Porous graphitic carbon based chromatography hyphenated with mass spectrometry: A new strategy for profiling thiopurine nucleotides in patients with inflammatory bowel diseases.

Thiopurine (TP) treatment is discontinued in up to 30% of patients suffering from inflammatory bowel diseases (IBD) due to various adverse effects. Therapeutic drug monitoring of biologically active TP metabolites, i.e. thiopurine nucleotides (TPN), can help to optimize the efficacy and safety of the TP treatment. In our work, a novel strategy for TPN profiling, based on a porous graphitic carbon (PGC) chromatography, was developed. The validated PGC-MS method was compared with ion-exchange LC-MS, a currently leading analytical approach established for the determination of TPN. The innovative approach enabled an enhancement of several key performance parameters demanded in a clinical routine use, namely (i) selectivity (time- and mass-recognition of all 12 TPN in one run), (ii) sensitivity (2-5-fold increase in intensities of the analytical signals), (iii) sample throughput (25% shorter analysis time). Application of the novel TPN profiling strategy to a pilot clinical study (12 patients) revealed significantly higher levels of 6-methylthioguanine 5'-diphosphate (MeTGDP) in non-responsive IDB patients treated with azathioprine. Some other TPN are very close to the critical level (p = 0.05) and they will need larger groups of IBD patients to confirm definitively their relevance. In conclusion, the developed PGC-MS method represents a significant improvement to currently available methods for detailed profiling of TPN and its use in bigger clinical studies should lead to a better understanding of the relationship between TPN profiles and therapeutic outcome.

Positive pharmacokinetic effect of azathioprine co-medication on infliximab trough levels is dose-dependent.

BACKGROUND: Thiopurines seem to have positive effect on the pharmacokinetics of anti-tumor necrosis factor biologics. It has been suggested that a reduced dose of thiopurines is sufficient to achieve this synergism. AIMS: To assess the differences of infliximab (IFX) trough levels according to the dose of concomitantly used azathioprine (AZA). PATIENTS & METHODS: All IBD patients treated with IFX (Remicade®) in two IBD centres between November 2015 and April 2017 were eligible. Infliximab trough levels were assessed by ELISA (Ridascreen®, R-Biopharm). The differences in IFX trough levels according to AZA dose were analyzed statistically. RESULTS: In total, 125 patients were included, 42 pts (33.6%) on infliximab monotherapy, 83 pts (66.4%) using combined immune suppression. The respective median IFX levels according to AZA dose were as follows: group 1 (no concomitant AZA) 2.83 µg/ml (range 0-12); group 2 (AZA dose less than 1 mg/kg) 4.91 µg/ml (range 0.09-15.36); group 3 (AZA dose 1 < 2 mg/kg) 5.67 (range 0.16-16.97); group 4 (AZA dose above 2 mg/kg) 7.53 µg/ml (range 1.15-8.73). The differences in IFX trough levels between the respective groups according to AZA dose were statistically significant (p = 0.0159). CONCLUSION: The positive effect of azathioprine on infliximab levels seems to be dependent on the dose of concomitantly used azathioprine.

Analytical and Sample Preparation Protocol for Therapeutic Drug Monitoring of 12 Thiopurine Metabolites Related to Clinical Treatment of Inflammatory Bowel Disease.

Thiopurines (TP) represent an important therapeutic tool for the treatment of inflammatory bowel diseases (IBD) in the current situation of rising incidence and health care costs. The results of multiple clinical studies aimed at finding correlations between levels of TP metabolites and response of IBD patients to the treatment are, however, often controversial due to variability in analytical and sample preparation procedures among these studies. In this work, therefore, an updated analytical and sample preparation procedure for therapeutic drug monitoring (TDM) of TP metabolites in blood samples obtained from patients with IBD was proposed to establish a unified protocol. An advanced analytical method based on ion-exchange liquid chromatography hyphenated with tandem mass spectrometry (IEC-ESI-MS/MS) was used for the determination of the profiles of 12 individual TP metabolites in the particular steps of sample preparation procedure including blood collection, red blood cells (RBC) isolation, lysis, and storage. Favorable performance parameters of the IEC-ESI-MS/MS method (LLOQs 1⁻10 nmol/L, accuracy 95⁻105%, intra-day and inter-day precision < 10%, selectivity demonstrated via no sample matrix interferences) and acceptable stability (peak area fluctuations < 15%) of clinical samples under the proposed sample preparation conditions {(i) EDTA anticoagulant tube for the blood collection; (ii) 4 °C and 4 h between the sample collection and RBC isolation; (iii) phosphate-buffered saline for RBC washing and re-suspendation; (iv) -20 °C for RBC lysis and short-term storage; (v) 50 mmol/L phosphate buffer, pH 7.4, 10 mmol/L DTT as a stabilizing medium for TPN in RBC lysates} demonstrated the suitability of such protocol for a well-defined and reliable routine use in studies on thiopurines TDM.