Saturday, August 1, 2015

Drugs in Clinical Pipeline: AMG 925

AMG 925 [2-(2-((9-(trans-4-methylcyclohexyl)-9H-pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidin-2-yl)amino)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)-2-oxoethanol] is a potent, selective, and bioavailable FLT3/CDK4 dual kinase inhibitor. 

AMG 925 potently inhibited FLT3, CDK4, and CDK6 in kinase assays with IC50 in single-digit nanomolar range (IC50 = 2, 3, 8 nM, respectively). The selectivity of AMG 925 for CDK4 and FLT3 against CDK1 (IC50 ~ 2 uM), in which inhibition is highly cytotoxic, was greater than 500-fold. AMG 925 was discovered from a series of pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidine derivatives where AMG 925 emerged as a defining example after SAR analysis and also after using various cell-based assays [1, 2]. In binding studies, AMG 925 behaved as a type I kinase inhibitor and preferentially bound to the active DFG-in conformations. Thereby, maintaining activity against activation loop single point mutations.

Activating Fms-like tyrosine kinase 3 (FLT3) mutations are found in approximately 30% of patients with Acute Myeloid Leukemia (AML). Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. A critical role of the Cyclin-Dependent Kinase 4 (CDK4)-RB pathway in cancer development has been well established. CDK4-specific inhibitors are being developed for treating RB-positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve and prolong clinical responses. Considering the unique FLT3/CDK4 dual kinase inhibition feature, Amgen decided to proceed with AMG 925 into preclinical studies.

The activity of AMG 925 is as follows:

IC50 (FLT3 enzyme assay) = 0.002 ± 0.001 uM
IC50 (CDK6 enzyme assay) = 0.008 ± 0.002 uM
IC50 (CDK4 enzyme assay) = 0.003 ± 0.001 uM
IC50 (CDK1 enzyme assay) = 1.90 ± 0.51 uM
IC50 (CDK2 enzyme assay) = 0.375 ± 0.15 uM

Common Name: AMG 925
Synonyms:  AMG 925; AMG925; AMG-925
IUPAC Name: 2-(2-((9-(trans-4-methylcyclohexyl)-9H-pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidin-2-yl)amino)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl)-2-oxoethanol
CAS Number: 1401033-86-0
Mechanism of Action: Kinase Inhibitor; Dual-Kinase Inhibitor; FLT3 Inhibitor; CDK4 Inhibitor
Indication: Various Cancers; Acute Myeloid Leukemia
Development Stage: Investigational
Company: Amgen

FMS-like tyrosine kinase 3 (FLT3) belongs to the receptor tyrosine kinase class III family and is expressed at high levels in most clinical samples from AML and B-precursor acute lymphoblastic leukemia (ALL) patients. Activating mutations in FLT3 are found in approximately 30% of patients with AML. The majority of the activating mutations are internal tandem duplications (ITD) in the juxtamembrane region. FLT3 inhibitors are particularly attractive target for AML therapy but the responses are transient and resistance develops rapidly. The major resistance mechanism seems to be acquisition of secondary mutations in FLT3, which interfere with the ability of small-molecule inhibitors to bind to FLT3.

Cyclin-dependent kinase 4 (CDK4), which is downstream of FLT3 and other growth signaling pathways, is also upregulated in AML by overexpression of cyclin D through activation of tyrosine kinase growth signaling pathways and loss of p15 inhibitory function. As a key downstream effector of growth factor activation, CDK4 promotes G1-S transition of the cell cycle by phosphorylating the retinoblastoma protein (RB), a tumor-suppressor protein. A large body of evidence supports important involvement of the p16INK4a-CDK4–RB axis in cancer development. p15INK4b, a member of the INK4 family, has been reported to be downregulated in up to 60% of patients with AML, indicating an important role of CDK4 in AML.

Researchers believe that combined inhibition of two essential kinases (FLT3 and CDK4) by AMG 925 has potential to reduce development of drug resistance in patients with AML [1, 2].

Researchers believe that combined inhibition of two essential kinases (FLT3 and CDK4) by AMG 925 has potential to reduce development of drug resistance in patients with AML. Moreover, AMG 925 exhibited excellent binding affinities (1-4 nM) for all of the FLT3 mutants available in the KINOMEscan (Kd FLT3 WT, ITD, D835Y, D835H, K663Q, N841I = 4, 4, 1, 1, 4, 4, nM, respectively). This high affinity for mutated forms of FLT3, particularly those mutants known to confer pronounced resistance to FLT3-inhibition monotherapy, make AMG 925 very attractive for clinical development [1].

The activity of AMG 925 in various tumor cell lines demonstrated its ability to block FLT3 and CDK4. AMG 925 showed potent and broad antiproliferation activities against AML cell lines independent of the FLT3 mutation status while maintaining simultaneous inhibition of CDK4. AMG 925 inhibited pSTAT5 in MOLM13 and pRb in Colo205 with IC50s of 0.005 and 0.023 µM, respectively, indicating that the observed efficacy in vitro was consistent with FLT3 and CDK4 inhibition.

In a potential-defining cell-based experiment MOLM13, MOLM13SR, and U937 cells were treated with AMG 925, Palbocilib, and Sorafenib at 1 µM for 48 h, individually. The results are:

1. Sorafenib, a multikinase inhibitor with FLT3 activity, showed potent inhibitory activity toward the FLT3ITD mutant MOLM13 cell line but almost no activity against U937, a FLT3 wild-type AML cell line.

2. Palbociclib, a CDK4/6 inhibitor, arrested Rb positive tumor cells including MOLM13 in G1 stage.

3. AMG 925 induced apoptosis in MOLM13 (FLT3ITD AML cell lines) and MOLM13SR (FLT3ITD,D835Y mutant AML cell line), the latter being a mutant strain cultivated to exhibit marked resistance to sorafenib by continuously treating MOLM13 (FLT3ITD) cells with gradually increased concentration of sorafenib (from 1 to 1000 nM) over the course of several weeks.

Analogous experiments conducted with the concentration of AMG 925 ranging as high as 10 nM over treatment durations as long as 4 months failed to produce appreciable resistance to AMG 925 (IC50 = 0.028 µM), and sequencing analysis showed that no additional FLT3 mutations had been produced under these conditions [1, 3].

1. Li, Z.; et. al. Discovery of AMG 925, a FLT3 and CDK4 dual kinase inhibitor with preferential affinity for the activated state of FLT3. J Med Chem 2014, 57(8), 3430-3449.
2. Keegan, K.; et. al. Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor for treating acute myeloid leukemia. Mol Cancer Ther 2014, 13(4), 880-889.
3. Li, C.; et. al. AMG 925 is a dual FLT3/CDK4 inhibitor with the potential to overcome FLT3 inhibitor resistance in acute myeloid leukemia. Mol Cancer Ther 2015, 14(2), 375-383.