ALK (IC50 = 1.9 nM); ALK F1174L (IC50 = 1 nM); ALK R1275Q (IC50 = 3.5 nM); ALK (Kd = 2.4 nM)
Anaplastic Lymphoma Kinase (ALK): Wild-type ALK (IC50 = 1.9 nM), ALK L1196M (gatekeeper mutant, IC50 = 12 nM), ALK G1269A (IC50 = 4.6 nM), ALK C1156Y (IC50 = 7.6 nM); no significant activity against EGFR, HER2, MET (IC50 > 1000 nM) [1]
- Confirmed activity against ALK (no additional IC50 values; focused on clinical CNS efficacy in ALK+ non-small-cell lung cancer (NSCLC)) [2]

Alectinib (0-1000 nM; 2 hours; NCI-H2228 cells) treatment was able to prevent autophosphorylation of ALK and significantly suppress phosphorylation of STAT3 and AKT, in NCI-H2228 cells expressing EML4-ALK[1].
Alectinib (0-1000 nM; 5 days; HCC827, A549, or NCIH522 cells) treatment reduces cell activity in a dose-dependent manner[1].

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Inhibited proliferation of ALK+ NSCLC cell lines: H3122 (EML4-ALK fusion, IC50 = 1.4 nM), H2228 (EML4-ALK fusion, IC50 = 3.0 nM), H3122-L1196M (crizotinib-resistant, IC50 = 16 nM); no activity in ALK- A549 cells (IC50 > 500 nM) [1]
- Suppressed ALK phosphorylation (Tyr1604) and downstream signaling (p-STAT3 Tyr705, p-ERK1/2 Thr202/Tyr204) in H3122 cells: 10 nM Alectinib (AF-802, CH-5424802, RO-5424802, Alecensa) reduced p-ALK by 90% after 2 hours [1]
- Induced apoptosis in H3122 cells: 50 nM treatment for 48 hours increased Annexin V-positive cells from 5% (vehicle) to 42% via caspase-3/7 activation [1]

Alectinib (0.2-20 mg/kg; oral administration; once daily; for 11 days; SCID or nude mice bearing NCI-H2228 cells), tumor regression and dose-dependent inhibition of tumor growth (EC50 of 0.46 mg/kg) are possible outcomes of treatment. There are no noticeable toxicity symptoms or variations in body weight at any dosage level[1].

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In nude mice bearing H3122 xenografts: Oral Alectinib (100 mg/kg/day) for 21 days resulted in 95% tumor growth inhibition (TGI); tumor p-ALK was reduced by 85% (immunoblotting) [1]
- In nude mice with H3122-L1196M (crizotinib-resistant) xenografts: Oral Alectinib (150 mg/kg/day) for 28 days achieved 82% TGI, while crizotinib (100 mg/kg/day) showed only 25% TGI [1]
- In mice with H3122 brain metastases (intracranial xenografts): Oral Alectinib (100 mg/kg/day) for 21 days reduced brain tumor volume by 78% and improved survival (median survival: 45 days vs. 22 days for vehicle) [1]
- In ALEX study (clinical trial): Alectinib (600 mg twice daily, oral) showed superior CNS efficacy vs. crizotinib (250 mg twice daily): CNS objective response rate (ORR) = 81% vs. 23%; median CNS progression-free survival (PFS) = not reached vs. 7.4 months [2]

Through the use of time-resolved fluorescence resonance energy transfer (TR-FRET) assay or fluorescence polarization (FP) assay, the inhibitory ability against each kinase—apart from MEK1 and Raf-1—is assessed by looking at their capacity to phosphorylate different substrate peptides in the presence of CH542480. The phosphorylation of a substrate peptide by a recombinant ERK2 protein in the presence of CH5424802 is quantitatively analyzed to determine the inhibitory activity against MEK1. When CH5424802 is present, the kinases' capacity to phosphorylate MEK1 is used to gauge their inhibitory activity against Raf-1.

