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Belinostat

別名: PXD101,NSC726630, PX-105684 中文名稱:貝利司他

Belinostat是一種新型HDAC抑制劑,無(wú)細(xì)胞試驗(yàn)中IC50為27 nM,對(duì)耐Cisplatin的腫瘤具有活性。Belinostat (PXD101) 可誘導(dǎo)自噬。

Belinostat Chemical Structure

Belinostat Chemical Structure

CAS: 866323-14-0

規(guī)格 價(jià)格 庫(kù)存 購(gòu)買數(shù)量
10mM (1mL in DMSO) 1317.13 現(xiàn)貨
10mg 981.69 現(xiàn)貨
50mg 3030.32 現(xiàn)貨
100mg 4653.78 現(xiàn)貨
200mg 7950.07 現(xiàn)貨
1g 12039.3 現(xiàn)貨
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400-668-6834

info@selleck.cn

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Belinostat相關(guān)產(chǎn)品

相關(guān)信號(hào)通路圖

細(xì)胞實(shí)驗(yàn)數(shù)據(jù)示例

細(xì)胞系 實(shí)驗(yàn)類型 給藥濃度 孵育時(shí)間 活性描述 文獻(xiàn)信息
PC9 Function Assay 500?nM 24?h decreases EGFR expression 23515752
H460 Function Assay 0.5/1/2 μM 4 h inhibits the levels of Akt (p-Akt) and EGFR 23515752
H1650 Function Assay 0.5/1/2 μM 4 h inhibits the levels of Akt (p-Akt) and EGFR 23515752
PC9 Function Assay 0.5/1/2 μM 4 h inhibits the levels of Akt (p-Akt) and EGFR 23515752
Panc0327 Apoptosis Assay 1 μM 24 h induces apoptosis 23475695
Panc1005 Apoptosis Assay 1 μM 24 h induces apoptosis 23475695
Panc0403 Apoptosis Assay 1 μM 24 h induces apoptosis 23475695
AsPc1 Function Assay 1/10 μM 24 h induces growth arrested in G2/M 23475695
MiaPaCa2 Function Assay 1/10 μM 24 h induces growth arrested in G2/M 23475695
T3M4 Growth Inhibition Assay 0-800 nM 48 h inhibits cell proliferation in a dose dependent manner 22681698
AsPC-1 Growth Inhibition Assay 0-800 nM 48 h inhibits cell proliferation in a dose dependent manner 22681698
Panc-1? Growth Inhibition Assay 0-800 nM 48 h inhibits cell proliferation in a dose dependent manner 22681698
T3M4 Apoptosis Assay 100/500/1000 nM 48 h induces dose dependent apoptosis 22681698
AsPC-1 Apoptosis Assay 100/500/1000 nM 48 h induces dose dependent apoptosis 22681698
Panc-1? Apoptosis Assay 100/500/1000 nM 48 h induces dose dependent apoptosis 22681698
HCT116 Function Assay 0.9?μM? 24 h down-regulats TS protein levels after 6?h incubation 17124594
H1650 Function Assay 500?nM 24?h decreases EGFR expression 23515752
H460 Function Assay 500?nM 24?h decreases EGFR expression 23515752
PANC-1 Function Assay 10?μM 2/4 h increases intracellular ROS level 23743198
PANC-1 Cell Viability Assay 1/10?μM 48 h decreases cell viability in a dose dependent manner 23743198
PANC-1 Function Assay 10?μM 2/4/6 h induces AMPK activation 23743198
HL-60? Function Assay 0.2?μM 24/48/72 h enhances RA-induced granulocytic differentiation 25864732
NB4 Function Assay 0.2?μM 24/48/72 h enhances RA-induced granulocytic differentiation 25864732
HL-60? Function Assay 2?μM 24/48 h blocks cell cycle in S phase 25864732
NB4 Function Assay 2?μM 24/48 h blocks cell cycle in S phase 25864732
HL-60? Cell Viability Assay 0.2/2?μM 24/48/72 h decreases cell viability in both time and dose dependent manner 25864732
NB4 Cell Viability Assay 0.2/2?μM 24/48/72 h decreases cell viability in both time and dose dependent manner 25864732
Huh-luc/neo7 Function assay 1 uM 1 to 3 hrs Inhibition of HDAC class 1 in human Huh-luc/neo7 cells assessed as histone H3 acetylation at 1 uM after 1 to 3 hrs by Western blotting analysis 25937017
PC3 Function assay 0.3 uM 48 hrs Inhibition of HDAC in human PC3 cells assessed as increase in amount of acetylated histone H3 at 0.3 uM after 48 hrs by Western blot analysis 27344487
HCT116 Function assay 0.3 uM 48 hrs Inhibition of HDAC in human HCT116 cells assessed as increase in amount of acetylated histone H3 at 0.3 uM after 48 hrs by Western blot analysis 27344487
