Drugs in Clinical Pipeline: Pacritinib | Dual Kinase Inhibitor | JAK2 Inhibitor | FLT3 Inhibitor

Pacritinib [11-(2-pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7,26-triaza-tetracyclo(19.3.1.1(2,6).1(8,12))heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene] is a novel low-molecular weight pyrimidine-based macrocycle with potent inhibitory activities against Janus Kinase 2 (JAK2) and Fms-Like Tyrosine Kinase-3 (FLT3). 

Pacritinib shows a unique kinase profile with selective inhibition of Janus Kinase-2 with IC₅₀ values of 23 and 19 nM against JAK2^WT and JAK2^V617F respectively and an IC₅₀ value of 22 nM gainst FLT3. Within the JAK family, Pacritinib has IC₅₀ values of 50, 520 and 1280 nM for TYK, JAK3 and JAK2, respectively, showing high preference for JAK2. The rest of the evaluated kinases showed less than 30% inhibition when tested against 100 nM Pacritinib at ATP concentrations equivalent to its Michaelis constant (K_m) [1].

Pacritinib: 2D and 3D Structure

The activity of Pacritinib is as follows:

IC₅₀ (JAK1 enzyme assay) = 1280 ± 370 nM
IC₅₀ (JAK2 enzyme assay) = 23 ± 6 nM
IC₅₀ (JAK2^V617F enzyme assay) = 19 nM
IC₅₀ (JAK3 enzyme assay) = 520 ± 110 nM
IC₅₀ (TYK2 enzyme assay) = 50 ± 6 nM
IC₅₀ (FLT3 enzyme assay) = 22 ± 6 nM
IC₅₀ (FLT3^D835Y enzyme assay) = 6 nM

1H NMR (Estimated) for Pacritinib

References:

1. Hart, S.; et. al. SB1518, a novel macrocyclic pyrimidine-based JAK2 inhibitor for the treatment of myeloid and lymphoid malignancies. Leukemia 2011, 25(11), 1751-1759.

Drugs in Clinical Pipeline: Quizartinib

Quizartinib [(N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea] is a once-daily, orally administered, potent and selective inhibitor of FMS-like tyrosine kinase 3 (FLT3) kinase (K_d = 1.6 ± 0.7 nM).

Quizartinib is a novel compound expressly optimized as a FLT3 inhibitor for the treatment of Acute Myeloid Leukemia (AML). Quizartinib inhibits FLT3 with low nanomolar potency in cellular assays and is highly selective when screened against the majority of the human protein kinome [1, 2].

Quizartinib is currently in development for the treatment of relapsed or refractory FLT3-ITD positive and negative AML patients and as a maintenance therapy. Though patients of any age may be FLT3 positive, over 35% of AML patients over age 55 are estimated to harbour a genetic mutation in FLT3, known as the FLT3 internal tandem duplication (FLT3-ITD) mutation. The FLT3-ITD mutation acts like a “power switch” that causes leukemic cells, or blasts, to spread more aggressively and grow back more rapidly following chemotherapy, conferring an especially poor survival outcome. Quizartinib is designed to turn off this switch.

This novel second generation class III receptor tyrosine kinase inhibitor is currently under clinical investigation for wildtype AML and also those bearing FLT3 ITD mutation. Quizartinib with superior pharmaceutical properties and an excellent pharmacokinetic profile is a clear leader as compared to other agents in clinical trials. A study using recombinant enzyme in in vitro kinase assays identified that Quizartinib targets related class III RTKs, such as wildtype and gain-of-function mutant KIT and PDGFR isoforms. This implies that patients with CBF AML with KIT D816Y or exon 11 mutations or patients with solid tumors associated with KIT and PDGFR mutations, such as GIST too might benefit from this agent. This would not include the group of mutant-KIT CBF AML that have KIT D816V mutations [1].

