CDK4 (IC50 = 1.2 nM); CDK6 (IC50 = 1.3 nM)
Cyclin-dependent kinase 4 (CDK4) (IC50 1.2 nM)
Cyclin-dependent kinase 6 (CDK6) (IC50 1.3 nM)[2]

The metabolite M2 exhibited similar potency to the parent drug abemaciclib in enzyme binding biochemical assays against CDK4 and CDK6[2].
In cell inhibition assays using various cancer cell lines (e.g., A549, Colo205), the cell inhibitory IC50 of M2 ranged between 0.3x and 1.1x that of abemaciclib, indicating comparable in vitro cellular activity[2].
The M2 metabolite, along with metabolite M20, was assessed in a bacterial mutation (Ames) test and an in vitro chromosome aberration test for genotoxicity[2].
In vitro assessment using Madin-Darby canine kidney (MDCK) cell monolayers transfected with human MDR1 gene indicated that M2 is a substrate for both P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP)[2].
In vitro studies using human recombinant CYPs indicated that the CYP-catalysed metabolism of M2 is almost entirely catalysed by CYP3A4[2].

The metabolite M2 (LSN2839567) of Abemaciclib binds to tethering proteins in humans, dogs, and gold at a rate of 83-92%, whereas Abemaciclib binds at a rate of 95-99% [2]. The most notable (active and significant) transporter in humans is abelacil metabolite M2 [2].

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In xenograft rodent models based on cancer cell line injections, the parent drug abemaciclib showed efficacy[2].
In the mass balance study in humans, M2 was identified as a prominent plasma metabolite following administration of abemaciclib[2].

The molecular affinity of M2 for the target molecules CDK4 and CDK6 was estimated using cell-free kinase assays. The assay measured the inhibitory concentration (IC50) required for 50% inhibition of kinase activity[2].

The in vitro cellular effect concentration for M2 was measured in cell inhibition assays using various human cancer cell lines. The assay determined the concentration causing 50% inhibition of cell growth (IC50)[2].

The assessment report does not describe a specific animal experiment protocol conducted solely with the M2 metabolite. Toxicokinetic data for M2 were obtained as part of repeat-dose toxicity studies of abemaciclib in rats and dogs, where exposure to M2 was monitored[2].

Following administration of abexicillin, M2 is one of the major active metabolites in human plasma, accounting for approximately 13% of total plasma radioactivity after a single dose [2]. The plasma protein binding rate of M2 is slightly lower than that of abexicillin, ranging from 83% to 92% across different species, with the lowest binding rate in canine plasma (83%) [2]. M2 is primarily generated from abexicillin via N-deethylation, which is the consistent major elimination pathway in rats, dogs, and humans [2]. M2 is further metabolized primarily by CYP3A4 and can also be excreted in feces, possibly mediated by P-gp/BCRP efflux [2]. In a human mass balance study, M2 and its associated metabolites were primarily excreted in feces [2].

M2 was evaluated according to standard genotoxicity study protocols (Ames test and in vitro chromosome aberration test). The Ames test result was negative [2]. In the in vitro chromosome aberration test (3-hour test, with or without metabolic activation), M2 resulted in a significant increase in the number of internally replicating cells. The applicant believes this may be due to its effect on mitotic processes or cell cycle disruption [2]. The affinity of the M2 metabolite for hERG channels (the potential for QT interphase prolongation) is lower than that of its parent compound, abexicillin (>10 µM) [2]. Exposure to M2 was evaluated in repeated-dose toxicity studies of abexicillin in rats and dogs. In rats, the exposure to M2 (AUC0–24h) was higher than in humans and was therefore identified by toxicology [2].

[1]. Abstract 2830: The major human metabolites of abemaciclib are inhibitors of CDK4 and CDK6. Cancer Research. July 2016, 76 (14).

[2]. CHMP. Assessment report Verzenios. 26 July 2018 EMA/551438/2018.

