Cdk4/cyclin D1 (IC50 = 2 nM); CDK6/cyclinD1 (IC50 = 10 nM); CDK9/cyclinT1 (IC50 = 57 nM); CDK5/p35 (IC50 = 287 nM); Cdk5/p25 (IC50 = 355 nM); CDK2/cyclinE (IC50 = 504 nM); CDK7/Mat1/cyclinH1 (IC50 = 3910 nM); CDK1/cyclinB1 (IC50 = 1627 nM); PIM1 (IC50 = 39 nM); PIM2 (IC50 = 3400 nM); HIPK2 (IC50 = 31 nM); DYRK2 (IC50 = 61 nM); CK2 (IC50 = 117 nM); GSK3b (IC50 = 192 nM); JNK3 (IC50 = 389 nM); FLT3 (D835Y) (IC50 = 403 nM); FLT3 (IC50 = 3960 nM); DRAK1 (IC50 = 659 nM)

Abemaciclib exhibits both single-agent antitumor activity and durable cell-cycle inhibition in a colorectal cancer xenograft model that was used to create an integrated pharmacokinetic/pharmacodynamic model. Abemaciclib can be dosed orally on a continuous schedule to achieve sustained target inhibition. Numerous other human cancer xenograft models, such as those derived from melanoma, glioblastoma, mantle cell lymphoma, and non-small cell lung cancer (NSCLC), show tumor growth inhibition. In an intracranial glioblastoma xenograft model, bemaciclib diffuses across the blood–brain barrier and increases survival time. Abemaciclib's pharmacokinetics in humans indicate a slow absorption phase, with a median of 4 to 6 hours between the oral dose and the maximum plasma concentration (tmax). It is distributed and cleared thoroughly. The average terminal elimination half-life (t1/2) varied between 17.4 and 38.1 hours, and there was no discernible shift in clearance that was dose-dependent[2].

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Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. [3]

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Researchers evaluated the safety, pharmacokinetic profile, pharmacodynamic effects, and antitumor activity of abemaciclib, an orally bioavailable inhibitor of cyclin-dependent kinases (CDK) 4 and 6, in a multicenter study including phase I dose escalation followed by tumor-specific cohorts for breast cancer, non-small cell lung cancer (NSCLC), glioblastoma, melanoma, and colorectal cancer. A total of 225 patients were enrolled: 33 in dose escalation and 192 in tumor-specific cohorts. Dose-limiting toxicity was grade 3 fatigue. The maximum tolerated dose was 200 mg every 12 hours. The most common possibly related treatment-emergent adverse events involved fatigue and the gastrointestinal, renal, or hematopoietic systems. Plasma concentrations increased with dose, and pharmacodynamic effects were observed in proliferating keratinocytes and tumors. Radiographic responses were achieved in previously treated patients with breast cancer, NSCLC, and melanoma. For hormone receptor-positive breast cancer, the overall response rate was 31%; moreover, 61% of patients achieved either response or stable disease lasting ≥6 months. [2]

LY2835219 (abemaciclib) was identified via compound and biochemical screening by scientists at Eli Lilly and Company Research Laboratories and selected for its biological activity and highly selective inhibition of the complexes CDK4/ cyclin D1 (IC50 =2 nmol/L) and CDK6/cyclin D1 (IC50 =10 nmol/L), with no activity against other CDK/cyclin complexes or cell-cycle-related kinases within the nanomolar ranges, except for inhibition of CDK9 at IC50 at least five times higher (Figure 2).23 The compound was shown to act as a competitive inhibitor of the ATP-binding domain of the CDK4 and CDK6 and to be 14 times more potent against CDK4 than against CDK6.24 In comparison to palbociclib and ribociclib, abemaciclib shows higher selectivity for the complex CDK4/cyclin D1, with IC50 values five times lower than those of the two other compounds [1].

The 96-well plate is seeded with cells, which are then left to adhere for an entire night before being treated for 72 hours with either the indicated compounds or DMSO control (0.1% v/v). A Cell Counting Kit is used, as directed by the manufacturer, to assess the viability and proliferation of cells. CompuSyn is used to analyze the relationship between mTOR inhibitor and abemaciclib. An additive drug interaction is indicated by a combination index (CI) value of 1, while a synergistic or antagonistic drug interaction is indicated by a CI value of <1 or >1.

