Development and Validation of a Sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Assay for the Simultaneous Quantification of Midostaurin (PKC412) and Its Two Major Metabolites: A Suitable Tool for the Monitoring of the Drug in Patients Suffering From Advanced Systemic Mastocytosis

Among tyrosine kinase inhibitors, midostaurin (PKC412) takes place in the treatment of patients presenting with advanced systemic mastocytosis and, specifically aggressive systemic mastocytosis (ASM) or mast cell leukemia (MCL) with or without associated clonal hematological non-mast cell lineage disease (AHNMD). Our knowledge of the pharmacokinetic (PK) behavior of this major drug remains unfortunately sporadic. We developed a powerful liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, which is able to sensitively detect PKC412 as its two major metabolites CGP52421 and CGP62221 in human plasma. The method was validated in clinical situation and, especially in the aim to monitor plasma concentrations of PKC412 in 11 patients, already enrolled in a Phase II clinical trial (Novartis Lab.). In addition, we also determined the PK profile of PKC412 at steady-state for one specific patient.

A sensitive LC-MS/MS assay was developed for the simultaneous quantification of the product PKC412 and its two major metabolites in human plasma. Midostaurin-D5 was used as internal standard. The method was then validated in accordance with international guidelines and within the bioanalytical ±15% acceptance limits.

In any case, PKC412 regimen was 100 mg bid. A total of 61 blood samples were collected from 11 patients and, between 15 min to 12 hrs following oral administration. Sampling took place over a period of 1 to 924 days following the beginning of treatment. PKC412 residual concentration at steady-state, range and mean concentration and, intra- and inter-individual variability coefficients were also calculated. On the other hand, a model independent method was used to determine the PK profile of PKC412 for a 12^th patient (using 22 samples collected within 24 hrs): maximal concentration post-dosing (C_max – ng/mL), time to Cmax (T_max - hrs) were experimental values; terminal half-life (t_1/2lz - hrs) and AUC_0–12 were calculated during each 12 hrs interval post-dosing. Data are expressed as means ± SD.

This LC-MS/MS method is satisfactory for the key validation criteria i. e. trueness, bias, precision (repeatability and intermediate precision), accuracy and, linearity, over a range of concentrations of 75 to 2500 ng/mL for both PKC412, CGP52421 and, CGP62221. Patients monitored (8/11 males) were 57±14 yrs-old. All except one were treated for ASM±AHNMD (the last one had MCL), 10/11 patients carrying the ckit D816V mutation. The main characteristics at inclusion were as followed: body weight = 65±11 kg, BMI = 22±3 kg/m², albumin = 31±6 g/L, GFR = 64±12 mL/min, bilirubin = 14±6 mg/L, GGT = 103±78 UI/L, ALP = 264±158 UI/L, tryptase = 412±411 μg/L. At the last follow-up visit i. e. between 54 to 924 day after initiation therapy, 8/11 patients were either in major or partial response with the following clinical picture: body weight 74±16 kg, BMI 24±4 kg/m², albumin 40±2 g/L, DFG 59±11 mL/min, bilirubin 12±8 mg/L, GGT 56±57 UI/L, ALP 128±59 UI/L, tryptase 256±264 μg/L. It is important to point out that the tolerability profile was very good. The PKC412 residual concentrations value determined at steady-state were between 487 to 3254 ng/mL (1333±867 ng/mL). The intra- and inter-individual coefficients of variations were 18 and 65% respectively. About our 12^th subject, major PK parameters were: C_max = 3692±447 ng/mL, T_max = 2, 5±2 hrs, t_1/2lz = 95±88 hrs, AUC_0–12= 34326±2197 ng/mL. hrs.

The LC-MS/MS method is efficient and enables a sensitive and discriminative analysis of PKC412 and its major metabolites in human plasma. These first results highlight the high intra- and inter-individual variability of the PK behavior of the drug and, suggest the interest of its routine therapeutic monitoring vs. both adherence to the treatment and, its tolerability profile. Further, it has been shown that the 2 major metabolites of PK412 are active; under these conditions, we are working to the simultaneous therapeutic drug monitoring of the 3 compounds.

No relevant conflicts of interest to declare.