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| 25mg |
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Purity: ≥98%
Litronesib (formerly also known as LY2523355; LY-2523355; KF-8961) is a novel, potent and selective inhibitor of the kinesin-related motor protein Eg5 with potential anticancer activity. Litronesib specifically inhibits the activity of Eg5, which in actively dividing tumor cells may cause disruption of mitosis, apoptosis, and ultimately cell death. Additionally referred to as KIF11 or kinesin spindle protein-5, the ATP-dependent Eg5 kinesin-related motor protein is a plus-end directed kinesin motor protein that is crucial for the regulation of spindle dynamics, including assembly and maintenance, during mitosis. Look for open or closed clinical trials that are utilizing this agent. (NCI Lexicon).
| Targets |
Eg5 (IC50 = 14 μM)
LY2523355 (Litronesib) is a selective allosteric inhibitor of human Eg5 (kinesin-5), a mitotic kinesin essential for bipolar spindle formation during mitosis. The biochemical studies established it as a potent and selective inhibitor [1] |
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| ln Vitro |
Litronesib (LY-2523355; KF-89617) is a selective Eg5 inhibitor. Litronesib (25 nM) induces cancer cells to death during mitotic arrest, and this needs sustained activation of spindle-assembly checkpoint (SAC)[1].
LY2523355 exhibits broad-spectrum antiproliferative activity against 21 cancer cell lines with IC50 values ranging from 0.4 nmol/L to 14 nmol/L. It induces dose- and time-dependent mitotic arrest (maximal at 10 nmol/L) with monopolar spindles, as shown by increased phospho-histone H3 (pHH3) and DNA condensation. The compound causes apoptosis following sustained mitotic arrest, evidenced by PARP cleavage, caspase-3/7 activation, and decreased Mcl1 and Bcl2 levels. Abrogation of the spindle assembly checkpoint (SAC) via CDK1 inhibition or Mad2 knockdown prevents mitotic arrest and apoptosis. [1] |
| ln Vivo |
Litronesib (LY2523355; KF89617; 1.1, 3.3, 10, and 30 mg/kg, i.v.) shows antitumor activity in a dose-dependently, and causes a dramatic increase in cancer cells immuno-positive for histone H3 phosphorylation in Colo205 xenograft tumors[1].
LY2523355 shows schedule-dependent antitumor activity in subcutaneous xenograft models (including patient-derived xenograft, PDX). Daily dosing for 3 consecutive days or dosing every 4 days induced tumor regression or suppression in multiple models (colorectal, NSCLC, ovarian, etc.). Activity correlates with increased pHH3 in tumor and proliferating skin cells. In the p388 ascites model, a threshold plasma exposure (~10 ng/mL) is required for maximal G2/M arrest and survival extension. [1] |
| Enzyme Assay |
Biochemical assays confirmed LY2523355 as a potent and selective inhibitor of human Eg5 ATPase activity. The assay measured ATPase activity of recombinant Eg5 in the presence of the compound. [1]
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| Cell Assay |
In 96-well plates coated with poly-d-lysine, cancer cells are plated and incubated for an entire night. Following that, cells are given different lengths of time to respond to the indicated concentrations of lidocaine. After that, the cells are permeabilized for 10 minutes with cold methanol, fixed for 45 minutes with 3.7% formaldehyde in PBS or 30 minutes with 1× Prefer fixative solution at room temperature, and then fixed for another 10 minutes with 0.2% Triton X-100 in PBS. PBS is used to wash the cells three times. Following an hour of incubation with 100 μg/mL DNase-free RNase and 10 μg/mL propidium iodide, the cells are scanned to determine the mitotic index (MI), which is calculated as the percentage of cells with condensed DNA. Cells are cultivated with anti-phospho-histone H3 antibody or anti-phospho-histone H2AX at 1:1,000 dilution with 5% bovine serum albumin (BSA) in PBS for an overnight period at 4°C in MI based on histone H3 phosphorylation or apoptosis analysis inspection. Alexa 488 secondary antibody (1:1,000 in PBS-2% BSA) is incubated on cells for 60 minutes at room temperature after they have been washed three times with 0.2% Triton-X 100 in PBS. The cells are staining for 15 minutes in PBS containing 10 μg/mL propidium iodide and 100 μg/mL RNaseA after being rinsed three times with purified water. In Acumen Explorer eX3 microplate cytometer, the stained cells are scanned. Phospho-histone H3-Ser10 or phospho-histone H2AX positivity as a percentage of cells is used to express the results[1].
For mitotic index (MI) measurement, cells were treated with LY2523355, fixed, permeabilized, and stained with propidium iodide or anti-pHH3 antibody. MI was quantified using high-content imaging (Acumen). Apoptosis was assessed by caspase-3/7 activity (Apo-ONE assay) and viability by CellTiter-Glo. Immunoblotting for pHH3, BubR1, cyclin B, PARP, Mcl1, and Bcl2 was performed to monitor mitotic arrest and apoptosis. siRNA knockdown of Mad2 was used to assess SAC involvement. [1] |
| Animal Protocol |
In nude mice, primary human tumor xenograft models are created and kept alive. Tumor volume is measured serially using caliper measurements to determine the antitumor efficacy in subcutaneous xenograft tumor-bearing mice (10 mice per treatment group) derived from either established cancer cell lines or fragments of human tumor explants. Female BDF1 mice weighing 20–23 g are used to create the p388 syngeneic tumor model for high-content imaging analysis. The mice are house-acclimated for a week prior to use in experiments. The STR assay is used to validate the p388 murine lymphocytic leukemia cells, which are then cultured in RPMI1640 medium with 10% FBS. Three rounds of serum-free medium washing precede the intraperitoneal injection of 1.25 million cells into mice for inoculation. Litronesib is administered to mice on the fifth post-implantation day, either as an intravenous bolus or as an intravenous infusion, at the prescribed doses and times. Following the death of the mice, the ascitic (intraperitoneal) fluid containing the p388 tumor cells is extracted and subjected to acumen, flow cytometry, and TUNEL assays for apoptosis, G2-M, and phospho-histone H3. Blood samples are obtained via cardiac puncture for pharmacokinetic analysis. EDTA is used to create plasma, and the amount of lidotrexeb in the plasma is measured[1].
