RESUMO
The mitogen-activated protein kinase (MEK) inhibitor zapnometinib is in development to treat acute viral infections like COVID-19 and influenza. While the antiviral efficacy of zapnometinib is well documented, further data on target engagement/pharmacodynamics (PD) and pharmacokinetics (PK) are needed. Here, we report zapnometinib PK and PD parameters in mice, hamsters, dogs, and healthy human volunteers. Mice received 25 mg/kg/day zapnometinib (12.5 mg/kg p. o. twice daily, 8 h interval). Syrian hamsters received 30 mg/kg (15 mg/kg twice daily) or 60 mg/kg/day once daily. Beagle dogs were administered 300 mg/kg/day, and healthy human volunteers were administered 100, 300, 600 and 900 mg zapnometinib (once daily p. o.). Regardless of species or formulation, zapnometinib maximum plasma concentration (Cmax) was reached between 2-4 h after administration with an elimination half-life of 4-5 h in dogs, 8 h in mice or hamsters and 19 h in human subjects. Doses were sufficient to cause up to 80% MEK inhibition. Across all species approximately 10 µg/ml zapnometinib was appropriate to inhibit 50% of peripheral blood mononuclear cells (PBMC) MEK activity. In mice, a 50%-80% reduction of MEK activity was sufficient to reduce influenza virus titer in the lungs by more than 90%. In general, while >50% MEK inhibition was reached in vivo at most doses, 80% inhibition in PBMCs required significantly higher doses and appeared to be the practical maximal level obtained in vivo. However, the period of reduced phosphorylated extracellular-signal regulated kinase (pERK), a measure of MEK inhibition, was maintained even after elimination of zapnometinib from plasma, suggesting a sustained effect on MEK consistent with regulatory effects or a slow off-rate. These data suggest a target plasma Cmax of at least 10 µg/ml zapnometinib in further clinical studies.
RESUMO
The Raf/MEK/ERK signaling pathway plays a key role in regulating cellular proliferation, differentiation, apoptosis, cytokine production, and immune responses. However, it is also involved in diseases such as cancer, and numerous viruses rely on an active Raf/MEK/ERK pathway for propagation. This pathway, and particularly MEK1/2, are therefore promising therapeutic targets. Assessment of target engagement is crucial to determine pharmacodynamics or the efficacy of a MEK1/2 inhibitor. In the field of infectious diseases, this is usually first determined in clinical trials with healthy volunteers. One method to detect MEK1/2 inhibitor target engagement is to assess the degree of ERK1/2 phosphorylation, as ERK1/2 is the only known substrate of MEK1/2. As healthy subjects, however, only feature a low baseline MEK1/2 activation and therefore low ERK1/2 phosphorylation in most tissues, assessing target engagement is challenging, and robust methods are urgently needed. We hence developed a method using PBMCs isolated from whole blood of healthy blood donors, followed by ex vivo treatment with the MEK1/2 inhibitor zapnometinib and stimulation with PMA to first inhibit and then induce MEK1/2 activation. As PMA cannot activate MEK1/2 upon MEK1/2 inhibition, MEK1/2 inhibition results in impaired MEK1/2 activation. In contrast, PMA stimulation without MEK1/2 inhibition results in high MEK1/2 activation. We demonstrated that, without MEK1/2 inhibitor treatment, MEK1/2 stimulation with PMA induces high MEK1/2 activation, which is clearly distinguishable from baseline MEK1/2 activation in human PBMCs. Furthermore, we showed that treatment with the MEK1/2 inhibitor zapnometinib maintains the MEK1/2 activation at approximately baseline level despite subsequent stimulation with PMA. As our protocol is easy to follow and preserves the cells in an in vivo-like condition throughout the whole handling process, this approach can be a major advance for the easy assessment of MEK1/2 inhibitor target engagement in healthy probands for clinical drug development.