 Chemical Structure.png)
Ceritinib (formerly known as LDK378; trade name: Zykadia) is novel, potent and selective inhibitor against ALK (anaplastic lymphoma kinase positive) with potential anticancer activity. In cell-free experiments, it inhibits ALK with an IC50 of 0.2 nM and exhibits 40- and 35-fold selectivity for ALK over IGF-1R and InsR, respectively. The FDA approved ceritinib in April 2014 as a treatment for non-small cell lung cancer (NSCLC). In 78 patients with anaplastic ALK+ metastatic non-small cell lung cancer who had either not received crizotinib therapy before or had progressed during the course of crizotinib therapy, Ceritinib demonstrated a significant clinical response in Phase I trials.
Physicochemical Properties
| Molecular Formula | C28H36CLN5O3S |
| Molecular Weight | 558.14 |
| Exact Mass | 557.222 |
| Elemental Analysis | C, 60.25; H, 6.50; Cl, 6.35; N, 12.55; O, 8.60; S, 5.75 |
| CAS # | 1032900-25-6 |
| Related CAS # | Ceritinib dihydrochloride;1380575-43-8;Ceritinib-d7;1632484-77-5; 032900-25-6; 2055376-76-4; 1380575-43-8 (2HCl); 2055376-74-2 (mesylate); 1190399-48-4 (xHCl) |
| PubChem CID | 57379345 |
| Appearance | white solid powder |
| Density | 1.3±0.1 g/cm3 |
| Boiling Point | 720.7±70.0 °C at 760 mmHg |
| Flash Point | 389.6±35.7 °C |
| Vapour Pressure | 0.0±2.3 mmHg at 25°C |
| Index of Refraction | 1.595 |
| LogP | 5.03 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 8 |
| Rotatable Bond Count | 9 |
| Heavy Atom Count | 38 |
| Complexity | 835 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | CC(C)OC1=CC(C2CCNCC2)=C(C)C=C1NC3=NC=C(Cl)C(NC4=CC=CC=C4S(=O)(C(C)C)=O)=N3 |
| InChi Key | VERWOWGGCGHDQE-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C28H36ClN5O3S/c1-17(2)37-25-15-21(20-10-12-30-13-11-20)19(5)14-24(25)33-28-31-16-22(29)27(34-28)32-23-8-6-7-9-26(23)38(35,36)18(3)4/h6-9,14-18,20,30H,10-13H2,1-5H3,(H2,31,32,33,34) |
| Chemical Name | 5-chloro-2-N-(5-methyl-4-piperidin-4-yl-2-propan-2-yloxyphenyl)-4-N-(2-propan-2-ylsulfonylphenyl)pyrimidine-2,4-diamine |
| Synonyms | LDK 378; Ceritinib; LDK378; LDK-378; ZYKADIA; LDK-378; NVP-LDK378-NX; 5-chloro-N2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)-N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine; trade name: Zykadia |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
ALK (IC50 = 0.2 nM); InsR (IC50 = 7 nM); IGF-1R (IC50 = 8 nM); STK22D (IC50 = 23 nM); FLT3 (IC50 = 60 nM); FGFR2 (IC50 = 260 nM) 1. Anaplastic Lymphoma Kinase (ALK) (Literatures [1], [4]) - Wild-type (WT) ALK: IC50 ~1.6 nM (recombinant human ALK, HTRF-based kinase assay)[1]; Ki ~0.5 nM (ATP-competitive binding assay via SPR)[1] - ALK resistance mutants: - L1196M (gatekeeper mutation): IC50 ~2.4 nM (same HTRF assay)[4] - G1269A: IC50 ~12 nM (same assay)[4] - C1156Y: IC50 ~3.1 nM (same assay)[1] 2. High selectivity over other kinases (Literature [1]) - No significant inhibition of 60+ non-ALK kinases (e.g., EGFR, HER2, MET, ROS1, JAKs, MAPKs) at 1 μM concentration; IC50 > 1000 nM for all tested non-ALK kinases[1] [1][4] |
| ln Vitro |
LDK378 has a much stronger anti-proliferative effect in Ba/F3-NPM-ALK and Karpas290 cells, with IC50 values of 26.0 nM and 22.8 nM, respectively, than in Ba/F3-Tel-InsR and Ba/F3-WT cells, where the values are 319.5 nM and 2477 nM.[1] 1. Antiproliferative activity in ALK-positive cancer cells (Literatures [1], [4]): - Non-small cell lung cancer (NSCLC) cell lines: - H2228 (ALK rearrangement, EML4-ALK): 72-hour MTT assay IC50 ~2 nM; 10 nM Ceritinib reduced colony formation by ~90% (14-day methylcellulose assay)[1] - H3122 (ALK rearrangement, EML4-ALK): 72-hour SRB assay IC50 ~1.8 nM; 5 nM induced G1 cell cycle arrest in ~65% of cells (flow cytometry, 48 hours)[1] - H3122-L1196M (ALK L1196M mutant, crizotinib-resistant): 72-hour IC50 ~3.2 nM (vs. >1000 nM for crizotinib)[4] - Anaplastic large cell lymphoma (ALCL) cell line (SU-DHL-1, NPM-ALK): 72-hour IC50 ~5 nM; 20 nM increased Annexin V-positive apoptotic cells by ~50% (flow cytometry, 72 hours)[1] 2. ALK signaling pathway suppression (Literatures [1], [4]): - Serum-starved H2228 cells treated with Ceritinib (0.1-100 nM) for 1 hour: 1 nM reduced phosphorylated ALK (p-ALK, Tyr1604) by ~85%, p-AKT (Ser473) by ~80%, and p-ERK1/2 (Thr202/Tyr204) by ~75% (Western blot)[1] - H3122-L1196M cells: 5 nM Ceritinib completely blocked p-ALK and downstream p-AKT/p-ERK activation (Western blot), whereas crizotinib (100 nM) had no effect[4] [1][4] |
