Identification of Degradation Products of the New Anticancer Drug Substance ONC201 by Liquid Chromatography–High-Resolution Multistage Mass Spectrometry

Abstract

ONC201 (dordaviprone) is a new drug substance used in a compassionate manner to treat patients with glioblastoma. Given the clinical context and the particularly promising preclinical results, we have been asked by the medical authorities to make a first treatment available throughout France as a hospital preparation to allow access to treatment and to conduct clinical trials. However, to control the quality and safety conditions inherent in this academic manufacturing process, while there is virtually no data available to date to understand the stability of ONC201, we had to determine the stability profile of ONC201, i.e., its sensitivity to different stressors and the types of impurities that could form during its degradation. We found that ONC201 was sensitive to oxidation in the presence of hydrogen peroxide or under light irradiation. Both conditions resulted in the formation of 20 degradation products detected and identified by liquid chromatography–high-resolution mass spectrometry. Their structural elucidation required an in-depth study of the fragmentation pattern of protonated ONC201, described for the first time. The product ions of the degradation products were compared to those of ONC201 protonated ion to assign the most plausible structures for all the detected degradation products. Of these degradation products, those that were rapidly produced, of high intensity and/or identified as potentially having a different toxicity profile to ONC201 by in silico studies, were selected to be monitored during batch release testing and stability studies.

Molecular Mechanisms Involved in the Chemical Instability of ONC201 and Methods to Counter Its Degradation in Solution

“…Thanks to the convergence of previously published structural data [3] and these ab initio calculation data (Table 1), we were able to propose degradation schemes linking ONC201 to its degradation products (Figures 2-4). As shown in Figure 1b, most degradation products detected during ONC201 stress studies exhibit an imidazole group instead of, initially, a tetrahydroimidazole group.…”

“…From the structures of the degradation products of ONC201 that have been identified via mass spectrometry (Figure 1) [3], it appears that two sites are primarily involved in degradation upon exposure to light, as the observed changes occurred in the vicinity of the imidazole imine nitrogen and/or the tetrahydropyridine nitrogen (positions (1), (2), ( 3) and (4), according to captions in Figure 1a). Indeed, the product arbitrarily named ONC201-DP-1 may exclusively be the result of the attacks in (1), whereas the other products reported in Figure 1b would be the result of both the attacks in (1) and those perpetrated in (2), (3) or (4).…”

“…The results of the conformational analysis and of the coordinates of the two lowest energy conformers are provided in the Supplementary Materials (Figure S1 and Tables S1 and S2). To understand the mechanisms involved, as a crucial step to guide the subsequent stages of pharmaceutical development, we jointly used the capital of knowledge acquired from the structures of the photoproducts [3] and the data which make it possible to highlight the molecular reactivity of ONC201 based on the DFT.…”

“…However, it appears that the departure of the hydrogen present in the alpha position from the radical site is a more rapid process, contributing to the termination reaction by leading to the formation of the imidazole group through the creation of additional unsaturation, as was the case for DP-1, 1 , 3, 3 , 4 and 4 (Figures 1b and 2). In competition with this termination process, the addition of singlet oxygen to the radical carbon would also have taken place as the latter is known to have fast kinetics and this helps to explain the formation in part of DP-2 and 2 [3]. Simultaneously or sequentially, changes were also highlighted in the tetrahydropyridine moiety of the ONC201 molecule under the action of light.…”

“…Previously, we had written a chapter as part of a program to study the pre-formulation of ONC201. The aim of this first part of the work was to gain access to the structure of the main degradation products of ONC201 formed following its exposure to stress conditions [3]. The degradation products identified were thus described for the first time, bearing in mind that they only appeared under conditions of oxidative and photolytic stress.…”