Examination of the metabolism of both MCT-imaging PET radioligands [18F]FACH and [18F]FACH-d6 in vitro by means of LC-MS/MS - support for understanding preclinical in vivo data from different species

Objectives: Positron Emission Tomography (PET) is a non-invasive molecular imaging technique, which continues to find increasing application and further development for diagnosis of a broad range of disorders and dysfunctions. Since their involvement in pathophysiological processes that lead to cancer, for example, monocarboxylate transporters (MCTs) are of interest for the development of new radioligands for PET, such as [18F]FACH [1, 2] and its deuterated isotopologue [18F]FACH-d6. Both candidates have been already examined previously in preclinical PET studies in healthy mice and piglets, also with regard to their biodistribution and metabolic fate [3]. However, the general finding that in piglets both radioligands showed significant lower metabolic stability than in mice left some important unanswered questions, especially those relevant to potential clinical application.
Therefore, this study aimed to further explore the metabolic properties of both radioligands by means of in vitro examinations, which besides discovering species dependency in some respects, might allow predictions for their metabolic stability in humans.
Methods: [18F]FACH, [18F]FACH-d6 as well as their corresponding non-radioactive references were incubated with murine (mouse MLM and rat RLM) and human (HLM) liver microsomes and with pig and human S9 fractions, in presence of NADPH, but also other co-factors (e.g. UDPGA). After collection of samples at defined time points, subsequently prepared samples were investigated by radio-HPLC, UV-HPLC or LC-MS/MS. Obtained data allowed calculation of basic metabolic parameters and identification of metabolites formed in vitro.
Results: [18F]FACH, incubated in presence of NADPH at 37°C for up to 60 min, showed highest stability in MLM (t1/2= 29913 min), whereas lowest stability was found in RLM (t1/2= 201 min). A moderate stability was found for both HLM (t1/2= 948 min) and pig S9 fraction (t1/2= 522 min). However, in a recent head-to-head comparison using S9 fractions only, [18F]FACH showed 38 % unchanged radioligand for pig, but 80 % unchanged radioligand for human after 60 min. The deuterated radioligand [18F]FACH-d6 showed a marginal increase of stability only in S9 fraction from pig (t1/2= 675 min). Species differences were also found regarding in vitro degradation pathways, whereby in the molecule the dialkylamino moiety was affected exclusively. By means of non-radiolabeled FACH and LC-MS/MS measurements, dealkylation of one or both of the alkyl chains was found as the main pathway in MLM and HLM. Moreover, single hydroxylation and, to a lesser extent, direct defluorination occurred. In contrast, in RLM and pig S9 fraction single hydroxylation reactions at one of the alkyl chains were predominant, followed by dealkylation and defluorination-hydroxylation. Furthermore, during analysis by acetonitrile-based gradient RP-HPLC, the use of methanol as an additive enabled a high chromatographic resolution of structurally similar hydroxylated metabolites.
Conclusion: Significant species differences could be demonstrated with regard to metabolic stability of both radioligands, as well as their metabolic pathways. In contrast to other examples from literature, the isotopologue [18F]FACH-d6 did not appear to be more stable than [18F]FACH. In this regard, in particular results from HLM and human S9 fraction indicate that the metabolic stability in human might be considerably higher for [18F]FACH than results from previous preclinical PET studies would suggest. Some final investigations are needed in terms of its ADME properties to prove that [18F]FACH is an appropriate candidate for a first in-human study and subsequent clinical translation.

Acknowledgments: This research was funded by the Alexander von Humboldt Foundation, grant number 7211172142.

References:

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