Plasma Inhibitory Activity Assay Research Articles

First in Human (FIH) FLT3 and SYK Inhibitor HM43239 Shows Single Agent Activity in Patients (pts) with Relapsed or Refractory (R/R) FLT3 Mutated and Wild-Type Acute Myeloid Leukemia (AML)

First in Human (FIH) FLT3 and SYK Inhibitor HM43239 Shows Single Agent Activity in Patients (pts) with Relapsed or Refractory (R/R) FLT3 Mutated and Wild-Type Acute Myeloid Leukemia (AML)

Phase 1 first-in-human study of irreversible FLT3 inhibitor FF-10101-01 in relapsed or refractory acute myeloid leukemia.

7008 Background: FF-10101-01 is a selective and irreversible FMS-like tyrosine kinase 3 (FLT3) inhibitor with potent in vitro activity against FLT3-mutated AML. FF-10101-01 is highly active against FLT3 internal tandem duplication (ITD) mutations associated with high relapse and low survival/remission rates, as well as resistance-conferring D835 and F691 tyrosine kinase domain (TKD) and non-canonical FLT3 activating mutations. Here we report on a Phase 1 dose escalation trial examining the safety, efficacy, pharmacokinetics, and pharmacodynamics of FF-10101-01 in patients (pts) with relapsed/refractory primary or secondary AML. Methods: To determine the recommended Phase 2 dose, pts with or without a FLT3 mutation received FF-10101-01 orally once (QD) or twice (BID) daily until unacceptable toxicity was observed or pts had no further clinical benefit (1 cycle = 28 days). Composite complete remission (CRc) and partial remission (PR) rates were assessed. Inhibition of FLT3 phosphorylation was evaluated using a plasma inhibitory activity assay and was correlated with associated FF-10101-01 exposure. Results: Fifty-two pts [median age 61 (range, 21-84); 52% female; FLT3: ITD [22 (42%)], TKD [5 (10%)], ITD+TKD [1 (2%)], Wt [24 (46%)] received continuous dosing of FF-10101-01 at 10 - 225 mg QD or 50 - 100 mg BID. Median number of prior therapies was 3 (range, 0-6) and the majority [23/28 (82%)] of pts with known FLT3 mutations had received prior FLT3 inhibitors. The median duration on study was 5.7 (range, 0.1-36) weeks. FF-10101-01 was generally well-tolerated up to total daily doses of 150 mg. The most common treatment related adverse events included nausea [n = 18 (35%)] diarrhea [14 (27%), 2 Grade (Gr) 3/4], elevations in creatine kinase [CK; 14 (27%), 4 Gr 3/4], vomiting [10 (19%)] and increased AST [10 (19%), 2 Gr 3]. Grade 3/4 differentiation syndrome (n = 4, 8%) was observed at 75 - 150 mg/day. Dose-limiting cardiac toxicity (heart failure with reduced ejection fraction; Gr 3 increased troponin/CK) was observed at total daily doses ≥200 mg. The CRc rate was 13% (4/30 pts evaluable for response): 1 CR at 75 mg BID (FLT3-ITD); 1 CRp at 100 mg BID (Wt-FLT3); and 2 CRi’s at 50 mg BID, one that previously progressed on gilteritinib. The median time to overall response was ̃13.3 weeks. Four pts achieved a PR (≥50% decrease in BM blasts to 5 - 25% abnormal cells) at total daily doses of 50 - 150 mg; 2 had ITD mutations, and all had received prior FLT3 kinase inhibitors. At ≥75 mg BID, trough plasma concentrations were > 90 ng/ml and associated with > 90% p-FLT3 inhibition maintained over the dosing interval. Conclusions: The FF-10101-01 FLT3 inhibitor has shown activity in pts with refractory/relapsed AML, including those with activating FLT3-ITD mutations resistant to gilteritinib and other FLT3 kinase inhibitors. Doses of 50-75 mg BID were well tolerated and resulted in sustained FLT3 inhibition. Clinical trial information: NCT03194685.

A Novel Combination Regimen of BET and FLT3 Inhibition for FLT3-ITD Acute Myeloid Leukemia