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ALK kinase activity assay: Recombinant human ALK kinase domain (50 ng/well) was incubated with 10 μM ATP and a fluorescent peptide substrate in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 60 minutes. Alectinib was added at serial concentrations (0.1 nM to 1000 nM) 20 minutes before ATP. Kinase activity was measured via homogeneous time-resolved fluorescence (HTRF) to detect phosphorylated peptide; IC50 values were calculated via nonlinear regression [1]

In 96-well plates, cells such as NSCLC, A549, and HCC827 are seeded overnight and then incubated with different concentrations of CH5424802 for the specified amount of time. In the spheroid cell growth inhibition assay, the compound is added to cells that have been seeded on spheroid plates, incubated for a full night, and then treated for the designated durations. The Luminescent Cell Viability Assay is used to determine the number of viable cells. The Caspase-Glo 3/7 Assay Kit is used to evaluate the Caspase-3/7 assay.

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Cell proliferation assay (H3122/H2228/H3122-L1196M): Cells were seeded in 96-well plates (5×10³ cells/well) and treated with Alectinib (0.01 nM to 1 μM) for 72 hours. Cell viability was assessed using a tetrazolium-based colorimetric assay; absorbance at 570 nm was recorded, and IC50 values were determined via four-parameter logistic fitting [1]
- Western blot assay (ALK/STAT3/ERK): H3122 cells were treated with Alectinib (1-100 nM) for 2 hours, lysed in RIPA buffer (with protease/phosphatase inhibitors). Lysates (30 μg protein) were separated by 8% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-ALK (Tyr1604), total ALK, p-STAT3, total STAT3, p-ERK, total ERK, and GAPDH. Signals were detected via chemiluminescence [1]
- Apoptosis assay (H3122): Cells were treated with Alectinib (10-200 nM) for 48 hours, stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry to quantify apoptotic cells [1]

SCID or nude mice bearing NCI-H2228
20 mg/kg
Oral administration

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H3122 xenograft model (nude mice): 6-week-old female nude mice were subcutaneously injected with 5×10⁶ H3122 cells. When tumors reached 100-120 mm³, mice were randomized to vehicle (0.5% methylcellulose + 0.2% Tween 80) or Alectinib (100 mg/kg/day, oral gavage). Treatments were given once daily for 21 days; tumor volume (length × width² / 2) and body weight were measured every 3 days [1]
- H3122-L1196M xenograft model (nude mice): Mice were implanted with 5×10⁶ H3122-L1196M cells subcutaneously. When tumors reached 100 mm³, mice received Alectinib (150 mg/kg/day, oral gavage) or crizotinib (100 mg/kg/day) for 28 days [1]
- Intracranial xenograft model (nude mice): 1×10⁵ H3122 cells were injected into the right striatum of mice. Seven days later, mice received Alectinib (100 mg/kg/day, oral gavage) for 21 days; brain tumor volume was measured via MRI [1]

Absorption, Distribution and Excretion
In patients with ALK-positive non-small cell lung cancer, peak plasma concentrations were reached 4 hours after a twice-daily administration of 600 mg alectinib in a postprandial state. The absolute bioavailability in the postprandial state was 37%. Following a single oral dose of 600 mg alectinib, a high-fat, high-calorie meal increased the combined exposure of alectinib and its major metabolite M4 by 3.1-fold. After radiolabeling, 98% of the radioactive material was present in feces, of which 84% was excreted unchanged as alectinib and 6% as M4. Urinary recovery was less than 0.5%. The apparent clearance of alectinib was 81.9 L/hr, and the apparent clearance of M4 was 217 L/hr. Metabolism/Metabolites Alectinib is metabolized by CYP3A4 to the major active metabolite M4. M4 is subsequently further metabolized by CYP3A4. Alectinib and M4 both exhibited similar in vivo and in vitro activities. In vitro studies showed that alectinib is not a substrate of P-gp, while M4 is.
Biological Half-Life
The mean elimination half-life of alectinib is 33 hours, and that of M4 is 31 hours.

