Raymond G. Morris

Therapeutic monitoring of mycophenolic acid: A consensus panel report

Biochem 1998;5:317–22. 4. Oellerich M, Shipkova M, Schütz E, Weber L, Tönshoff B, Armstrong VW, et al. Pharmacokinetic and metabolic investigations of mycophenolic acid in pediatric patients after renal transplantation: implications for therapeutic drug monitoring. Ther Drug Monit 2000;22:20–6. 5. Pescovitz MD, Conti D, Dunn J, Gonwa T, Halloran P, Sollinger H, et al. Intravenous mycophenolate mofetil: safety, tolerability, and pharmacokinetics. Clin Transplant 2000:14;179–88. 6. Tsina I, Kaloostian M, Lee R, Tarnowski T, Wong B. High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma. J Chromatogr B 1996;681:347–53. 7. McBride JH, Kim S, Reyes A, Rodgerson DO. Measurement of plasma mycophenolic acid in pediatric renal transplant recipients. Clin Chem 1998;44(Suppl 6):A93. 8. Shipkova M, Niedmann PD, Armstrong VW, Schütz E, Wieland E, Oellerich M. Simultaneous determination of mycophenolic acid and its glucuronide in human plasma using a simple high-performance liquid chromatographic procedure. Clin Chem 1998;44:1481–8. 9. Shipkova M, Schütz E, Armstrong VW, Niedmann PD, Oellerich M, Wieland, E. Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit. Clin Chem 2000;46:365–72. 10. Stamm D. A new concept for quality control of clinical laboratory investigations in the light of clinical requirements and based on reference method values. J Clin Chem Clin Biochem 1982;20:817–24. 11. Hyneck ML, Munafo A, Benet LZ. Effect of pH on acyl migration and hydrolysis of tolmetin glucuronide. Drug Metab Dispos 1988;16:322–4.

Role of MRP2 in the hepatic disposition of mycophenolic acid and its glucuronide metabolites : Effect of cyclosporine

Mycophenolic acid (MPA) is part of the immunosuppressant therapy for transplant recipients. This study examines the role of the canalicular transporter, Mrp2, and the effect of cyclosporin A (CsA), on the biliary secretion of the ether (MPAGe) and acyl (MPAGa) glucuronides of MPA. Isolated livers from Wistar rats (n = 6), or Wistar TR– rats (n = 6) were perfused with MPA (5 mg/l). A third group of Wistar rats (n = 6) was perfused with MPA and CsA (250 μg/l). There was no difference in the half-life, hepatic extraction ratio (EH), clearance or partial clearance of MPA to MPAGe, but there was a difference in partial clearance to MPAGa between control and CsA groups (0.9 ± 0.4 versus 0.5 ± 0.1 ml/min). TR– rats had a lower EH (0.59 ± 0.30 versus 0.95 ± 0.30), a lower clearance (18 ± 8 versus 29 ± 7 ml/min), and a longer half-life (19.5 ± 10.3 versus 10.1 ± 2.4 min) than controls. Compared to controls, MPAGe and MPAGa biliary excretion was reduced by 99% and 71.8%, respectively, in TR– rats, and 17.5% and 53.8%, respectively, in the MPA-CsA group. The biliary excretion of MPAGe is mediated by Mrp2, whereas that of MPAGa seems to depend on both Mrp2 and another unidentified canalicular transporter. Although CsA can inhibit Mrp2, our data suggest that it may also inhibit the hepatic glucuronidation of MPA in Wistar rats.

Comparison of trough, 2-hour, and limited AUC blood sampling for monitoring cyclosporin (Neoral) at day 7 post-renal transplantation and incidence of rejection in the first month.

The use of alternative strategies to the traditional pre-dose/trough (C0) blood sampling for cyclosporine (CsA) therapeutic drug monitoring has the potential to revolutionize analytical practices which have, in many centers, been established for some 20 years. While the C0 sample has previously been recommended, current attitudes are increasingly proposing alternatives for assessing CsA exposure, including various limited sampling strategies of the AUC (lssAUC) in the early postdose period, or alternative single-point nontrough samples, such as a 2-hour postdose sample (C2). The present study has reviewed a series of consecutive renal transplant recipients over 18 months where CsA was the primary immunosuppressant. The lssAUC performed at around day 7 posttransplantation included drawing blood at 0, 2, and 4 hours postdose, giving AUC(0–4). The aim of this study was to review the occurrence of acute biopsy-proven rejection in the first month and consider which of (simultaneously measured) C0, C2 or AUC(0–4) was a better early indicator of this adverse outcome. The result was best described by comparing the data from rejectors (n = 13) and nonrejectors (n = 42) for these 3 indices of CsA exposure (i.e., C0, C2 or AUC(0–4)). There was no evidence that C0 predicted the likelihood of such adverse clinical outcomes. In contrast, rejectors tended to have lower mean C2 CsA concentrations, and the incidence of rejection was 0.0 when C2 exceeded 1200 &mgr;g/L (n = 10). While the data are limited in the higher C2 CsA concentration range, it is nevertheless consistent with more recent recommendations suggesting that the CsA at C2 should target 1700 &mgr;g/L in this first month posttransplantation. As 64% of the patients were also receiving a CsA-sparing agent (diltiazem [DTZ]), the relationships were also investigated to determine whether any affect of concomitant DTZ therapy could be demonstrated. However, in this small sample, no significant affect of DTZ was seen.