Background: Acute myeloid leukemia patients with FLT3-ITD mutations have a high risk of relapse and death. FLT3 tyrosine kinase inhibitors such as quizartinib and gilteritinib improve overall survival in relapsed patients, but their efficacy is limited and most such patients die of the disease. This is because even with potent FLT3 inhibition, the disease persists within the bone marrow microenvironment, mainly due to bone marrow stroma activating parallel signaling pathways that maintain pro-survival factors. BET inhibitors suppress pro-survival factors such as c-Myc and Bcl2, but these drugs thus far have shown only limited single-agent clinical potential. PLX51107 is a novel BET inhibitor designed to inhibit BET activity in intermittent daily fashion to allow for greater tolerability. We investigated whether the addition of PLX51107 to potent FLT3 inhibition with quizartinib could overcome the protective effect of the bone marrow stroma through inhibition of c-Myc expression. Methods: We developed a plasma inhibitory activity assay for c-Myc (c-Myc PIA) to assess in vivo efficacy of the PLX51107 in patients and to identify c-Myc-inhibitory doses of the drug. We tested PLX51107 alone and in combination with quizartinib in murine models of AML and against primary FLT3-ITD AML cells co-cultured with human bone marrow stroma. In addition, we analyzed gene expression patterns in the treatment models to explore the basis of any observed synergistic cytotoxic effect. Results: In a murine xenograft model of AML using MV4-11 cells, PLX51107 alone induced suppression of tumor growth in association with a 90% decrease in c-Myc gene expression. The combination of PLX51107 and quizartinib induced complete tumor regression in 5 out of 7 animals after 14 days of treatment (Figure 1). Animals treated with a 5-day course of quizartinib alone displayed tumor regression persisting until day 26 of treatment, while the addition of PLX51107 resulted in tumor regression until day 39. In patients treated with a dose of 120 mg/day of PLX51107, the c-Myc PIA demonstrated a robust suppression of c-Myc expression for roughly 6 hours, returning to baseline between 7 and 9 hours post-treatment (Figure 2). The mean plasma concentration to achieve this inhibition was 3.3 uM, which, accounting for the difference in protein drug binding between plasma and culture medium with 10% FBS, corresponded to a concentration of 250 nM PLX51107 in culture medium. With this same concentration and schedule (i.e., at concentrations and exposure times equivalent to what human patients would experience taking 120 mg daily of PLX51107 and 60 mg daily of quizartinib), the combination induced synergistic cytotoxicity in a series of 10 different FLT3-ITD AML patient blast samples co-cultured with bone marrow stroma (Figure 3). C-Myc RNA and protein were directly suppressed in these primary samples, and ingenuity pathway analysis of RNA expression confirmed that c-Myc associated genes displayed the highest level of down-regulation. Conclusions: These studies suggest that combination therapy with approximately 120 mg/day PLX51107 and 60 mg/day quizartinib will be a more effective therapy for relapsed FLT3-ITD AML than 60 mg/day of quizartinib alone. The combination of FLT3 inhibition and BET inhibition may represent an attractive therapeutic option for FLT3-ITD AML. Disclosures Hizukuri: Daiichi Sankyo Co, Ltd: Employment. Severson:Plexxikon Inc.: Employment. Powell:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Ma:Plexxikon Inc: Employment. Narahara:Daiichi Sankyo Co, Ltd: Employment. Sumi:Daiichi Sankyo, Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Levis:FUJIFILM: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Menarini: Consultancy, Honoraria.

A Phase II Study of Midostaurin and 5-Azacitidine for Untreated Elderly and Unfit Patients With FLT3 Wild-type Acute Myelogenous Leukemia

BackgroundMidostaurin, a multikinase inhibitor, is approved for treatment of FLT3-mutant acute myeloid leukemia (AML). A phase I study established that midostaurin 75 mg orally twice daily for 14 days with standard dose azacitidine was safe and tolerable in elderly patients with AML. Herein, we report the phase II expansion cohort of previously untreated elderly or unfit patients with AML. Patients and MethodsPrimary objectives were to further describe the toxicity profile and determine the response rate in untreated patients with AML. Patients received midostaurin 75 mg orally twice daily on days 8 to 21 in combination with intravenous azacitidine at 75 mg/m2 on days 1 to 7. Plasma inhibitory activity assay for FLT3 was performed pretreatment and on day 8 and day 15 of each cycle. ResultsTwenty-six patients (median age, 74 years; range, 59-85 years) with FLT3 wild-type AML were accrued. Patients received a median of 2 cycles of therapy (range, 1-10 cycles). Seven (29%) of 24 evaluable patients achieved a clinical response (4 complete response; 1 complete response with incomplete count recovery; and 2 partial response). The median overall survival was 244 days (95% confidence interval, 203-467 days). Hematologic, infectious, and gastrointestinal toxicities were comparable to similar studies. Peripheral blood FLT3 wild-type phosphorylation declined to 8% to 55% of pretreatment by day 15 of cycle 1 (7 patients) and declined with subsequent cycles (< 10% baseline) in 2 patients who were analyzed after cycle 3. ConclusionMultiple cycles of azacitidine and midostaurin were not well-tolerated, but persistent inhibition of FLT3 wild-type phosphorylation suggest intermittent dosing of midostaurin should be considered in future low-intensity regimens for FLT3-mutant AML.

A Prospective Study of Peritransplant Sorafenib for Patients with FLT3-ITD Acute Myeloid Leukemia Undergoing Allogeneic Transplantation

FLT3-ITD-mutated acute myeloid leukemia (AML) remains a therapeutic challenge. FLT3 inhibition in the setting of minimal residual disease and a new immune system via allogeneic transplantation offers a promise of improved survival for these patients. We performed a prospective study of patients with FLT3-ITD AML undergoing allogeneic transplant that was conducted to evaluate the safety, tolerability, and outcome of sorafenib administered peritransplant. Sorafenib dosing was individualized, starting at 200 mg twice a day (BID), and titrated based on tolerability or toxicities until a tolerable dose was identified. Forty-four patients, with a median age of 52 years, undergoing allogeneic transplant were started on sorafenib in the peritransplant period (21 pretransplant). The median duration of post-transplant follow-up was 27.6 months (range, 5.2 to 60.4). Overall survival was 76% at both 24 and 36 months. Event-free survival at 24 and 36 months was 74% and 64%, respectively. Ten patients died in the post-transplant period, with 6 deaths due to relapsed leukemia and 4 from transplant-associated toxicity. Tolerable doses ranged from 200 mg every other day to 400 mg BID with similar exposure. Correlative studies evaluating FLT3 inhibition via a plasma inhibitory activity assay showed consistent inhibition of FLT3 at all tolerability-determined dosing levels. Sorafenib is well tolerated in the peritransplant setting irrespective of the conditioning intensity or the donor source. Our findings indicate that sorafenib dosing can be individualized in the post-transplantation setting according to patient tolerability. This approach results in effective in vivo FLT3 inhibition and yields encouraging survival results.