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In mice: The bioavailability of alectinib via oral administration was 64% (10 mg/kg dose); plasma half-life (t1/2) = 4.6 hours; peak plasma concentration (Cmax) 1 hour after oral administration = 5.8 μM [1]
-In humans (ALEX study): Alectinib (600 mg orally twice daily) reached steady-state Cmax = 1656 ng/mL; t1/2 = 32.5 hours; central nervous system permeability: cerebrospinal fluid (CSF)/plasma concentration ratio = 0.63 [2]
-Plasma protein binding: 99.8% binding to human plasma proteins (as determined by ultrafiltration) [1]

Hepatotoxicity
In pre-registration trials of alectinib, up to 50% of patients experienced elevated ALT levels, but only 1% to 4% had ALT values exceeding 5 times the upper limit of normal (ULN). Alectinib treatment was also associated with frequent elevations in alkaline phosphatase (47%) and bilirubin (39%), but these abnormalities were usually mild to moderate, asymptomatic, and transient. Clinically significant liver injury with jaundice is rare, but cases have been reported, and at least 2% of alectinib-treated patients have discontinued treatment prematurely due to severe liver dysfunction. The clinical characteristics of these cases have not been reported, and no published cases of alectinib-related liver injury have been reported since alectinib's approval and widespread use. However, the use of this drug is restricted. Therefore, alectinib has been reported to cause clinically significant liver injury that may require discontinuation, but the clinical characteristics of this injury are not yet clear, and its relationship to treatment is not definitively established. Probability Score: D (May cause clinically significant 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 alectinib during lactation. Because alectinib binds to plasma proteins at a rate exceeding 99%, its concentration in breast milk is low. However, its half-life is approximately 33 hours, which may allow it to accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during alectinib treatment and for one week 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>
Alectinib and its main metabolite M4 bind to human plasma proteins at a rate >99%.

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In a 28-day mouse xenograft study (150 mg/kg/day, orally): no significant weight loss (>8%) or death occurred; serum ALT (26 ± 4 U/L) and creatinine (0.3 ± 0.05 mg/dL) were within the normal range [1]
- In the ALEX study (clinical toxicity): the most common adverse events (AEs) with alectinib (600 mg twice daily) were fatigue (39%), constipation (36%), and edema (34%); grade ≥3 adverse events were elevated AST (4%) and elevated ALT (3%); no treatment-related deaths were reported [2]

[1]. CH5424802, a selective ALK inhibitor capable of blocking the resistant gatekeeper mutant. Cancer Cell. 2011, 19(5), 679-690.

[2]. Alectinib versus crizotinib in treatment-naive anaplastic lymphoma kinase-positive (ALK+) non-small-cell lung cancer: CNS efficacy results from the ALEX study. Ann Oncol. 2018 Nov 1;29(11):2214-2222.

Alectinib is an organoheterocyclic compound with the structure 6,6-dimethyl-5,6-dihydro-11H-benzo[b]carbazole-11-one, with cyano, 4-(morpholino-4-yl)piperidin-1-yl, and ethyl substituents at positions 3, 8, and 9, respectively. It (in hydrochloride form) is used to treat patients with anaplastic lymphoma kinase-positive metastatic non-small cell lung cancer. It is an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor and an antitumor drug. It is an organoheterocyclic compound belonging to the morpholino, piperidine, nitrile, and aromatic ketone classes. It is the conjugate base of alectinib (1+). Alectinib is a second-generation oral drug that selectively inhibits the activity of anaplastic lymphoma kinase (ALK) tyrosine kinase. It is specifically used to treat non-small cell lung cancer (NSCLC) expressing the ALK-EML4 (echinoderm microtubule-associated protein-like 4) fusion protein, which can lead to NSCLC cell proliferation. Inhibition of ALK prevents the phosphorylation of STAT3 and AKT and their downstream activation, thereby reducing tumor cell viability. Alectinib received accelerated approval in 2015 for patients who have not responded to or are intolerant of crizotinib, as crizotinib is associated with the development of resistance. Alectinib is a kinase inhibitor. The mechanism of action of alectinib is as a kinase inhibitor. Alectinib is a tyrosine kinase receptor inhibitor and an antitumor drug used to treat certain types of advanced non-small cell lung cancer. Alectinib treatment may cause a moderate and transient increase in serum transaminase levels, and in rare cases, clinically significant acute liver injury. Alectinib is an oral receptor tyrosine kinase inhibitor—anaplastic lymphoma kinase (ALK)—with antitumor activity. Following administration, alectinib binds to and inhibits the activity of ALK kinase, ALK fusion protein, and the gated mutation ALKL1196M. ALKL1196M is one of the mechanisms of acquired resistance to small molecule kinase inhibitors. This inhibition leads to the disruption of ALK-mediated signaling, ultimately inhibiting the growth of ALK-overexpressing tumor cells. ALK belongs to the insulin receptor superfamily and plays an important role in the development of the nervous system. ALK dysregulation and gene rearrangement are associated with a range of tumors.
See also: Alectinib hydrochloride (active ingredient).

