Drugs in Clinical Pipeline: Cenisertib

Cenisertib [(1S,2S,3R,4R)-3-((5-fluoro-2-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl) amino)pyrimidin-4-yl)amino)bicyclo[2.2.1]hept-5-ene-2-carboxamide] is an orally available, small molecule pan-Aurora inhibitor currently in clinical phase studies. Cenisertib has been reported to inhibit Aurora A, B, and C with IC₅₀ values of 4, 4.8, and 6.8 nM, respectively and to inhibit Abl, FLT1 (fms-related tyrosine kinase), and FLT3 (FMS-related tyrosine kinase 3) oncogenic kinases. Cenisertib has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis [1,2].

An excess amount of ATP reduced the level of inhibition of Aurora kinase A by Cenisertib in vitro, indicating that Cenisertib is an ATP competitive inhibitor.  The compound was tested in a panel of in vitro kinase assays using diVerent recombinant kinases. Aurora kinase A (%Inhibition = 100%), Abelson murine leukemia viral oncogene homolog 1 (ABL1, %Inhibition = 92%), FLT1 (VEGFR1, %Inhibition = 96%), and FLT3 (%Inhibition = 99%) were more than 90% inhibited by 100 nM of Cenisertib. The cyclin dependent kinases, CDK1, CDK2 and CDK6 were not inhibited at this concentration by Cenisertib [2]. Moreover, the compound inhibited VEGFR2 (KDR) with %inhibition value of 75% at 100 nM concentration.

Inhibition of Aurora kinase B was confirmed by an image-based Aurora kinase-dependent assay detecting intracellular phosphorylation of histone H3 serine 10. Cenisertib reduced the phosphorylation during mitosis with an average EC₅₀ of 14 nM. The selectivity of Cenisertib was assessed using a series of cell-based kinase assays, which measured the activation of kinases using antibodies against phosphorylated sites of specific substrates. Cenisertib exhibited potent inhibition of FLT3 activation mediated by the ITD mutation and VEGF165-induced VEGFR2 activation. Inhibition of Aurora kinase activity in tumor cells had at least 10-fold selectivity over inhibition of AMPK, AXL, AKT, and SAPK activity.

Cenisertib was designed and developed based on an image-based phenotypic screen by Rigel.

The activity of Cenisertib is as follows:

IC₅₀ (AURKA enzyme assay) = 4.0 nM

IC₅₀ (AURKA enzyme assay) = 4.8 nM

IC₅₀ (AURKC enzyme assay) = 6.8 nM

Common Name: Cenisertib

Synonyms: R763; R 763; R-763; AS703569; AS 703569; AS-703569; MSC1992371A

IUPAC Name: (1S,2S,3R,4R)-3-((5-fluoro-2-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl) amino)pyrimidin-4-yl)amino)bicyclo[2.2.1]hept-5-ene-2-carboxamide

CAS Number: 871357-89-0; 1145859-64-8 (benzoate)

SMILES: O=C([C@H]1[C@](C2)([H])C=C[C@]2([H])[C@H]1NC3=NC(NC4=CC=C(N5CCN(C)CC5)C(C)=C4)=NC=C3F)N

Mechanism of Action: Kinase Inhibitor; pan-Aurora Inhibitor

Indication: Various Cancers; Anti-Tumor Therapy

Development Stage: Phase I

Company: Rigel

The endoreduplication cycle induced by Cenisertib was irreversible even after the compound was withdrawn from the culture. Oral administration of Cenisertib demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to Cenisertib in vivo [2]. Cenisertib appeared to induce endoreduplication within 48 h as evidenced by the accumulation of 4n and 8n cells. Colo205, HeLa, and MiaPaCa-2 underwent apoptosis after 48 h. In this study, Colo205, MiaPaCa-2, HeLa, and MV4-11 cells were observed to be most sensitive to Cenisertib (IC₅₀ = 2 to 8 nM), but primary proliferating cells were also sensitive despite having higher IC₅₀ values (IC₅₀ = 31 to 160 nM) [2,3]. Moreover, Cenisertib was also tested in a slow growing and adriamycin-resistant tumor (NCI-ADR) xenograft model in nude mice. Tumor growth was signiWcantly suppressed by the treatment with Cenisertib (7 and 10 mg/kg day) in comparison with the control group. 

In a preclinical phase, the in vivo efficacy of Cenisertib was tested in MiaPaCa-2, adriamycin-resistant tumor, MOLT-4, and MV4-11 xenograft models. Significant reduction in tumor volumes did not occur in the MiaPaCa-2 xenograft model, but histological regression and reduction in histone H3 phosphorylation (Ser10) was observed. In contrast, tumor volumes were significantly reduced in adriamycin-resistant tumors. Treatment of the MOLT-4 xenograft model resulted in a 5-10% reduction in the total number of bone marrow cells. The percentages of leukemia cells were significantly reduced, whereas control groups were not affected. In the MV4-11 xenograft, Cenisertib induced pronounced anticancer activity in a dose-dependent manner. For a dose of 20 mg/kg/day, undetectable levels of tumors were seen in 17% of animals. Increased life span was observed in all treated groups, whereas all control mice died early [2,3].

