Clinical Pharmacokinetics and FLT3 Phosphorylation of AC220, a Highly Potent and Selective Inhibitor of FLT3
Authors: James Joyce , Pratz Keith , Stine Adam , Apuy Julius L , Insko Darren E , Armstrong Robert C , Zarrinkar Patrick P , Small Donald , Wierenga Wendell , Levis Mark J. 
Published: April 2020

Abstract
Several different FLT3 inhibitors have now been tested as monotherapy for AML patients and have met with only limited success. To date, none of these inhibitors have resulted in complete, sustained inhibition of phosphorylated FLT3 in vivo, as measured by immunoblotting techniques. AC220 is a novel FLT3 kinase inhibitor with greater potency and kinase selectivity than others previously characterized. To establish a dose of AC220 that will lead to complete, sustained inhibition of phosphorylated FLT3, a Phase I open-label, sequential dose escalation study investigating safety, tolerability, pharmacokinetics, and pharmacodynamics in patients with relapsed or refractory acute myeloid leukemia is being conducted. AC220 is administered once daily as an oral solution for 14 days followed by a 14-day rest period, (one cycle) with a starting dose of 12 mg. Patients showing clinical benefit may continue to receive further cycles of therapy. The study is a standard 3+3 dose escalation design with 50% dose increments. Currently 52 patients have been enrolled and AC220 has been well tolerated and escalated to 450 mg daily (10th dose cohort) on this intermittent dosing regimen and PK has been evaluated up to 300 mg. AC220 is orally bio-available and has a long effective half-life, estimated to be 2.5 days, exhibiting minimal peak and trough variation of plasma levels. AC220 plasma exposure in AML patients is sustained between dose intervals and continues to increase in a dose-proportional manner from 12 mg to 300 mg daily, with steady-state plasma concentrations achieving greater than 1,500 nM at 300 mg. In addition, a pharmacologically active metabolite, AC886, has been identified in patient urine and plasma samples, which has similar potency and kinase selectivity to AC220 as determined by in vitro kinase binding and cell assays. The IC50s for AC220 and AC886 in FLT3-ITD-expressing cell lines and in primary FLT3-ITD AML patient blast samples are in the low nanomolar range (1.7 and 0.3 nM, respectively) and are in good agreement with cytotoxicity (Annexin V and MTT) assays. Both drugs display a high degree of selectivity for FLT3 in an IL-3 rescue assay. AC220 and AC886 also potently inhibit wild type FLT3 (3.6 and 4.2 nM, respectively). In standard immunoblot curves using AC220 and AC886 spiked into plasma, FLT3-ITD phosphorylation was completely inhibited at 50 and 200 nM, respectively. As predicted from the standard curve data, plasma from AC220-treated AML patients completely suppresses FLT3 phosphorylation on Day 1 and at steady-state in an ex vivo plasma inhibitory activity (PIA) assay, even at the lowest dose of 12 mg daily. AC220, with its metabolite AC886, is a potent, selective inhibitor of both mutant and wild type FLT3. It is orally bio-available with sustained plasma exposure, and the pharmacological impact of AC220 is further enhanced by the presence of AC886. In vivo, therefore, AC220 is expected to completely inhibit FLT3-ITD at &gt;100 nM (achieved at the lowest AC220 dose of 12 mg) and FLT3-WT at 500 nM (achieved by the 60 mg dose). AC220 is the most potent and selective FLT3 inhibitor tested using the PIA assay thus far, and, unlike other FLT3 inhibitors that have been assessed in this assay, is the first to completely suppress FLT3 phosphorylation ex vivo at doses that are easily achievable and sustainable in the clinic. Therefore, AC220 should be able to effect substantial and prolonged target inhibition in patients. This makes it an ideal compound with which to test the hypothesis that complete, sustained inhibition of FLT3 activity will lead to clinical benefit in FLT3 mutant AML patients.