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In Vitro Treatment of Human Breast Carcinoma and T Cells [3]
Breast cancer cell lines MCF-7 and MDA-MB-46 were treated with DMSO and abemaciclib (500 nM) for 8 days. RNA was isolated as described above. Jurkat T cells or primary human T cells were stimulated with anti-CD3/CD28/CD2 or thapsigargin and incubated with abemaciclib as indicated. Primary human T cells were isolated from whole blood by negative selection using RosetteSep kits. T cells were cultured in RPMI1640 supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES, and 2 mM L-glutamine and stimulated with CD3/CD28 DynaBeads at a 1:3 T cell/bead ratio. Abemaciclib was added at 0.3 μM final concentration. Cells were counted and fed fresh media every 2–3 days.

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In Vitro Tumor Cell Viability Assay [3]
Tumor cells were cultured for 4 hr alone at 37°C, and then abemaciclib, palbociclib, or DMSO control was added at indicated concentrations for 96 hr at 37°C. Cell viability was then assessed using CellTiter-Glo. Cell viability inhibition (%) was calculated according to the formula [1 − (mean luminosity of treated sample/mean luminosity of untreated control)] × 100. Fifty-percent inhibitory concentration (IC50) for growth or viability inhibition was calculated using a four-parameter logistic curve fit formula.

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NFAT Reporter Assay [3]
Jurkat T cells containing an NFAT-luc reporter gene were incubated with abemaciclib at the indicated concentrations for 30 min and then stimulated with 250 ng/mL of anti-CD3/CD28/CD2 for 6 hr. NFAT-luc activity was monitored using Luciferase Assay Reagent in a Wallac 1420 Victor2 Multilabel Counter. NFAT stimulation (%) was calculated using untreated control samples.

Subcutaneous injections of OSC-19 (1×10⁶) cells are given to six-week-old BALB/c female nude mice. Mice are randomized by tumor size and given each treatment when tumor sizes approach 100 mm³. Each treatment group comprises a minimum of 5 mice. Every group of mice receives a daily oral gavage dose of either RAD001 (5 mg/kg/d), Abemaciclib (45 mg/kg/d or 90 mg/kg/d), or a combination of both. In 20 mM phosphate buffer (pH 2.0), 1% HEC is used to dissolve the Abemaciclib. Weight and tumor size are measured twice a week. V=(L×W²)/2 is the formula used to compute tumor volumes. On day 14, mice undergo one last gavage before being sacrificed the next day. In order to perform immunohistochemistry and Western blot, the tumors are removed.

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In Vivo Tumor Studies [2]
BALB/c or C57BL/6 mice were implanted into the flank subcutaneously with 1 × 106 CT26, 5 × 105 MC38, or 5 × 105 EMT6 tumor cells per mouse on day 0. Mice were randomized into individual treatment groups (n = 5–15 mice per group) as indicated. A separate cohort of animals (n = 5 animals per time point) was allocated for mechanistic analyses in some cases. Abemaciclib was used. Rat anti-mouse anti-PD-L1 was generated from a rat of Lou/WS1 strain immunized with recombinant mouse PD-L1-Fc protein. 178G7 was identified on the basis of its binding to PD-L1 (half maximal effective concentration [EC50] = 0.1 nM) and blocking activities against PD-L1 interactions with PD-1 and CD80 (IC50 = 1.5 nM and 2.5 nM, respectively). Tumor volume (TV) was calculated as TV (mm3) = π/6 × length × width2. Animals were sacrificed because of progressive disease if tumor burden was greater than 2,500 mm3 or growth would surpass 2,500 mm3 before the next measurement. For experiments with secondary tumor re-challenge, mice were re-challenged with 1 × 106 CT26 tumors on the opposite flank of the original tumor injection site. Secondary challenge tumor growth was followed for up to 22 days.

Absorption, Distribution and Excretion
The plasma concentration of the drug increases in a dose-proportional manner. Following a single oral dose administration of 200 mg abemaciclib, the mean peak plasma concentration (Cmax) of 158 ng/mL is reached after 6 hours. The median time to reach maximum plasma concentration (Tmax) ranges from 4-6 hours following an oral administration of abemaciclib over a range of 50–275 mg, but may range up to 24 hours. The absolute bioavailability of the drug is reported to be 45%.
Following a single oral dose of 150mg radiolabeled abemaciclib, approximately 81% of the total dose was recovered in feces while 3% of the dose was detected in urine. The majority of the drug is exceted as metabolites.
The geometric mean systemic volume of distribution is approximately 690.3 L (49% CV).
The geometric mean hepatic clearance (CL) of abemaciclib in patients was 26.0 L/h (51% CV).