AsPc1 Growth Inhibition Assay 48 h EC50=0.3 μM 23475695
Panc0327 Growth Inhibition Assay 48 h EC50=0.5 μM 23475695
MiaPaCa2 Growth Inhibition Assay 48 h EC50=0.7 μM 23475695
BxPc3 Growth Inhibition Assay 48 h EC50=1.0 μM 23475695
Panc0403 Growth Inhibition Assay 48 h EC50=1.1 μM 23475695
Panc1005 Growth Inhibition Assay 48 h EC50=1.1 μM 23475695
PL45 Growth Inhibition Assay 48 h EC50=20.8 μM 23475695
Panc0203 Growth Inhibition Assay 48 h EC50=22.2 μM 23475695
HBL-2 Growth Inhibition Assay 24 h IC50=0.4 μM 20068080
Jeko-1 Growth Inhibition Assay 24 h IC50=0.2 μM 20068080
Granta-519 Growth Inhibition Assay 24 h IC50=56.3 μM 20068080
HCT116 Growth Inhibition Assay 48 h EC50=0.28 μM 17124594
HCC4006 Growth Inhibition Assay 72?h IC50=0.46 μM 23515752
HCC2935 Growth Inhibition Assay 72?h IC50=0.97 μM 23515752
HCC827 Growth Inhibition Assay 72?h IC50=0.29 μM 23515752
HCC2279 Growth Inhibition Assay 72?h IC50=0.4 μM 23515752
PC9 Growth Inhibition Assay 72?h IC50=0.29 μM 23515752
H820 Growth Inhibition Assay 72?h IC50=0.4 μM 23515752
H1650 Growth Inhibition Assay 72?h IC50=0.88 μM 23515752
H1975 Growth Inhibition Assay 72?h IC50=0.68 μM 23515752
H520 Growth Inhibition Assay 72?h IC50=0.75 μM 23515752
H1299 Growth Inhibition Assay 72?h IC50=1.2 μM 23515752
H460 Growth Inhibition Assay 72?h IC50=0.86 μM 23515752
H1666 Growth Inhibition Assay 72?h IC50>10 μM 23515752
RAW264.7 Anti-inflammatory assay 1 hr Anti-inflammatory activity in LPS-stimulated mouse RAW264.7 cells assessed as suppression of IL6 production pre-incubated for 1 hr before LPS stimulation for 24 hrs by ELISA method, IC50 = 0.000059 μM. 25113875
HeLa Function assay 30 mins Inhibition of HDAC in human HeLa cells nuclear extracts incubated for 30 mins by fluorescent assay, IC50 = 0.0264 μM. 25113875
MDA-MB-231 Antiproliferative assay 72 hrs Antiproliferative activity against human MDA-MB-231 cells after 72 hrs by resazurin dye based fluorescence assay, IC50 = 0.062 μM. 29456804
Jurkat Antiproliferative assay 48 hrs Antiproliferative activity against human Jurkat cells after 48 hrs by MTT assay, IC50 = 0.07 μM. 29533873
A549 Antiproliferative assay 72 hrs Antiproliferative activity against human A549 cells after 72 hrs by resazurin dye based fluorescence assay, IC50 = 0.077 μM. 29456804
HeLa Antiproliferative assay 72 hrs Antiproliferative activity against human HeLa cells after 72 hrs by resazurin dye based fluorescence assay, IC50 = 0.087 μM. 29456804
MCF7 Antiproliferative assay 72 hrs Antiproliferative activity against human MCF7 cells after 72 hrs by resazurin dye based fluorescence assay, IC50 = 0.096 μM. 29456804
HEL Antiproliferative assay 48 hrs Antiproliferative activity against human HEL cells after 48 hrs by MTT assay, IC50 = 0.1 μM. 29533873
Huh7 Antiviral assay 3 days Antiviral activity against HCV genotype 1b infected in human Huh7 cells after 3 days by luciferase reporter gene assay, EC50 = 0.12 μM. 25490700
HCT116 Antiproliferative assay 48 hrs Antiproliferative activity against human HCT116 cells after 48 hrs by SRB assay, GI50 = 0.13 μM. 27344487
MOLT4 Antiproliferative assay 48 hrs Antiproliferative activity against human MOLT4 cells after 48 hrs by MTT assay, IC50 = 0.14 μM. 29533873
SK-N-BE(2) Antiproliferative assay 48 hrs Antiproliferative activity against human SK-N-BE(2) cells after 48 hrs by MTT assay, IC50 = 0.31 μM. 29533873
PC3 Antiproliferative assay 48 hrs Antiproliferative activity against human PC3 cells after 48 hrs by SRB assay, GI50 = 0.39 μM. 27344487