The activity of Quizartinib is as follows:

K_d (FLT3 binding affinity) = 1.6 ± 0.7 nM

K_d (FLT1 binding affinity) = 41 nM

K_d (FLT3(D835H) binding affinity) = 3.7 nM

K_d (FLT3(D835Y) binding affinity) = 7.1 nM

K_d (FLT3(ITD) binding affinity) = 8.8 nM

K_d (FLT3(K663Q) binding affinity) = 2.2 nM

K_d (FLT3(N841I) binding affinity) = 4.1 nM

K_d (FLT4 binding affinity) = 41 nM

                                                                                                                      

Common Name: Quizartinib

Synonyms: AC220; AC 220; AC-220

IUPAC Name: (N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea

CAS Number: 950769-58-1; 1132827-21-4 (dihydrochloride)

SMILES:O=C(NC1=CC=C(C(N=C2SC3=C4)=CN2C3=CC=C4OCCN5CCOCC5)C=C1)NC6=NOC(C(C)(C)C)=C6

Mechanism of Action: Kinase Inhibitor; FLT3 Inhibitor

Indication: Acute Myeloid Leukemia

Development Stage: Phase III

Company: Ambit Biosciences

To assess the potential of Quizartinib to inhibit off-target kinases, and to compare its selectivity with that of the other FLT3 inhibitors, it was screened against a KinomeScan panel of 402 kinase binding assays, representing almost 80% of “typical” human protein kinases. Quizartinib was screened against the panel at a single concentration of 10 µM in a primary screen, and dissociation constants (Kds) were determined for all kinases identified as targets in the primary screen. For Quizartinib, researchers also measured a K_d for every kinase not found to bind in the primary screen to ensure that no targets were missed.

The highest affinity target identified for Quizartinib was FLT3. The only other kinases with binding constants within 10-fold that for FLT3 were the closely related receptor tyrosine kinases (RTKs) KIT, PDGFRA, PDGFRB, RET, and CSF1R (K_d = 4.8, 11, 7.7, 9.9 and 12 nM, respectively) and only 4 additional kinases, also related RTKs (FLT1, FLT4, DDR1, VEGFR2 K_d = 41, 41, 81, 87 nM, respectively), bound with K_ds within 100-fold of that for FLT3. In follow-up cellular assays the activity of Quizartinib against KIT, RET, CSF1R, and PDGFR was at least 10-fold less potent than the cellular activity against FLT3, confirming the selectivity observed in the biochemical assays. The kinase interaction pattern for Quizartinib was therefore highly focused [1].

To determine the ability of Quizartinib to inhibit FLT3 in the cellular environment, researchers measured inhibition of FLT3 autophosphorylation in the human leukemia cell lines MV4-11 (IC₅₀ = 0.56 ± 0.3 nM), which harbors a homozygous FLT3-ITD mutation and is FLT3 dependent and RS4;11, which expresses wild-type FLT3. Furthermore, to determine the effect of FLT3-ITD inhibition on cell growth, researchers measured MV4-11 cell proliferation in the presence of Quizartinib. The IC₅₀s in this assay tracked very well with FLT3 autophosphorylation inhibition, with Quizartinib having values of 4.2 ± 0.3 and 1.1 ± 0.1 for wild type and ITD respectively [1].

References:

1. Zarrinkar, P. P.; et. al. AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML). Blood 2009, 114(14), 2984-2992.

2. Bhagwat, S. S.; et. al. Identification of N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea dihydrochloride (AC220), a uniquely potent, selective, and efficacious FMS-like tyrosine kinase-3 (FLT3) inhibitor. J Med Chem 2009, 52(23), 7808-7816.

Drugs in Clinical Pipeline: KW-2449

KW-2449 [(E)-(4-(2-(1H-indazol-3-yl)vinyl)phenyl)(piperazin-1-yl)methanone] is an orally available multikinase inhibitor of FMS-like receptor tyrosine kinase (FLT3), ABL, ABL-T315I, and Aurora kinase. It is currently in Phase I trials for leukemia patients. KW-2449 inhibited FLT3 and ABL kinases with half-maximal inhibitory concentration (IC₅₀) values of 0.0066 and 0.014 µM, respectively. In addition, it potently inhibited ABL-T315I, which is associated with IM resistance, with an IC₅₀ value of 0.004 µM. On the other hand, KW-2449 had little effect on PDGFRβ, IGF-1R, EGFR, and various serine/threonine kinases even at a concentration of 1 µM. Among various serine/threonine kinases examined, KW-2449 inhibited Aurora A kinase with IC₅₀ of 0.048 µM and Aurora B kinase with the equivalent potency.