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Metabolism / Metabolites
Abemaciclib mainly undergoes hepatic metabolism mediated by CYP3A4. The major metabolite formed is N-desethylabemaciclib (M2), while other metabolites hydroxyabemaciclib (M20), hydroxy-N-desethylabemaciclib (M18), and an oxidative metabolite (M1) are also formed. M2, M18, and M20 are equipotent to abemaciclib and their AUCs accounted for 25%, 13%, and 26% of the total circulating analytes in plasma, respectively.

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Biological Half-Life
The mean plasma elimination half-life for abemaciclib in patients was 18.3 hours (72% CV).

Hepatotoxicity
In the large clinical trials, adverse events were common and led to dose reductions in up to one-half of patients and discontinuation in 9%. In preregistration clinical trials, ALT elevations occurred in 31% to 41% of abemaciclib treated subjects which were above 5 times the ULN in 3% to 5%. In one study, several recipients developed clinically apparent liver injury with jaundice and one recipient died of hepatic failure, but these outcomes were considered to be unrelated to abemaciclib therapy. Thus, there were no cases of clinically apparent liver injury that could be attributed to abemaciclib therapy during prelicensure studies. Since the approval and more widescale use of abemaciclib, there have been no published reports of its hepatotoxicity. Nevertheless, the high rate of serum enzyme elevations during therapy and the similarity of abemaciclib to ribociclib and palbociclib makes it an agent that should be suspected of causing rare instances of clinically significant liver injury.
Likelihood score: E* (unproved but suspected, rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of abemaciclib during breastfeeding. Because abemaciclib and its metabolites are over 90% bound to plasma proteins, the amount in milk is likely to be low. However, the manufacturer recommends that breastfeeding be discontinued during abemaciclib therapy and for 3 weeks after the final dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
According to in vitro models using animal brain tissues, the protein binding of abemaciclib is approximately 95-98%. While abemaciclib demonstrated *in vitro* binding to serum albumin, alpha-1-acid glycoprotein and other human plasma proteins in a concentration-depedent manner, its major metabolites are also shown to bind to plasms proteins as well. The approximate bound fractions of M2, M18 and M20 are 93.4%, 96.8% and 97.8%, respectively.

[1]. Drug Des Devel Ther . 2018 Feb 16:12:321-330.

[2]. Cancer Discov . 2016 Jul;6(7):740-53.

[3]. Cell Rep . 2018 Mar 13;22(11):2978-2994.

Pharmacodynamics
In combination with fulvestrant, the progression-free survival for patients with HR-positive, HER2-negative breast cancer was 16.4 months compared to 9.3 months for patients taking a placebo with fulvestrant. As a monotherapy, 19.7% of patients taking abemaciclib achieved complete or partial shrinkage of their tumors for a median 8.6 months after treatment. Abemaciclib induces cell cycle arrest and exerts an antitumor activity in human tumor xenograft models. In patient investigations and a healthy volunteer study, abemaciclib is not shown to induce any clinically significant changes in the QTc interval.

C27H32F2N8

506.59

506.271

C, 64.01; H, 6.37; F, 7.50; N, 22.12

1231929-97-7

Abemaciclib methanesulfonate;1231930-82-7;Abemaciclib-d8;2088650-53-5

46220502

White to off-white solid powder

1.3±0.1 g/cm3

689.3±65.0 °C at 760 mmHg

370.7±34.3 °C

0.0±2.2 mmHg at 25°C

1.656

2.74

1

9

7

37

723

0

CC1=NC2=C(F)C=C(C3=NC(NC4=NC=C(CN5CCN(CC)CC5)C=C4)=NC=C3F)C=C2N1C(C)C

UZWDCWONPYILKI-UHFFFAOYSA-N

InChI=1S/C27H32F2N8/c1-5-35-8-10-36(11-9-35)16-19-6-7-24(30-14-19)33-27-31-15-22(29)25(34-27)20-12-21(28)26-23(13-20)37(17(2)3)18(4)32-26/h6-7,12-15,17H,5,8-11,16H2,1-4H3,(H,30,31,33,34)

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: 3.33 mg/mL (6.57 mM) in 0.5% HEC (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.

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Solubility in Formulation 2: 5% DMSO+40% PEG 300+5%Tween80+ 50%ddH2O: 0.2mg/ml

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