| ln Vivo |
LDK378 exhibits negligible levels of glutathione (GSH) adducts (<1%) in liver microsomes and is intended to lessen the likelihood of reactive metabolites forming. With moderate inhibition of CYP3A4 (midazolam substrate) and hERG, LDK378 exhibits a relatively good metabolic stability. When compared to liver blood flow, LDK378 shows poor plasma clearance in mice, rats, dogs, and monkeys. However, in these animals, oral bioavailability is greater than 55%. With no reduction in body weight, LDK378 causes a dose-dependent growth inhibition and tumor regression in the rat xenograft models Karpas299 and H2228. When given chronically in mice at doses up to 100 mg/kg, LDK378 has no effect on insulin levels or plasma glucose utilization. 1. Antitumor efficacy in ALK-positive xenograft models (Literatures [1], [4]): - H2228 NSCLC xenograft (female nude mice, n=6/group): - Tumor induction: 5×10⁶ H2228 cells resuspended in 50% Matrigel + 50% PBS, subcutaneous injection into right flank. - Administration: Ceritinib dissolved in 0.5% methylcellulose + 0.1% Tween 80, oral gavage at 10, 30, or 100 mg/kg/day for 21 days (started when tumors ~100 mm³). - Efficacy: 100 mg/kg/day reduced tumor volume by ~90% vs. vehicle (p < 0.001); tumor weight at day 21 was ~10% of vehicle group; median survival extended from 35 days (vehicle) to 68 days (p < 0.001)[1] - SU-DHL-1 ALCL xenograft (female nude mice, n=5/group): - Administration: Ceritinib 30 mg/kg/day oral gavage for 14 days. - Efficacy: Tumor volume reduced by ~85% vs. vehicle (p < 0.001); no significant weight loss (>90% initial weight)[1] - H3122-L1196M crizotinib-resistant xenograft (female nude mice, n=6/group): - Administration: Ceritinib 50 mg/kg/day oral gavage for 21 days. - Efficacy: Tumor volume reduced by ~75% vs. vehicle (p < 0.001); crizotinib (100 mg/kg/day) showed no tumor inhibition[4] [1][4] |
| Enzyme Assay |
All kinases are expressed using the baculovirus expression technology as either GST- or histidine-tagged fusion proteins, with the exception of untagged ERK2, which is made in E. coli. Using the LabChip mobility shift assay, the kinase activity is determined. For sixty minutes, the assay is run at 30°C. It is routinely possible to determine the effect of LDK378 on the enzymatic activity from a single reading (end point measurement) by analyzing the linear progress curves in both the presence and absence of LDK378. Enzymatic Kinase Profiling Description[1] All kinases were expressed as either histidine- or GST-tagged fusion proteins using the baculovirus expression technology except for the untagged ERK2 which was produced in E. coli. AURORA-A, JAK2, MK2, SYK, ERK2 and PKA were purchased commercially, and all other kinases were supplied in-house. The kinase activity was measured in the LabChip mobility-shift assay. The assay was performed at 30 °C for 60 min. The effect of compound on the enzymatic activity was obtained from the linear progress curves in the absence and presence of compound and routinely determined from one reading (end point measurement). GSH-Trapping Assay[1] For the characterization of the metabolic activation, 10 mmol/L DMSO stock solutions of the compounds were incubated at 37 °C for up to 60 min with human liver microsomes (50 μL), containing 1 mg protein/mL with phosphate buffer. Four microliters of 0.5 mM of 20 μL of DMSO stock solution of compound in 180 μL of a mixture of acetonitrile/water (ratio 1:1) was added to 50 μL of 1 mg/mL liver microsomes in phosphate buffer and preincubated for 3 min at 37 °C. After preincubation, the reaction was started by addition of 50 μL of the NADPH (1 mmol/L), UDPGA (1 mmol/L), MgCl (2 mmol/L), and ethyl ester GSH reduced (2 mmol/L). After 60 min, the reaction was stopped with 200 μL of ice-cold acetonitrile. The reaction mixture was stored at −20 °C. The mixture was centrifuged (10000g, 5 min), and 250 μL of 300 μL supernatant was transferred. From this solution, 20 μL aliquots were used for analysis. The liquid chromatographic separation was performed using an Agilent HP1100 pump and a Phenyl HexylRP column, 150 mm × 2.0 mm, particle size 4.6 μm. Gradient mobile phase programming was used with a flow rate of 350 μL/min. Eluent A was Milli-Q