Src HuH7 (GI50 = 9 nM); Src PLC/PRF/5 (IC50 = 13 nM); Src Hep3B (IC50 = 26 nM); Src HepG2 (IC50 = 60 nM)

One Src inhibitor that targets the Src substrate pocket is tirbanibulin (KX2-391). When compared to four hepatic cell carcinoma (HCC) cell lines, namely Huh7 (GI50=9 nM), PLC/PRF/5 (GI50=13 nM), Hep3B (GI50=26 nM), and HepG2 (GI50=60 nM), tirbanibulin (KX2-391) exhibits steep dose-response curves[1]. It has been discovered that tirbanibulin (KX2-391) inhibits some leukemia cells, such as those originating from chronic leukemia cells with the T3151 mutation, that are resistant to currently marketed medications. In SYF/c-Src527F and NHH3T3/c-Src527F cells, tirbanibulin (KX2-391) is assessed in Src-driven cell growth assays and displays GI50 values of 39 nM and 23 nM, respectively[2].

Tirbanibulin (KX2-391) taken orally has been demonstrated in pre-clinical animal models of cancer to decrease primary tumor growth and to suppress metastasis[2].

Tirbanibulin (KX2-391) is a Src inhibitor that targets the substrate pocket of Src.
Tirbanibulin (KX2-391) exhibits sharp dose-response curves for four hepatic cell cancer (HCC) cell lines: Hep3B (GI50=26 nM), HepG2 (GI50=60 nM), PLC/PRF/5 (GI50=13 nM), and Huh7 (GI50=9 nM).

Regularly cultured and maintained in basal medium containing 2% fetal bovine serum (FBS) at 37°C and 5% CO₂, liver cell lines such as Huh7, PLC/PRF/5, Hep3B, and HepG2 (NutriCyte, Buffalo, NY) are used. 1.5% FBS-containing basal medium is used to seed cells at 4.0×10³/190 μL and 8.0×10³/190 μL per well of a 96-well plate. KX2-391, at concentrations ranging from 6,564 to 0.012 nM in triplicates, is added after these are cultivated for an additional night at 37°C and 5% CO₂. Incubation lasts three days for treated cells. Day 3 then sees the addition of 10 microliters of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) to each well, followed by a 4-hour incubation period. With 10% SDS in diluted HCl, the formazan product is dissolved. Using a BioTek Synergy HT multiplatform microplate reader, optical density at 570 nm is discovered. Parallel experiments using KX2-391 are carried out for comparison of potency and activity. A statistical program called GraphPad Prism 5 is used to calculate growth inhibition curves, 50% inhibition concentration (GI50), and 80% inhibition concentration (GI80). The data are presented in the optical density at wavelength of 570 nm (OD570) signal format and normalized to represent the percentage of maximum response.

Mouse bearing MDA-MB-231 tumors; Oral gavage; 1, 5mg/kg dose
Xenograft procedures and KX-01 oral dosing were as described (11). Briefly, mammary fat pad tumors were established by injecting 5×106 MDA-MB-231 cells in 150μl of PBS-Matrigel mixture (1:2) orthotopically and bilaterally into the mammary fat pads of female NUDE mice (two tumors/mouse). Treatments were started when tumors reached ∼80-100mm3. The first study used MDA-MB-231 xenografts and was performed using vehicle (ultra-pure water) and two doses of KX-01 (1, 5mg/kg) administered twice/day (BID) by oral gavage (using metal 22g feeding needle) for 28 days. A similar experiment was performed with MDA-MB-157 xenografts (another ER/PR/HER2 negative model) to assess KX-01 response. A second study was performed to test combination of KX-01 with paclitaxel on tumor growth. MDA-MD-231 tumor xenograft bearing mice were treated with vehicle or KX-01 (5mg/kg) BID, paclitaxel by intraperitoneal injection (IP) once/week, or combination of KX-0+paclitaxel. Treatments were for 40 days for all groups. A third study used MDA-MB-157 xenografts with the same combination treatment. A fourth study tested the effect of KX-01 or combination with paclitaxel for 24 days on larger MDA-MB-231 tumors (∼300mm3). Tumors were allowed to reach ∼300mm3 before beginning treatments. In this experiment mice were treated with KX-01 at a higher dose of 15mg/kg, and mice were treated once/day instead of twice/day. Paclitaxel was used at a dose of 20mg/kg IP once/week. In all experiments, tumor caliper measurements were taken twice/week and tumor volume was by calculated by the formula: 0.523×LM2 (where L-large diameter, M-small diameter). At the end the experiments animals were sacrificed and tumors and mouse organs removed. Tissues were either stored in 10% neutral buffered formalin for paraffin embedding, or snap frozen for measurement of chromosome-17 by real-time PCR, and embedded for frozen sectioning for CD-31 staining. Immunohistochemistry (IHC) was performed as described on paraffin-embedded tumor tissues [3].

[1]. Lau GM, et al. Expression of Src and FAK in hepatocellular carcinoma and the effect of Src inhibitors on hepatocellular carcinoma in vitro. Dig Dis Sci, 2009, 54(7), 1465-1474.
[2]. Fallah-Tafti A, et al. Thiazolyl N-benzyl-substituted acetamide derivatives: synthesis, Src kinase inhibitory and anticancer activities. Eur J Med Chem, 2011, 46(10), 4853-4858.

[3]. Peptidomimetic Src/pretubulin inhibitor KX-01 alone and in combination with paclitaxel suppresses growth, metastasis in human ER/PR/HER2-negative tumor xenografts. Mol Cancer Ther. 2012 Sep; 11(9): 1936–1947.

C26H29N3O3

431.53

431.22089

C, 71.92; H, 6.52; N, 10.06; O, 11.50

897016-82-9

Tirbanibulin dihydrochloride;1038395-65-1;Tirbanibulin Mesylate;1080645-95-9

White to off-white solid powder

2.9

63.7Ų

O=C(C1=CC=C(/C=C/C2=NNC3=C2C=CC=C3)C=C1)N4CCNCC4

YYLKKYCXAOBSRM-JXMROGBWSA-N

InChI=1S/C20H20N4O/c25-20(24-13-11-21-12-14-24)16-8-5-15(6-9-16)7-10-19-17-3-1-2-4-18(17)22-23-19/h1-10,21H,11-14H2,(H,22,23)/b10-7+

(E)-(4-(2-(1H-indazol-3-yl)vinyl)phenyl)(piperazin-1-yl)methanone

KX 01, KX2-391; KX-01, KX-2-391; Tirbanibulin; KX2391; KX-2391; Tirbanibulin free base; KXO1; KX 2-391; KX 2391

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: ≥ 2.08 mg/mL (4.82 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 20.8 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: ≥ 2.08 mg/mL (4.82 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 20.8 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: ≥ 2.08 mg/mL (4.82 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 4% DMSO+30% PEG 300+ddH2O:5 mg/mL

---

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