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 (K_d 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 FLT3^ITD 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 (FLT3^ITD AML cell lines) and MOLM13SR (FLT3^ITD,D835Y mutant AML cell line), the latter being a mutant strain cultivated to exhibit marked resistance to sorafenib by continuously treating MOLM13 (FLT3^ITD) 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].

References:

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.

Drugs in Clinical Pipeline: Abemaciclib

Abemaciclib [N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine] is an orally administered Cyclin Dependent Kinase (CDK) inhibitor, designed to block the growth of cancer cells by specifically inhibiting CDK-4 and -6. The G1 restriction point is critical for regulating the cell cycle and is controlled by the retinoblastoma protein (Rb) pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). This pathway is important because of its inactivation in a majority of human tumors. Transition through the restriction point requires phosphorylation of Rb by CDK4/6, which are highly validated cancer drug targets. 

In biochemical assays, Abemaciclib inhibits CDK4/cyclin D1 and CDK6/cyclin D1 with IC50 = 2 nM and 10 nM, respectively, and shows selectivity over closely related cell cycle kinases. Ki(ATP) constants were determined through kinetic studies, showing Ki(ATP) = 0.6 nM and 2.4 nM for CDK4/cyclin D1 and CDK6/cyclin D1, respectively, indicating Abemaciclib is a competitive ATP inhibitor [1]. 

Out of all these biochemical profiling activities, it is particularly important to observe approximately 2-3 orders of magnitude in measured IC50s in biochemical kinase selectivity against CDK1/cyclin B1, CDK2/cyclin E, and CDK7/Mat1/cyclin H. Complete lack of activity against other important cell-cycle related kinases for which inhibition could also lead to potentially confounding cell cycle arrest in G2/M such as Aurora A, B, and PLK1 was noted [1].

On Oct 2015, USFDA granted Breakthrough Therapy Designation to Abemaciclib for patients with refractory hormone-receptor-positive (HR+) advanced or metastatic breast cancer.  This designation is based on data from the breast cancer of the company’s Phase I trial, JPBA, which studied the efficacy and safety of Abemaciclib in advanced or metastatic breast cancer.

Common Name: Abemaciclib

Synonyms:  LY2835219; LY-2835219; LY 2835219

IUPAC Name: N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine

CAS Number: 1231929-97-7; 1231930-82-7 (mesylate)

Mechanism of Action: Kinase Inhibitor; Cyclin Dependent Kinase Inhibitor; CDK Inhibitor; CDK4 Inhibitor; CDK6 Inhibitor

Indication: Various Cancers

Development Stage: Phase I

Company: Eli Lilly

An integrated semi-mechanistic pharmacokinetic/pharmacodynamic model successfully described Abemaciclib-mediated CDK4/6 inhibition, cell-cycle arrest, and TGI in colo-205, and was validated in A375. The model also demonstrated that a chronic dosing strategy achieving minimum steady-state trough plasma concentrations of 200 ng/mL is required to maintain durable cell-cycle arrest. Quiescence and cell death can be induced by further increasing Abemaciclib plasma concentrations [2].

Abemaciclib inhibits CDK4 and CDK6 with low nanomolar potency, inhibits Rb phosphorylation resulting in a G1 arrest and inhibition of proliferation, and its activity is specific for Rb-proficient cells. In vivo target inhibition studies show Abemaciclib is a potent inhibitor of Rb phosphorylation, induces a complete cell cycle arrest and suppresses expression of several Rb-E2F-regulated proteins 24 hours after a single dose. 

References:

1. Gelbert, T. M.; et. al. Preclinical characterization of the CDK4/6 inhibitor LY2835219: in-vivo cell cycle-dependent/independent anti-tumor activities alone/in combination with gemcitabine. Invest New Drugs 2014, 32(5), 825-837.
2. Tate, S. C.; et. al. Semi-mechanistic pharmacokinetic/pharmacodynamic modeling of the antitumor activity of LY2835219, a new cyclin-dependent kinase 4/6 inhibitor, in mice bearing human tumor xenografts. Clin Cancer Res 2014, 20(14), 3763-3774.