Abstract 3727: Plasma inhibitory activity (PIA) is a possible pharmacodynamic marker for clinical development of a next generation pan-TRK inhibitor, ONO-5390556

Abstract Background: Colorectal cancer and Mammary analogue secretory carcinoma harboring Neurotrophic tyrosine receptor kinase (NTRK/TRK) gene rearrangements developed resistance to Entrectinib, ALK/ROS/TRK inhibitor. We previously demonstrated that the next generation pan-TRK inhibitor,ONO-5390556 may potently overcome Entrectinib-resistance mutations. Furthermore, inhibition of Trk phosphorylation in tumors has excellent correlation with the in vivo anti-tumor effect by ONO-5390556 (AACR2016, Kozaki et al). Purpose: Accurate measurement of target inhibition in a phase 1 clinical trial is critical to informing selection of appropriate doses for ONO-5390556 in more advanced clinical trials. Plasma inhibitory activity (PIA) assay has recently been performed as a surrogate assay in clinical trials of FLT3, MET and ALK kinase inhibitors. The assay employs the incubation of Tyrosine Kinase expressing cell lines in aliquots of plasma collected at various time points from patients treated with TKI. Herein, we report the preclinical evaluation of PIA assay in accordance with an inhibition of TRK phosphorylation in tumors. Methods: KM12 cells, human colorectal cancer cell lines expressing TPM3-TRKA, were implanted subcutaneously into nude mice. Mice were randomized when the mean tumor volume was 150-600 mm3. Tumors and blood were collected from mice, 2, 4, 7 and 24 hours after the single treatment of ONO-5390556 with the doses of 0.06 and 0.6 mg/kg. The collected tumors were disrupted and phosphorylated TrkA in tumors was detected by Electrochemiluminescence (ECL). In a PIA assay, KM12 was incubated in plasma from the collected blood for 4 hours and phosphorylated TrkA in the cells was detected by ECL.. Results: Treatment with ONO-5390556 at doses of 0.06, 0.6 mg/kg resulted in a significant inhibition of Trk phosphorylation in tumors up to 24 hours. Compared to inhibition of P-TRK in tumors, the inhibition of P-TRK in PIA sustained until 7 hours but rapidly decreased at 24 hours after administration. The levels of both tumors and PIA showed in a dose-dependent inhibition and an excellent correlation until 7 hours. Inhibition of TRK phosphorylation (PIA/Tumor, %)0.06 mg/kg (2,4,7,24h) : 56.3/41.4 (PIA/Tumor, %), 51.9/53.5, 43.0/66.5, -5.5/51.30.6 mg/kg (2,4,7,24h) : 90.2/84.3, 88.4/93.0, 87.4/95.7, 8.3/83.3Conclusion: We have validated the PIA assay that correlates with the inhibition of P-TRK in tumors. Our results demonstrated that the potential utility of PIA as a PD marker may contribute to determining the effective dose of ONO-5390556 in the clinical development. Citation Format: Ryohei Kozaki, Ryu Fujikawa, Hikaru Kato, Natsuka Goto, Toshio Yoshizawa. Plasma inhibitory activity (PIA) is a possible pharmacodynamic marker for clinical development of a next generation pan-TRK inhibitor, ONO-5390556 [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 3727. doi:10.1158/1538-7445.AM2017-3727

A randomized assessment of adding the kinase inhibitor lestaurtinib to first-line chemotherapy for FLT3-mutated AML

AbstractThe clinical benefit of adding FMS-like tyrosine kinase-3 (FLT3)-directed small molecule therapy to standard first-line treatment of acute myeloid leukemia (AML) has not yet been established. As part of the UK AML15 and AML17 trials, patients with previously untreated AML and confirmed FLT3-activating mutations, mostly younger than 60 years, were randomly assigned either to receive oral lestaurtinib (CEP701) or not after each of 4 cycles of induction and consolidation chemotherapy. Lestaurtinib was commenced 2 days after completing chemotherapy and administered in cycles of up to 28 days. The trials ran consecutively. Primary endpoints were overall survival in AML15 and relapse-free survival in AML17; outcome data were meta-analyzed. Five hundred patients were randomly assigned between lestaurtinib and control: 74% had FLT3-internal tandem duplication mutations, 23% FLT3–tyrosine kinase domain point mutations, and 2% both types. No significant differences were seen in either 5-year overall survival (lestaurtinib 46% vs control 45%; hazard ratio, 0.90; 95% CI 0.70-1.15; P = .3) or 5-year relapse-free survival (40% vs 36%; hazard ratio, 0.88; 95% CI 0.69-1.12; P = .3). Exploratory subgroup analysis suggested survival benefit with lestaurtinib in patients receiving concomitant azole antifungal prophylaxis and gemtuzumab ozogamicin with the first course of chemotherapy. Correlative studies included analysis of in vivo FLT3 inhibition by plasma inhibitory activity assay and indicated improved overall survival and significantly reduced rates of relapse in lestaurtinib-treated patients who achieved sustained greater than 85% FLT3 inhibition. In conclusion, combining lestaurtinib with intensive chemotherapy proved feasible in younger patients with newly diagnosed FLT3-mutated AML, but yielded no overall clinical benefit. The improved clinical outcomes seen in patients achieving sustained FLT3 inhibition encourage continued evaluation of FLT3-directed therapy alongside front-line AML treatment. The UK AML15 and AML17 trials are registered at www.isrctn.com/ISRCTN17161961 and www.isrctn.com/ISRCTN55675535 respectively.