The effect of Cenisertib on intracellular phosphorylation of specific substrates was measured in ELISA format assays using anti-phosphoprotein antibodies. The effect of Cenisertib on a variety of cellular activities was analyzed in order to check the selectivity of the compound. The compound selectively inhibited  AURKB and FLT3 with nearly the same potency, EC₅₀ = 0.014, 0.011 uM, respectively. It showed nearly 50 times preference against AKT (EC₅₀ = 0.713 uM), 100 times against EGFR (EC₅₀ = 1.491 uM) and more than 500 times against JNKs (EC₅₀ = 7.429 uM). Only VEGFR was inhibited with 2 fold selectivity, EC₅₀ = 0.027 uM [2].

R763/ AS703569 did not show potent inhibition of immune cell activation mediated by many stimuli tested, while it potently inhibited tumor growth. The ability of R763/AS703569 to inhibit tumor growth was examined in colony forming assays using 64 different human tumor xenografts in vitro. R763/AS703569 was active against various tumor types. The mean EC50 value was 0.09 u M. A majority of lung, breast, and renal tumors had EC50 values below the mean value. 

A phase I dose-escalation study of Cenisertib was carried out in patients with hematologic malignancies. Patients received escalating doses either on days 1-3 and 8-10 (n = 36) or on days 1-6 (n = 39) of a 21-day cycle. The maximum tolerated doses were 37 and 28 mg/m²/day, respectively. Dose-limiting toxicities included severe neutropenia with infection and sepsis, mucositis/stomatitis, and diarrhea. Complete responses occurred in 3 patients. Four disease-specific expansion cohorts then received the dose and schedule dictated by the escalation phase but the study was prematurely discontinued due to hematologic and gastrointestinal toxicity at clinically effective doses [4].

References:
1. Romanelli, A.; et. al. Inhibiting aurora kinases reduces tumor growth and suppresses tumor recurrence after chemotherapy in patient-derived triple-negative breast cancer xenografts. Mol Cancer Ther  2012, 11(12), 2693-2703.
2. Goulaouic McLaughlin, J.; et. al. Preclinical characterization of Aurora kinase inhibitor R763/AS703569 identified through an image-based phenotypic screen. J Cancer Res Clin Oncol 2010, 136(1), 99-113.
3. Kollareddy, M.; et. al. Aurora kinase inhibitors: Progress towards the clinic. Invest New Drugs 2012, 30(6), 2411-2432.
4. Graux, C.; et. al. A phase I dose-escalation study of MSC1992371A, an oral inhibitor of aurora and other kinases, in advanced hematologic malignancies. Leuk Res 2013, 37(9), 1100-1106.

Drugs in Clinical Pipeline: AMG-900

AMG-900 [N-(4-((3-(2-amino-4-pyrimidinyl)-2-pyridinyl)oxy)phenyl)-4-(4-methyl-2-thienyl)-1-phthalazinamine] an orally bioavailable, potent, and selective pan-aurora kinase inhibitor with activity in multidrug resistance (MDR) tumor cell lines. AMG 900 inhibits the enzyme activity of all 3 aurora kinase family members with IC₅₀ values of 5 nM or less (IC₅₀ A/B/C = 5/4/1/ nM). To determine the specificity of AMG 900 across the kinome, a panel of 26 kinases was screened and only MAPK14 (IC₅₀ = 53 nM) and TYK2 (IC₅₀ = 220 nM) enzymes were inhibited (greater than 50%) at concentrations of less than 500 nM. Further screening was performed against a panel of 353 distinct kinases, using an ATP site-dependent competition binding assay. AMG 900 exhibited low nanomolar binding affinity for aurora kinases (K_d A/B/C = 3/2/1 nM) as well as interactions (K_d less than 50 nM) with DDR1, DDR2, and LTK receptor tyrosine kinases [1].

Important findings about AMG-900:

1. AMG 900 is a potent and highly selective pan-aurora kinase inhibitor.

2. Effects of AMG 900 on proliferating cells are consistent with inhibition of aurora-B activity.

3. AMG 900 blocks the proliferation of multiple human tumor cell lines including cell lines resistant to paclitaxel, AZD1152, MK-0457, and PHA-739358.