The researchers at Kyowa Hakko Kirin (previously Kyowa Hakko Kogyo) aimed at designing an orally available and highly potent FLT3 inhibitor with low toxicity profile for leukemia patients. For this goal, they screened the in-house chemical libraries using several leukemia cells, which have several activated mutations in FLT3 or BCR-ABL translocation. As a result, they identified several chemo-types with different kinase inhibition profiles, intensively studied the structures of the identified chemo-types to improve the potency and selectivity, and then finally generated KW-2449 [1].

Preclinical studies revealed that KW-2449 is converted by monoamine oxidase-B (MAO-B) and aldehyde oxidase into its major metabolite M1. Metabolite M1 is 3.6-fold less potent than the parent drug. The combination of KW-2449 and metabolite can successfully inhibit FLT3 in patients, and FLT3 mutant patients treated with KW-2449 on a twice daily schedule display the typical, short-lived reduction in peripheral blasts that has been seen with the other FLT3 inhibitors in development.

The activity of KW-2449 is as follows:

IC₅₀ (FLT3 enzyme assay) = 0.0066 uM

IC₅₀ (FLT3-D835Y enzyme assay) = 0.001 uM

IC₅₀ (KIT enzyme assay) = 0.3 uM

IC₅₀ (PDFGRα enzyme assay) = 1.7 uM

IC₅₀ (ABL enzyme assay) = 0.014 uM

IC₅₀ (ABL-T315I enzyme assay) = 0.004 uM

IC₅₀ (SRC enzyme assay) = 0.4 uM

IC₅₀ (JAK2 enzyme assay) = 0.15 uM

IC₅₀ (FGFR1 enzyme assay) = 0.036 uM

IC₅₀ (AURKA enzyme assay) = 0.048 uM

Common Name: KW-2449

Synonyms: KW2449; KW-2449; KW 2449

IUPAC Name: (E)-(4-(2-(1H-indazol-3-yl)vinyl)phenyl)(piperazin-1-yl)methanone

CAS Number: 1000669-72-6

Mechanism of Action: Kinase Inhibitor; FLT3 Inhibitor; ABL Inhibitor; Aurora Kinase Inhibitor; MultiKinase Inhibitor

Indication: Various Cancers; Leukemia

Development Stage: Phase I

Company: Kyowa Hakko Kirin Pharmaceuticals

FMS-like receptor tyrosine kinase (FLT3) is a class III receptor tyrosine kinase together with cKIT, FMS, and PDGFR. FLT3 mutations were first reported as internal tandem duplication (FLT3/ITD) of the juxtamembrane domain-coding sequence; subsequently, a missense point mutation at the Asp835 residue and point mutations, deletions, and insertions in the codons surrounding Asp835 within a tyrosine kinase domain of FLT3 (FLT3/KDM) have been found. FLT3 mutation is the most frequent genetic alteration in acute myeloid leukemia (AML) and involved in the signaling pathway of proliferation and survival in leukemia cells. Several large-scale studies have confirmed that FLT3/ITD is strongly associated with leukocytosis and a poor prognosis. In addition to FLT3 mutation, overexpression of FLT3 is an unfavorable prognostic factor for overall survival in AML, and it has been revealed that overexpressed FLT3 had the same sensitivity to the FLT3 inhibitor as FLT3/ITD [1]. Acute leukemia is a complex multigenetic disorder, a simultaneous inhibition of multiple protein kinases is thought to be advantageous over the increasing potency against the selective kinases.

In vitro kinase inhibition profile of KW-2449 indicated its extreme potency against FLT3 kinase. KW-2449 showed growth inhibitory activities against FLT3/ITD-, FLT3/D835Y-, and wt-FLT3/FL-expressing 32D cells, MOLM-13 and MV4;11 with half-maximal growth inhibitory concentration (GI₅₀) values of 0.024, 0.046, 0.014, 0.024, and 0.011 µM, respectively [1].

