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)
Cyclin - dependent kinase 4 (CDK4) and cyclin - dependent kinase 6 (CDK6), with IC50 values of 2 nM and 10 nM respectively [1]

Cyclin-Dependent Kinase 4 (CDK4) [1]
Cyclin-Dependent Kinase 6 (CDK6) [1]
Cyclin-Dependent Kinase 4 (CDK4) [2]
Cyclin-Dependent Kinase 6 (CDK6) [2]
Cyclin-Dependent Kinase 4 (CDK4) [3]
Cyclin-Dependent Kinase 6 (CDK6) [3]

In vitro activity: Abemaciclib (formerly known as LY2835219) is a potent and selective, orally available dual inhibitor of CDK4 (cyclin-dependent kinase) and CDK6 with IC50 of 2 nM and 10 nM in cell-free assays, respectively. LY2835219 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 can cause cell cycle deregulation as seen in certain types of cancer.

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Kinase Assay: Cells (5 × 103) are plated in 96 well plates. Cells are treated the next day for 24 to 48 hours and then assessed for caspase-3 activity by Caspase-Glo-3/7 Assay, as per manufacturers instructions and a luminescence plate reader.

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Cell Assay: Cells are seeded in a 96-well plate, allowed to adhere overnight, and treated with DMSO control (0.1% v/v) or the indicated compounds for 72 h. Cell viability and proliferation are determined using a Cell Counting Kit according to the manufacturers instructions. The interaction between LY2835219 and mTOR inhibitor is determined using CompuSyn. Combination index (CI) values of 1 indicates and additive drug interaction, whereas a CI of < 1 is synergistic and a CI of > 1 is antagonistic.

Treats HNSCC cell lines (OSC - 19, FaDu, YD - 10B) with Abemaciclib at concentrations of 0.01 - 10 μM for 72 h, which inhibits cell growth and induces apoptosis. The IC50 values for reducing cell viability range from 0.5 μM to 0.7 μM. It also activates the pro - survival autophagy pathway and the ERK pathway in HNSCC cells through generating reactive oxygen species (ROS) [1]

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Abemaciclib inhibits the CDK4/cyclin D1 complex, which is responsible for the phosphorylation of the Rb tumor-suppressor gene product; this inhibition induces cell-cycle arrest, reduces cell viability, and blocks the cell cycle progression from G1 to S phase [1]
- Treatment with Abemaciclib results in increased activation of human T cells and upregulates the expression of antigen presentation genes in MCF-7 breast cancer cells [3]

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]

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Administers Abemaciclib (45 or 90 mg/kg) by oral gavage to BALB/c nude mice bearing HNSCC xenografts once daily for 14 days. It can significantly inhibit tumor growth. Co - inhibition of CDK4/6 and autophagy shows a synergistic effect in inhibiting tumor growth [1]

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In murine syngeneic tumor models, Abemaciclib monotherapy leads to tumor growth delay, which is associated with an increased T cell inflammatory signature in tumors; combination with anti-PD-L1 therapy results in complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and improved cell cycle control [3]
- In a multicenter clinical study including patients with advanced solid tumors, Abemaciclib shows single-agent antitumor activity; radiographic responses are achieved in previously treated patients with breast cancer, non-small cell lung cancer (NSCLC), and melanoma; for hormone receptor-positive breast cancer, the overall response rate is 31%, and 61% of patients achieve either response or stable disease lasting ≥6 months [2]
- The combination of Abemaciclib with standard endocrine therapies exhibits synergic antitumor activity in hormone receptor-positive breast cancer, showing superiority over endocrine therapy alone [1]

To measure CDK4/6 inhibitory activity, recombinant CDK4/cyclin D1 and CDK6/cyclin D3 complexes are incubated with varying concentrations of Abemaciclib (LY2835219) and a fluorescently labeled peptide substrate. The reaction is monitored for kinase activity, and IC50 values are calculated as the concentration required to reduce activity by 50%. Abemaciclib (LY2835219) shows selective inhibition of CDK4 and CDK6 over other CDKs [1] [3]
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.