Signaling Adaptation to TKI Treatment Reactivates ERK Signaling in FLT3/ITD Leukemia

FMS-like tyrosine kinase-3 (FLT3) is one of the most commonly mutated genes in acute myeloid leukemia (AML). The internal tandem duplication (FLT3/ITD) is an established driver mutation in AML and results in constitutive activation of the receptor, activating downstream signaling pathways including STAT5, PI3K/AKT, and RAF/MEK/ERK. The use of FLT3 tyrosine kinase inhibitors (TKIs) for the treatment of FLT3/ITD AML has been explored as a promising strategy for over a decade, but clinical responses have remained limited.In some cancers driven by mutated oncogenes, small molecule inhibition of the target is known to result in reactivation of downstream signaling pathways. This response has been observed in the context of PI3K/AKT/mTOR and BRAF inhibition, but it remains unclear whether this phenomenon extends to cancers driven by receptor tyrosine kinase (RTK) activation, such as FLT3/ITD AML. We hypothesized that FLT3/ITD leukemia cells exhibit mechanisms of intrinsic signaling adaptation to FLT3 TKI treatment that are associated with an incomplete biologic response.To test this, we treated FLT3/ITD AML cell lines (Molm14 and MV4;11) with FLT3 TKIs for up to 48 hours. We observed a rebound in ERK phosphorylation beginning as early as 6 hours and continuing for the duration of treatment, while no such rebound was observed in the phosphorylation of downstream targets AKT or STAT5. When these cells were treated with inhibitors of both FLT3 and MEK in combination, little to no pERK rebound was observed, and the anti-leukemia effects were more pronounced compared to either drug alone, both in vitro and in vivo. In vitro, the addition of low dose MEK inhibitor to FLT3 TKI treatment synergistically increased cell death and decreased cell proliferation. In a xenograft transplant model of leukemia, the addition of low-dose MEK inhibitor to FLT3 TKI treatment resulted in a significant reduction of both peripheral blood and bone marrow blasts (p < 0.05).We next sought to determine whether rebound in ERK signaling following FLT3 inhibition was likely to occur in patients as well. We first examined whether primary leukemic cells from patients with FLT3/ITD AML also exhibit the same phenomenon observed in FLT3/ITD cell lines. After 24 hours of sorafenib treatment, the majority of samples revealed a similar rebound in ERK phosphorylation, despite sustained inhibition of phospho-STAT5. As observed with the cell lines, the addition of a MEK inhibitor to sorafenib treatment resulted in sustained pERK inhibition after 24 hours. Next, we explored whether signaling adaptation observed in culture conditions would be recapitulated by the concentrations of sorafenib present in human plasma from patients being treated with this FLT3 TKI. To replicate these conditions, we cultured Molm14 cells in plasma samples obtained from AML patients treated with sorafenib for a prolonged period. Using a variant of the plasma inhibitory activity assay, Molm14 cells were treated in either normal human plasma for one hour or patient plasma for either one or 24 hours. For all plasma samples, profound pERK rebound was observed by 24 hours after treatment, despite maximal inhibition at one hour and persistent inhibition of FLT3 and STAT5 phosphorylation over the duration.Finally, we sought to determine whether TKI-mediated rebound in ERK activity was restricted to FLT3/ITD AML or may also occur in the context of other RTK-driven cancers, such as those driven by EGFR mutation or HER2 amplification. We treated an EGFR mutant lung cancer (PC-9) and a HER2 amplified breast cancer (SKBR3) cell line with the EGFR/HER2 inhibitor lapatinib. As was seen for FLT3/ITD driven AML, pERK rebound was observed following 24 hours of TKI treatment despite persistent inhibition of the receptor and downstream target AKT. Again, the rebound could be overcome with the addition of low concentrations of MEK inhibitor.Together, these studies reveal that FLT3/ITD and other RTK driven cancer cells demonstrate an adaptive feedback mechanism capable of reactivating ERK signaling in response to upstream target inhibition. This adaptation limits the efficacy of TKI treatment and can be abrogated by the addition of a MEK inhibitor. Our data suggest that the addition of low-dose MEK inhibitor to TKI treatment as a means to overcome signaling adaptation may improve outcomes for patients with FLT3/ITD AML and possibly other RTK-driven cancer types. DisclosuresLevis:Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria.

Differential Inhibition of FLT3-ITD, FLT3-WT, and KIT By Quizartinib As Assessed By Modified PIA Assay

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

Pharmacodynamic Analysis Of The Inhibitory Potency Of The Tyrosine Kinase Inhibitor Midostaurin In Combination With Intensive Chemotherapy Including Allogeneic Hematopoietic Stem Cell Transplantation Followed By Maintenance Therapy In FLT3-ITD Positive Acute Myeloid Leukemia In The Ongoing AMLSG 16-10 Trial