PC3 Antiproliferative assay 96 hrs Antiproliferative activity against human PC3 cells after 96 hrs by celltiter 96 assay, IC50 = 0.45 μM. 21634430
HeLa Antiproliferative assay 48 hrs Antiproliferative activity against human HeLa cells after 48 hrs by MTT assay, IC50 = 0.51 μM. 29533873
HCT116 Antiproliferative assay 96 hrs Antiproliferative activity against human HCT116 cells after 96 hrs by celltiter 96 assay, IC50 = 0.6 μM. 21634430
A2780 Antiproliferative assay 96 hrs Antiproliferative activity against human A2780 cells after 96 hrs by celltiter 96 assay, IC50 = 0.67 μM. 21634430
HuH7 Cytotoxicity assay 3 days Cytotoxicity against human HuH7 cells assessed as inhibition of cell viability after 3 days by CellTiter 96 assay, CC50 = 0.68 μM. 25490700
COLO205 Antiproliferative assay 96 hrs Antiproliferative activity against human COLO205 cells after 96 hrs by celltiter 96 assay, IC50 = 0.7 μM. 21634430
A549 Antiproliferative assay 48 hrs Antiproliferative activity against human A549 cells after 48 hrs by SRB assay, GI50 = 0.78 μM. 27344487
HL60 Antiproliferative assay 48 hrs Antiproliferative activity against human HL60 cells after 48 hrs by SRB assay, GI50 = 1.09 μM. 27344487
K562 Antiproliferative assay 48 hrs Antiproliferative activity against human K562 cells after 48 hrs by MTT assay, IC50 = 1.1 μM. 29533873
PC3 Antiproliferative assay 48 hrs Antiproliferative activity against human PC3 cells after 48 hrs by MTT assay, IC50 = 1.3 μM. 29533873
NFF Cytotoxicity assay 72 hrs Cytotoxicity against human NFF cells after 72 hrs by SRB assay, IC50 = 1.4 μM. 28241112
HEK293 Cytotoxicity assay 48 hrs Cytotoxicity against HEK293 cells after 48 hrs by resazurin assay, IC50 = 1.4 μM. 28241112
NFF Cytotoxicity assay 72 hrs Cytotoxicity against human NFF cells after 72 hrs by sulforhodamine B assay, IC50 = 1.42 μM. 30245402
HEK293 Cytotoxicity assay 48 hrs Cytotoxicity against HEK293 cells after 48 hrs by resazurin dye based assay, IC50 = 1.42 μM. 30245402
RAW264.7 Anti-inflammatory assay 1 hr Anti-inflammatory activity in LPS-stimulated mouse RAW264.7 cells assessed as suppression of nitric oxide production pre-incubated for 1 hr before LPS stimulation for 24 hrs by Griess reagent based assay, IC50 = 2.2 μM. 25113875
RAW264.7 Anti-inflammatory assay 1 hr Anti-inflammatory activity in LPS-stimulated mouse RAW264.7 cells assessed as suppression of TNFalpha production pre-incubated for 1 hr before LPS stimulation for 24 hrs by ELISA method, IC50 = 4.7 μM. 25113875
RAW264.7 Anti-inflammatory assay 1 hr Anti-inflammatory activity in LPS-stimulated mouse RAW264.7 cells assessed as suppression of PGE2 production pre-incubated for 1 hr before LPS stimulation for 24 hrs by enzyme immunoassay method, IC50 = 8.28 μM. 25113875
HEK293 Function assay Inhibition of HDAC6 in HEK293 cells, IC50 = 0.015 μM. 18308563
HEK293 Function assay Inhibition of HDAC1 in HEK293 cells, IC50 = 0.018 μM. 18308563
HeLa Function assay Inhibition of HDAC in human HeLa cells using Fluor de Lys as substrate by fluorescence assay, IC50 = 0.027 μM. 23639537
HeLa Function assay Inhibition of HDAC from human HeLa cells, IC50 = 0.028 μM. 18247554
HEK293 Function assay Inhibition of HDAC3 in HEK293 cells, IC50 = 0.046 μM. 18308563
HCT116 Antiproliferative assay Antiproliferative activity against human HCT116 cells assessed as growth inhibition, IC50 = 0.16 μM. 21650221
HCT116 Antiproliferative assay Antiproliferative activity against human HCT116 cells, IC50 = 0.16 μM. 21742496
H1299 Antiproliferative assay Antiproliferative activity against human H1299 cells, IC50 = 0.46 μM. 21650221
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生物活性