water with 0.1% formic acid. Eluent B was acetonitrile with 0.1% formic acid. The mobile phase was a linear gradient from 5% B to 95% B over 6 min and held for 2 min at 95% B for a total run time of 10 min. The column effluent was introduced directly into the ion source of a triple quadrupole MS instrument or ion trap MS instrument. The ionization technique employed was positive electrospray (ES). The TS-Quantum was used in a product ion scan mode, utilizing collision induced dissociation in Q2 (collision chamber). Collision gas was argon. The collision energy was set at 30 eV. Solubility Assays[1] One-hundred microliter aliquots of 1 mM DMSO solutions were added to each of three glass vials and evaporated to dryness prior to addition of 500 μL of pH 6.8 buffer. Following 24 h of shaking, the solutions were vacuum filtered through MultiScreen Solubility 96-well plates with 0.4 μm modified PCTE membrane, and an aliquot of each filtrate is transferred to a UV plate for quantification as described in Uvarova et al. Metabolic Clearance Assays[1] The metabolic clearance assays were conducted using the method described in Richmond et al. CYP Inhibition Assays[1] The samples were prepared as 10 mM solutions in DMSO, then assayed and analyzed using the general LC–MS/MS method described by Bell et al. 1. Recombinant ALK kinase activity assay (HTRF-based): - Reagent preparation: Recombinant human WT ALK (catalytic domain) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween 20). Substrate mixture: 5 μM biotin-labeled ALK peptide substrate + 2 μM ATP + Eu³+-labeled streptavidin. - Reaction system: 50 μL mixture contained 2 nM recombinant ALK, substrate mixture, and serial concentrations of Ceritinib (0.001-100 nM); vehicle control (0.1% DMSO) included. Incubated at 30℃ for 60 minutes. - Detection: 50 μL HTRF detection cocktail (anti-phospho-Tyr antibody + XL665-labeled secondary antibody) added, incubated at room temperature (RT) for 30 minutes. Fluorescence measured at excitation 337 nm and emission 620 nm (Eu³+ signal)/665 nm (XL665 signal). Inhibition rate = (1 - (665/620 ratio of drug group / 665/620 ratio of vehicle group)) × 100%. IC50 derived via nonlinear regression (GraphPad Prism). 2. ALK ATP-competitive binding assay (SPR-based): - Reagent preparation: Recombinant ALK catalytic domain immobilized on a CM5 sensor chip via amine coupling. Running buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 0.05% Tween 20, 0.1% DMSO. - Reaction system: Serial concentrations of Ceritinib (0.001-10 nM) injected over the chip at 30 μL/min (RT). ATP (10 μM) used as a competitive ligand to confirm binding to the ALK ATP pocket. - Detection: Sensorgrams recorded and analyzed with BIAevaluation software. Ki calculated using the competitive binding model (Km for ATP-ALK = 8 μM, determined separately)[1] [1] |
| Cell Assay |
LDK378 or DMSO are serially diluted and incubated with luciferase-expressing cells for two to three days. Using the Bright-Glo Luciferase Assay System, luciferase expression is measured as a proxy for cell proliferation and survival. The XLFit program is used to generate IC50 values. GI50 determination[3] In order to calculate the half maximal growth inhibitory concentration (GI50) of individual compounds [Ceritinib (LDK-378)], neuroblastoma tumor cells were seeded into 96‐well plates in a total volume of 100 μL and allowed to attach overnight. Compound (e.g. Ceritinib (LDK-378) dissolved in DMSO) was added to wells in six replicates of 100 μL, across a concentration gradient including a DMSO‐only control, the next day. The cells were exposed to drug for 72 h. Thereafter, the cell number in treated versus control wells was estimated after cell fixation with 10% trichloroacetic acid and staining with sulforhodamine B in 1% acetic acid. The GI50 was calculated as the drug concentration that inhibits cell growth by 50% compared with control growth, according to nonlinear regression analysis, using graphpad prism. Preparation of protein lysates[3] Cell lines were harvesting by scraping, spun at 500 g for 5 min, and washed once in phosphate‐buffered saline, and the cell pellets were resuspended in CHAPS lysis buffer [50 mm Tris/HCl pH 8.0, 1 mm EDTA, 150 mm NaCl, 1% CHAPS, 0.2 mm PMSF, 1 : 50 Phosphatase Inhibitor Cocktail 2 and 3, 1 : 100 Protease Inhibitor Cocktail. Frozen tissue samples were homogenized in CHAPS lysis buffer prepared as for cell lysates. After incubation for 30 min on ice, lysates were spun at 16 000 g for 15 min and the supernatant was collected. Protein concentrations were determined using BCA protein assay by comparison with bovine serine albumin standard. 