CDK4 (IC50 = 2 nM); CDK6 (IC50 = 10 nM); 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)

It was observed that abemaciclib monotherapy could enhance immune infiltration, especially CD8+ T cell and B cell infiltration, in the ID8 murine ovarian cancer model. Immunophenotyping analysis showed that abemaciclib induced a proinflammatory immune response in the tumor microenvironment. PCR array analysis suggested the presence of a Th1-polarized cytokine profile in abemaciclib-treated ID8 tumors. In vitro studies showed that abemaciclib-treated ID8 cells secreted more CXCL10 and CXCL13, thus recruiting more lymphocytes than control groups. Combination treatment achieved better tumor control than monotherapy, and the activities of CD8+ and CD4+ T cells were further enhanced when compared with monotherapy. The synergistic antitumor effects of combined abemaciclib and anti-PD-1 therapy depended on both CD8+ T cells and B cells. Conclusion: These findings suggest that combined treatment with CDK4/6i and anti-PD-1 antibody could improve the efficacy of anti-PD-1 therapy and hold great promise for the treatment of poorly immune-infiltrated ovarian cancer.[3]

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LY2835219 has an unbound plasma IC50 of approximately 95 nM, which saturates BBB efflux. The percentage of LY2835219-MsOH dose in the brain ranges from 0.5-2.9%. LY2835219-MsOH, when used alone or in conjunction with temozolomide, inhibited tumor growth in a dose-dependent manner in both a subcutaneous and intracranial human glioblastoma model (U87MG).[1]

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].

In vitro migration of CD8+ T cells and B cells was evaluated in 24-well plates with a polyethylene terephthalate hanging cell culture insert (5.0 μm). The bottom chamber contained 600 μL of supernatants of abemaciclib- or PBS-treated ID8 cells as the chemoattractant. For transwell assays utilizing blocking antibodies, 30 μg/mL of anti-CXCL10 or 40 μg/mL anti-CXCL13 were added to the lower chamber containing supernatants of abemaciclib-treated ID8 cells. Rat IgG or goat IgG were used as isotype control antibodies. Freshly isolated CD8+ T cells or B cells in 100 μL were seeded in the upper chamber. After a 3-hour incubation at 37 °C in a standard 5% CO2 incubator, cells that migrated into the lower chamber were counted with a hemocytometer.[3]

Female C57BL/6 mice
50 mg/kg
i.p.

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In this study, researchers first assessed the antitumor efficacy of abemaciclib, an FDA-approved CDK4/6i, in a syngeneic murine ovarian cancer model. Then, immunohistochemistry, immunofluorescence and flow cytometry were performed to evaluate the number, proportion, and activity of tumor-infiltrating lymphocytes. Cytokine and chemokine production was detected both in vivo and in vitro by PCR array analysis and cytokine antibody arrays. The treatment efficacy of combined abemaciclib and anti-PD-1 therapy was evaluated in vivo, and CD8+ and CD4+ T cell activities were analyzed using flow cytometry. Lastly, the requirement for both CD8+ T cells and B cells in combination treatment was evaluated in vivo, and potential cellular mechanisms were further analyzed by flow cytometry. [3]

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Mouse cytokine and chemokine array[3]
ID8 cells seeded at equal numbers were cultured in complete medium for 24 hours, washed twice with PBS and cultured in FBS-free DMEM plus 10 µmol/L abemaciclib or PBS for 24 hours. Then, the supernatants were collected and processed for mouse cytokine array analysis according to the manufacturer's protocols. Membranes were scanned using an LAS-500 imager. Relative cytokine levels were obtained by grayscale analysis using ImageJ.[3]

Absorption
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%.

Route of Elimination
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.

Volume of Distribution
The geometric mean systemic volume of distribution is approximately 690.3 L (49% CV).

Clearance
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]. Nat Med . 2021 Feb;27(2):289-300.

[3]. Theranostics . 2020 Aug 25;10(23):10619-10633.

Abemaciclib Mesylate is the mesylate salt of abemaciclib, an orally available cyclin-dependent kinase (CDK) inhibitor that targets the cyclin D1-CDK4 and cyclin D3-CDK6 cell cycle pathway, with potential antineoplastic activity. Abemaciclib specifically inhibits CDK4 and 6, thereby inhibiting retinoblastoma (Rb) protein phosphorylation in early G1. Inhibition of Rb phosphorylation prevents CDK-mediated G1-S phase transition, thereby arresting the cell cycle in the G1 phase, suppressing DNA synthesis and inhibiting cancer cell growth. Overexpression of the serine/threonine kinases CDK4/6, as seen in certain types of cancer, causes cell cycle deregulation.