產(chǎn)品描述 Belinostat是一種新型HDAC抑制劑,無(wú)細(xì)胞試驗(yàn)中IC50為27 nM,對(duì)耐Cisplatin的腫瘤具有活性。Belinostat (PXD101) 可誘導(dǎo)自噬。
特性 Belinostat是Topotarget的領(lǐng)先藥物,已經(jīng)進(jìn)行過(guò)多次臨床試驗(yàn)。
靶點(diǎn)
HDAC [1]
(Cell-free assay)
27 nM
體外研究(In Vitro)
體外研究活性 Belinostat抑制腫瘤細(xì)胞生長(zhǎng)(包括A2780, HCT116, HT29, WIL, CALU-3, MCF7, PC3,及HS852),IC50為0.2到0.66 μM。Belinostat 作用于A2780/cp70 和2780AD細(xì)胞時(shí)活性很低, 這兩個(gè)細(xì)胞是抗cisplatin和doxorubicin的A2780細(xì)胞衍生的。Belinostat通過(guò)PARP分裂和組蛋白H3/H4的乙酰化而誘導(dǎo)細(xì)胞凋亡。[1] Belinostat抑制膀胱癌細(xì)胞生長(zhǎng),尤其是5637細(xì)胞,細(xì)胞在G0-G1期積累, 在S期下降,在 G2-M期上升。[2] Belinostat抑制細(xì)胞生長(zhǎng)的活性不受多重耐藥表現(xiàn)型的影響, 但是docetaxel的活性明顯受影響。Belinostat 可以增強(qiáng)docetaxel或carboplatin 抑制OVCAR-3和A2780細(xì)胞的活性。Belinostat作用于卵巢癌細(xì)胞系也增強(qiáng)微管乙?;饔?。[3] 最新研究顯示 Belinostat在TGF-β信號(hào)依賴機(jī)制中激活蛋白激酶A 和降低survivin mRNA。[4]
激酶實(shí)驗(yàn) 組蛋白脫乙?;富钚詫?shí)驗(yàn)
匯合培養(yǎng),用冷PBS中沖洗2遍,按200×g 轉(zhuǎn)速離心5分鐘。 細(xì)胞懸浮在2體積的溶解 buffer 中,60 mm Tris buffer (pH 為7.4)包含30%甘油和450 mm NaCl,用干冰凍結(jié),然后30oC水浴溶解,循環(huán)3次。細(xì)胞碎片按1.2×104g轉(zhuǎn)速離心5分鐘, 然后上清液儲(chǔ)存于?80oC中。使用 [3H]乙酰 CoA,通過(guò)p300的重組蛋白包括次黃嘌呤-氨基喋呤-胸腺嘧啶域乙?;M蛋白H4肽段 (序列為SGRGKGGKGLGKGGAKRHRK)。100 μg H4 肽段與次黃嘌呤-氨基喋呤-胸腺嘧啶buffer(buffer包含50 mM Tris HCl pH為8.0, 5% 甘油, 50 mM KCl, 和0.1 mM EDTA),1 mM DTT, 1 mM 4-(2-氨乙基) ,苯磺?;? 1×蛋白酶抑制劑, 50 μL純化的p300, 及1.85 m [3H]乙酰 CoA (4.50Ci/mmol)混合,最終體積為300 μL,在30oC溫育45分鐘。p300蛋白 和 20 μL 50% Ni-瓊脂糖在4oC下溫育1小時(shí),然后離心分離。上清液上樣到2 mL Sephadex G15 柱中。加入1毫升蒸餾水,收集三滴樣片,重復(fù)加入蒸餾水直到體積為4-5 mL,收集40滴樣片。用2 mL 閃爍液稀釋3微升樣片,在閃爍計(jì)數(shù)板上計(jì)數(shù),用于鑒定包含標(biāo)記肽段的樣片。合并樣片,測(cè)定1 μL組合樣本,用于測(cè)定每個(gè)肽段的放射性。在150 μL buffer,2 μL 細(xì)胞抽提物,和2 μL Belinostat的混合液中進(jìn)行反應(yīng)。加入2 μL [3H] 標(biāo)記的底物開(kāi)始反應(yīng)。樣本在 37oC下溫育45分鐘,加入HCl 和乙酸(終濃度分別為0.72和0.12 M)終止反應(yīng)。釋放的[3H]乙酸鹽加到750 μL of 乙酸乙酯中, 按1.2×104g轉(zhuǎn)速 離心5分鐘。上層 (600 μL) 轉(zhuǎn)移到3 mL閃爍液,然后計(jì)數(shù)。
細(xì)胞實(shí)驗(yàn) 細(xì)胞系 A2780, A2780/cp70, 2780AD, HCT116, HT29, WIL, CALU-3, MCF7, PC3,和HS852細(xì)胞
濃度 0.016到10 μM
孵育時(shí)間 24小時(shí)