BackgroundActivating mutations in the receptor tyrosine kinase FLT3 occur in roughly 30% of acute myeloid leukemia (AML) patients (pts), implicating FLT3 as a potential target for kinase inhibitor therapy. The multi-targeted kinase inhibitor midostaurin (PKC412) shows potent activity against FLT3 as a single agent but also in combination with intensive chemotherapy. Besides its mere presence, the allelic ratio as well as ITD insertion site within the FLT3 gene had been reported as prognostic factors in FLT3-ITD positive AML. Furthermore, pharmacokinetic analyses revealed clinically important interactions between potent CYP3A4 inhibitors, such as azoles, and midostaurin. AimsTo evaluate the pharmacodynamic activity of midostaurin measured as inhibition of the degree of phosphorylated FLT3 (pFLT3) in correlation to co-medication and outcome data. MethodsThe study includes intensively treated adults (age 18-70 years) with newly diagnosed FLT3-ITD positive AML enrolled in the ongoing single-arm phase-II AMLSG 16-10 trial (NCT: NCT01477606). Pts with acute promyelocytic leukemia are not eligible. The presence of FLT3-ITD is analyzed by Genescan-based fragment-length analysis (allelic ratio >0.05 required to be FLT3-ITD positive). Induction therapy consists of daunorubicin (60 mg/m², d1-3) and cytarabine (200 mg/m², continuously, d1-7); midostaurin 50 mg twice daily is applied from day 8 onwards until 48h before start of the next treatment cycle. For consolidation therapy, pts proceed to allogeneic hematopoietic stem cell transplantation (HSCT) as first priority; if allogeneic HSCT is not possible pts receive three cycles of age-adapted high-dose cytarabine in combination with midostaurin from day 6 onwards. In all pts maintenance therapy for one year is intended. A total sample size of n=142 is planned to show an improvement in event-free survival from 25% after 2 years to 37.5%. Plasma inhibitory activity assay (PIA) for pFLT3 is performed as previously described (Levis MJ, et al. Blood 2006; 108:3477-83). For PIA, measured time points include day 15 of induction therapy, the end of each treatment cycle and every three months during maintenance therapy. ResultsTo date, 72 pts (median age, 54.5 years; range, 29-69 years) have been included and PIA was performed so far in 37 pts during induction therapy. Median pFLT3 inhibition after one week of midostaurin intake measured on day 15 of cycle 1 (C1D15) was 57.5% (range, 14.2-93.7%) with 2 of 31 pts showing inhibition >85%. At the end of the first induction cycle (C1end), median inhibition was 60.3% (range, 0-99.8%); here, 6 of 37 pts had an inhibition >85%. Co-medication with azoles was present in 7 of 23 pts at C1D15 and 13 of 28 pts at C1end. There was no significant difference in pFLT3 inhibition either on C1D15 (p=0.79) or at C1end (p=0.70) between pts on (median pFLT3 inhibition: 52.5%) or off (median pFLT3 inhibition 57.5%) azoles. Response data were available in 56 pts: complete remission (CR) was achieved in 78.5%; rates of early death and refractory disease (RD) were 9% and 12.5%, respectively. In first analyses, there was no difference in pFLT3 inhibition in pts achieving CR (n=30) as compared to those with RD (n=3; p=0.99). In contrast to our previously published data from three historical trials without a FLT3 inhibitor which showed that high allelic ratio was associated with low CR rates (Kayser S, et al. Blood 2009;114:2386-92), in the current trial CR rates remained high (81.5%) despite of a high allelic ratio above the median (>0.58). In addition, we did not see a negative prognostic impact of ITD insertion site within the tyrosine kinase domain of the FLT3 gene (p=0.99). Analyses are currently ongoing, measurement of FLT3 ligand levels and evaluation of pharmacokinetics of midostaurin are also intended. ConclusionsThe addition of 50 mg midostaurin twice daily to intensive induction therapy resulted in a moderate pFLT3 inhibition during induction therapy. Nonetheless, CR rates are promising, especially in pts with unfavorable FLT3-ITD characteristics. Concomitant azoles do not appear to significantly influence pFLT3 inhibitory activity of midostaurin. Disclosures:Levis:Ambit Biosciences: Consultancy. Schlenk:Ambit: Honoraria; Chugai: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Amgen: Research Funding.

Safety and biological activity of the FLT3 inhibitor lestaurtinib in infant MLL-rearranged (MLL-r) ALL: Children's Oncology Group protocol AALL0631.

9548 Background: MLL-r infant ALL overexpresses activated FLT3. Lestaurtinib potentiates chemotherapy(chemo)-induced cytotoxicity in MLL-r ALL. AALL0631 therapy is stratified based on age and MLL status: high risk (HR) &lt;90 days, MLL-r; intermediate risk (IR) ≥ 90 days, MLL-r; standard risk any age, not MLL-r. IR and HR patients (pts) get intensive chemo and are eligible for lestaurtinib. A safety/activity (S/A) phase to determine a safe and biologically active dose of lestaurtinib is followed by an efficacy phase randomizing MLL-r patients to chemo +/- lestaurtinib. The S/A phase proceeds independently for IR &amp; HR pts, as optimal lestaurtinib dosing may differ between neonates and older infants. We previously reported successful completion of the S/A phase for IR pts; here, we report results of the S/A phase for HR pts. Methods: Lestaurtinib-related dose limiting toxicities (DLTs) were defined as grade ≥ 3 non-heme toxicities excluding those expected with chemo alone (e.g., F&amp;N/infection); a dose of lestaurtinib was “safe” if 0 or 1 DLTs were seen in 5 evaluable pts. FLT3 inhibition was assessed using a plasma inhibitory activity (PIA) assay that measures % inhibition of phospho-FLT3 at 5 trough time points relative to pre-treatment baseline. A dose of lestaurtinib was “biologically active” if PIA was &gt; 90% for the majority (≥3 of 5) of troughs in at least 3 of 5 pts. Results: 11 eligible HR pts received lestaurtinib, 6 at dose level 1 (DL1, 3.5 mg/kg/day) and 5 at DL2 (4.25 mg/kg/day). There were no DLTs in 5 evaluable pts at DL1. The inevaluable pt discontinued lestaurtinib due to parental refusal, not toxicity. There was 1 DLT in 5 evaluable pts at DL2. The DLT was grade 4 intestinal perforation secondary to severe typhlitis during neutropenia following reinduction, possibly related to lestaurtinib. The pt was taken off protocol and chemo and died of progressive disease. By PIA assay, 2 of 5 pts treated at DL1, and 4 of 5 at DL2 demonstrated FLT3 inhibitory biologic activity. Conclusions: DL2 is safe and biologically active in HR pts. The efficacy phase of AALL0631 is expanding such that HR pts will join IR pts in being randomized (1:1) post-induction to chemo +/- lestaurtinib at DL2.