International Federation of Clinical Chemistry/International Association of Therapeutic Drug Monitoring and Clinical Toxicology working group on immunosuppressive drug monitoring.

Issues surrounding the measurement and interpretation of immunosuppressive drug concentrations have been summarized in a number of consensus documents. The Scientific Division of the International Federation of Clinical Chemistry has formed a working group in collaboration with the International Association of Therapeutic Drug Monitoring and Clinical Toxicology. This paper sets out the goals of the working group in light of the developments that have occurred in the field of immunosuppressive drug monitoring since the publication of the last consensus documents.

Pharmacokinetic interaction between tacrolimus and diltiazem: dose-response relationship in kidney and liver transplant recipients.

ObjectiveTo study the dose-response relationship of the pharmacokinetic interaction between diltiazem and tacrolimus in kidney and liver transplant recipients.DesignNonrandomised seven-period stepwise pharmacokinetic study.PatientsStable kidney (n = 2) and liver (n = 2) transplant recipients maintained on oral tacrolimus twice daily but not taking diltiazem.MethodsPatients were treated with seven incremental dosages of diltiazem (0 to 180 mg/day) at ≥ 2-weekly intervals. At the end of each interval, 13 blood samples were taken over a 24-hour period to allow determination of morning (AUC12), evening (AUC12–24) and 24-hour (AUC24) areas under the concentration-time curve for tacrolimus, as well as AUC24 for diltiazem and three of its metabolites.ResultsThere was considerable interpatient variability in tacrolimus-sparing effect. In the two kidney transplant recipients, an increase in tacrolimus AUC24 occurred following the 20 mg/day dosage of diltiazem (26 and 67%). The maximum increase in tacrolimus AUC24 occurred at the maximum diltiazem dosage used (180 mg/day), when the increase was 48 and 177%. In the two liver transplant recipients, an increase in tacrolimus AUC24 did not occur until a higher diltiazem dosage (60 to 120 mg/day) was given. The increase at the maximum diltiazem dosages used (120 mg/day in one and 180 mg/day in the other) was also lower (18 and 22%) than that exhibited by the kidney transplant recipients. The increase in tacrolimus AUC12 was similar to the increase in AUC12–24 when diltiazem was given in the morning only (dosages ≤60 mg/day). Hence, diltiazem affects blood tacrolimus concentrations for longer than would be predicted from the half-life of diltiazem in plasma.ConclusionsThe mean tacrolimus-sparing effect of diltiazem was similar in magnitude to the cyclosporin-sparing effect previously reported. Whether the lesser tacrolimus-sparing effect with diltiazem seen in the liver transplant recipients was due to functional differences in the transplanted liver is not known, but it was not due to lower plasma diltiazem concentrations. Diltiazem makes a logical tacrolimus-sparing agent because of the potential financial savings and therapeutic benefits. Because of interpatient variability, the sparing effect should be demonstrated in each patient and not merely assumed.

Comparison of blood sirolimus, tacrolimus and everolimus concentrations measured by LC-MS/MS, HPLC-UV and immunoassay methods

OBJECTIVES An LC-MS/MS method was developed for simultaneous quantitation of tacrolimus, sirolimus and everolimus in whole blood, and compared to HPLC-UV and immunoassay methods. DESIGN AND METHODS Blood (0.1mL) was analysed following solid-phase extraction and chromatographic resolution using a C18 column (45°C) and mobile phase of methanol/40mM ammonium acetate/glacial acetic acid (83/17/0.1) at 200μL/min, with positive electrospray ionisation and multiple reaction monitoring. RESULTS Intra- and inter-day imprecision and inaccuracy were ≤12.2% over a 1.5-40μg/L calibration range. An external quality assurance programme confirmed acceptable inaccuracy and imprecision of the LC-MS/MS method, but highlighted problems with immunoassay quantitation, particularly for everolimus, showing a >30% bias in FPIA everolimus concentrations measured in pooled patient samples versus spiked drug-free whole blood. CONCLUSIONS LC-MS/MS provides significant accuracy and precision advantages compared to HPLC and immunoassays. Discrepancies in everolimus concentrations measured by the Seradyn FPIA immunoassay require further investigation.

