Evofosfamide (TH-302)

製品コード：S2757 純度：99.82%

Evofosfamide (TH-302)は、19nMのIC50で固形腫瘍の低酸素部位を目標としている選択的な低酸素を起動するプロドラッグです。

CAS No. 918633-87-1

サイズ	価格(税別)	在庫状況
10mM (1mL in DMSO)	JPY 50900	国内在庫あり
5mg	JPY 46800	国内在庫あり
10mg	JPY 63400	国内在庫あり

代表番号: 045-509-1970\|電子メール：[email protected] よく尋ねられる質問

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製品安全説明書

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99.82

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Cell Data

Cell Lines	Assay Type	Incubation Time	活性情報	PMID
H460	Cytotoxicity assay	2 hrs	Cytotoxicity against human H460 cells under hypoxic condition after 2 hrs by clonogenic assay, IC90=0.1μM.	18257544
H460	Cytotoxicity assay	2 hrs	Cytotoxicity against human H460 cells under hypoxic condition after 2 hrs by Alamar blue staining assay, IC50=0.019μM.	18257544
HT29	Cytotoxicity assay	2 hrs	Cytotoxicity against human HT29 cells under hypoxic condition after 2 hrs by clonogenic assay, IC90=0.2μM.	18257544
H460	Cytotoxicity assay	2 hrs	Cytotoxicity against human H460 cells under normoxic condition after 2 hrs by Alamar blue staining assay, IC50=5.1μM.	18257544
H460	Cytotoxicity assay	2 hrs	Cytotoxicity against human H460 cells under normoxic condition after 2 hrs by clonogenic assay, IC90=30μM.	18257544
HT29	Cytotoxicity assay	2 hrs	Cytotoxicity against human HT29 cells under normoxic condition after 2 hrs by clonogenic assay, IC90=40μM.	18257544
NCI-H460	Cytotoxicity assay	24 hrs	Cytotoxicity against human NCI-H460 cells pretreated for 24 hrs under hypoxic condition followed by compound washout measured after 72 hrs by MTT assay, IC50=9.08μM.	28350997
HT-29	Cytotoxicity assay	24 hrs	Cytotoxicity against human HT-29 cells pretreated for 24 hrs under hypoxic condition followed by compound washout measured after 72 hrs by MTT assay, IC50=49.51μM.	28350997
NCI-H460	Cytotoxicity assay	24 hrs	Cytotoxicity against human NCI-H460 cells after 24 hrs under hypoxic condition by luminescence-based Cell-Titer Glo assay, IC50=0.0093μM.	29079474
NCI-H460	Cytotoxicity assay	24 hrs	Cytotoxicity against human NCI-H460 cells after 24 hrs under normoxic condition by luminescence-based Cell-Titer Glo assay, IC50=6.65μM.	29079474
DU145	Cytotoxicity assay	2 hrs	Cytotoxicity against human DU145 cells incubated for 2 hrs under hypoxic condition measured after 72 hrs by Alamar blue assay, IC50=4.14μM.	29259746
PC3	Cytotoxicity assay	2 hrs	Cytotoxicity against human PC3 cells incubated for 2 hrs under hypoxic condition measured after 72 hrs by Alamar blue assay, IC50=6.49μM.	29259746
HEMC-SS	Antiproliferative assay	24 hrs	Antiproliferative activity against human HEMC-SS cells treated for 24 hrs under hypoxic condition followed by compound wash-out and incubated under normoxic condition for 48 hrs by alamar blue assay, IC50=0.13μM.	30199705
HEMC-SS	Antiproliferative assay	24 hrs	Antiproliferative activity against human HEMC-SS cells treated for 24 hrs under normoxic condition followed by compound wash-out and incubated under normoxic condition for 48 hrs by alamar blue assay, IC50=2.9μM.	30199705
MDA-MB-468	Cytotoxicity assay	4 hrs	Cytotoxicity against human MDA-MB-468 cells incubated for 4 hrs under hypoxic condition followed by compound washout and measured after 5 days by SRB assay, IC50=0.0062μM.	30885680
SW620	Cytotoxicity assay	4 hrs	Cytotoxicity against human SW620 cells incubated for 4 hrs under hypoxic condition followed by compound washout and measured after 5 days by SRB assay, IC50=0.052μM.	30885680
MDA-MB-468	Cytotoxicity assay	4 hrs	Cytotoxicity against human MDA-MB-468 cells incubated for 4 hrs under aerobic condition followed by compound washout and measured after 5 days by SRB assay, IC50=4.403μM.	30885680
SW620	Cytotoxicity assay	4 hrs	Cytotoxicity against human SW620 cells incubated for 4 hrs under aerobic condition followed by compound washout and measured after 5 days by SRB assay, IC50=20.6μM.	30885680
NCI-H460	Antiproliferative assay		Antiproliferative activity against human NCI-H460 cells in presence of N2, IC50=2μM.	30295477
NCI-H460	Antiproliferative assay		Antiproliferative activity against human NCI-H460 cells in presence of O2, IC50=25μM.	30295477
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生物活性