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Great progress has been made in the field of tumor immunotherapy in the past decade. However, the therapeutic effects of immune checkpoint blockade (ICB) against ovarian cancer are still limited. Recently, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6i) has been reported to enhance antitumor immunity in preclinical models. The combined use of CDK4/6i and ICB may be beneficial, but the effects of CDK4/6is on the tumor immune microenvironment and whether they can synergize with ICB in treating ovarian cancer remain unknown. Methods: In this study, we first assessed the antitumor efficacy of abemaciclib, an FDA-approved CDK4/6i, in a syngeneic murine ovarian cancer model. Then, immunohistochemistry, immunofluorescence and flow cytometry were performed to evaluate the number, proportion, and activity of tumor-infiltrating lymphocytes. Cytokine and chemokine production was detected both in vivo and in vitro by PCR array analysis and cytokine antibody arrays. The treatment efficacy of combined abemaciclib and anti-PD-1 therapy was evaluated in vivo, and CD8+ and CD4+ T cell activities were analyzed using flow cytometry. Lastly, the requirement for both CD8+ T cells and B cells in combination treatment was evaluated in vivo, and potential cellular mechanisms were further analyzed by flow cytometry. Results: We observed that abemaciclib monotherapy could enhance immune infiltration, especially CD8+ T cell and B cell infiltration, in the ID8 murine ovarian cancer model. Immunophenotyping analysis showed that abemaciclib induced a proinflammatory immune response in the tumor microenvironment. PCR array analysis suggested the presence of a Th1-polarized cytokine profile in abemaciclib-treated ID8 tumors. In vitro studies showed that abemaciclib-treated ID8 cells secreted more CXCL10 and CXCL13, thus recruiting more lymphocytes than control groups. Combination treatment achieved better tumor control than monotherapy, and the activities of CD8+ and CD4+ T cells were further enhanced when compared with monotherapy. The synergistic antitumor effects of combined abemaciclib and anti-PD-1 therapy depended on both CD8+ T cells and B cells. Conclusion: These findings suggest that combined treatment with CDK4/6i and anti-PD-1 antibody could improve the efficacy of anti-PD-1 therapy and hold great promise for the treatment of poorly immune-infiltrated ovarian cancer.[3]

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Although early breast cancer (BC) is highly curable, advanced or metastatic disease poses numerous challenges in terms of medical management and treatment decisions and is associated with significantly worse prognosis. Among the new targeted agents, anticancer drugs exploiting the cell-cycle machinery have shown great potential in preclinical studies. CDK4/6 inhibitors target the cyclin D/CDK/retinoblastoma signaling pathway, inducing cell-cycle arrest, reduced cell viability and tumor shrinking. As the cyclin D/CDK complex is activated downstream of estrogen signaling, the combination of CDK4/6 inhibitors with standard endocrine therapies represents a rational approach to elicit synergic antitumor activity in hormone receptor-positive BC. The results of clinical trials have indeed confirmed the superiority of the combination of CDK4/6 inhibitors plus endocrine therapies over endocrine therapy alone. Currently approved are three compounds that exhibit similar structural characteristics as well as biological and clinical activities. Abemaciclib is the latest CDK4/6 inhibitor approved by the US Food and Drug Administration (FDA) in view of the results of the MONARCH 1 and 2 trials. Further trials are ongoing as other important questions await response. In this review, we focus on abemaciclib to examine preclinical and clinical results, describing current therapeutic indications, open questions and ongoing clinical trials.[1]

C28H36F2N8O3S

602.7

602.259

C, 55.80; H, 6.02; F, 6.30; N, 18.59; O, 7.96; S, 5.32

1231930-82-7

Abemaciclib;1231929-97-7

71576678

White to yellow solid powder

5.47

2

12

7

42

815

0

CC(N1C2=CC(C3=NC(NC4=NC=C(CN5CCN(CC)CC5)C=C4)=NC=C3F)=CC(F)=C2N=C1C)C.CS(=O)(O)=O

NCJPFQPEVDHJAZ-UHFFFAOYSA-N

InChI=1S/C27H32F2N8.CH4O3S/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;1-5(2,3)4/h6-7,12-15,17H,5,8-11,16H2,1-4H3,(H,30,31,33,34);1H3,(H,2,3,4)

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;methanesulfonic acid

Abemaciclib; LY-2835219 mesylate; LY2835219; Abemaciclib methanesulfonate; 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 methanesulfonate; LY2835219 Mesylate; LY-2835219 methanesulfonate; Abemaciclib (methanesulfonate); KKT462Q807; LY 2835219; Abemaciclib mesylate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 2.5 mg/mL (4.15 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 (4.15 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), suspension solution; with ultrasonication.
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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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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Solubility in Formulation 4: 2 mg/mL (3.32 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 5: ≥ 2 mg/mL (3.32 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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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Solubility in Formulation 6: Water: 100 mg/mL (~165.9 mM)

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Solubility in Formulation 7: 25 mg/mL (41.48 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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Solubility in Formulation 8: 12.5 mg/mL (20.74 mM) in 0.5% HEC (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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