Adaptation of the plasma inhibitory activity assay to detect Aurora, ABL and FLT3 kinase inhibition by AT9283 in pediatric leukemia

Non-invasive assessment of biomarker modulation is important for evaluating targeted therapeutics, particularly in pediatrics. The plasma inhibitory activity (PIA) assay is used clinically to assess FLT3 inhibition ex vivo and guide dosing. AT9283 is a novel Aurora kinase inhibitor with secondary activity against FLT3 and ABL. We adapted the PIA assay to simultaneously detect inhibition of Aurora and FLT3 in AML, and Aurora and ABL in CML by AT9283. Furthermore, we optimized the assay for children, where limited blood volumes are available for pharmacodynamic studies. Simultaneously detecting multiple kinase inhibition may identify important mechanisms of action for novel anti-leukemic drugs.

Adaptation and Validation of the Plasma Inhibitory Activity (PIA) Assay to Detect Inhibition of Aurora, ABL and FLT3 Kinases by AT9283 In Children and Adolescents with Leukaemia

AbstractAbstract 1818Non-invasive assessment of biomarker modulation is critically important when conducting early phase trials of targeted therapeutics, particularly in children. Measuring target modulation using primary leukaemic blasts is challenging, since obtaining bone marrow is invasive and invariably requires a general anaesthetic in children. Furthermore, in many cases only a limited number of circulating blasts can be detected in peripheral blood and these may be rapidly cleared upon commencement of effective therapy. For these two reasons, pharmacodynamic (PD) assays using surrogate biological material can be invaluable. The plasma inhibitory activity (PIA) assay was initially developed for FLT3 inhibitors and is currently used in clinical trials to assess ex vivo inhibition of FLT3 kinase and guide optimal dosing (Levis M et al, Blood 2006). Briefly, the assay involves incubating reference leukaemic cell lines in plasma from patients treated with FLT3 inhibitors then assessing the degree of FLT3 inhibition by western blotting. Inhibition of FLT3 in leukemia cell lines has been shown to correlate with inhibition in primary leukaemic blasts. Aurora kinase inhibitors are emerging as promising new agents with activity in leukaemia (Moore AS et al, Leukemia 2010). AT9283 is a novel Aurora kinase inhibitor with activity against the secondary kinase targets FLT3 and ABL (Howard S et al, J Med Chem 2009). We therefore hypothesised that the PIA assay would be applicable as an ex vivo PD assay to simultaneously detect inhibition of these target kinases in leukaemic cell lines: Aurora with FLT3 and Aurora with ABL. Furthermore, we hypothesised that the assay could be adapted to paediatric patients, where limited volumes of blood are available for PD studies. Here we report the validation of the PIA assay for use with AT9283 using leukaemic cell lines incubated in human plasma spiked with clinically relevant concentrations of AT9283 known to inhibit Aurora kinase in primary leukaemic blasts. The PIA assay proved to be a robust means of semi-quantitatively detecting concentration-dependent inhibition of Aurora kinase in the FLT3-ITD positive AML cell line MOLM-13 and the CML cell line K-562. The PIA assay was also effective at qualitatively detecting the inhibition of FLT3 signaling in MOLM-13 cells and ABL signaling in K-562 cells. Of importance to paediatric patients, the assay was successfully performed using 0.5 mL of plasma, half the volume described in the original PIA assay for FLT3 inhibitors. It will be used to assess target kinase modulation in a phase I trial of AT9283 in children and adolescents with relapsed and refractory acute leukaemia (EudraCT No. 2009–016952-36). In conclusion, the PIA assay is applicable not only to FLT3 inhibitors, but also Aurora kinase inhibitors and potentially, other multi-kinase inhibitors. By simultaneously detecting multiple kinase inhibition, the PIA assay may help delineate important mechanisms of action for novel anti-leukaemic drugs. Disclosures:Podesta:The Institute of Cancer Research: Employment, The Institute of Cancer Research (ICR) has a commercial interest in drug development programs (www.icr.ac.uk). Authors employed by ICR are subject to a Rewards to Inventors Scheme which may reward contributors to a program that is subsequently licensed. Squires:Astex Therapeutics Ltd: Employment. Linardopoulos: The Institute of Cancer Research: Employment, The Institute of Cancer Research (ICR) has a commercial interest in drug development programs (www.icr.ac.uk). Authors employed by ICR are subject to a Rewards to Inventors Scheme which may reward contributors to a program that is subsequently licensed. Pearson:The Institute of Cancer Research: Employment, The Institute of Cancer Research (ICR) has a commercial interest in drug development programs (www.icr.ac.uk). Authors employed by ICR are subject to a Rewards to Inventors Scheme which may reward contributors to a program that is subsequently licensed. Moore:The Institute of Cancer Research: Employment, The Institute of Cancer Research (ICR) has a commercial interest in drug development programs (www.icr.ac.uk). Authors employed by ICR are subject to a Rewards to Inventors Scheme which may reward contributors to a program that is subsequently licensed.

Increased Plasma FLT3 Ligand Levels Following Chemotherapy May Interfere with the Clinical Efficacy of FLT3 Inhibitors.