References:

1. Shiotsu, Y.; et. al. KW-2449, a novel multikinase inhibitor, suppresses the growth of leukemia cells with FLT3 mutations or T315I-mutated BCR/ABL translocation. Blood 2009, 114(8), 1607-1617.

2. ClinicalTrials.gov Safety, Tolerability, and Pharmacokinetic/Pharmacodynamic Study of KW-2449 in Acute Myelogenous Leukemia (AML) (Protocol Number: 2449-US-002). NCT00779480. Retrieved (01-09-2015)

3. ClinicalTrials.gov An Ascending Dose Study of KW-2449 in Acute Leukemias, Myelodysplastic Syndromes, and Chronic Myelogenous Leukemia. NCT00346632. Retrieved (01-09-2015)

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 (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: Gilteritinib

Gilteritinib [6-ethyl-3-((3-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazine-2-carboxamide] an orally available FLT3/AXL inhibitor, showed potent antileukemic activity against Acute Myeloid Leukaemia (AML) with either or both FLT3-ITD and FLT3-D835 mutations. These findings support the development of Gilteritinib for the potential use in treating AML.

Of the 78 tyrosine kinases tested, Gilteritinib inhibited FLT3, LTK, ALK, and AXL kinases by over 50% at 1 nM with an IC₅₀ value of 0.29 nM for FLT3, approximately 800-fold more potent than for c-KIT (IC₅₀ = 230 nM), the inhibition of which is linked to a potential risk of myelosuppression. Inhibition values for other kinases are, TRKA (IC₅₀ = 1.1 nM), RET (IC₅₀ = 1.8 nM), ROS (IC₅₀ = 1.5 nM) and MER (IC₅₀ = 2.9 nM).

The activity of Gilteritinib is as follows:

IC₅₀ (FLT3 enzyme assay) = 0.29 nM

IC₅₀ (LTK enzyme assay) = 0.25 nM

IC₅₀ (ALK enzyme assay) = 0.42 nM

IC₅₀ (AXL enzyme assay) = 0.70 nM 

Common Name: Gilteritinib

Synonyms:  ASP2215; ASP-2215; ASP 2215

IUPAC Name: 6-ethyl-3-((3-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazine-2-carboxamide

CAS Number: 1254053-43-4; 1254053-84-3 (hemifumarate)

Mechanism of Action: Kinase Inhibitor; Fms-like Tyrosine Kinase 3 Inhibitors; Proto Oncogene Protein c-KIT Inhibitors

Indication: Treatment of Acute Myeloid Leukaemia; AML Therapy

Development Stage: Phase I/II

Company: Astellas Pharma

Gilteritinib inhibited the growth of MV4-11 cells, which harbor FLT3-ITD, with an IC₅₀ value of 0.92 nM, accompanied with inhibition of pFLT3, pAKT, pSTAT5, pERK, and pS6. Gilteritinib also inhibited the growth of Ba/F3 cells expressing FLT3-ITD and/or FLT3-D835 mutation with similar activity. Colony formation of human granulocyte-macrophage decreased to 58% in response to Gilteritinib at 100 nM, more than 100-fold higher than required for MV4-11. In MV4-11 xenografted-mice, the concentration of Gilteritinib in tumors was more than 20-fold higher than that in plasma with oral administration of Gilteritinib at 10 mg/kg for 4 days. Treatment of Gilteritinib for 28 days resulted in dose-dependent inhibition of MV4-11 tumor growth and induced complete tumor regression at more than 6 mg/kg. Further, Gilteritinib decreased tumor burden in bone marrow and prolonged the survival of mice intravenously transplanted with MV4-11 cells [1].

References:

1. Ueno, Y.; et. al. ASP2215, a novel FLT3/AXL inhibitor: Preclinical evaluation in acute myeloid leukemia (AML). J Clin Oncol 2014 32(suppl; abstr 7070).