Abecitabine (LY2835219) is an ATP-competitive cyclin-dependent kinase 4 and 6 (CDK4 and CDK6) inhibitor currently undergoing clinical evaluation for the treatment of breast and lung cancer. A radiolabeled in vivo distribution study following a single oral dose of 150 mg [14C]LY2835219 in healthy subjects showed that, in addition to the parent drug, five metabolites were present in plasma, labeled M1, M2, M18, M20, and M22. Abecitabine (34%), M20 (26%), M2 (13%), and M18 (5%) constituted the majority of plasma exposure. This study investigated the in vitro bioactivity of these circulating metabolites, excluding the trace metabolite M1, and compared their potency with that of the parent drug, abecitabine. This study evaluated the effects of abecitabine and its metabolites on growth inhibition, cell cycle inhibition, and biomarker expression in non-small cell lung cancer (NSCLC) cells, colorectal cancer (CRC) cells, and breast cancer cell lines. Furthermore, metabolites were analyzed and compared with abexicillin in cell-free biochemical kinase activity assays for the inhibition of CDK4, CDK6, CDK1, and CDK9. Metabolites M2, M18, and M20 (but not M22) exhibited IC50 values between 1 and 3 nM for the inhibition of CDK4 and CDK6, showing efficacy nearly equivalent to abexicillin. Similarly, metabolites M2, M20, and M18 inhibited cell growth and cell cycle progression in a concentration-dependent manner, consistent with the inhibition of CDK4 and CDK6, as these results were associated with concentration-dependent inhibition of multiple biomarkers such as phosphorylated serine 780-Rb (pRb), topoisomerase II-α (Topo IIα), and phosphorylated serine 10-histone H3 (pHH3)). In this regard, metabolites M2 and M20 showed nearly identical potency to abexicillin in the evaluated cancer cell lines, while M18 was approximately 3–20 times less potent than abexicillin, depending on the measured endpoints. M22 exhibited the lowest inhibitory potency against cell growth at concentrations below 2 μM, with little or no inhibition of biomarker expression or cell cycle progression. Although cell-free kinase activity assays suggested that M2, M18, and M20, like abexicillin, possessed the potential to inhibit CDK9, measurable inhibition of CDK9 by these compounds was not observed in cancer cells, suggesting that the primary targets for cell cycle inhibition by these metabolites in cancer cells are CDK4 and CDK6, rather than CDK9. Studies in breast cancer cells using abexicillin, M2, or M20 showed that treatment with these three compounds at concentrations of 200 nM and 500 nM for 6–8 days, in addition to inhibiting cell growth, induced cellular senescence. Overall, the results indicate that the major human metabolites of abexicillin, M2 and M20, are potent CDK4 and CDK6 inhibitors, and they are very similar to abexicillin in their effects on cancer cell growth, senescence, and other phenotypic responses. [1] M2 (LSN2839567) is the major human metabolite of the CDK4/6 inhibitor abexicillin. [2] It is pharmacologically active and has similar inhibitory efficacy against CDK4 and CDK6 as the parent drug. [2] M2, together with another major metabolite, M20, contributes to the overall pharmacological activity of abexicillin therapy. [2]

C25H28F2N8

478.540230751038

478.24

1231930-57-6

Abemaciclib metabolite M2-d6

59376686

Light yellow to yellow solid powder

3

2

9

6

35

680

0

FC1=CC(C2C(=CN=C(N=2)NC2=CC=C(C=N2)CN2CCNCC2)F)=CC2=C1N=C(C)N2C(C)C

IXGZDCRFGCEEBU-UHFFFAOYSA-N

InChI=1S/C25H28F2N8/c1-15(2)35-16(3)31-24-19(26)10-18(11-21(24)35)23-20(27)13-30-25(33-23)32-22-5-4-17(12-29-22)14-34-8-6-28-7-9-34/h4-5,10-13,15,28H,6-9,14H2,1-3H3,(H,29,30,32,33)

5-fluoro-4-(7-fluoro-2-methyl-3-propan-2-ylbenzimidazol-5-yl)-N-[5-(piperazin-1-ylmethyl)pyridin-2-yl]pyrimidin-2-amine

1231930-57-6; Abemaciclib Metabolites M2; Abemaciclib metabolite M2; 5-Fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)-N-[5-(1-piperazinylmethyl)-2-pyridyl]pyrimidin-2-amine; 5-fluoro-4-(7-fluoro-2-methyl-3-propan-2-ylbenzimidazol-5-yl)-N-[5-(piperazin-1-ylmethyl)pyridin-2-yl]pyrimidin-2-amine; Des-Et-Abemaciclib; MFCD32067988; LSN2839567;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~2 mg/mL (~4.18 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.22 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.22 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)