Abstract 937FLT3 mutations are relatively common in AML and are associated with a poor prognosis. A large number of different small molecule inhibitors of FLT3 tyrosine kinase activity have been identified and many are being studied in clinical trials. There are currently several trials in the planning stage or actively accruing in which the FLT3 inhibitor is administered along with a chemotherapy regimen. While FLT3 inhibitors have some degree of clinical activity when given as monotherapy, their efficacy in the setting of chemotherapy is not well understood. We examined in vivo FLT3 inhibition in AML patients treated with chemotherapy followed by the FLT3 inhibitor lestaurtinib (CEP-701), comparing newly-diagnosed AML patients (enrolled on the MRC AML15 trial) with relapsed patients enrolled on the Cephalon 204 trial. We noted that in vivo FLT3 inhibition by lestaurtinib (as measured by a plasma inhibitory activity assay) was less effective in the relapsed patients (204 trial) compared with the newly-diagnosed patients (AML15 trial). It has been noted by others that FLT3 ligand (FL) levels increase dramatically following myelosuppressive therapy. We hypothesized that FL could be interfering with the ability of lestaurtinib to inhibit FLT3 autophosphorylation, particularly in the patients enrolled on the 204 trial. Using an ELISA assay, we measured plasma FL levels in the two patient populations at Day 15 following the start of intensive chemotherapy (34 patients from the AML15 trial, 78 patients from the 204 trial). FL levels averaged 2.7 pg/mL in newly-diagnosed AML and 15.6 pg/mL in AML patients at first relapse (prior to therapy). FL levels rose to a mean of 402 pg/mL (range 3-3183 pg/mL) on day 15 of induction therapy for the AML15 trial (newly diagnosed) patients, while they rose to a mean of 1174 pg/mL (range 18.6-3818) in the 204 trial (relapsed) patients. In many patients on the 204 (relapsed) trial, FL levels remained elevated at day 42 of therapy (mean 641 pg/mL, range 4.4-5767) pg/mL). Following successive courses of consolidation chemotherapy, FL levels continued to rise higher with each course of chemotherapy in patients on AML15, with a mean of 3151 pg/mL after the 4th course. On comparing individual samples from both trials, we noted that samples with similar levels of drug but different levels of FL showed different degrees of FLT3 inhibition, with FL levels seeming to exert a negative influence on inhibition. To explore this phenomenon in vitro, we tested the influence of exogenous FL on FLT3 inhibition and cytotoxic effects exerted by lestaurtinib and 4 other FLT3 inhibitors- AC220, KW-2449, PKC-412, and sorafenib. In culture medium using FLT3 mutant cell lines (Molm14, TF/ITD), the IC50 for cytotoxic effect of all inhibitors shifted upwards 2-4-fold in the presence of exogenous FL at concentrations of 1000-3000 pg/mL (the levels we observed in patients following chemotherapy). A similar degree of shift was observed in immunoblot assays of FLT3 autophosphorylation using the same cell lines. In primary samples from AML patients that expressed predominantly the mutant allele (i.e., hemi- or homozygous FLT3/ITD), exogenous FL had similar effect as that seen in the cell lines: a modest effect on FLT3 autophosporylation, and a 2-4-fold shift upward of the cytotoxicity IC50 of FLT3 inhibitors. However, a somewhat different pattern was observed in samples from patients with heterozygous FLT3/ITD mutations (i.e., both mutant and wild type allele present). The addition of exogenous FL increased the degree of FLT3 autophosphorylation, and dramatically increased cell survival in culture medium. The enhanced survival was abrogated by FLT3 inhibition. These findings have important implications regarding trial design for FLT3 mutant AML. It is possible that any FLT3 inhibitor administered concomitantly with chemotherapy could be affected by this phenomenon, with only the most potent inhibitors being able to overcome the effects of FL in vivo. Furthermore, newly diagnosed FLT3/ITD patients (in whom the mutations are commonly heterozygous) might be predicted to respond differently to FLT3 inhibition according to the context in which the inhibitor is given- i.e., in the presence of absence of chemotherapy. Finally, we might speculate that FL is an important driver in FLT3 mutant AML, and may have a strong influence on prognosis. This hypothesis should be tested prospectively by examining FL levels in ingoing clinical trials in which FLT3 mutant AML patients are enrolled. Disclosures:Sato:Kyowa Hakko-Kirin: Employment. Knapper:Cephalon: Served on scientific advisory board. Levis:Cephalon: Member, clinical advisory board.

Clinical Pharmacokinetics and FLT3 Phosphorylation of AC220, a Highly Potent and Selective Inhibitor of FLT3

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 >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.

A Phase I Dose Escalation Study of KW-2449, An Oral Multi-Kinase Inhibitor against FLT3, Abl, FGFR1 and Aurora in Patients with Relapsed/Refractory AML, ALL and MDS or Resistant/Intolerant CML

