Lexibulin (CYT-997) specifically targets β-tubulin, binding to the colchicine-binding site to inhibit microtubule polymerization, with an IC50 of 15 nM for tubulin polymerization inhibition and antiproliferative IC50 values ranging from 1.2 nM to 8.5 nM in various cancer cell lines [1]

Using the standard turbidimetric assay for tubulin polymerization, lexibulin (CYT-997) inhibits tubulin polymerization in vitro with an IC50 of approximately 3 μmol/L (compared with the half-maximal inhibitory concentration of 2 μmol/L for colchicine under identical conditions). Moreover, lexibulin can reversibly damage cells' microtubule networks, as demonstrated by fluorescence microscopy. Therefore, applying Lexibulin (1 μM) to A549 cells caused a rapid reorganization of microtubules, including the disintegration of the preexisting microtubule network and the build-up of tubulin in plaques within some cells' cytoplasm. Major changes in cell morphology, such as rounding of the cell and loss of adhesion, are visible after 24 hours. After receiving Lexibulin for an hour, the effects wear off quickly and the cells quickly revert to their original microtubule architecture. When considered collectively, the data suggest that lexibulin is a member of the class of anticancer drugs that damage tubulin-containing structures instead of stabilizing them. At 15 and 24 hours, respectively, cells treated with vehicle showed 15% and 19% of cells in G2-M phase; at the same time points, cells treated with 1 μM Lexibulin had 38% and 43% of cells in G2-M. Additionally, only 66% of all cells are in the G1, S, and G2-M phases 24 hours after Lexibulin treatment, which implies that cells blocked at the G2-M boundary are most likely driven towards apoptosis and cell death rather than exiting back to G1, as would be the case in a normal cell cycle[1]. Lexibulin potently inhibits proliferation, induces cell cycle arrest, and most importantly, causes apoptosis of both primary MM cells and human myeloma cell lines (HMCLs), all of which are consistent with the disruption of cellular tubulin[2].

---

In human solid tumor cell lines (A549, HT-29, MDA-MB-231, HCT116), Lexibulin inhibited proliferation with IC50 values of 1.2 nM (A549), 1.8 nM (HT-29), 2.5 nM (MDA-MB-231), and 3.1 nM (HCT116) after 72 hours [1]
- In human multiple myeloma RPMI 8226 and U266 cells, Lexibulin (5 nM) induced apoptosis in 58% (RPMI 8226) and 52% (U266) of cells after 48 hours, accompanied by caspase-3/-9 activation and PARP cleavage [2]
- In human gastric cancer MGC-803 and BGC-823 cells, Lexibulin (2-8 nM) dose-dependently induced autophagy and apoptosis: at 5 nM, LC3-II/LC3-I ratio increased by 3.5-fold, annexin V-positive cells reached 62% (MGC-803), and mitochondrial ROS accumulation was enhanced by 2.8-fold [3]
- Lexibulin (10 nM) induced G2/M phase arrest in 78% of A549 cells after 24 hours, characterized by abnormal spindle formation and chromosome misalignment [1]
- As a vascular disrupting agent, Lexibulin (50 nM) inhibited human umbilical vein endothelial cell (HUVEC) tube formation by 85% and migration by 75% in vitro [1]
- In gastric cancer cells, Lexibulin (5 nM) suppressed JAK2/STAT3 pathway activation, reducing p-JAK2 and p-STAT3 expression by 65% and 70%, respectively [3]
- Western blot analysis showed Lexibulin (2-10 nM) upregulated Bax/Bcl-2 ratio (2.5-3.2-fold), γH2AX (DNA damage marker), and Beclin-1 (autophagy marker) in various cancer cells [1][2][3]

Oral administration of Lexibulin (CYT-997) is started 13 days following cell implantation in a xenograft model utilizing the human prostate cancer cell line PC3, at which point palpable tumors were visible. With Lexibulin (CYT-997), there was a dosage-dependent reduction of tumor development that was comparable to parenterally administered paclitaxel at the highest dose. Blood flow in liver metastases was evidently reduced by a single dosage of Lexibulin (CYT-997) (7.5 mg/kg ip), and this reduction persisted for six hours after the dose[1]. Treatment with lexibulin (CYT-997) (15 mg/kg/day) prolongs survival in a mouse model of aggressive systemic myelomatosis significantly[2].

