A novel: Plasma apoptosis assay to demonstrate CLL cell target engagement in first-in-human study of a highly selective oral bcl-2 inhibitor lonitoclax (ZE50-0134)

Abstract Background. B-cell lymphoma 2 (Bcl-2) is a member of the anti-apoptotic Bcl-2 family. Sensitivity to pharmaceutical disruption of Bcl-2 BH3 domain interactions with pro-apoptotic proteins characterizes diseases overtly responsive to this treatment. To date, clinical trials with the first generation Bcl-2 inhibitor, venetoclax, have shown single agent or combination activity in a variety of B-cell malignancies (chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), select Non-Hodgkin's lymphoma subtypes, multiple myeloma) and myeloid diseases (acute myeloid leukemia). To mitigate the hematologic effects, immune suppression and drug-drug interactions (CYP3A4) observed with venetoclax, we developed lonitoclax, a structurally unique chemotype with a shorter half-life, high selectivity to Bcl-2and reduced P4503A4 inhibition. Lonitoclax has demonstrated monotherapy activity in pre-clinical models, as well as synergistic activity when combined with azacytidine, FLT3 and menin inhibitors in AML xenograft models. Unlike venetoclax, lonitoclax had minimal immunosuppressive activity on B cells, CD8 T cells, and NK cells in preclinical models1of AML To optimize effective transition of lonitoclax to patients with several types of blood cancers, we developed a novel plasma apoptosis assay (PAA) utilizing ex vivo CLL cells to assess the efficacious free drug plasma exposure of lonitoclax in a Phase 1 healthy volunteer study. Application of healthy volunteer studies with agents having a broad therapeutic index such as Ionitoclax offer the opportunity to de-risk therapeutics early and begin administration of the agent in the subsequent cancer clinical trial at a dose where on target effect is expected. Methods We performed single-center, prospective, randomized, double-blind placebo-controlled study of lonitoclax. Lonitoclax (n=6/cohort) or placebo (n=2/cohort) was administered orally as a single dose to healthy adult participants. This interim analysis focuses on the 8 single ascending dose (SAD) cohorts (100, 200, 400 mg, 800 mg, 400 mg with food, 400 mg BID, 200 mg with DDI, and 400 mg with PPI). The primary objective of this trial was pharmacokinetic (PK) evaluation of lonitoclax; secondary objective was safety, and the exploratory objective was pharmacodynamics (immune cell changes over time and PAA). The PAA was performed by taking plasma from patients at corresponding PK time points, adding this ex vivo to human CLL cells, and measuring apoptosis by caspase 3 activity or annexin-V/PI after 4-hour exposure. Plasma derived from venetoclax treated patients with this assay demonstrated reproducible apoptosis. Results. A total of 48 healthy subjects has been treated to date in the trial in 8 cohorts (100, 200, 400 mg, 800 mg, 400 mg BID, 400 mg with food, 200 mg with DDI, and 400 mg with PPI). Lonitoclax was well tolerated with no significant treatment-related safety signals reported in any of the 8 SAD cohorts. The PK of lonitoclax was linear with mean plasma exposure (AUC0-inf) values that increased proportionally with dose escalation. There was a rapid absorption with a Tmax at 2 hours and a half-life of approximately 9-10 hours. Target engagement of Bcl-2 in healthy human subjects was confirmed using an ex vivo PAA towards CLL cells in a Cmax-driven manner in a dose-dependent fashion. For cohorts 1 and 2 rapid activation of caspase occurred at 2 hours and was diminished by 6 hours and at the higher cohort's caspase activation persisted from 2 to 6 hours which is consistent with the short half-life of lonitoclax. There was no change in normal B-cells, CD8 T-cells or NK cells with lonitoclax treatment. Pharmacokinetic AUC0-inf was increased (3.5-fold) with food but minimally influenced by itraconazole (+3%) or PPI administration (-27%). Conclusion. Lonitoclax demonstrates favorable PK and safety profiles when administered orally as a single dose of up to 800 mg, with dose-proportional increases in systemic exposure, food but not DDI or PPI effect, target engagement using the novel PAA, and no treatment-related adverse events. By using the PAA to demonstrate therapeutic exposures, the phase 1B study in R/R CLL patients was initiated with expectations that clinical activity could be observed at the first dose level.