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，無細胞試驗中IC50為27 nM，對耐Cisplatin的腫瘤具有活性。Belinostat (PXD101) 可誘導自噬。
Belinostat Chemical Structure
CAS: 866323-14-0
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純度：
99.74%
99.74
Belinostat相關產品
相關信號通路圖
細胞實驗數據示例
| 細胞系 | 實驗類型 | 給藥濃度 | 孵育時間 | 活性描述 | 文獻信息 |
|---|---|---|---|---|---|
| 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 | ||
| 點擊查看更多細胞系數據 | |||||
生物活性
| 產品描述 | Belinostat是一種新型HDAC抑制劑，無細胞試驗中IC50為27 nM，對耐Cisplatin的腫瘤具有活性。Belinostat (PXD101) 可誘導自噬。 | ||
|---|---|---|---|
| 特性 | Belinostat是Topotarget的領先藥物，已經進行過多次臨床試驗。 | ||
| 靶點 |
|
| 體外研究(In Vitro) | ||||
| 體外研究活性 | Belinostat抑制腫瘤細胞生長(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細胞時活性很低, 這兩個細胞是抗cisplatin和doxorubicin的A2780細胞衍生的。Belinostat通過PARP分裂和組蛋白H3/H4的乙?；T導細胞凋亡。[1] Belinostat抑制膀胱癌細胞生長，尤其是5637細胞,細胞在G0-G1期積累, 在S期下降，在 G2-M期上升。[2] Belinostat抑制細胞生長的活性不受多重耐藥表現型的影響, 但是docetaxel的活性明顯受影響。Belinostat 可以增強docetaxel或carboplatin 抑制OVCAR-3和A2780細胞的活性。Belinostat作用于卵巢癌細胞系也增強微管乙?；饔谩?sup>[3] 最新研究顯示 Belinostat在TGF-β信號依賴機制中激活蛋白激酶A 和降低survivin mRNA。[4] | |||
|---|---|---|---|---|
| 激酶實驗 | 組蛋白脫乙?；富钚詫嶒?/td> | |||
| 匯合培養(yǎng)，用冷PBS中沖洗2遍，按200×g 轉速離心5分鐘。 細胞懸浮在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，然后30oC水浴溶解，循環(huán)3次。細胞碎片按1.2×104g轉速離心5分鐘, 然后上清液儲存于?80oC中。使用 [3H]乙酰 CoA，通過p300的重組蛋白包括次黃嘌呤-氨基喋呤-胸腺嘧啶域乙酰化組蛋白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，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，在閃爍計數板上計數，用于鑒定包含標記肽段的樣片。合并樣片，測定1 μL組合樣本，用于測定每個肽段的放射性。在150 μL buffer，2 μL 細胞抽提物，和2 μL Belinostat的混合液中進行反應。加入2 μL [3H] 標記的底物開始反應。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。釋放的[3H]乙酸鹽加到750 μL of 乙酸乙酯中, 按1.2×104g轉速 離心5分鐘。上層 (600 μL) 轉移到3 mL閃爍液，然后計數。 | ||||
| 細胞實驗 | 細胞系 | A2780, A2780/cp70, 2780AD, HCT116, HT29, WIL, CALU-3, MCF7, PC3，和HS852細胞 | ||
| 濃度 | 0.016到10 μM | |||
| 孵育時間 | 24小時 | |||
| 方法 | 腫瘤細胞系(A2780, A2780/cp70, 2780AD, HCT116, HT29, WIL, CALU-3, MCF7, PC3，和HS852) 按8×104個細胞/25 cm2 瓶接種在5 mL 培養(yǎng)基中，溫育48小時。用Belinostat (0.016 到 10 μm)處理細胞24小時。1 mL 胰蛋白酶/EDTA加到培養(yǎng)瓶中。細胞分離后，加入1 mL培養(yǎng)基，細胞再次懸浮。稀釋細胞，按0.5-2×103個細胞/皿移到6cm Petri皿中。37oC下溫育10到15天。用PBS沖洗細胞，溶于甲醇，用結晶紫染色，計數大于50個細胞的群落。通過IC50值計算敏感度。 | |||
| 實驗圖片 | 檢測方法 | 檢測指標 | 實驗圖片 | 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 |
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24155971 | |
| Growth inhibition assay | Cell viability IC50 |
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24155971 | |
| 體內研究(In Vivo) | ||
| 體內研究活性 | Belinostat按10mg/kg劑量處理A2780和A2780/cp70 移植瘤，明顯延遲腫瘤生長，但是對動物體重沒有影響。[1] 在鼠膀胱細胞中，Belinostat也誘導p21WAF1, HDAC 核心和細胞通訊基因。[2] Belinostat按100mg/kg劑量單獨處理A2780移植瘤，產生抗癌功效，腫瘤抑制率（TGI）達47% ，這種抑制存在劑量依賴性。100 mg/kg Belinostat和40 mg/kg Carboplatin聯用可以延遲腫瘤生長，從18.6 天到22.5 天。 [3] Bortezomib和Belinostat聯用，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] | |
|---|---|---|
| 動物實驗 | Animal Models | 右側腹皮下注射A2780, A2780/cp70和HCT116細胞的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 |
化學信息&溶解度
| 分子量 | 318.35 | 分子式 | C15H14N2O4S |
| CAS號 | 866323-14-0 | SDF | Download Belinostat SDF |
| Smiles | C1=CC=C(C=C1)NS(=O)(=O)C2=CC=CC(=C2)C=CC(=O)NO | ||
| 儲存條件(自收到貨起) | |||
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體外溶解度 |
DMSO : 64 mg/mL ( (201.03 mM) ；DMSO吸濕會降低化合物溶解度，請使用新開封DMSO) Water : Insoluble Ethanol : Insoluble |
摩爾濃度計算器 |
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體內溶解度 現配現用，請按從左到右的順序依次添加，澄清后再加入下一溶劑 |
動物體內配方計算器 | ||||
實驗計算
動物體內配方計算器（澄清溶液）
第一步：請輸入基本實驗信息（考慮到實驗過程中的損耗，建議多配一只動物的藥量）
mg/kg
g
μL
只
第二步：請輸入動物體內配方組成（配方適用于不溶于水的藥物；不同批次藥物配方比例不同，請聯系Selleck為您提供正確的澄清溶液配方）
% DMSO
%
% Tween 80
% ddH2O
%DMSO
%
計算結果：
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，注：如該濃度超過該批次藥物DMSO溶解度，請先聯系Selleck）；
體內配方配制方法：取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。
體內配方配制方法：取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。
技術支持
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如果有其他問題，請給我們留言。
* 必填項
常見問題及建議解決方法
問題 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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