In xenograft studies, tumor-bearing mice were treated with LY2523355 via intravenous infusion or bolus at doses ranging from 1.1 to 30 mg/kg. Dosing schedules included daily (q1d), every 4 days (q4d), and every 7 days (q7d). Tumor volume and body weight were monitored. For PDX models, tumor fragments were implanted subcutaneously and treated similarly. In the p388 ascites model, mice were inoculated intraperitoneally and treated with LY2523355; ascitic fluid was analyzed for pHH3 and apoptosis. [1] |
| ADME/Pharmacokinetics |
In the p388 model, steady-state plasma exposure of LY2523355 was measured by continuous infusion. The threshold exposure required to achieve maximal mitotic arrest in tumors and skin is approximately 10 ng/mL. [1]
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| Toxicity/Toxicokinetics |
In vivo studies showed that daily administration for three consecutive days resulted in significant weight loss and partial death. Dosing every four days resulted in minimal toxicity (no significant weight loss). Myelosuppression and gastrointestinal toxicity are expected to be the dose-limiting toxicities of EG5 inhibitors. [1]
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| References | |
| Additional Infomation |
Litronesib has been used in clinical trials for the treatment of various cancers, including solid tumors, ovarian cancer, gastric cancer, prostate cancer, and acute leukemia. Litronesib is an Eg5 kinesin inhibitor with potential antitumor activity. Litronesib selectively inhibits Eg5 activity, which may lead to mitotic disturbances, apoptosis, and ultimately the death of actively dividing tumor cells. ATP-dependent Eg5 kinesin (also known as KIF11 or kinesin spindle protein-5) is a positive-end guided kinesin that plays a crucial role in mitosis, particularly in the regulation of spindle dynamics, including assembly and maintenance. Sustained activation of the spindle assembly checkpoint (SAC) and subsequent mitotic slippage are required to induce apoptosis. Phosphorylation of histone H3 in proliferating skin cells can serve as a pharmacodynamic biomarker for target binding and antitumor activity. The efficacy of this compound is highly dependent on the dosing regimen; therefore, the importance of threshold exposure duration rather than the maximum tolerated dose is emphasized. [1]
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| Molecular Formula |
C23H37N5O4S2
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|---|---|
| Molecular Weight |
511.7010
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| Exact Mass |
511.229
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| Elemental Analysis |
C, 53.99; H, 7.29; N, 13.69; O, 12.51; S, 12.53
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| CAS # |
910634-41-2
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| Related CAS # |
Litronesib Racemate;546111-97-1
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| PubChem CID |
25167017
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| Appearance |
White to off-white solid powder
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| Density |
1.23 g/cm3
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| LogP |
4.499
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
34
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| Complexity |
865
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C([C@]1(SC(NC(=O)C(C)(C)C)=NN1C(=O)C(C)(C)C)C1C=CC=CC=1)NS(=O)(=O)CCNCC
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| InChi Key |
YVAFBXLHPINSIK-QHCPKHFHSA-N
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| InChi Code |
InChI=1S/C23H37N5O4S2/c1-8-24-14-15-34(31,32)25-16-23(17-12-10-9-11-13-17)28(19(30)22(5,6)7)27-20(33-23)26-18(29)21(2,3)4/h9-13,24-25H,8,14-16H2,1-7H3,(H,26,27,29)/t23-/m0/s1
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| Chemical Name |
N-[(5R)-4-(2,2-dimethylpropanoyl)-5-[[2-(ethylamino)ethylsulfonylamino]methyl]-5-phenyl-1,3,4-thiadiazol-2-yl]-2,2-dimethylpropanamide
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| Synonyms |
LY2523355; KF 89617; LY-2523355; KF89617; LY 2523355; KF-89617; Eg5 KinesinRelated Motor Protein Inhibitor
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO: ≥ 50 mg/mL (~97.7 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.89 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.89 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 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.89 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9543 mL | 9.7714 mL | 19.5427 mL | |
| 5 mM | 0.3909 mL | 1.9543 mL | 3.9085 mL | |
| 10 mM | 0.1954 mL | 0.9771 mL | 1.9543 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT01358019 | Completed | Drug: LY2523355 | Solid Tumors | Kyowa Kirin Co., Ltd. | May 2011 | Phase 1 |
| NCT01416389 | Completed | Drug: LY2523355 Drug: ixabepilone |
Metastatic Breast Cancer | Eli Lilly and Company | May 25, 2018 | Phase 2 |
| NCT01214629 | Completed | Drug: LY2523355 Drug: pegfilgrastim |
Advanced Cancer Metastatic Cancer |
Eli Lilly and Company | July 2007 | Phase 1 |
| NCT01059643 | Completed | Drug: LY2523355 Drug: pegfilgrastim |
Ovarian Cancer Prostate Cancer |
Eli Lilly and Company | April 2011 | Phase 2 |
| NCT01214642 | Completed | Drug: pegfilgrastim Drug: LY2523355 |
Advanced Cancer | Eli Lilly and Company | May 2008 | Phase 1 |
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