製品説明	Evofosfamide (TH-302)は、19nMのIC50で固形腫瘍の低酸素部位を目標としている選択的な低酸素を起動するプロドラッグです。

In Vitro
In vitro	TH-302 is selectively potent under hypoxia and stable to liver microsomes. Substitution of the chlorine with bromine on the phosphorus mustard in 3b increases the potency by 10-fold and maintaines the high hypoxic selectivity [Hypoxia cytotoxicity ratio (HCR) = 270]. In both human lung cancer H460 cells and human colon cancer HT29 cells, potent cytotoxicity of TH-302 is observed under N₂. TH-302 inhibits H460 cells and HT29 cells with IC90 of 0.1 μM and 0.2 μM, respectively. ^[1] TH-302 shows much enhanced potency in H460 spheroids compared to H460 monolayer cells under normoxia. ^[2] TH-302 exhibits potent cytotoxicity to MM cells with hypoxic selectivity and dose dependency. TH-302 can induce G0/G1 cell-cycle arrest under hypoxic conditions. The effect of TH-302 on cell-cycle machinery is mediated by down-regulating cyclin D1/2/3, CDK4/6, p21cip-1, p27kip-1, and pRb expression, whereas CDK2 expression remained undisturbed. TH-302 can induce dose-dependent apoptosis in both human and murine MM cells in hypoxic conditions. TH-302-activated apoptosis is mediated through down-regulating the antiapoptotic proteins BCL-2 and BCL-xL, as well as up-regulating the expression of cleaved proapoptotic protein caspase-3, -8, and -9 and poly ADP-ribose polymerase. In contrast to the hypoxia-specific toxicity, TH-302 shows very low toxicity in normoxic condition, even at high concentrations. ^[3]
細胞実験	細胞株	Human H460 or HT29 cells
	濃度	0.01 -1 μM
	反応時間	2 hours
	実験の流れ	Exponentially growing human H460 or HT29 cells are seeded into 60 mm notched glass plates at 3 × 10⁵ cells per plate and grown in RPMI medium supplemented with 10% fetal bovine serum for 2 days prior to initiating treatment. On the day of the test, TH-302 stocks of known concentrations are prepared in complete medium and 2 mL of the desired stock is added to each plate. The plates are placed in either an anaerobic chamber or a standard tissue-culture incubator. The anaerobic chamber is evacuated and gassed with the anaerobic gas mixture (90% N₂/5% CO₂/5% H₂) to create a hypoxic environment. Cells are then incubated with TH-302 for 2 hours at 37 °C. At the end of treatment, plates are removed from each vessel and washed with phosphate-buffered saline and a solution of trypsin-EDTA and then trypsinized for 5 min at 37 °C. Detached cells are neutralized with medium plus serum and spun for 5 min at 100g. Cells are resuspended at approximately 1 × 10⁶ cells/mL and diluted 10-fold for plating. The exact concentration of each stock is determined. Known numbers of cells are plated and placed undisturbed in an incubator for between 9 and 13 days. Colonies are fixed and stained with a solution of 95% ethanol with 0.25% crystal violet stain. Colonies of greater than 50 cells are counted, and the surviving fraction is determined. Plating efficiencies (PEs) are determined by dividing the number of colonies by the actual number of cells plated. Surviving fractions are calculated by dividing the PEs of treated cells by the PEs of untreate

