Induction of cytokine release by the acyl glucuronide of mycophenolic acid: a link to side effects?

OBJECTIVES: We have identified an acyl glucuronide (M-2) of the immunosuppressant mycophenolic acid (MPA). Acyl glucuronides have toxic potential and may contribute to drug toxicity. Whether acyl glucuronides are able to induce release of proinflammatory cytokines is unknown. Gastrointestinal disturbances have been observed during MPA therapy and may involve an inflammatory reaction. This study investigated whether M-2 can induce IL-6 and TNF-alpha release as well as gene expression of these cytokines in leukocytes. DESIGN AND METHODS: M-2 was produced by incubation of MPA with human liver microsomes. Human mononuclear leukocytes were incubated in the presence of M-2. Concentrations of IL-6 and TNF-alpha were measured by ELISA. Expression of mRNA was determined by quantitative RT-PCR. RESULTS: Incubation of 3 x 10(6) cells with M-2 resulted in a time and dose dependent release of cytokines, whereas MPA or its phenolic glucuronide MPAG were without effect. Cytokine liberation depended on mRNA induction. Response to M-2 showed much inter individual variability (30-fold for IL-6, 3-fold for TNF-alpha). CONCLUSIONS: If M-2 promotes release of cytokines in vivo, these may mediate some of the toxic actions of MPA.

Cyclosporin whole blood immunoassays (AxSYM, CEDIA, and Emit): a critical overview of performance characteristics and comparison with HPLC.

Assays with different specificity are used for cyclosporin monitoring in clinical transplantation. A recent survey of 35 centers showed that 86% used immunoassays for cyclosporin A (CsA). In consensus documents the following performance criteria were recommended: (a) imprecision < or = 10% at 50 microg/L and < or = 5% at 300 microg/L; and (b) comparison with the reference method (HPLC) should yield a slope of 0.9-1.1, an intercept of -15 to 15 microg/L, and S(y/x) < or = 15 microg/L. The newly developed CsA assays for the AxSYM (Abbott) and the CEDIA (Boehringer Mannheim) as well as the Emit assay (Behring Diagnostica) were evaluated. Results from samples of heart, kidney, and liver recipients (100 specimens each) were compared with a validated HPLC-ultraviolet detection method. Between-series imprecision (CV) with commercial controls was 5.8% and 1.7% for AxSYM (70 and 300 microg/L), 11% and 5.5% for CEDIA (90 and 200 microg/L), and 8.1% and 4.5% for Emit (63 and 172 microg/L). In the presence of 300 microg/L parent CsA, cross-reactivities were (for AxSYM, CEDIA, and Emit, respectively) 7%, 4%, and none for AM1 (1 mg/L) and 12.6%, 25%, and 6% for AM9 (0.5 mg/L). Comparison with HPLC showed in heart and kidney recipients an average overestimation with the Emit and the CEDIA of approximately 22%, with overestimation in the AxSYM of 32%. In liver recipients, the most challenging patient group, the CEDIA and the AxSYM showed a mean overestimation of 43% and 47%, respectively, and the Emit differed by 31% compared with HPLC. None of the immunoassays fully satisfied the performance criteria recommended in the consensus documents. In terms of specificity, Emit ranks before CEDIA, which ranks before AxSYM. Regarding imprecision, the ranking is AxSYM < Emit < CEDIA. These limitations must be considered when using these assays for therapeutic drug monitoring of CsA in clinical transplantation.

A simple and reliable reverse-phase high-performance liquid chromatographic procedure for determination of paclitaxel (taxol) in human serum.

A simple, fast, and reliable isocratic (mobile phase: acetonitrile/methanol/water [48/11/41], reverse-phase (C18 column) high-performance liquid chromatography method for the determination of paclitaxel concentration in human serum is presented. The procedure uses a new and convenient one-step sample-purification procedure that requires only 400 microliters of sample and uses N-heptylbenzamide as an internal standard. Paclitaxel is detected by UV absorbance measurement at 227 nm. The method has a broad linear range (0.01 to 10 mg/l, or 0.012 to 11.7 mumol/l; r > 0.999), and the detection limit is 0.01 mg/l (0.012 mumol/l). The deviation from target value is < or = 1.5%, and coefficients of variation are < or = 13.8% within runs and < or = 15.3% between runs. Recovery paclitaxel is > or = 92.6%. No interferences were observed from endogenous compounds or from more than 30 drugs that may be administered with paclitaxel. Docetaxel, which is not concurrently administered, coeluted with paclitaxel. Compared with previously published high-performance liquid chromatography procedures for the determination of paclitaxel, the particular advantage of the method presented here is its simple and rapid single-step sample-purification procedure, which makes a high recovery of paclitaxel from serum samples possible and results in a pure extract, avoiding interferences from endogenous compounds. The method is suitable for pharmacological studies and routine analysis.

Prenatal diagnosis of the Pallister-Killian mosaic aneuploidy syndrome by CVS.

Prenatal cytogenetic analysis at 11 weeks of gestation revealed an abnormal karyotype 47,XX,+mar in all metaphases obtained from a chorionic villi sample after 24 h culture. Karyotyping of amniotic fluid cells in the second trimester showed mosaicism 47,XX,+i(12p)/46,XX with 10% aneuploid cells. The pregnancy was terminated at 20 weeks of gestation on the patient's request. The aborted fetus showed typical manifestations of the Pallister-Killian mosaic aneuploidy syndrome. The identity of the supernumerary isochromosome 12p was proven by LDH isozyme electrophoresis using cultured fibroblasts and by nonradioactive in situ hybridization using a biotinylated set of chromosome 12-specific DNA probes.