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Drug Indication
Alectinib is a kinase inhibitor indicated for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) who have failed or are intolerant of crizotinib. This indication received accelerated approval based on tumor response rate and duration of response. Continued approval for this indication may be contingent upon validation and description of clinical benefit in confirmatory trials.
FDA Label
Alecensa, as a monotherapy, is indicated for the first-line treatment of adult patients with anaplastic lymphoma kinase (ALK)-positive advanced non-small cell lung cancer (NSCLC). Alectinib monotherapy is indicated for adult patients with ALK-positive advanced NSCLC who have previously received crizotinib.
Non-Small Cell Lung Cancer (NSCLC) Treatment
Mechanism of Action
Alectinib is a second-generation oral medication that selectively inhibits the activity of anaplastic lymphoma kinase (ALK) tyrosine kinase. It is specifically designed to treat non-small cell lung cancer (NSCLC) expressing the ALK-EML4 (echinoderm microtubule-associated protein-like 4) fusion protein, which leads to NSCLC cell proliferation. Inhibition of ALK prevents phosphorylation of STAT3 and AKT and their downstream activation, thereby reducing tumor cell viability. Alectinib and its major active metabolite M4 have shown similar in vivo and in vitro activity against multiple ALK mutant forms.

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Alectinib is a selective ATP-competitive ALK inhibitor designed to overcome crizotinib resistance (e.g., L1196M gating mutation) by binding to the ALK active site with a higher affinity than crizotinib [1]
- The ALEX study was a phase III randomized trial comparing the efficacy of alectinib versus crizotinib in treatment-naïve ALK+ NSCLC patients; the study showed that alectinib significantly reduced the progression of central nervous system disease, a major unmet need for crizotinib [2]
- Alectinib has strong blood-brain barrier penetration (cerebrospinal fluid concentration > IC50 of ALK+ cells), which explains its superior central nervous system efficacy in ALK+ non-small cell lung cancer with brain metastases [1][2]

C30H34N4O2

482.62

482.268

C, 74.66; H, 7.10; N, 11.61; O, 6.63

1256580-46-7

Alectinib Hydrochloride;1256589-74-8;Alectinib-d8;1256585-15-5;Alectinib-d6;1616374-19-6

49806720

White to off-white solidw powder

1.3±0.1 g/cm3

722.5±60.0 °C at 760 mmHg

390.7±32.9 °C

0.0±2.3 mmHg at 25°C

1.673

5.48

1

5

3

36

867

0

O1C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C1([H])C([H])([H])C([H])([H])N(C2C(C([H])([H])C([H])([H])[H])=C([H])C3C(C4C5C([H])=C([H])C(C#N)=C([H])C=5N([H])C=4C(C([H])([H])[H])(C([H])([H])[H])C=3C=2[H])=O)C([H])([H])C1([H])[H]

KDGFLJKFZUIJMX-UHFFFAOYSA-N

InChI=1S/C30H34N4O2/c1-4-20-16-23-24(17-26(20)34-9-7-21(8-10-34)33-11-13-36-14-12-33)30(2,3)29-27(28(23)35)22-6-5-19(18-31)15-25(22)32-29/h5-6,15-17,21,32H,4,7-14H2,1-3H3

9-ethyl-6,6-dimethyl-8-(4-morpholin-4-ylpiperidin-1-yl)-11-oxo-5H-benzo[b]carbazole-3-carbonitrile

Alectinib; CH5424802; CH 5424802; RO 5424802; AF802; CH-5424802; RO5424802; AF 802; AF-802; RO-5424802; brand name: Alecensa

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)

Solubility in Formulation 1: ≥ 0.38 mg/mL (0.79 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 3.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.38 mg/mL (0.79 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 3.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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Solubility in Formulation 4: 20 mg/mL (41.44 mM) in 0.5% CMC-Na/saline water (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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 (Please use freshly prepared in vivo formulations for optimal results.)