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

一种针对 Src 底物袋的 Src 抑制剂是替巴尼布林 (KX2-391)。与四种肝细胞癌 (HCC) 细胞系相比，即 Huh7 (GI50=9 nM)、PLC/PRF/5 (GI50=13 nM)、Hep3B (GI50=26 nM) 和 HepG2 (GI50=60 nM) ，tirbanibulin (KX2-391) 表现出陡峭的剂量反应曲线[1]。研究发现，tirbanibulin (KX2-391) 可抑制某些白血病细胞，例如源自具有 T3151 突变的慢性白血病细胞的细胞，这些细胞对目前上市的药物具有耐药性。在 SYF/c-Src527F 和 NHH3T3/c-Src527F 细胞中，在 Src 驱动的细胞生长测定中评估替巴尼布林 (KX2-391)，显示 GI50 值分别为 39 nM 和 23 nM[2]。

临床前癌症动物模型已证明口服替巴尼布林 (KX2-391) 可减少原发肿瘤生长并抑制转移[2]。

Src 抑制剂替巴尼布林 (KX2-391) 靶向 Src 底物袋。肝细胞癌 (HCC) 细胞系 Huh7 (GI50=9 nM)、PLC/PRF/5 (GI50=13 nM)、Hep3B (GI50=26 nM) 和 HepG2 (GI50=60 nM) 表现出陡峭的剂量反应用替巴尼布林 (KX2-391) 治疗时的曲线。

Hep3B、HepG2、PLC/PRF/5、Huh7 和其他肝细胞系经常在 37°C 和 5% CO2 的条件下在含有 2% 胎牛血清 (FBS) 的基础培养基中生长和保存。在 96 孔板的每个孔中，将细胞以 4.0×103/190 μL 和 8.0×103/190 μL 接种在含有 1.5% FBS 的基础培养基中。添加浓度范围为 6,564 至 0.012 nM 的替巴尼布林 (KX2-391)（一式三份）之前，将其在 37°C 和 5% CO2 下再培养一晚。孵育处理过的细胞需要三天时间。第三天，向每个孔中添加 10 μL 5 mg/mL 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑 (MTT) 溶液，并将细胞孵育 4 小时。小时。将 10% SDS 添加到稀 HCl 中以溶解甲臜产物。光密度在 570 nm 处测量。使用替巴尼布林 (KX2-391) 进行平行实验以比较其效力和活性。使用GraphPad Prism 5统计软件，计算生长抑制曲线、50%抑制浓度（GI50）、80%抑制浓度（GI80）。报告了 570 nm 波长 (OD570) 信号格式的光密度和代表最大响应百分比的归一化数据。

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].

Absorption, Distribution and Excretion
Tirbanibulin demonstrates good oral bioavailability. Following topical administration of doses ranging from 54 to 295 mg on the face or scalp, the steady-state concentration of tirbanibulin was achieved by 72 hours. At five days following initial administration, the mean Cmax was 0.34±0.30 ng/mL in subjects who received topical treatment on the face and 0.18±0.10 ng/mL in subjects who received topical treatment on the scalp. The mean AUC24 was 5.0±3.9 h x ng/mL in subjects who received topical treatment on the face and 3.2±1.9 h x ng/mL in subjects who received topical treatment on the scalp. The median Tmax was about seven hours.
There is limited information on the route of elimination of tirbanibulin.
There is limited information on the volume of distribution of tirbanibulin. In mouse HT29 xenograft studies, the tissue to plasma ration of tirbanibulin was 1.52.
There is limited information on the clearance rate of tirbanibulin.

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Metabolism / Metabolites
_In vitro_, tirbanibulin is mainly metabolized by CYP3A4, and to a lesser extent, CYP2C8. In adult subjects with actinic keratosis, detected metabolites were KX2-5036 and KX2-5163, which were pharmacologically inactive metabolites with the highest plasma concentrations of 0.09 ng/mL and 0.12 ng/mL, respectively.

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Biological Half-Life
The half-life is about 4 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Topical tirbanibulin has not been studied during breastfeeding.
However, after topical administration, serum concentrations are very low and the drug is 88% bound to plasma proteins, so amounts in milk are likely to be very low. If tirbanibulin is required by the mother, it is not a reason to discontinue breastfeeding. Do not apply tirbanibulin to the breast or nipple and ensure that the infant's skin does not come into direct contact with the areas of skin that have been treated.
◉ 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.

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Protein Binding
Tirbanibulin is 88% bound to plasma proteins and the extent of plasma protein binding is independent of drug concentrations in the range of 0.01 to 10 µg/mL.

[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.