Background: Activating mutations of FMS-like tyrosine kinase 3 (FLT3) occur in 30% of patients with de novo AML and confer a poor prognosis. KW-2449 is an oral multi-kinase inhibitor which is highly potent against mutant FLT3 (IC50=0 1–7 nmol/L) and other tyrosine kinases including FGFR1, TrkA, Abl (including T315I) and Aurora A serine threonine kinase. Based on the activity of KW-2449 and its metabolite (M1) in both in vitro and in vivo preclinical leukemia models, KW-2449 was evaluated in patients with leukemia and MDS in this first-in-man study. M1 is formed via monoamine oxidase-B and aldehyde oxidase mediated oxidation of KW-2449.Methods: The objectives were to assess the safety, tolerability, pharmacokinetic (PK) and pharmacodynamic effects of KW-2449 in patients with refractory/relapsed AML, ALL and MDS, or resistant/intolerant CML. A range of daily doses of KW-2449 (12.5–250 mg twice daily, i.e. 25–500 mg/day) on 2 treatment schedules (14 or 28 days) with a recovery period of 7–28 days between cycles were evaluated. Dose limiting toxicity (DLT) and maximum tolerated dose (MTD) were assessed for the 1st cycle. The 28-day schedule was later eliminated. The plasma concentration of KW-2449 and M1 were analyzed by LC-MS/MS method. A plasma inhibitory activity (PIA) assay [Blood 108(10) 3477–83] for P-FLT3 and P-STAT5 was used to measure FLT3 inhibition.Results: 37 patients aged 26–88 years (16 male) were treated at 7 dose levels: 25, 50, 100, 200, 300, 400, and 500 mg daily. Thirty-one patients had AML, 5 CML and 1 ALL. The mean duration of therapy was 2 cycles in AML patients and 4 cycles in CML patients. KW-2449 was rapidly absorbed and metabolized to M1. The half-lives for KW-2449 and M1 were not dose-related and ranged from 2.4 to 4.9 hours and 2.6 to 6.6 hours, respectively. Administration of KW-2449 in a BID regimen led to minor accumulation and was consistent with the short half-life. The PIA assay demonstrated the near complete down-regulation of P-FLT3 and P-STAT5 2 hours post-dose at a dosing level of 400 mg daily. The extent of inhibition was lower at 8 hours and generally absent at 12 hours post-dose. The most frequently reported adverse events (AEs; any grade, regardless of causality) were nausea (70.3%), vomiting (48.6%), fatigue (45.9%), diarrhea (32.4%), dyspnea (29.7%), febrile neutropenia (29.7%), pain in extremity (29.7%), and arthralgia (27.0%). Febrile neutropenia (24.3%), pneumonia (10.8%), and thrombocytopenia (10.8%) were the most frequently reported Grade 3/4 AEs. DLTs occurred in 2 patients: Grade 3 atrial fibrillation (100 mg daily) and Grade 3 nausea and vomiting (500 mg daily). A total of 70 SAEs were reported in 27 patients including 11 on-study deaths; only atrial fibrillation and pleural effusion were considered possibly related to KW-2449. Eight of 31 patients with AML (26%) (FLT3 mutation: 5 positive and 3 negative) and 1 of 5 patients with CML (20%) exhibited a ³ 50% reduction in peripheral blasts and/or bone marrow blasts from baseline to the end of Cycle 1. One patient (500 mg daily) with AML exhibited a > 50% decrease in peripheral blasts, increased platelets, and ANC, and decreased WBC count. A patient with CML (Bcr-Abl T315I +) lost the mutant clone while on KW-2449 treatment.Conclusions: KW-2449 was safe and well tolerated at the dose levels evaluated. There were no complete or partial responses, but transient decreases in peripheral blood and bone marrow blasts were observed, justifying continued investigation of this agent. Sustained inhibition of P-STAT5 and P-FLT3 was not achieved at trough at the highest BID dose evaluated. TID and QID dosing schedules should be evaluated to accommodate the short t1/2 and to achieve sufficient target inhibition.

Incorporating a FLT3 inhibitor into AML therapy

7060 Background: Activating mutations of FLT3 occur in 30% of adults with AML and are associated with worse survival. Lestaurtinib (CEP-701), an oral FLT3 kinase inhibitor, has activity as monotherapy in relapsed/refractory AML patients with FLT3 mutations. Using the results of pre-clinical models, a trial was designed in which patients with AML and FLT3 mutations in first relapse were randomized 1:1 to receive chemotherapy alone or chemotherapy followed by treatment with lestaurtinib. The primary endpoint was complete remission (CR), with a target accrual of 220 patients. We present here correlative data from this trial. Methods: Efficacy of FLT3 inhibition was measured using the plasma inhibitory activity (PIA) assay for FLT3. This assay method has been previously published (Blood 108(10):3477–83). Prior to initiation of chemotherapy, leukemia cells of the patients randomized to lestaurtinib were tested for in vitro sensitivity to lestaurtinib using a cytotoxicity assay. Plasma samples were obtained at baseline and at trough time points on Days 15 and 42. PIA assay and in vitro cytotoxicity results were correlated with the clinical results from the first 40 patients enrolled. In addition, plasma levels of lestaurtinib and alpha-1 acid glycoprotein (AAG; the principle plasma binding protein for this drug) were measured. Results: Accrual is ongoing. Twenty-three of 35 patients treated with lestaurtinib achieved a PIA result of 85% or greater (i.e., successful FLT3 inhibition). Of the first 40 patients enrolled, all those who achieved a FLT3 PIA results of 85% or greater and whose pretreatment leukemia cells were sensitive in vitro to lestaurtinib achieved a clinical response. Conversely, patients with insensitive cells or inadequate PIA did not respond. Ten of 17 patients randomized to lestaurtinib showed response (5 CR, 3 CRp, 2 PR). Four of 17 patients randomized to receive chemotherapy alone responded (2 CR, 2 CRp). Conventional pharmacokinetic analysis showed no correlation whatsoever with response, due to the high degree to which lestaurtinib is bound to AAG. Conclusions: These results demonstrate that the PIA assay for FLT3 is able to accurately predict response to lestaurtinib, and provides important evidence that targeting FLT3 remains a valid therapeutic approach for AML with FLT3 activating mutations. No significant financial relationships to disclose.

Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors

AbstractWe have developed a useful surrogate assay for monitoring the efficacy of FLT3 inhibition in patients treated with oral FLT3 inhibitors. The plasma inhibitory activity (PIA) for FLT3 correlates with clinical activity in patients treated with CEP-701 and PKC412. Using the PIA assay, along with in vitro phosphorylation and cytotoxicity assays in leukemia cells, we compared PKC412 and its metabolite, CGP52421, with CEP-701. While both drugs could effectively inhibit FLT3 in vitro, CEP-701 was more cytotoxic to primary samples at comparable levels of FLT3 inhibition. PKC412 appears to be more selective than CEP-701 and therefore less effective at inducing cytotoxicity in primary acute myeloid leukemia (AML) samples in vitro. However, the PKC412 metabolite CGP52421 is less selective than its parent compound, PKC412, and is more cytotoxic against primary blast samples at comparable levels of FLT3 inhibition. The plasma inhibitory activity assay represents a useful correlative tool in the development of small-molecule inhibitors. Our application of this assay has revealed that the metabolite CGP52421 may contribute a significant portion of the antileukemia activity observed in patients receiving oral PKC412. Additionally, our results suggest that nonselectivity may constitute an important component of the cytotoxic effect of FLT3 inhibitors in FLT3-mutant AML.