed several AcMPAG target proteins that could be related to ultrastructural abnormalities, metabolic effects, and altered oxidative stress/detoxification responses. Predominately cytosolic proteins such as selenium binding protein, protein disulfide isomerase, aldehyde dehydrogenase, triosephosphate isomerase, and kidney aminoacylase were involved in adduct formation. Two cytoskeletal proteins tropomyosin 1 and 4 as well as the antioxidant proteins peroxiredoxin 3 and 6 were also targets of AcMPAG. Functional consequences from these protein modifications remain to be demonstrated.