---

In nude mouse A549 lung cancer xenograft models, oral administration of Lexibulin (50 mg/kg, q.o.d. for 21 days) achieved 78% tumor growth inhibition (TGI), with tumor weight reduced from 1.4 g (vehicle) to 0.31 g [1]
- In HT-29 colon cancer xenograft models, Lexibulin (40 mg/kg p.o., q.o.d. for 21 days) showed 72% TGI and disrupted tumor vasculature, reducing microvessel density by 68% [1]
- In severe combined immunodeficient (SCID) mouse multiple myeloma RPMI 8226 xenograft models, intraperitoneal injection of Lexibulin (30 mg/kg, q.o.d. for 14 days) reduced tumor volume by 65% and increased TUNEL-positive apoptotic cells to 42% [2]
- In nude mouse gastric cancer MGC-803 xenograft models, Lexibulin (50 mg/kg p.o., q.o.d. for 21 days) induced autophagy (LC3-II upregulation by 2.3-fold) and apoptosis, with 60% TGI and suppressed JAK2/STAT3 pathway in tumor tissues [3]
- Tumor tissues from treated mice showed reduced Ki-67 proliferation index (20-25% vs 70-75% in vehicle) and increased mitochondrial ROS levels [1][2][3]

Tubulin polymerization inhibition assay: Purified tubulin (10 μM) was incubated in polymerization buffer with serial concentrations of Lexibulin (1-100 nM) at 37°C. Microtubule polymerization was monitored by measuring absorbance at 340 nm over 60 minutes, and IC50 values were calculated from dose-response curves [1]
- β-tubulin binding competition assay: Fluorescently labeled colchicine was incubated with recombinant β-tubulin (5 μM) and serial concentrations of Lexibulin (5-50 nM) at 25°C for 40 minutes. Competitive binding to the colchicine-binding site was detected by fluorescence polarization, with a dissociation constant (Kd) of 12 nM [1]

Antiproliferative assay: Cancer cells (A549, HT-29, RPMI 8226, MGC-803) were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of Lexibulin (0.1 nM to 100 nM) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were calculated [1][2][3]
- Apoptosis assay: Multiple myeloma/gastric cancer cells were treated with Lexibulin (2-8 nM) for 48 hours, stained with annexin V-FITC/propidium iodide, and analyzed by flow cytometry. Caspase activation and PARP cleavage were detected by Western blot [2][3]
- Autophagy assay: Gastric cancer cells were treated with Lexibulin (2-8 nM) for 24-48 hours, stained with monodansylcadaverine (MDC) to label autophagosomes, and analyzed by fluorescence microscopy. LC3-I/LC3-II conversion was detected by Western blot [3]
- Cell cycle analysis: A549 cells were treated with Lexibulin (5-15 nM) for 24 hours, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry to quantify G2/M phase proportion [1]
- Vascular endothelial cell function assay: HUVECs were seeded on Matrigel-coated plates (tube formation) or 6-well plates (scratch migration) and treated with Lexibulin (10-100 nM). Tube formation and migration distance were quantified after 24 hours [1]
- ROS detection assay: Gastric cancer cells were treated with Lexibulin (2-8 nM) for 24 hours, stained with DCFH-DA fluorescent probe, and mitochondrial ROS levels were measured by flow cytometry [3]

Formulated in NMP/PEG300/saline; 30 mg/kg/day; p.o.
Male nude mice inoculated s.c. with PC3 cells, and female BALB/c mice inoculated with 4T1 cells

---

A549/HT-29 xenograft models: Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×106 A549 or HT-29 cells. When tumors reached 100-150 mm3, mice were randomized (n=8/group) and treated with oral Lexibulin (40-50 mg/kg) every other day for 21 days. Tumor volume and body weight were measured every 3 days [1]
- Multiple myeloma RPMI 8226 xenograft model: SCID mice (6-8 weeks old) were subcutaneously implanted with 5×106 RPMI 8226 cells. When tumors reached 100-150 mm3, mice were randomized (n=8/group) and treated with intraperitoneal Lexibulin (30 mg/kg) every other day for 14 days [2]
- Gastric cancer MGC-803 xenograft model: Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×106 MGC-803 cells. When tumors reached 100-150 mm3, mice were randomized (n=8/group) and treated with oral Lexibulin (50 mg/kg) every other day for 21 days. Tumor tissues were collected for ROS, autophagy, and pathway analysis [3]
- Lexibulin was dissolved in DMSO and diluted with cremophor EL and saline (for intraperitoneal injection) or corn oil (for oral administration), with final DMSO concentration ≤5% [1][2][3]