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Seeds HNSCC cells (OSC - 19, FaDu, YD - 10B) in 96 - well plates, and after overnight adhesion, treats them with Abemaciclib at different concentrations or DMSO control for 72 h. Then uses a cell counting kit to measure cell viability, and determines the IC50 value. Also, detects apoptosis - related proteins and autophagy - related proteins by Western blot, and observes the change of ROS level by using a corresponding reagent [1]
- Animal Protocol：Subcutaneously injects HNSCC cells into the right - flank of 6 - week - old female BALB/c nude mice. When the tumor volume reaches about 100 mm³, randomly divides the mice into groups. Dissolves Abemaciclib in 1% HEC in 20 mM phosphate - buffer (pH 2.0), and administers it to the mice by oral gavage at a dose of 45 mg/kg or 90 mg/kg once a day. Measures the tumor size and body weight twice a week. After 14 - day treatment, sacrifices the mice and removes the tumors for further analysis [1]

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CDK4/cyclin D1 complex inhibition assay: Cultivate cells expressing the CDK4/cyclin D1 complex, treat with Abemaciclib, and detect the phosphorylation level of Rb protein to evaluate the inhibitory effect of the drug on the complex, as well as observe cell-cycle progression to assess cell-cycle arrest [1]
- T cell activation and antigen presentation gene expression assay: Culture human T cells and MCF-7 breast cancer cells separately, treat with Abemaciclib, then detect the activation status of T cells and the expression level of antigen presentation genes in MCF-7 cells to analyze the in vitro biological activity of the drug [3]

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.

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Murine syngeneic tumor model assay: Establish murine syngeneic tumor models; administer Abemaciclib as monotherapy or in combination with anti-PD-L1 therapy according to a specific schedule; monitor tumor growth dynamically, analyze the tumor microenvironment (including T cell inflammatory signature and antigen presentation level) after treatment, and evaluate the antitumor efficacy and related mechanisms of the drug [3]

Absorption, Distribution and Excretion
Plasma concentrations of the drug increase proportionally to the dose. Following a single oral dose of 200 mg abexicillin, the mean peak plasma concentration (Cmax) is reached at 158 ng/mL after 6 hours. Following oral doses of 50–275 mg abexicillin, the median time to reach maximum plasma concentration (Tmax) is 4–6 hours, but can be as long as 24 hours. The absolute bioavailability of the drug has been reported to be 45%. Following a single oral dose of 150 mg of radiolabeled abexicillin, approximately 81% of the total dose is recovered in feces, and 3% is detectable in urine. The drug is largely excreted as metabolites. The geometric mean volume of distribution is approximately 690.3 L (coefficient of variation 49%). The geometric mean hepatic clearance (CL) of abexicillin in patients is 26.0 L/h (coefficient of variation 51%).

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Metabolism/Metabolites
Abecitabine is primarily metabolized in the liver via CYP3A4-mediated metabolism. The major metabolite is N-deethylabecidine (M2), and other metabolites are also generated, such as hydroxyabecidine (M20), hydroxy-N-deethylabecidine (M18), and an oxidative metabolite (M1). M2, M18, and M20 are comparable in potency to abecitabine, representing 25%, 13%, and 26% of the total circulating analyte in plasma, respectively.

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Biological Half-Life
The mean plasma elimination half-life of abecitabine in patients is 18.3 hours (72% CV).

Hepatotoxicity
Adverse events are relatively common in large clinical trials, leading to dose reductions in up to half of the patients and discontinuation of treatment in 9%. In premarketing clinical trials, 31% to 41% of abeciclib-treated subjects experienced elevated ALT levels, with 3% to 5% of these elevations exceeding five times the upper limit of normal. In one study, several subjects experienced clinically significant liver injury (with jaundice), and one subject died of liver failure; however, these outcomes were considered unrelated to abeciclib treatment. Therefore, no cases of clinically significant liver injury attributable to abeciclib treatment were observed in premarket studies. Since abeciclib's approval and widespread use, no published reports of its hepatotoxicity have been received. However, given the high incidence of elevated serum enzymes during abeciclib treatment and its similarity to ribociclib and palbociclib, rare, clinically significant liver injury should be suspected. Probability Score: E (Unproven but suspected, rare, clinically significant cause of liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the clinical use of abecitabine during lactation. Because abecitabine and its metabolites bind to plasma proteins at a rate exceeding 90%, its concentration in breast milk may be low. However, the manufacturer recommends discontinuing breastfeeding during abecitabine treatment and for 3 weeks after the last dose.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
Based on in vitro models using animal brain tissue, the protein binding rate of abecitabine is approximately 95-98%. Although abecitabine binds to serum albumin, α-1-acid glycoprotein, and other human plasma proteins in an in vitro concentration-dependent manner, its major metabolite has also been shown to bind to plasma proteins. The approximate binding fractions for M2, M18, and M20 were 93.4%, 96.8%, and 97.8%, respectively. In the Phase I dose-escalation study, the dose-limiting toxicity of abexilide was grade 3 fatigue; the maximum tolerated dose was 200 mg every 12 hours [2]. The most common treatment-related adverse events associated with abexilide involved fatigue and gastrointestinal, renal, or hematopoietic system issues [2].

