Abstract 160: High target binding affinity with long lasting cellular target engagement and high dose intermittent schedule of PI3K inhibitor copanlisib contribute to the potent anti-tumor activity and good safety profile
Authors: Fernández-Montalván Amaury E. , Georgi Victoria , Vasta James , Glaeske Sarah , Puetter Vera , Robers Matthew B. , Moenning Ursula , Sturz Andrea , Lefranc Julien , Ziegelbauer Karl , Brands Michael , Stegmann Christian , Scott William J. , Liu Ningshu 
Published: July 2017

Abstract
Introduction: Several generations of PI3K inhibitors have been tested in clinic. However, thus far, clinical activity has been moderate. Different from other oral PI3K inhibitors dosed continuously, copanlisib (BAY 80-6946) is an intravenous PI3K inhibitor given intermittently to patients. Copanlisib dosed once weekly demonstrated clinical benefit with an improved safety profile, and therefore challenges the concept of default continuous dosing of PI3K inhibitors. However, it is still unclear if this concept can be generalized and whether ‘micropharmacokinetic parameters’ also contributed to the potent anti-tumor profile of copanlisib. Here, we report the characterization of binding kinetics for copanlisib, as well as the functional consequence in vivo.
Methods: A set of PI3K inhibitors were characterized in 1) a kinetic probe competition assay (kPCA); 2) a cellular nanoBRET target engagement assay; 3) a cellular washout study with the assessment on pathway engagement; and 4) in vivo pharmacokinetics analysis.
Results: Copanlisib showed nearly diffusion-controlled on- and relatively slow off-rates with kon = 3.45E+7 [M-1*s-1] and koff = 1.67E-3 [s-1] to PI3Kα. Consequently, it exhibited very high affinity to PI3Kα ( Ki ePCA = 9.31E-11[M] and KD kPCA = 4.77E-11 [M]). In a cellular nanoBRET target engagement assay, the apparent half-life (t1/2) of ca. 2 hours greatly surpassed the 6.9 min measured using kPCA. The high affinity to PI3Kα also translated into potent cellular pathway engagement demonstrated by inhibition of p-AKT and p-PRAS40 in the PIK3CAmut KPL4 cell line. In a cellular washout study, p-AKT and p-PRAS40 were assessed till 168 h after incubation with copanlisib for 1 h followed by a washout step. A dose- and time-dependent pathway engagement was observed even at 72 h post washout. This result indicated that in cells, copanlisib engages PI3Kα for an extremely long time, likely due to rebinding effects facilitated by the fast equilibration kinetics of the compound and its micropharmacokinetic properties. Interestingly, in vivo, BAY 80-6946 levels were approximately 100-fold higher in the tumor than in plasma at 48 hours and drug clearance from the tumor occurred more slowly than from plasma. This high and prolonged tumor exposure might be explained, at least in part, by the high expression of PI3Kα and long lasting target occupancy of copanlisib in tumors.
Conclusion: Copanlisib demonstrated high affinity to PI3Kα with protracted target engagement at cellular and in vivo levels. This ‘micropharmacokinetic feature’ not only supports intermittent dosing but likely also explains the high exposure in tumors vs plasma, potent anti-tumor activity and good safety profiles.
Citation Format: Amaury E. Fernández-Montalván, Victoria Georgi, James Vasta, Sarah Glaeske, Vera Puetter, Matthew B. Robers, Ursula Moenning, Andrea Sturz, Julien Lefranc, Karl Ziegelbauer, Michael Brands, Christian Stegmann, William J. Scott, Ningshu Liu. High target binding affinity with long lasting cellular target engagement and high dose intermittent schedule of PI3K inhibitor copanlisib contribute to the potent anti-tumor activity and good safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 160. doi:10.1158/1538-7445.AM2017-160