The primary molecular target of lexibulin hydrochloride is tubulin, the protein subunit of microtubules. It acts as a tubulin polymerization inhibitor by binding to the colchicine binding site on β-tubulin. This inhibits microtubule polymerization, disrupting the mitotic spindle and leading to cell cycle arrest in G2/M phase and apoptosis. It also has vascular disrupting activity, causing rapid collapse of tumor vasculature and reduction of tumor blood flow. This dual mechanism contributes to its antitumor activity.

In vitro, lexibulin (CYT-997) inhibits tubulin polymerization with an IC50 of roughly 3 μmol/L by traditional turbidimetry (as opposed to the half-maximum inhibitory concentration of colchicine, which is 2 μmol/L in the same circumstances). Tuberculin polymerization. Furthermore, as demonstrated by fluorescent microscopy, lexibulin has the capacity to reversibly damage the microtubule network within cells. The application of Lexibulin (1 μM) to A549 cells causes a fast reconfiguration of microtubules, causing some cells to accumulate tubulin in intracytoplasmic plaques and others to break the preexisting microtubule network. The morphology of the cells underwent notable alterations after 24 hours, such as rounding and lack of adhesion. Following a one-hour Lexibulin treatment, cells quickly recovered their original microtubule structure. These effects were reversible. All of the information points to lexibulin as an anticancer medication that upsets tubulin-containing structures rather than stabilizes them. The G2-M phase was observed in 15% and 19% of vehicle-treated cells at 15 and 24 hours (respectively), but 38% and 43% of cells treated with 1 μM Lexibulin displayed G2-M phase at the same time point. phase G2-M. Further evidence that cells halted at the G2-M boundary may not exit back to G1, as in the normal cell cycle, but are instead likely to be driven to apoptosis and cell death comes from the fact that only 66% of total cells were in G1, S, and G2-M phases 24 hours after Lexibulin administration [1]. Lexibulin efficiently reduces proliferation, produces cell cycle arrest, and—most importantly—induces apoptosis in human myeloma cell lines (HMCL) and genuine MM cells, all of which are consistent with the breakdown of cellular tubulin [2].

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In vitro, lexibulin hydrochloride inhibits tubulin polymerization with an IC50 in the micromolar range. It inhibits the proliferation of various cancer cell lines, including those from breast, prostate, colon, and lung cancers, with IC50 values in the nanomolar range. It induces cell cycle arrest in G2/M phase and apoptosis. Its in vitro activity is measured using tubulin polymerization assays and cell proliferation assays. It also inhibits endothelial cell proliferation and tube formation.

Oral administration of Lexibulin (CYT-997) was started 13 days following cell engraftment in a xenograft model using the human prostate cancer cell line PC3, at which point tumors were evidently obvious. The tumor development is significantly inhibited by lexibulin (CYT-997) at doses up to and including parenterally administered paclitaxel at the highest dose. In liver metastases, a single dosage of lexibulin (CYT-997) (7.5 mg/kg ip) dramatically decreased blood flow; this effect persisted six hours after treatment [1]. A mouse model of aggressive systemic myeloma showed a significant increase in survival with lexibulin (CYT-997) treatment (15 mg/kg/day) [2].

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In vivo, lexibulin hydrochloride inhibits tumor growth in various xenograft models. It has vascular disrupting activity, leading to rapid reduction in tumor blood flow and tumor necrosis. Its in vivo efficacy is dose-dependent and correlates with tubulin inhibition and vascular disruption. It is orally active with good pharmacokinetic properties. It is well tolerated with no significant toxicity observed at therapeutic doses. Its in vivo efficacy is assessed in mouse models of solid tumors.

For tubulin polymerization assays, purified tubulin is incubated with lexibulin hydrochloride at various concentrations in the presence of GTP. The reaction is incubated at 37°C for 30-60 minutes, and tubulin polymerization is measured by turbidity at 340 nm. IC50 values are determined by nonlinear regression analysis. For colchicine binding assays, tubulin is incubated with [3H]colchicine and lexibulin hydrochloride, and competition binding is measured.

For in vitro cell experiments, cancer cell lines (e.g., MCF-7, PC-3, HCT116, or A549) are cultured and treated with lexibulin hydrochloride at concentrations ranging from 0.001-10 μM for 24-72 hours. Cell viability is assessed by MTT or SRB assays. Cell cycle analysis is performed by flow cytometry after propidium iodide staining. Apoptosis is evaluated by Annexin V/PI staining or caspase-3/7 activity assays. For endothelial cell studies, tube formation assays are performed.

For in vivo animal experiments, nude mice bearing xenograft tumors are treated with lexibulin hydrochloride by oral gavage at doses ranging from 1-50 mg/kg/day for 2-4 weeks. Tumor volume is measured regularly. For vascular disrupting activity studies, tumor blood flow is measured using laser Doppler flowmetry or contrast-enhanced ultrasound. At necropsy, tumors are excised and processed for histology and immunohistochemistry. Pharmacokinetic parameters are determined by LC-MS/MS.

[1]. CYT997: a novel orally active tubulin polymerization inhibitor with potent cytotoxic and vascular disrupting activity in vitro and in vivo. Mol Cancer Ther. 2009 Nov;8(11):3036-45.

[2]. CYT997 causes apoptosis in human multiple myeloma. Invest New Drugs. 2011 Apr;29(2):232-8.

[3]. Ya Cao, wt al. Mitochondrial ROS Accumulation Inhibiting JAK2/STAT3 Pathway Is a Critical Modulator of CYT997-induced Autophagy and Apoptosis in Gastric Cancer. J Exp Clin Cancer Res. 2020 Jun 23;39(1):119.

C₂₄H₃₂CL₂N₆O₂

507.46

506.196

917111-49-0

Lexibulin;917111-44-5

56928415

Typically exists as solid at room temperature

0

101

5

6

9

34

565

1

CCC[C@@H](C1=CN=CC=C1)NC2=NC(=NC=C2C)C3=CC(=C(C=C3)NC(=O)NCC)OC.Cl.Cl

HGDCRTLMRFHNOZ-TXEPZDRESA-N

InChI=1S/C24H30N6O2.2ClH/c1-5-8-19(18-9-7-12-25-15-18)28-22-16(3)14-27-23(30-22)17-10-11-20(21(13-17)32-4)29-24(31)26-6-2;;/h7,9-15,19H,5-6,8H2,1-4H3,(H2,26,29,31)(H,27,28,30);2*1H/t19-;;/m0../s1

1-ethyl-3-[2-methoxy-4-[5-methyl-4-[[(1S)-1-pyridin-3-ylbutyl]amino]pyrimidin-2-yl]phenyl]urea;dihydrochloride

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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