In Vivo
In Vivo	TH302 inhibits primary tumor growth by 41% on day 25 after implantation, whereas TH302 plus gemcitabine (a nucleoside analog) inhibits primary tumor growth by 96% on day 25. ^[1] When TH-302 is administered at 6.25, 12.5, 25, or 50 mg/kg in the H460 NSCLC xenograft model QD × 5/wk × 2 wks (once a day for 5 days per week for 2 weeks) i.p., the tumor growth inhibition at Day 22 is 43%, 51%, 75%, and 89%, respectively. TH-302 at 100 mg/kg shows a decrease in blood cell counts 3 days after treatment end, but is totally recovered 7 days post-treatment. TH-302 under all tested regimens exhibits efficacy metrics ranging from 58% to 89% tumor growth inhibition. TH-302 induced cell killing is breathing oxygen concentration dependent, with the greatest cytotoxicity occurring when the tumor-bearing mice are exposed to low oxygen concentrations. Tumor growth is significantly reduced by TH-302 in animals breathing 10% O₂ compared with 95% O₂ breathing. After TH-302 treatment, the pimonidazole-positive area is significantly decreased at 48 hours after dosing (6.3 % in vehicle vs. 1.8 % in the TH-302 treatment group). ^[4]
動物実験	動物モデル	H460, Calu-6, PC-3, H82, A375, Stew2, 786-O, PLC/PRF/5, Hs766t, BxPC-3, and SU.86.86 xenografts are established in NCI SCID female mice.
	投与量	50 mg/kg
	投与経路	Intraperitoneally

NCT Number	Recruitment	Conditions	Sponsor/Collaborators	Start Date	Phases
臨床試験 (data from https://clinicaltrials.gov, updated on 2024-01-11)
NCT02020226	Unknown status	Solid Tumors	Threshold Pharmaceuticals	November 2013	Phase 1
NCT01833546	Completed	Solid Tumor\|Pancreatic Cancer	Merck KGaA Darmstadt Germany\|Threshold Pharmaceuticals	April 18 2013	Phase 1

参考
[1] Duan JX, et al. J Med Chem, 2008, 51(8), 2412-2420. [2] Meng F, et al. Mol Cancer Ther, 2012, 11(3), 740-751. [3] Hu J, et al. Blood, 2010, 116(9), 1524-1527. [4] Sun JD, et al. Clin Cancer Res, 2012, 18(3), 758-770. + 展開

参考

[4] Sun JD, et al. Clin Cancer Res, 2012, 18(3), 758-770.

+ 展開

化学情報

分子量	449.04	化学式	C₉H₁₆Br₂N₅O₄P
CAS No.	918633-87-1	SDF	Download Evofosfamide (TH-302) SDFをダウンロードする
Smiles	CN1C(=CN=C1[N+](=O)[O-])COP(=O)(NCCBr)NCCBr
保管	3年-20°C粉	溶液の保管冷凍と解凍の繰り返しを避けるため小分けにしてください	1年-80°Cin solvent
保管	3年-20°C粉	溶液の保管冷凍と解凍の繰り返しを避けるため小分けにしてください	1个月-20°Cin solvent

In vitro
Batch:

DMSO : 90 mg/mL ( (200.42 mM)；吸湿したDMSOは溶解度を減少させます。新しいDMSOをご使用ください。)

Ethanol : 90 mg/mL

Water : 10 mg/mL

モル濃度計算器

in vivo
Batch:

Add solvents to the product individually and in order.

投与溶液組成計算機

実験計算

モル濃度計算器

質量	濃度	体積	分子量
=	×	×

希釈計算器分子量計算器

投与溶液組成計算機(クリア溶液)

ステップ１：実験データを入力してください。（実験操作によるロスを考慮し、動物数を1匹分多くして計算・調製することを推奨します）

投与量 mg/kg 動物平均体重 g 投与体積（動物毎）μL 動物数匹

ステップ２：投与溶媒の組成を入力してください。（ロット毎に適した溶解組成が異なる場合があります。詳細については弊社までお問い合わせください）

% DMSO + % + % Tween 80 + % ddH₂O

%DMSO+ %

計算結果：

投与溶媒濃度： mg/ml;

DMSOストック溶液調製方法： mg 試薬を μL DMSOに溶解する（濃度 mg/mL, 注：濃度が当該ロットのDMSO溶解度を超える場合はご連絡ください。 )

投与溶媒調製方法：Take μL DMSOストック溶液に μL PEG300，を加え、完全溶解後μL Tween 80，を加えて完全溶解させた後 μL ddH₂O，を加え完全に溶解させます。

投与溶媒調製方法：μL DMSOストック溶液に μL Corn oil，を加え、完全溶解。

注意：１.ストック溶液に沈殿、混濁などがないことをご確認ください；
２.順番通りに溶剤を加えてください。次のステップに進む前に溶液に沈殿、混濁などがないことを確認してから加えてください。ボルテックス、ソニケーション、水浴加熱など物理的な方法で溶解を早めることは可能です。

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Handling Instructions

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* 必須

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