方法 腫瘤細(xì)胞系(A2780, A2780/cp70, 2780AD, HCT116, HT29, WIL, CALU-3, MCF7, PC3,和HS852) 按8×104個(gè)細(xì)胞/25 cm2 瓶接種在5 mL 培養(yǎng)基中,溫育48小時(shí)。用Belinostat (0.016 到 10 μm)處理細(xì)胞24小時(shí)。1 mL 胰蛋白酶/EDTA加到培養(yǎng)瓶中。細(xì)胞分離后,加入1 mL培養(yǎng)基,細(xì)胞再次懸浮。稀釋細(xì)胞,按0.5-2×103個(gè)細(xì)胞/皿移到6cm Petri皿中。37oC下溫育10到15天。用PBS沖洗細(xì)胞,溶于甲醇,用結(jié)晶紫染色,計(jì)數(shù)大于50個(gè)細(xì)胞的群落。通過(guò)IC50值計(jì)算敏感度。
實(shí)驗(yàn)圖片 檢測(cè)方法 檢測(cè)指標(biāo) 實(shí)驗(yàn)圖片 PMID
Western blot p-H2AX(Ser139) / KU70 / KU80 / RAD51 / RAD52 / ERCC1 Acetyl Histone H3 / Acetyl Histone H4 / Acetyl tubulin p21 / p27 SOS1 / SOS2 PARP / p-ERK / p-p38 / p38 / p-BRAF / p-MEK / MEK 24155971
Growth inhibition assay Cell viability IC50 24155971
體內(nèi)研究(In Vivo)
體內(nèi)研究活性 Belinostat按10mg/kg劑量處理A2780和A2780/cp70 移植瘤,明顯延遲腫瘤生長(zhǎng),但是對(duì)動(dòng)物體重沒(méi)有影響。[1] 在鼠膀胱細(xì)胞中,Belinostat也誘導(dǎo)p21WAF1, HDAC 核心和細(xì)胞通訊基因。[2] Belinostat按100mg/kg劑量單獨(dú)處理A2780移植瘤,產(chǎn)生抗癌功效,腫瘤抑制率(TGI)達(dá)47% ,這種抑制存在劑量依賴性。100 mg/kg Belinostat和40 mg/kg Carboplatin聯(lián)用可以延遲腫瘤生長(zhǎng),從18.6 天到22.5 天。 [3] Bortezomib和Belinostat聯(lián)用,明顯抑制腫瘤,此外,作用于攜帶抗Bortezomib UMSCC-11A移植瘤的鼠顯示腸胃毒性 。[5]
動(dòng)物實(shí)驗(yàn) Animal Models 右側(cè)腹皮下注射A2780, A2780/cp70和HCT116細(xì)胞的CD1 nu/nu鼠
Dosages ≤40 mg/kg
Administration 腹腔注射
NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT06406465 Not yet recruiting
Carcinoma Neuroendocrine|Tumor Neuroendocrine|Tumors Neuroendocrine|Neuroendocrine; Carcinoma|Small Cell; Receptors
National Cancer Institute (NCI)|National Institutes of Health Clinical Center (CC)
May 15 2024 Phase 2
NCT04315233 Recruiting
Metastatic Breast Cancer|Recurrent Ovarian Carcinoma
University of Utah|Novartis|Acrotech Biopharma
May 3 2021 Phase 1
NCT04703920 Recruiting
Metastatic Breast Cancer|Metastatic Castration-resistant Prostate Cancer|Metastatic Ovarian Carcinoma
University of Michigan Rogel Cancer Center|Pfizer|Acrotech Biopharma Inc.
March 4 2021 Phase 1
NCT03772925 Active not recruiting
Recurrent Acute Myeloid Leukemia|Recurrent Myelodysplastic Syndrome|Refractory Acute Myeloid Leukemia|Refractory Myelodysplastic Syndrome
National Cancer Institute (NCI)
June 20 2019 Phase 1

