A Novel Method for Overcoming Plasma Protein Inhibition of FLT3 Tyrosine Kinase Inhibitors

Background: The FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in acute myeloid leukemia (AML) of adults, and also results in poor prognosis for pediatric AML patients, and thus represents an attractive target for tyrosine kinase inhibitors (TKI) for the treatment of AML. This led to the recent approval of the staurosporine (STS) derivative, midostaurin (Rydapt, PKC-412) as the first therapy specifically indicated for FLT3-mutated AML harboring either the more common internal tandem duplication (FLT3/ITD) or kinase domain mutations (FLT3/KD).

Our lab has previously shown that the activity of all FLT3-targeted TKI are inhibited by plasma protein binding. In a prior study of a series of pediatric patients, we demonstrated that the STS-derived TKI are inhibited almost exclusively by the plasma protein alpha-1 acid glycoprotein (AGP), an acute-phase reactant, whereas other TKI are subject to inhibition by different proteins. This inhibition appears responsible for the failure of the previous STS-derivative, lestaurtinib (CEP-701) in clinical trials. Therefore, we have studied the impact of AGP binding on the other STS-derivatives and report the development of a novel method to overcome this binding.

Results: Using purified human AGP, we assayed the impact of human plasma upon the activity of the STS-derived TKI (midostaurin, lestaurtinib, and the novel agent TTT-3002) against proliferation of the FLT3-ITD dependent cell line MOLM-14, as well as upon the parent compound (staurosporine) against the non-FLT3-dependent cell line HL-60. The TKI are inhibited in a linear AGP-dependent manner (fold change increase of IC₅₀ per mg/dL AGP: midostaurin 3.00-fold, lestaurtinib 11.73-fold, TTT-3002 0.33-fold, P < 2 x 10^-16) across the range of concentrations observed in human plasma. Furthermore, staurosporine is completely inhibited by AGP, demonstrating no activity until AGP is saturated by drug. Assays using bovine and murine plasma, serum or purified AGP do not demonstrate similar drug inhibition.

Using a model of first-order kinetics of interaction, we have demonstrated that these results correspond to the drug-protein binding constants for the TKI: midostaurin 12.6 µM^-1, lestaurtinib 49.2 µM^-1, TTT-3002 1.41 µM^-1. These results were validated using classical competitive fluorescence displacement assays. Furthermore, these results predict in vivo IC₅₀ values significantly higher than in vitro : midostaurin 4.7 µM, lestaurtinib 4.8 µM^-1, TTT-3002 34 nM.

Out of a series of known AGP bound drugs, we have identified mifepristone (Mifeprex, RU-486) as binding AGP stronger than that of the STS-derived TKIs (2-10 fold greater competitive fluorescence displacement). Furthermore, co-treatment with 10 µM mifepristone restores the IC₅₀ of TTT-3002 from 12 nM with AGP to < 0.1 nM. Although activity is not fully restored, similar disinhibition is seen for lestaurtinib (>1000-fold increase in IC₅₀ reduced to 50-fold) and midostaurin (300-fold increase reduced to 80-fold). For both midostaurin (850 nM) and lestaurtinib (400 nM), this results in a clinically relevant predicted in vivo IC₅₀. Alone, mifepristone had no effect upon FLT3-depedent cell growth.

Conclusions: Although potent inhibitors of FLT3 activity, as a class the STS-derivatives suffer from significant inhibition by plasma protein binding. In the case of lestaurtinib, our data provide a mechanism for its failure in previous clinical trials. Additionally, the shift in activity seen for midostaurin to a predicted in vivo IC₅₀ slightly higher than the reported steady state levels (3-5 µM) suggests that much of its activity observed in the clinical trial leading to its approval may be through non-FLT3 mechanisms. However, the ability of mifepristone to disinhibit TKIs in vitro suggests a novel approach by which the combination of TKIs with already-approved and well-tolerated drugs may restore TKI activity. We are currently undertaking a broader screen for other TKI-drug combinations with similar synergy.