Differential Inhibition of FLT3-ITD, FLT3-WT, and KIT By Quizartinib As Assessed By Modified PIA Assay
Authors: Nepomuceno Ronald , Rooks Allison M , Gardner Michael , Armstrong Robert 
Published: October 2019

Abstract
The plasma inhibitory activity (PIA) assay has been used to determine if sufficient exposure of FLT3 kinase inhibitors have been achieved to inhibit their target following clinical administration. Traditionally, clinical plasma samples have been used undiluted to determine inhibitory activity. In the case of quizartinib (AC220), undiluted plasma often results in full inhibition of ex vivo FLT3 activity, regardless of dose administered. To more closely examine the inhibitory properties of circulating quizartinib in a clinical trial, we modified the western blot PIA assay to utilize MSD-based phospho (p)FLT3 and pKIT ELISAs. This modification allows for the comparison of inhibitory activity between samples, resolution of activity against multiple targets from a single subject sample, as well as demonstrates the differences between administered quizartinib doses.
A total of 76 relapsed/refractory FLT3-ITD AML subjects were enrolled in a Phase 2b study. Of those, 56 subjects (30 at 30 mg/day; 26 at 60 mg/day) had sufficient pre-dose and either day 8 or 15 plasma samples available for evaluation. Samples were tested undiluted and serially 4-fold diluted into normal human plasma. FLT3 inhibitory activity was determined in Ba/F3 cells exogenously expressing human FLT3 with an internal tandem duplication (ITD) mutation (ITD cells), and in THP-1 cells, where wild-type (WT) FLT3 phosphorylation was FLT3 ligand induced. Inhibitory activity on c-KIT in H256 cells with SCF-induced phosphorylation was also determined. Percent inhibition was calculated by comparing post-dose samples to equivalently diluted pre-dose samples.
As in previous western blot-based PIA assays, undiluted plasma samples contained sufficient quizartinib and its active metabolites to inhibit FLT3 phosphorylation in ITD cells by 98.0 ± 3.3% and 100.4 ± 4.0% at 30 and 60 mg doses, respectively, suggesting exposures that completely inhibit cellular FLT3-ITD activity. Diluting the plasma samples allowed for determination of the IT50 (the plasma dilution titer resulting in 50% inhibition of kinase activity) to assess the excess active drug in each sample. For subjects receiving 30 mg, the median IT50 was 21.0 (range 7.3-45.9) (inverse dilution). For subjects receiving 60 mg, the median IT50 was 44.8 (range 19.1-135.5), indicating the expected 2-fold difference in inhibitory activity. The percent inhibition levels were significantly different between 30 and 60 mg doses at all dilutions in the ITD cells. For FLT3-WT in THP-1 cells, the mean inhibitions of ligand-induced phosphorylation in the undiluted samples were 77.6 ± 9.6% and 90.1 ± 5.6% with the two doses, indicating that FLT3-WT is not as well inhibited by quizartinib. The median IT50s were 4.3 (range 3.0-8.0) and 6.6 (range 3.7-13.6) for the 30 and 60 mg doses, consistent with less excess drug relative to FLT3-WT inhibition. KIT inhibitory activity was weaker at these doses of quizartinib. Undiluted plasmas had a mean inhibition of 43.3 ± 16.1% and 63.9 ± 14.1% at 30 and 60 mg, so IT50s could only be calculated for one subject at 30 mg and 10 subjects at 60 mg. KIT inhibition levels were still significantly different between the two doses in the undiluted and 1:4 diluted plasma samples, but were largely eliminated by the 1:16 dilution.
Measured plasma levels of quizartinib and its metabolite AC886 were used to calculate the plasma IC50 values for each cell line. For ITD cells, the median IC50 was 20.5 nM (range 6.9-64.6 nM). In the THP-1 cells, it was 129 nM (range 52.1-552 nM). For KIT inhibition, the median IC50 was 442 nM (range 167-1577 nM). Of note, the median quizartinib + AC886 levels were 370 nM (range 205-802 nM) for 30 mg subjects, and 917 nM (range 291-4234 nM) for 60 mg subjects, indicating on average, the 30 mg subjects were 18-fold above the the IC50 for FLT3-ITD inhibition, and the 60 mg subjects were 45-fold above this IC50, and drug levels were 3-fold and 7-fold above the IC50 for FLT3-WT in the two doses. For KIT inhibition, most 30 mg subjects had drug levels below the KIT IC50, while 60 mg subjects had drug levels only 2-fold above it.
These results suggest that quizartinib, when dosed at 30 mg or 60 mg achieved peripheral exposures sufficient for complete inhibition of FLT3-ITD as assessed by the PIA assay. At these same doses the degree of inhibition of FLT3-WT, and especially KIT is reduced. Implications of this data for patient response and myelosuppression will be available for presentation.

Disclosures
Nepomuceno: Ambit Biosciences Corporation: Employment. Rooks:Ambit Biosciences Corporation: Employment. Gardner:Ambit Biosciences Corporation: Employment. Armstrong:Ambit Biosciences Corporation: Employment, Equity Ownership.