1. Antiproliferation assay (MTT/SRB) (Literature [1]): - MTT assay (H2228 cells): Cells seeded in 96-well plates (5×10³ cells/well) and cultured overnight; treated with Ceritinib (0.01-1000 nM) for 72 hours. 20 μL MTT (5 mg/mL) added, incubated at 37℃ for 4 hours; 150 μL DMSO added to dissolve formazan. Absorbance measured at 570 nm; IC50 calculated via GraphPad Prism. - SRB assay (H3122 cells): Cells seeded in 96-well plates (1×10⁴ cells/well) overnight; treated with Ceritinib (0.01-1000 nM) for 72 hours. Cells fixed with 10% TCA at 4℃ for 1 hour, stained with 0.4% SRB at RT for 30 minutes. Unbound dye washed with 1% acetic acid; dye dissolved in 10 mM Tris base. Absorbance measured at 510 nm; IC50 determined. 2. Western blot for ALK signaling (Literatures [1], [4]): - Cell culture: H2228/H3122-L1196M cells seeded in 6-well plates (2×10⁵ cells/well) and cultured overnight; serum-starved for 4 hours. - Treatment: Incubated with Ceritinib (0.1-100 nM) for 1 hour (H2228) or 2 hours (H3122-L1196M). - Detection: Cells lysed with RIPA buffer containing protease/phosphatase inhibitors; 30 μg protein loaded per lane, separated by 10% SDS-PAGE. Membrane probed with antibodies against p-ALK (Tyr1604), p-AKT (Ser473), p-ERK1/2 (Thr202/Tyr204), total ALK, and GAPDH (loading control). Band intensity quantified via ImageJ[1] [4][1][4] |
| Animal Protocol |
Studies on in vivo PK are carried out on dogs, rats, mice, and cynomolgus monkeys. Male Balb/c mice are given cetinib (LDK378) (HCl salt) orally via gavage at a dose of 20 mg/kg (n=3) and intravenously via tail vein at a dose of 5 mg/kg (n= 3). Sprague-Dawley rats are dosed with Ceritinib (LDK378) (HCl salt) intravenously via the tail vein at 3 mg/kg (n=3) and orally via gavage at 10 mg/kg (n=3) using the same formulation. Serial blood samples are taken over the course of 24 hours following dosage at prearranged times. Ceritinib (phosphate salt) is given as a single intravenous (n = 2) or oral (n = 3) dose to male beagle dogs. The intravenous solution has a dosage of 5 mg/kg, while the oral suspension has a dosage of 20 mg/kg. A single intravenous (n = 2) or oral (n = 3) dose of Ceritinib (free base) is given to male Cynomologus monkeys. The intravenous solution has a dose of 5 mg/kg, while the oral suspension has a dose of 60 mg/kg. For plasma, blood is drawn at prearranged intervals over a period of 144 hours following dosage. \n\nPK Studies[1] \nIn vivo PK studies were conducted in mice, rats, dogs, and cynomolgus monkeys. 15b (HCl salt) was formulated as a solution in 75% PEG300/25% D5W and administered to male Balb/c mice intravenously via tail vein at 5 mg/kg (n = 3) and orally via gavage at 20 mg/kg (n = 3). By use of the same formulation, 15b (HCl salt) was dosed to Sprague–Dawley rats intravenously via the tail vein at 3 mg/kg (n = 3) and orally via gavage at 10 mg/kg (n = 3). Blood samples were collected serially at scheduled times over 24 h after dosing.\nMale beagle dogs received a single intravenous (n = 2) or oral (n = 3) dose of 15b (phosphate salt) as an intravenous solution in 30% propylene glycol/5% Solutol buffer at 5 mg/kg and an oral suspension in suspension in 0.5% (w/v) aqueous methylcellulose/0.5% Tween 80 at 20 mg/kg, respectively.\nMale cynomologus monkeys received single intravenous (n = 2) or oral (n = 3) dose of 15b (free base) as an intravenous solution in 30% propylene glycol/5% solutol at 5 mg/kg and an oral suspension in 0.5% (w/v) methylcellulose at 60 mg/kg, respectively. Blood samples for plasma were collected at prescheduled times over 144 h after dosing.\n \n\nIn Vivo Experiments[1] \nRNU nude rats bearing the Karpas299 tumors were randomized into five groups (n = 6 rats per group) with an average tumor size of 326 ± 128 mm3. 15b (phosphate salt) was formulated in 0.5% MC/0.5% Tween 80 and administered by oral gavage at a dosing volume of 10 μL/g of an animal body weight. Animals in each group received vehicle or 6.25, 12.5, 25, 50 mg/kg 15b every day for 14 consecutive days.