[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.

The cyclin-D-CDK4/6-RB pathway is often abnormally regulated in head and neck squamous cell carcinoma (HNSCC). Abeciclib can inhibit CDK4/6, arrest the cell cycle, and inhibit the growth of HNSCC cells, providing a potential targeted therapy strategy for HNSCC [1]. Pharmacodynamics: The progression-free survival of patients with HR-positive, HER2-negative breast cancer treated with abeciclib in combination with fulvestrant was 16.4 months, while the progression-free survival of patients in the fulvestrant combined with placebo group was 9.3 months. In the abeciclib monotherapy group, 19.7% of patients experienced complete or partial tumor shrinkage within a median of 8.6 months after treatment. Abeciclib can induce cell cycle arrest and exert anti-tumor activity in human tumor xenograft models. In patient studies and healthy volunteer studies, abeciclib did not show any clinically significant changes in the QTc interval. Abeciclib is an anti-tumor drug, a dual inhibitor of cyclin-dependent kinases 4 (CDK4) and 6 (CDK6). CDK4 and CDK6 are involved in cell cycle regulation and promote cancer cell growth when their activity is dysregulated. On September 28, 2017, the FDA approved abeciclib under the brand name Verzenio for the treatment of hormone receptor-positive, HER2-negative advanced or metastatic breast cancer in patients whose disease has progressed after failure of endocrine therapy. Abeciclib can be used alone in patients with metastases after endocrine therapy and chemotherapy, or in combination with [DB00947]. In patients with hormone receptor-positive, HER2-negative breast cancer, oral abeciclib improved progression-free survival and objective response rates. Abeciclib has been used in trials for the treatment of melanoma, lymphoma, tumors, solid tumors, and glioblastoma. Abeciclib is a kinase inhibitor. The mechanism of action of abeciclib is as a kinase inhibitor.
Abeciclib is a unique cyclin-dependent kinase inhibitor used in combination with anti-estrogens to treat postmenopausal metastatic breast cancer. Elevated serum transaminases during abeciclib treatment occur at a moderate rate and are suspected as a rare cause of clinically significant liver injury.
Abeciclib is an orally administered cyclin-dependent kinase (CDK) inhibitor that targets the CDK4 (cyclin D1) and CDK6 (cyclin D3) cell cycle pathways and has potential antitumor activity. Abeciclib specifically inhibits CDK4 and CDK6, thereby inhibiting the phosphorylation of retinoblastoma protein (Rb) in the early G1 phase. Inhibition of Rb phosphorylation prevents CDK-mediated G1-S phase transition, thereby arresting the cell cycle in the G1 phase, inhibiting DNA synthesis, and suppressing cancer cell growth. Overexpression of serine/threonine kinases CDK4/6 is observed in some cancers, leading to cell cycle dysregulation.
Abemaciclib is a small molecule drug whose clinical trial phase is up to Phase IV (covering all indications). It was first approved in 2017 and currently has 6 approved indications and 48 investigational indications.

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Abemaciclib is a CDK4/6 inhibitor with structural features, biological and clinical activity similar to palbociclib and ribociclib; based on the results of the MONARCH 1 and 2 trials, abemaciclib is the latest CDK4/6 inhibitor approved by the U.S. Food and Drug Administration (FDA) for the treatment of hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer[1].
-Abemaciclib is the first CDK4 and CDK6 selective inhibitor with a safety profile that allows for continuous administration to achieve target inhibition[2].
- Abecil can induce an inflammatory tumor microenvironment in T cells, thereby enhancing the efficacy of PD-L1 checkpoint blockade; this supports clinical research on the combined use of abecil with drugs that regulate T cell anti-tumor immunity, such as anti-PD-L1 [3].

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.)