Palbociclib

Common name: Palbociclib; PD-0332991; PD0332991; PD 0332991; Ibrance
Trademarks: Ibrance
Molecular Formula: C24H29N7O2
CAS Registry Number: 571190-30-2

CAS Name: 6-Acetyl-8-cyclopentyl-5-methyl-2-{[5-(1-piperazinyl)-2-pyridinyl]amino} pyrido[2,3-d]pyrimidin-7(8H)-one

Molecular Weight: 447.532
SMILES:CC1=C(C(=O)N(C2=NC(=NC=C12)NC3=NC=C(C=C3)N4CCNCC4)C5CCCC5)C(=O)C
InChI Key: AHJRHEGDXFFMBM-UHFFFAOYSA-N
InChI:InChI=1S/C24H29N7O2/c1-15-19-14-27-24(28-20-8-7-18(13-26-20)30-11-9-25-10-12-30)29-22(19)31(17-5-3-4-6-17)23(33)21(15)16(2)32/h7-8,13-14,17,25H,3-6,9-12H2,1-2H3,(H,26,27,28,29)
Activity: Breast Cancer Drug; Cancer Drug; CDK4 Inhibitor; CDK6 Inhibitor; Dual Kinase Inhibitor; Protein Kinase Inhibitor; Cyclin Dependent Kinase Inhibitor; Treatment of Metastatic Breast Cancer
Status: Launched 2015
Originator: Pfizer


Palbociclib is an inhibitor of cyclin-dependent kinase (CDK) 4 and 6. In vitro palbociclib reduced cellular proliferation of ER-positive breast cancer cell lines by blocking progression of cells from G1 into S phase of the cell cycle [1].

PD 0332991 is a highly specific inhibitor of cyclin-dependent kinase 4 (Cdk4) (IC50, 0.011 micromol/L) and Cdk6 (IC50, 0.016 micromol/L), having no activity against a panel of 36 additional protein kinases. It is a potent antiproliferative agent against retinoblastoma (Rb)-positive tumor cells in vitro, inducing an exclusive G1 arrest, with a concomitant reduction of phospho-Ser780/Ser795 on the Rb protein. Oral administration of PD 0332991 to mice bearing the Colo-205 human colon carcinoma produces marked tumor regression. Therapeutic doses of PD 0332991 cause elimination of phospho-Rb and the proliferative marker Ki-67 in tumor tissue and down-regulation of genes under the transcriptional control of E2F. The results indicate that inhibition of Cdk4/6 alone is sufficient to cause tumor regression and a net reduction in tumor burden in some tumors [2].

On February 3, 2015, the U. S. Food and Drug Administration granted accelerated approval to palbociclib (IBRANCE, Pfizer) for use in combination with letrozole for the treatment of postmenopausal women with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer as initial endocrine-based therapy for their metastatic disease.

The approval of palbociclib is based on a randomized, multicenter, open-label trial in postmenopausal women with ER-positive, HER2-negative, advanced (locally advanced or metastatic) breast cancer who had not received previous systemic treatment for advanced disease. The trial enrolled 165 patients randomly allocated to receive either palbociclib (125 mg orally daily for 21 consecutive days, followed by 7 days off treatment) plus letrozole (2.5 mg daily continuously throughout the 28-day cycle) or letrozole alone.

Among the 165 patients, 43% had received chemotherapy and 33% had received anti-hormonal therapy as a neoadjuvant or adjuvant treatment. Forty- nine percent of patients had no prior systemic therapy in the neoadjuvant or adjuvant setting. The majority of patients (98%) had metastatic disease; 48% had visceral disease, 75% had bone disease and 19% had bone only disease.

The major efficacy outcome measure was investigator-assessed progression-free survival (PFS) evaluated according to Response Evaluation Criteria in Solid Tumors Version 1.0 (RECIST). Median investigator-assessed PFS was 20.2 months (95% CI 13.8, 27.5) in the palbociclib plus letrozole arm and 10.2 months (95% CI 5.7, 12.6) in the letrozole alone arm [Hazard Ratio (HR) 0.488 (95% CI 0.319, 0.748)]. The treatment effect of the combination on PFS was also supported by a retrospective radiographic independent review [HR 0.621 (95% CI: 0.378, 1.019).]  Overall response rate in patients with measurable disease (investigator assessment) was higher in the palbociclib plus letrozole compared to the letrozole alone arm (55.4% versus 39.4%).

Most common adverse reactions (greater than or equal to 10%) were neutropenia, leukopenia, fatigue, anemia, upper respiratory infection, nausea, stomatitis, alopecia, diarrhea, thrombocytopenia, decreased appetite, vomiting, asthenia, peripheral neuropathy, and epistaxis. The most frequently reported serious adverse reactions in patients receiving palbociclib plus letrozole were pulmonary embolism (3 of 83; 4%) and diarrhea (2 of 83; 2%).

The recommended dose and schedule of palbociclib is 125 mg daily for 21 consecutive days followed by 7 days off treatment with letrozole 2.5 mg daily continuously throughout the 28-day cycle [1].

References:
1. Cadoo, K. A.; et. al. Palbociclib: an evidence-based review of its potential in the treatment of breast cancer. Breast Cancer (Dove Med Press) 2014, 6, 123-133. (activity)
2. Fry, D. W.; et. al. Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Mol Cancer Ther 2004, 3(11), 1427-1438.