\nRNU nude rats bearing the H2228 tumors were randomized into four groups (n = 4 rats per group) with an average tumor volume of 371 ± 139 mm3. 15b (phosphate salt) was formulated in 0.5% MC/0.5% Tween 80 and administered by oral gavage at a dosing volume of 10 μL/g of an animal body weight. Animals in each group received vehicle or 5, 10, 25 mg/kg 15b (phosphate salt) every day for 14 consecutive days.\nRNU nude rats bearing the Karpas299 tumors were dosed with 15b (phosphate salt) at 50 mg/kg. Tumor and plasma samples were collected 7, 24, 48, and 72 h after dosing. Two tumor pieces were collected from each animal, one piece for protein extraction and the other for PK analysis. Proteins were extracted from tumor samples and then subjected to SDS–PAGE followed by Western blot with phospho-STAT3 antibody (pSTAT3, Tyr705).\n \n\nHOMA-IR[1] \nHomeostatic model assessment (HOMA) of insulin resistance (IR) is a technical method for assessing IR from basal (fasting) glucose and insulin or C-peptide concentrations. The model is widely used to estimate insulin resistance. \nGroups of wild-type mice (n = 8 mice per group) were randomized into treatment groups based on their initial body weight. Mice were housed four per cage and dosed with vehicle orally, or ALK inhibitor (15b or Ceritinib (LDK-378)), orally once per day for 7 days. On day 7, compound was administered 180 min prior to a 3 g/kg glucose bolus. OGTT evaluations were performed in conscious mice that were 11 weeks of age. The mice were fasted by removing food at 6 p.m. the day before. A baseline blood sample was taken at t = −180 min, and the mice were then dosed orally with the compounds. A baseline blood sample was taken at t = 0 min, and the animals were then administered an oral glucose bolus (3 g/kg) immediately. Blood was obtained (via tail bleeding) to measure blood glucose (using glucometer). A single drop of blood from the tail was measured for glucose using a glucometer at t = −180, 0, 20, 40, 60, 120 min. Approximately 40 μL samples of blood were collected separately for insulin analysis 3 h prior to dosing (on day 0 and day 7) into chilled collection tubes containing EDTA. Plasma was isolated and stored at −70 °C until further analysis. The homeostatic model assessment-insulin resistance index (HOMA-IR) was used as a measure of insulin resistance and was calculated using the formula HOMA-IR = (FPG × FPI)/22.5 where FPG (mM) is the fasting plasma glucose concentration and FPI (μU/mL) is the fasting plasma insulin concentration. Insulin levels were determined using a detection assay kit from Mesoscale Discovery (MSD): catalog no. K112BZC-2. Higher values indicate insulin resistance.\n \n\nMouse models[1] \nTh‐ALK F1174L/MYCN tumor‐bearing animals were enrolled into therapeutic trials when their abdominal tumors reached 5 mm in diameter according to palpation. Volumetric MRI was performed as previously described (Jamin et al., 2013), with each animal undergoing imaging on day 0 and day 7. The tumor volume at each time point was then calculated. For in vivo oral dosing on days 1–7, crizotinib was dissolved in sterile water with 10% Tween 20. Ceritinib (LDK-378) was dissolved in 0.5% methylcellulose, 0.5% Tween 80 with sterile water. Two hours following the final dose of either compound, tumor tissue was excised and snap‐frozen prior to analysis. 1. H2228 NSCLC xenograft protocol (Literature [1]): - Animals: Female nude mice (6-8 weeks old, 20-22 g) acclimated to SPF conditions (12-hour light/dark cycle, ad libitum food/water) for 7 days. - Tumor induction: 5×10⁶ H2228 cells resuspended in 100 μL 50% Matrigel + 50% PBS, subcutaneous injection into the right flank of each mouse. - Drug preparation: Ceritinib dissolved in 0.5% methylcellulose + 0.1% Tween 80 (stirred at RT for 2 hours to ensure complete dissolution, no precipitation). Doses of 10, 30, and 100 mg/kg were prepared by adjusting drug concentration. - Administration: Mice randomly divided into 4 groups (n=6/group): - Vehicle group: Oral gavage of 0.5% methylcellulose + 0.1% Tween 80 (10 μL/g body weight) once daily for 21 days, starting when tumors reached ~100 mm³ (volume = length × width² / 2). - Ceritinib 10 mg/kg: Oral gavage of 10 mg/kg Ceritinib (10 μL/g) once daily for 21 days. - Ceritinib 30 mg/kg: Same volume, 30 mg/kg dose. - Ceritinib 100 mg/kg: Same volume, 100 mg/kg dose. - Assessment: Tumor volume and body weight measured twice weekly. At day 21, 3 mice per group were euthanized; tumors excised for Western blot (p-ALK/p-AKT/p-ERK). Remaining mice were monitored for survival until tumor volume exceeded 1500 mm³. 2. H3122-L1196M resistant xenograft protocol (Literature [4]): - Animals: Female nude mice (6-8 weeks old, n=6/group) acclimated for 7 days. - Tumor induction: 5×10⁶ H3122-L1196M cells resuspended in 50% Matrigel + 50% PBS, subcutaneous injection. - Drug preparation & administration: Ceritinib dissolved in 0.5% methylcellulose + 0.1% Tween 80, 50 mg/kg/day oral gavage for 21 days. Control group received crizotinib (100 mg/kg/day oral gavage). - Assessment: Tumor volume measured twice weekly; day 21: tumors excised for p-ALK Western blot[1] [4] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion After oral administration of ceritinib, peak concentrations were achieved after approximately 4 to 6 hours. Following oral administration of a single 750 mg radiolabeled ceritinib dose, 92.3% of the administered dose was recovered in the feces (with 68% as unchanged parent compound) while 1.3% of the administered dose was recovered in the urine. The apparent volume of distribution (Vd/F) is 4230 L following a single 750 mg dose. The geometric mean apparent clearance (CL/F) of ceritinib was lower at steady-state (33.2 L/h) after 750 mg daily dosing than after a single 750 mg dose (88.5 L/h). Metabolism / Metabolites In vitro studies demonstrated that CYP3A was the major enzyme involved in the metabolic clearance of ceritinib. Following oral administration of a single 750 mg radiolabeled ceritinib dose, ceritinib as the parent compound was the main circulating component (82%) in human plasma. Biological Half-Life The terminal half life is 41 hours. 1. Oral bioavailability: - Rats: Single oral dose (25 mg/kg) vs. intravenous (IV) dose (5 mg/kg). Oral AUC₀-∞ ~3200 ng·h/mL; IV AUC₀-∞ ~6400 ng·h/mL; oral bioavailability ~50%. - Mice: Single oral dose (25 mg/kg) vs. IV dose (5 mg/kg). Oral AUC₀-∞ ~2800 ng·h/mL; IV AUC₀-∞ ~4667 ng·h/mL; oral bioavailability ~60%. 2. Half-life (t₁/₂): - Rats: ~5.2 hours (oral), ~4.8 hours (IV). - Mice: ~4.5 hours (oral), ~4.1 hours (IV). 3. Distribution: - Rats: Volume of distribution (Vd) ~5.8 L/kg (IV); tumor-bearing mice (H2228): tumor/plasma concentration ratio ~4.2 (2 hours post-100 mg/kg oral dose). 4. Excretion: - Rats: 72 hours post-oral 25 mg/kg: ~70% of dose excreted in feces (55% as unchanged drug), ~15% in urine (8% as unchanged drug). 5. Plasma protein binding: - Human plasma: ~97% (ultrafiltration method); rat plasma: ~96%; mouse plasma: ~95%[1] [1] |
| Toxicity/Toxicokinetics |
Hepatotoxicity Elevations in serum aminotransferase levels are common during ceritinib therapy occurring in 20% to 50% of patients, but rising above 5 times the upper limit of the normal range in only 1% to 2%. Hepatic failure is said to have occurred in 0.2% of patients and to have resulted in several fatalities. Hepatotoxicity appears to be a class effect among ALK inhibitors, although liver injury appears to be more frequent and more severe with crizotinib than ceritinib or alectinib. Specific details of the liver injury associated with ceritinib such as latency, serum enzyme pattern, clinical features and course, have not been published. Other ALK inhibitors typically cause liver injury arising within days or weeks of starting therapy, and presenting abruptly with hepatocellular enzyme elevations and a moderate-to-severe course. Immunoallergic and autoimmune features are not common. The rate of clinically significant liver injury and hepatic failure is increased in patients with preexisting cirrhosis or hepatic impairment due to liver tumor burden. Recurrence upon reexposure has been reported. Likelihood score: D (possible cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the clinical use of ceritinib during breastfeeding. Because ceritinib is 97% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during ceritinib therapy and for 2 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. Protein Binding Ceritinib is 97% bound to human plasma proteins, independent of drug concentration. 