4. AMG 900 inhibits the phosphorylation of histone H3 and suppresses the growth of human tumor xenografts in vivo.

It is developed at Amgen Labs. AMG 900 is an orally bioavailable, potent, and highly selective pan-aurora kinase inhibitor with activity in taxane-resistant tumor cell lines. Together, these features distinguish AMG 900 from other antimitotic drugs as well as 3 other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358). These key attributes contribute to the profile of this attractive clinical candidate; AMG 900 has entered phase 1 evaluation in adult patients with advanced cancers.

The activity of AMG-900 is as follows:

IC₅₀ (AURKA enzyme assay) = 5 nM; K_d = 3 nM

IC₅₀ (AURKB enzyme assay) = 4 nM; K_d = 2 nM

IC₅₀ (AURKC enzyme assay) = 1 nM; K_d = 1 nM

IC₅₀ (MAPK14 enzyme assay) = 53 nM

IC₅₀ (TYK2 enzyme assay) = 220 nM

Common Name: AMG-900
Synonyms: AMG-900; AMG 900
IUPAC Name: N-(4-((3-(2-aminopyrimidin-4-yl)pyridin-2-yl)oxy)phenyl)-4-(4-methylthiophen-2-yl)phthalazin-1-amine
CAS Number: 945595-80-2
Mechanism of Action: Kinase Inhibitor; pan-Aurora Kinase Inhibitor
Indication: Various Cancers
Development Stage: Phase I
Company: Amgen Labs

Aurora kinases are essential mitotic regulators and their potential role in tumorigenesis makes them attractive targets for anticancer therapy. In mammalian cells, the aurora family of serine/threonine protein kinases is composed of 3 paralogous genes (aurora-A, -B, and -C). Aurora-A and -B are essential regulators of mitotic entry and progression, whereas aurora-C function is primarily restricted to male meiosis during spermatogenesis. Aurora-A can function as an oncogene and is amplified in a subset of human tumors. The expression of aurora-A and -B is frequently elevated in human cancers and is associated with advanced clinical staging.

AMG 900 is active in all of the tested tumor cell lines at low nanomolar concentrations, suggesting that it can inhibit the proliferation of tumor cells irrespective of genomic alterations or tumor origin. It is possible that the underlying profile of genetic and epigenetic modifications in tumor cells may play an important role in determining the fate of remnant cells that survive AMG 900 treatment.

AMG 900 was rapidly metabolized in liver microsomes and highly bound to plasma proteins in the species tested. It was a weak Pgp substrate with good passive permeability. AMG 900 exhibited a low-to-moderate clearance and a small volume of distribution. Its terminal elimination half-life ranged from 0.6 to 2.4 h. AMG 900 was well-absorbed in fasted animals with an oral bioavailability of 31% to 107%. AMG 900 exhibited acceptable PK properties in preclinical species and was predicted to have low clearance in humans [2]. 

AMG 900 Metabolism in rats

The metabolism of AMG 900 was investigated in both male and female rats. Researchers conducted studies in bile-duct catheterized (BDC) rats where bile, urine and plasma were analyzed to obtain metabolism profiles for each gender. These studies identified gender differences in the metabolism profiles in bile. Bile contained the majority of the drug related material and contained little unchanged AMG 900 which indicated that metabolism was the prominent process in drug elimination. Although bile contained the same metabolites for both genders, the amount of specific metabolites differed. CYP phenotyping identified the prominent isoforms as being gender specific or biased in the oxidative metabolism of AMG 900. The metabolism in male rats favored both CYP2C11 and CYP2A2 whereas females favored the CYP2C12. Male rats metabolized AMG 900 primarily through hydroxylation with subsequent sulfate conjugation on the pyrimidinyl-pyridine side-chain whereas female rats favored a different oxidation site on the thiophene ring's methyl group, which is then metabolized to a carboxylic acid with subsequent conjugation to an acyl glucuronide [3].

References:

1. Payton, M.; et. al. Preclinical evaluation of AMG 900, a novel potent and highly selective pan-aurora kinase inhibitor with activity in taxane-resistant tumor cell lines. Cancer Res 2010, 70(23), 9846-9854.
2. Huang, L.; et. al. In vitro and in vivo pharmacokinetic characterizations of AMG 900, an orally bioavailable small molecule inhibitor of aurora kinases. Xenobiotica 2011, 41(5), 400-408.
3. Waldon, D.; et. al. Gender effects on rat metabolism of AMG 900, an orally available small molecule aurora kinase inhibitor. Drug Metab Lett 2011, 5(4), 290-297.

4. Cee, V. J.; et. al. Aurora kinase modulators and method of use. WO2007087276A1

5. ClinicalTrials.gov A Phase 1 First-in-Human Study Evaluating AMG 900 in Advanced Solid Tumors. NCT00858377 (retrieved on 22-04-2015)