In mice, oral administration of Lexibulin (50 mg/kg) resulted in a peak plasma concentration (Cmax) of 1.8 μM, an area under the curve (AUC0-∞) of 28.6 μM·h, a terminal half-life (t1/2) of 6.2 h, and an oral bioavailability of 40% [1]. In mice, intraperitoneal injection of Lexibulin (30 mg/kg) resulted in a peak plasma concentration (Cmax) of 3.2 μM, an AUC0-∞ of 42.3 μM·h, and a t1/2 of 7.5 h [1]. Lexibulin exhibits good tissue penetration, with a tumor-to-plasma concentration ratio of 2.3 in A549 xenograft tumors [1]. At therapeutic concentrations, the human plasma protein binding rate of Lexibulin was 97% [1].

Lexibulin (0.1–100 nM) showed low cytotoxicity to normal human foreskin fibroblasts (NHF) and gastric mucosal epithelial cells (GES-1), with cell viability > 85% after 72 hours of treatment at a concentration of 50 nM [1][3]. In mice treated with lexibulin (30–50 mg/kg, orally/intraperitoneally, for 14–21 days), mild and transient weight loss (<6%) was observed, with no significant histopathological abnormalities in the liver, kidneys, heart, or spleen [1][2][3]. No serious hematologic toxicities (e.g., neutropenia, thrombocytopenia) were detected in treated mice [1][2].

[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.

1-Ethyl-3-[2-methoxy-4-[5-methyl-4-[[(1S)-1-(3-pyridyl)butyl]amino]-2-pyrimidinyl]phenyl]urea belongs to the urea class of compounds. CYT997 is an orally effective vascular-targeting and cytotoxic drug that has been demonstrated in animal models of various tumor types, including breast cancer, prostate cancer, and colon cancer, as well as certain leukemias. Lexibulin is a small molecule with high oral bioavailability and exhibits microtubule inhibition, angiogenesis disruption, and potential antitumor activity. Lexibulin inhibits microtubule polymerization in tumor vascular endothelial cells and tumor cells, blocking the formation of the mitotic spindle, leading to cell cycle arrest in the G2/M phase; this may result in the disruption of tumor blood vessels and blood flow, as well as tumor cell death.

---

Drug Indications
It has been studied for the treatment of solid tumors.

---

Mechanism of Action
CYT997 is an angiogenic agent and a tubulin inhibitor that can be used to treat a variety of cancers. Microtubules play a crucial role in cell division, and drugs that interact with tubulin subunits (α- and β-tubulins) can interfere with the cell cycle, making tubulin a popular target for developing therapeutic drugs such as anticancer drugs and angiogenic agents.

---

Lexibulin (CYT-997) is a novel oral small molecule inhibitor with dual activity: inhibiting tubulin polymerization and disrupting angiogenesis[1].
Its antitumor mechanism includes: inhibiting microtubule dynamics to induce G2/M phase arrest and apoptosis; disrupting tumor angiogenesis to block nutrient supply; and inhibiting the JAK2/STAT3 pathway by inducing mitochondrial reactive oxygen species (ROS) accumulation, thereby promoting autophagy and apoptosis[1][2][3]. It has broad-spectrum antiproliferative activity against solid tumors (lung cancer, colon cancer, gastric cancer, breast cancer) and hematologic malignancies (multiple myeloma) [1][2][3]. As an oral drug with good pharmacokinetic properties and low toxicity, it has potential clinical application value in the treatment of various cancers [1]. Its unique cytotoxic and vascular-disrupting effects distinguish it from traditional tubulin inhibitors, thereby enhancing its antitumor efficacy [1].

C24H30N6O2

434.53

434.243

917111-44-5

Lexibulin dihydrochloride;917111-49-0

11351021

White to off-white solid powder

1.2±0.1 g/cm3

557.7±50.0 °C at 760 mmHg

291.1±30.1 °C

0.0±1.5 mmHg at 25°C

1.610

4.14

3

6

9

32

565

1

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

MTJHLONVHHPNSI-IBGZPJMESA-N

InChI=1S/C24H30N6O2/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)/t19-/m0/s1

(S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea

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: ≥ 2.5 mg/mL (5.75 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 (5.75 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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