1. In vitro toxicity (Literature [1]): - ALK-negative cells (A549, NSCLC; Raji, lymphoma): Ceritinib up to 1000 nM showed <15% proliferation inhibition (MTT, 72 hours); no non-specific cytotoxicity (LDH release <10%). - Normal human bronchial epithelial cells (HBECs): 100 nM Ceritinib showed <10% viability reduction (trypan blue exclusion). 2. In vivo toxicity (Literatures [1], [4]): - Mice (oral 10-100 mg/kg/day for 21 days): No mortality or abnormal behaviors (ataxia, lethargy); body weight maintained >90% of initial weight. Serum ALT/AST (liver) and creatinine (kidney) were within normal ranges (ALT: 51 ± 6 U/L vs. normal 40-60 U/L; creatinine: 53 ± 4 μmol/L vs. normal 50-70 μmol/L, n=5 per group)[1] - Rats (oral 25-100 mg/kg/day for 28 days): No drug-induced histopathological damage in liver, kidney, spleen, or lung; hematological parameters (RBC, WBC, platelets) normal[1] - Clinical preclinical note (Literature [4]): No dose-limiting toxicity observed at doses up to 100 mg/kg/day in mice; potential mild gastrointestinal toxicity (transient diarrhea) at 100 mg/kg/day, but resolved without intervention[4] |
| References |
[1]. Synthesis, structure-activity relationships, and in vivo efficacy of the novel potent and selective anaplastic lymphoma kinase (ALK) inhibitor 5-chloro-N2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)-N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine (LDK378) currently in phase 1 and phase 2 clinical trials. J Med Chem. 2013 Jul 25;56(14):5675-90. [2]. LDK378: a promising anaplastic lymphoma kinase (ALK) inhibitor. J Med Chem. 2013 Jul 25;56(14):5673-4. [3]. Immunoassays for the quantification of ALK and phosphorylated ALK support the evaluation of on-target ALK inhibitors in neuroblastoma. Mol Oncol. 2017 Aug;11(8):996-1006. [4]. Rothschild SI. Ceritinib-a second-generation ALK inhibitor overcoming resistance in ALK-rearranged non-small celllung cancer. Transl Lung Cancer Res. 2014 Dec;3(6):379-81. |
| Additional Infomation |
Ceritinib is a member of the class of aminopyrimidines that is 2,6-diamino-5-chloropyrimidine in which the amino groups at positions 2 and 6 are respectively carrying 2-methoxy-4-(piperidin-4-yl)-5-methylphenyl and 2-(isopropylsulfonyl)phenyl substituents. Used for the treatment of ALK-positive metastatic non-small cell lung cancer. It has a role as an antineoplastic agent and an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor. It is an aminopyrimidine, an aromatic ether, an organochlorine compound, a secondary amino compound, a member of piperidines and a sulfone. Ceritinib is used for the treatment of adults with anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer (NSCLC) following failure (secondary to resistance or intolerance) of prior crizotinib therapy. About 4% of patients with NSCLC have a chromosomal rearrangement that generates a fusion gene between EML4 (echinoderm microtubule-associated protein-like 4) and ALK (anaplastic lymphoma kinase), which results in constitutive kinase activity that contributes to carcinogenesis and seems to drive the malignant phenotype. Ceritinib exerts its therapeutic effect by inhibiting autophosphorylation of ALK, ALK-mediated phosphorylation of the downstream signaling protein STAT3, and proliferation of ALK-dependent cancer cells. Following treatment with crizotinib (a first-generation ALK inhibitor), most tumours develop drug resistance due to mutations in key \"gatekeeper\" residues of the enzyme. This occurrence led to development of novel second-generation ALK inhibitors such as ceritinib to overcome crizotinib resistance. The FDA approved ceritinib in April 2014 due to a surprisingly high response rate (56%) towards crizotinib-resistant tumours and has designated it with orphan drug status. Ceritinib is a Kinase Inhibitor. The mechanism of action of ceritinib is as a Tyrosine Kinase Inhibitor, and Cytochrome P450 3A Inhibitor, and Cytochrome P450 2C9 Inhibitor. Ceritinib is a small molecule tyrosine kinase receptor inhibitor and antineoplastic agent that is used in the therapy of selected forms of advanced non-small cell lung cancer (NSCLC). Ceritinib is associated with a moderate rate of serum aminotransferase elevations during therapy and rare instances of clinically apparent acute liver injury. Ceritinib is an orally available inhibitor of the receptor tyrosine kinase activity of anaplastic lymphoma kinase (ALK) with antineoplastic activity. Upon administration, ceritinib binds to and inhibits wild-type ALK kinase, ALK fusion proteins and ALK point mutation variants. Inhibition of ALK leads to both the disruption of ALK-mediated signaling and the inhibition of cell growth in ALK-overexpressing tumor cells. ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development. ALK dysregulation and gene rearrangements are associated with a variety of tumor cell types. Drug Indication Ceritinib 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 progressed on or are intolerant to crizotinib. This indication is approved under accelerated approval based on tumor response rate and duration of response. An improvement in survival or disease-related symptoms has not been established. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials. FDA Label Zykadia is indicated for the treatment of adult patients with anaplastic lymphoma kinase (ALK) positive advanced non small cell lung cancer (NSCLC) previously treated with crizotinib. Mechanism of Action Ceritinib inhibits Anaplastic lymphoma kinase (ALK) also known as ALK tyrosine kinase receptor or CD246 (cluster of differentiation 246), which is an enzyme that in humans is encoded by the ALK gene. About 4-5% of NSCLCs have a chromosomal rearrangement that generates a fusion gene between EML4 (echinoderm microtubule-associated protein-like 4) and ALK (anaplastic lymphoma kinase), which results in constitutive kinase activity that contributes to carcinogenesis and seems to drive the malignant phenotype. Ceritinib exerts its therapeutic effect by inhibiting autophosphorylation of ALK, ALK-mediated phosphorylation of the downstream signaling protein STAT3, and proliferation of ALK-dependent cancer cells. Ceritinib has been shown to inhibit in vitro proliferation of cell lines expressing EML4-ALK and NPM-ALK fusion proteins and demonstrated dose-dependent inhibition of EML4-ALK-positive NSCLC xenograft growth in mice and rats. 1. Mechanism of action (Literatures [1], [4]): Ceritinib (LDK-378) is a second-generation ALK inhibitor that binds to the ATP-binding pocket of ALK (wild-type and most resistance mutants, e.g., L1196M, C1156Y). This binding blocks ALK autophosphorylation and subsequent activation of downstream signaling pathways (PI3K-AKT, RAS-ERK), which are critical for ALK-positive cancer cell proliferation and survival. Unlike first-generation ALK inhibitors (e.g., crizotinib), it maintains activity against ALK gatekeeper mutations (L1196M) that drive crizotinib resistance[1] [4] 2. Clinical significance (Literatures [1], [4]): - Ceritinib was in Phase 1/2 clinical trials at the time of Literature [1]/[4] publication, targeting ALK-rearranged non-small cell lung cancer (NSCLC) and ALK-positive anaplastic large cell lymphoma (ALCL), particularly in patients with crizotinib-resistant disease[1] [4] - Its favorable oral bioavailability (~50-60% in rodents) and high tumor penetration support oral administration in clinical settings; selectivity for ALK minimizes off-target toxicity[1] 3. Limitations (Literatures [1], [4]): - Ceritinib shows reduced activity against certain ALK resistance mutations, such as G1269A (IC50 > 50 nM) and F1174L (IC50 ~25 nM), limiting efficacy in patients with these mutations[4] - No data on combination with other anticancer agents (e.g., chemotherapy, immune checkpoint inhibitors) reported in the specified literatures[1] [1][4] 4. Literature notes: - Literature [2] is a brief commentary on Literature [1], confirming Ceritinib’s potential as a potent, selective ALK inhibitor but providing no additional experimental data[2] - Literature [3] describes immunoassays for ALK and phosphorylated ALK but does not involve Ceritinib, so no relevant information[3] [2][3] |
Solubility Data
| Solubility (In Vitro) |
|
|||
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.5 mg/mL (0.90 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 5.0 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. Solubility in Formulation 2: ≥ 0.5 mg/mL (0.90 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 5.0 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. Solubility in Formulation 3: ≥ 0.5 mg/mL (0.90 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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7917 mL | 8.9583 mL | 17.9167 mL | |
| 5 mM | 0.3583 mL | 1.7917 mL | 3.5833 mL | |
| 10 mM | 0.1792 mL | 0.8958 mL | 1.7917 mL |