Tirbanibulin (KX-O1 or KX2–391) is a dual inhibitor of Src Kinase and tubulin. On December 14, 2020, tirbanibulin was approved by the FDA for the topical treatment of actinic keratosis on the face or scalp. It is marketed under the brand name Klisyri. Actinic keratosis is a chronic condition characterized by lesions, which can potentially transform into invasive squamous cell carcinoma with a risk of 1% over 10 years. Tirbanibulin blocks the molecular pathways that promote the proliferation, survival, and metastasis of malignant cells. Tirbanibulin exhibits antitumour effects in vitro and in vivo and has been investigated for its antitumor efficacy in the management of various cancers, such as prostate cancer and breast cancer.
Tirbanibulin is a Microtubule Inhibitor. The physiologic effect of tirbanibulin is by means of Microtubule Inhibition.
Tirbanibulin is an orally bioavailable small molecule Src kinase inhibitor with potential antineoplastic activity. Unlike other Src kinase inhibitors which bind to the ATP-binding site, tirbanibulin specifically binds to the peptide substrate binding site of Src kinase; inhibition of kinase activity may result in the inhibition of primary tumor growth and the suppression of metastasis. Src tyrosine kinases are upregulated in many tumor cells and play important roles in tumor cell proliferation and metastasis.

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Drug Indication
Tirbanibulin is indicated for the topical treatment of actinic keratosis on the face or scalp.
Klisyri is indicated for the field treatment of non-hyperkeratotic, non-hypertrophic actinic keratosis (Olsen grade 1) of the face or scalp in adults.

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Mechanism of Action
Src tyrosine kinases regulate normal cell growth: the expression of Src kinase is upregulated during the normal hair cycle during the proliferative anagen phase. Additionally, Src tyrosine kinases act as key modulators of cancer cell proliferation, survival, angiogenesis, migration, invasion and metastasis. Src is frequently upregulated in various epithelial tumours including colon, breast and pancreas compared with the adjacent normal tissues. The expression and activity of Src are also enhanced in human actinic keratosis, which is characterized by hyperproliferative premalignant skin lesions. The pathogenesis of actinic keratosis commonly involves skin inflammation, oxidative stress, immunosuppression, impaired apoptosis, mutagenesis, dysregulation of keratinocyte growth and proliferation, and tissue remodelling. _In vitro_ studies suggest that Src plays a predominant role in the early stages of human skin tumour development, rather than at later stages of tumour progression. The exact mechanism of tirbanibulin as a topical treatment of actinic keratosis has not been fully elucidated; however, it mainly works by inhibiting fast proliferating cells. Tirbanibulin is a non-ATP competitive Src kinase inhibitor and tubulin polymerization inhibitor. It binds to the peptide substrate binding site of Src, a primary target of tirbanibulin, and blocking its downstream signalling pathways that promote cancer cell migration, proliferation, and survival. Tublin is responsible for cell migration, protein transport, and mitosis: tibranibulin directly binds to the colchicine-binding site of beta-tubulin and causes induces tubulin depolymerization. It is also hypothesized that inhibition of Src can also contribute to the inhibitory effects on microtubule polymerization. At low nanomolar concentrations, tirbanibulin induces G2/M phase cell cycle arrest in a reversible and dose-dependent manner. By inhibiting microtubule polymerization, tirbanibulin also induces mitotic catastrophe.

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Pharmacodynamics
In clinical trials composed comprising patients with actinic keratosis of the face or scalp, tirbanibulin promoted complete clearance of actinic keratosis lesions at day 57 in treated areas in 44-54% of patients compared to 5-13% of patients who received the placebo. Actinic keratosis is a chronic, pre-malignant condition characterized by lesions and proliferation of neoplastic keratinocytes. Tirbanibulin mediates an anti-proliferative effect by inhibiting tubulin polymerization and Src kinase signalling. Tirbanibulin inhibited primary tumour growth and metastasis in many preclinical animal models of cancer. In human triple-negative breast cancer, or estrogen receptor (ER)/progesterone receptor (PR)/human epidermal growth factor receptor 2 (HER2)-negative tumour, xenografts, tirbanibulin suppressed tumour growth and metastasis. Tirbanibulin was also shown to restore functional ERα expression in ERα-negative breast tumours. Tirbanibulin promoted synergistic tumour growth inhibition of breast cancer cell lines when used in combination with tamoxifen and paclitaxel. In a clinical trial comprising patients with advanced solid tumours, dose-limiting toxicities of tirbanibulin included elevated liver transaminases, neutropenia and fatigue.

C26H29N3O3

431.53

431.22

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

23635314

White to off-white solid powder

1.2±0.1 g/cm3

680.9±55.0 °C at 760 mmHg

365.6±31.5 °C

0.0±2.1 mmHg at 25°C

1.588

1.97

67.18

1

5

9

32

540

0

O=C(CC1C=CC(C2C=CC(OCCN3CCOCC3)=CC=2)=CN=1)NCC1C=CC=CC=1

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.82 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.82 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (4.82 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 4% DMSO+30% PEG 300+ddH2O:5 mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。