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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.

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Background Vedolizumab (VDZ) effectively induces and maintains remission in inflammatory bowel disease (IBD). The loss of response to VDZ has been shown to be recaptured with dose escalation but the data in this field are still scarce. In addition, data on pharmacokinetics (PK) of dose escalation are limited and it is unclear whether PK should used in decision-making algorithm in adjusting VDZ dose regimen. Therefore, the aim of our study was to assess clinical efficacy and pharmacokinetic profile of VDZ dose escalation. Methods All IBD patients treated with VDZ in one tertiary IBD centre were retrospectively retrieved from the database. According to local protocol, non-responders to standard dosing of 300 mg i.v. of VDZ every 8 weeks, received escalated dose of 300mg i.v. every 6 or 4 weeks. Disease activity was assessed by Harvey-Bradshaw index (HBI) and partial Mayo score in Crohn’s disease (CD) and ulcerative colitis (UC) pts; respectively. VDZ dose was escalated in case of clinically assessed primary non response by week 22 of the treatment or in case of secondary loss of response. Response to dose escalation was defined as a decrease of HBI of ≥2 points, partial Mayo score ≥3 points or endoscopic improvement. VDZ through levels were assessed at the completion of induction and in dose escalated patients after at least two VDZ administrations in shortened interval. Results In total, 75 IBD patients were included (mean age 47 years, range 20–90; 36 men; 35 CD/39UC/1 IBD-U). Fifty two pts (69%) were primary responders, out of these 23 pts (44%) required dose escalation at some point of the treatment due to secondary loss of response. Out of 23 primary non-responders, 10 stopped the treatment, the remaining 13 received escalated dose of VDZ. Altogether, dose escalation was used in 36 pts (48%). There were no differences in the proportion of CD and UC between conventional and escalated dose regimen groups. Among secondary loss of response, the response was recaptured in 15 out of 23 pts (65%} while only two out of thirteen primary non-responders responded to dose escalation. There were no significant differences in VDZ levels between pts requiring dose escalation and pts with stable response to conventional regimen (mean levels 9,97±1,276 vs. 12,79±1,771 µg/mL; p=n.s.). VDZ levels increased significantly in patients who responded to dose escalation (from 10,12±3,460 to 20,81±3,326 µg/mL; p=0.0497). Conclusion Response to vedolizumab can be successfully recaptured in two thirds of secondary non responders by dose escalation. Patients requiring dose escalation do not seem to differ from stable responders with regards to vedolizumab pharmacokinetics.