化學(xué)信息&溶解度

分子量 318.35 分子式

C15H14N2O4S

CAS號(hào) 866323-14-0 SDF Download Belinostat SDF
Smiles C1=CC=C(C=C1)NS(=O)(=O)C2=CC=CC(=C2)C=CC(=O)NO
儲(chǔ)存條件(自收到貨起)

體外溶解度
批次:

DMSO : 64 mg/mL ( (201.03 mM) ;DMSO吸濕會(huì)降低化合物溶解度,請(qǐng)使用新開(kāi)封DMSO)

Water : Insoluble

Ethanol : Insoluble

摩爾濃度計(jì)算器

體內(nèi)溶解度
批次:

現(xiàn)配現(xiàn)用,請(qǐng)按從左到右的順序依次添加,澄清后再加入下一溶劑

動(dòng)物體內(nèi)配方計(jì)算器

實(shí)驗(yàn)計(jì)算

摩爾濃度計(jì)算器

質(zhì)量 濃度 體積 分子量

動(dòng)物體內(nèi)配方計(jì)算器(澄清溶液)

第一步:請(qǐng)輸入基本實(shí)驗(yàn)信息(考慮到實(shí)驗(yàn)過(guò)程中的損耗,建議多配一只動(dòng)物的藥量)

mg/kg g μL

第二步:請(qǐng)輸入動(dòng)物體內(nèi)配方組成(配方適用于不溶于水的藥物;不同批次藥物配方比例不同,請(qǐng)聯(lián)系Selleck為您提供正確的澄清溶液配方)

% DMSO % % Tween 80 % ddH2O
%DMSO %

計(jì)算結(jié)果:

工作液濃度: mg/ml;

DMSO母液配制方法: mg 藥物溶于μL DMSO溶液(母液濃度mg/mL,:如該濃度超過(guò)該批次藥物DMSO溶解度,請(qǐng)先聯(lián)系Selleck);

體內(nèi)配方配制方法:μL DMSO母液,加入μL PEG300,混勻澄清后加入μL Tween 80,混勻澄清后加入μL ddH2O,混勻澄清。

體內(nèi)配方配制方法:μL DMSO母液,加入μL Corn oil,混勻澄清。

注意:1. 首先保證母液是澄清的;
2.一定要按照順序依次將溶劑加入,進(jìn)行下一步操作之前必須保證上一步操作得到的是澄清的溶液,可采用渦旋、超聲或水浴加熱等物理方法助溶。

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常見(jiàn)問(wèn)題及建議解決方法

問(wèn)題 1:
Could you please give some suggestions for the use of Belinostat in vivo (i.p. injection)?

回答:
For I.P. injection, S1085 Belinostat (PXD101) can be dissolved in 2% DMSO+30% PEG 300+ddH2O at 10 mg/ml clearly. When preparing the solution, please dissolve the compound in DMSO clearly first. Then add PEG, after they mixed well, then dilute with water. Hope this information is useful to you.

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