Nigel W. Brown

Monitoring mycophenolate in liver transplant recipients: Toward a therapeutic range

Predose plasma mycophenolic acid (MPA) concentrations measured with a semi‐automated enzyme‐multiplied immunoassay were related to adverse events (e.g., rejection, leukopenia, infection), drug dose, and clinical status in 147 adult and 63 pediatric liver allograft recipients receiving adjunctive immunosuppression with mycophenolate mofetil (MMF). In 12 of 13 acute rejection episodes, predose MPA levels were below the 1 mg/L cut‐off defined using receiver operating characteristic (ROC) curve analysis. The relative risk of developing infection or leukopenia increased more than 3‐fold above predose MPA levels of 3 to 4 mg/L. Plasma MPA levels correlated weakly (r2 = 0.081) with MMF dose and the dose / level relationship was variably influenced by age, the indication for MMF, concentrations of serum albumin and creatinine, and comedication with tacrolimus or cyclosporine. The median mycophenolate dose required per unit mycophenolate level was 50% lower in children than in adults. Comparable drug requirements were also decreased by renal dysfunction (by 40 and 43% in adults and children, respectively), and in patients prescribed MMF alone rather than with tacrolimus or cyclosporine. However, in patients with serum albumin less than 35g/L, MMF dose requirements were higher than in those with normal albumin levels (by 2.1‐ and 2.6‐fold in adults and children, respectively). In adults, 44.7% achieved clinically acceptable therapeutic MPA concentrations at a dose less than 1 g MMF twice daily and only 6.3% required 1.5 g twice daily as suggested by the manufacturer. The immunoassay was a rapid, reliable, and acceptably precise technique in which only 10.8% of measurements were unproductive. In conclusion, our data suggests that MPA predose level monitoring is both clinically‐ and cost‐effective and that a therapeutic range of 1 to 3.5mg/L (by immunoassay) is applicable in liver allograft recipients given adjunctive MMF. (Liver Transpl 2004;10:492–502.)

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 drugs major phenolic glucuronide metabolite (MPAG), cyclosporin (CsA), or tacrolimus (Tac) co-medication and liver and renal function. Trough concentrations (C0) most reliably predicted the area under the curve (AUC) of 0–7 hours MPA plasma concentrations (r2 = 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 C0 (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 C0 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.

Immunosuppression in pediatric solid organ transplantation: Opportunities, risks, and management

Abstract:  The pediatric transplant community stands at a time of unprecedented choice of immunosuppressive agents – and with a legacy of morbidity from those agents used in the previous two decades. This review considers the clinical utility and side‐effect profiles of immunosuppressants used widely in current practice (e.g., glucocorticoids, azathioprine, ciclosporin, tacrolimus, mycophenolate, and sirolimus) and those agents which are in increasing use or in evaluation (e.g., IL‐2 receptor antibodies, everolimus, FTY720, LEA29Y, and deoxyspergualin). Further consideration is given to the wider drug interactions likely during the use of new immunosuppressant regimens and to our growing awareness of the influences of genetic heterogeneity on drug efficacy and handling. Finally, we consider the new demands being placed on the use of drug monitoring to regulate dosage of this new repertoire of immunosuppressants.

Evidence for long-term pancreatic damage caused by laxative abuse in subjects recovered from anorexia nervosa.

OBJECTIVE This study examined whether a prior history of laxative abuse results in long-term changes in gastrointestinal function. METHOD The functioning of the enteroinsular axis was examined by measuring the insulin response to a standard meal. The study involved 18 subjects who had fully recovered from anorexia nervosa (AN) and an age and weight-matched control group. In the recovered group, 10 of 18 subjects had a history of laxative abuse. RESULTS Subjects with a prior history of laxative abuse show a more gradual increase and decrease in insulin secretion, but no differences in glucose response or hunger ratings. DISCUSSION Because there are no differences in the glucose response to the meal, it is hypothesized that the difference in insulin response is due to changes in the enteroinsular axis. These data indicate that chronic laxative abuse induces long-term changes in gastrointestinal function.

A direct method for the measurement of everolimus and sirolimus in whole blood by LC-MS/MS using an isotopic everolimus internal standard.

Background: Whole-blood concentrations of the immunosuppressant drugs everolimus and sirolimus should be monitored. A sensitive and selective method offering the detection of both analytes in small sample volumes would optimize the throughput of samples for sirolimus or everolimus analysis. This study reports the validation of a liquid chromatography tandem mass spectrometry method, including a stable isotope internal standard, for the simultaneous measurement of everolimus and sirolimus. Methods: Whole-blood samples (20 &mgr;L) were treated with ammonium bicarbonate (20 &mgr;L), zinc sulfate (20 &mgr;L), and internal standard solution (13C2D4-everolimus in acetonitrile, 100 &mgr;L). After centrifugation, 20 &mgr;L of the supernatant was injected onto a Waters Symmetry C18 high-performance liquid chromatography column. The aqueous and organic mobile phases were 2 mmole/L of ammonium acetate containing 0.1% (vol/vol) formic acid, in water and methanol, respectively. Analytes were detected using tandem mass spectrometry (Waters Acquity TQD). Results: Analytes were eluted at around 2 minutes within a 6-minute analytical run time. Detector response was linear for both analytes across the ranges studied (1–49 &mgr;g/L for sirolimus, 1–41 &mgr;g/L for everolimus), and a lower limit of quantitation of 1 &mgr;g/L was reliably attained. Intraassay and interassay imprecision and inaccuracy were <15% (coefficient of variation) in all cases. Analyte recovery was in the range of 72%–117%. The analytes were stable in blood after freezing and thawing, and for at least 12 hours after preparation while waiting to be injected. Ion suppression and interference from phospholipids were not significant. Conclusions: A straightforward, robust liquid chromatography tandem mass spectrometry assay has been developed and validated for the simultaneous measurement of everolimus and sirolimus in small volumes of whole blood.