Therapeutic Drug Monitoring of Everolimus: A Consensus Report

Abstract: In 2014, the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology called a meeting of international experts to provide recommendations to guide therapeutic drug monitoring (TDM) of everolimus (EVR) and its optimal use in clinical practice. EVR is a potent inhibitor of the mammalian target of rapamycin, approved for the prevention of organ transplant rejection and for the treatment of various types of cancer and tuberous sclerosis complex. EVR fulfills the prerequisites for TDM, having a narrow therapeutic range, high interindividual pharmacokinetic variability, and established drug exposure–response relationships. EVR trough concentrations (C0) demonstrate a good relationship with overall exposure, providing a simple and reliable index for TDM. Whole-blood samples should be used for measurement of EVR C0, and sampling times should be standardized to occur within 1 hour before the next dose, which should be taken at the same time everyday and preferably without food. In transplantation settings, EVR should be generally targeted to a C0 of 3–8 ng/mL when used in combination with other immunosuppressive drugs (calcineurin inhibitors and glucocorticoids); in calcineurin inhibitor-free regimens, the EVR target C0 range should be 6–10 ng/mL. Further studies are required to determine the clinical utility of TDM in nontransplantation settings. The choice of analytical method and differences between methods should be carefully considered when determining EVR concentrations, and when comparing and interpreting clinical trial outcomes. At present, a fully validated liquid chromatography tandem mass spectrometry assay is the preferred method for determination of EVR C0, with a lower limit of quantification close to 1 ng/mL. Use of certified commercially available whole-blood calibrators to avoid calibration bias and participation in external proficiency-testing programs to allow continuous cross-validation and proof of analytical quality are highly recommended. Development of alternative assays to facilitate on-site measurement of EVR C0 is encouraged.

Cyclosporin A Monitoring in Australia: Consensus Recommendations

Cyclosporin-A (CsA) therapeutic drug monitoring plays an integral role in therapeutic management of immunosuppressed patients, including those with organ transplants. This communication, prepared by an Australian team, presents recommendations for the routine monitoring of CsA, including blood sampling methodological approaches and interpretation of results generated. The consensus approach described is intended to address the diversity of attitudes to CsA monitoring demonstrated in a recent national survey of Australian laboratories that provide CsA analyses.

The effect of food on oral melphalan absorption.

SummaryFifteen patients receiving oral melphalan (4.2–5.3 mg/m2) for a variety of neoplastic disorders were studied. Ten patients received the drug on separate occasions, with and without a standardized breakfast. Eight of these patients also received an IV bolus dose (5 mg/m2) to determine bioavailability. Serial melphalan plasma samples were taken over 5 h after administration and assayed by high-performance liquid chromatography. The median area under the curve (AUC) when taken fasting was 179 (range 95–336) ng · h · ml-1, and when taken with food, 122 (47–227) ng · h · ml-1, the median reduction being 39% (P0.01). In one patient, who died before completing the study, the drug was not detectable at all after being taken with food. In the eight patients who were also given IV melphalan, the median terminal melphalan half-life (57 min, range 38–71) was no different from its oral half-life [55 (27–104) min fasting; 55 (30–72) min with food] (P>0.1). In these patients bioavailability was 85% (26–96)% when the drug was taken fasting and 58% (7–99)% when taken with food (P0.025). Median clearance following IV administration was 362 ml/min/m2 (range 104–694). It was found that the melphalan level in a single plasma sample drawn 1.5 h after administration was highly predictive of oral melphalan AUC (rs=0.915, P0.1). This study suggests that to ensure optimum absorption of the drug, melphalan should not be taken with food.

High-performance liquid chromatography quantitation of plasma lamotrigine concentrations: application measuring trough concentrations in patients with epilepsy.

Lamotrigine is a phenyltriazine anticonvulsant recently approved for clinical use. A high-performance liquid chromatographic (HPLC) method was developed using a silica column (5 microm) with an aqueous methanol mobile phase consisting of 94% methanol, 5.92% water, and 0.08% NH4H2PO4 adjusted to a final apparent pH of 4.0 and pumped at a flow rate of 1.0 ml/minute. Ultraviolet detection was carried out at a wavelength of 280 nm, and plasma samples were prepared for HPLC analysis by extraction into ethyl acetate after basification. Retention times for lamotrigine and its internal standard (BWA725C) were 10.3 and 11.2 minutes, respectively, and there was no chromatographic interference from other commonly coadministered anticonvulsants. Calibration curves were linear over a concentration range of 0.5 to 30 mg/l, with intra-assay and interassay coefficients of variation less than 8%. Assessment of assay performance in an international quality assurance program showed an average bias of 0.3% compared with the consensus mean. A review of 52 patient specimens showed that, if patients were grouped according to coadministered anticonvulsants, a significant correlation between lamotrigine dosage and concentration was evident in those coadministered valproate (in the absence of metabolic inducers) and in those coadministered a combination of valproate and inducers, but not in patients coadministered inducers alone. Mean (SD) trough concentrations were 9.2 (5.2), 2.8 (1.3), and 3.8 (2.8) mg/l in the valproate, inducer, and combination groups, respectively.