An Investigation Into the Bias Between Liquid Chromatography-Tandem Mass Spectrometry and an Enzyme Multiplied Immunoassay Technique for the Measurement of Mycophenolic Acid

Mycophenolic acid is now the second most widely used immunosuppressant in solid organ transplantation. Overestimation of mycophenolic acid concentration is a recognized problem of immunoassay, and high-performance liquid chromatography with ultraviolet detection methods have long analysis times and a risk of analyte coelution which may compromise high sample throughput in a clinically meaningful time frame. A novel liquid chromatography-tandem mass spectrometry assay for mycophenolic acid was developed using very small (10 μL) sample volumes and evaluated in comparison with an established immunological assay. The enzyme mediated immunoassay showed a median positive bias compared with liquid chromatography-tandem mass spectrometry of 14.6%. Linear regression analysis showed a significant positive impact of bilirubin (r2 = 0.230) on bias with further increases of r2 to 0.261, 0.286, and 0.294 with the stepwise addition of creatinine, hematocrit, and γ-glutamyl transpeptidase, respectively. The impact of comedication and transplant type depended on the patient population: analysis of all samples showed opposing effects to analysis of those samples lacking data with biochemical variables above. The liquid chromatography-tandem mass spectrometry method described in this report is capable of measuring mycophenolic acid concentrations in very small sample volumes and in a timely fashion without the significant overestimates characterizing enzyme mediated immunoassay measurements in patients with serologic features characterizing liver or renal graft rejection.

Immunohistochemical phenotyping of the inflammatory infiltrate in de novo autoimmune hepatitis after liver transplantation in children

Hadžić N, Quaglia A, Cotoi C, Hussain MJ, Brown N, Vergani D, Mieli‐Vergani G. Immunohistochemical phenotyping of the inflammatory infiltrate in de novo autoimmune hepatitis after liver transplantation in children.

Application of an isocratic methanol-based HPLC method for the determination of iohexol concentrations and glomerular filtration rate in patients with cirrhosis

Background Glomerular filtration rate (GFR) and mortality is more accurately determined by gold standard measures than serum creatinine-based estimates in cirrhosis. No formal validation of any gold standard method has been reported. Methods An isocratic methanol-based method incorporating the reference standard iohexol-related compound C was developed and validated in 12 patients with cirrhosis by simultaneously determining GFR using iohexol and chromium-51 labelled ethylenediamine tetraacetic acid (51Cr-EDTA) clearance. Iohexol pharmacokinetics was also studied with the collection of blood and ascitic fluid at intervals following an iohexol bolus. Results Triplicate assays produced a linear calibration curve (R2 = 0.99, N = 5) over an iohexol concentration range of 23.6–755 µg/L. Mean (range) extraction recovery of iohexol from serum was greater than 95% (94–97%), with an intra-day coefficient of variation less than 3%. Twelve patients with cirrhosis with mean Child-Pugh score of 9 displayed a mean difference (bias) −1.3 mL/min/1.73 m2 (−18 to + 16) comparing iohexol with 51Cr-EDTA. Iohexol equilibrated between blood and ascitic compartments after 4 h. Conclusion A simple, cheap, and accurate isocratic, methanol-based method for the determination of iohexol concentrations is described, validated according to Food and Drug Administration guidance. Iohexol demonstrated comparable performance with 51Cr-EDTA in determining GFR. Delayed equilibrium of iohexol between blood and ascitic compartments suggests sampling beyond 4 h would improve accuracy of GFR determinations in patients with cirrhosis.

Concentration Monitoring of Plasma Ribavirin: Validation of a Liquid Chromatography-Mass Spectrometric Method and Clinical Sample Collection.

Background: A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for routine measurement of ribavirin concentrations in EDTA-anticoagulated plasma. Methods: After protein precipitation, we used a bridged ethylene hybrid (hydrophilic interaction) chromatography column, 0.1 mmol/L ammonium formate pH 3.0, and a gradient of 85%–96% acetonitrile to achieve baseline separation of ribavirin from isobaric uridine. Quantitation was assured using both primary (m/z 245.3 > 113.0) and secondary transitions (m/z 245.3 > 96.0) of the protonated species. Chromatographic separation and column washing also negated interference from major phospholipid species. Results: There was a linear relationship between concentration and response to 10 mg/L, with a minimum detectable level and a minimum level of quantitation both of 0.1 mg/L. Imprecision within the assay was <10% at 0.1 mg/L and <6% between assays for concentrations >0.4 mg/L. Bias was <4%. In clinical samples (n = 12), there was no difference in ribavirin concentrations obtained by an established liquid chromatographic assay with ultraviolet detection. Ribavirin concentrations were stable in plasma stored at room temperature for 3 days but then decreased significantly on day 7. Plasma concentrations were stable for 15 weeks at −20°C. Concentrations in plasma separated from whole blood at room temperature fell by a median of 19.4% at 4 hours and then rose substantially (median 251% by 3 days). Dose-normalized ribavirin concentrations reached a steady state after a mean of >6 weeks treatment in 76 patients with hepatitis C. Conclusions: A hydrophilic interaction liquid chromatography-tandem mass spectrometric method to measure ribavirin in plasma was developed. Samples for ribavirin estimation should be kept at 4°C, separated within 2 hours of collection and stored at 4°C before analysis, with long-term storage at −20°C. This method was applied to a study of the ribavirin therapeutic monitoring in patients with hepatitis C.