Butyzamide

JAK2; STAT3/5

在 Ba/F3-hMpl 细胞中，Butyzamide（3 μM；15 分钟）磷酸化 JAK2、STAT3、STAT5 和 MAPK [1]。Butyzamide（3 μM；48 小时）刺激人 CD34+ 造血祖细胞产生多倍体巨核细胞和形成集落形成单位的巨核细胞 [1]。

Butyzamide（10 mg/kg、50 mg/kg；侧向；每日一次，持续 20 天）可提高植入人胎肝源性 CD34+ 细胞的 NOG 的人类预期水平 [1]。
Butyzamide增加FL-CD34+移植NOG小鼠血小板[1]
为了评估Butyzamide在体内产生人血小板的能力，我们将FL-CD34细胞移植到没有淋巴细胞或自然杀伤细胞、补体活性低的免疫缺陷NOG小鼠体内一些研究者已经使用NOG小鼠来分析干细胞、肥大细胞和巨核细胞的造血功能，因为人类细胞在NOG小鼠中的植入率高于NOD/SCID小鼠。26,29当Butyzamide胺以10或50 mg/kg的剂量给予移植了flcd34细胞的NOG小鼠，每天一次，持续20天，它增加了人血小板的数量，但不影响小鼠血小板的数量(图4A, B)。在人血小板增加之前，经噻唑橙染色的网状人CD41血小板也被Butyzamide增加了(图4C)。Butyzamide治疗21 d后处死小鼠，冲洗股骨收集骨髓细胞。Butyzamide能增加骨髓中人CD41巨核细胞的数量(图4D)。另一侧股骨用抗人CD42b抗体染色，石蜡切片可见人巨核细胞(图4E)。与对照组相比，Butyzamide处理增加了人CD42b细胞，这表明Butyzamide刺激了人巨核细胞在体内的增殖和成熟。这些数据表明，口服Butyzamide可有效增加体内人类血小板的数量。[1]

为了建立Ba/F3-hMpl、Ba/F3-mMpl和Ba/F3-h Mpl（H499L）细胞，通过电穿孔用每个构建的质粒转染Ba/F3细胞。在2mg/mL遗传素存在下，通过限制性稀释法克隆细胞。为了建立Ba/F3 mMpl（L490H）细胞，根据制造商的方案，通过Fugene 6用pQCXIP mMpl和pVSV-G载体（Invitrogen）转染293GP2包装细胞。收集培养上清液并用于感染Ba/F3细胞。这些建立的稳定细胞系对rmIL-3以及亲代Ba/F3细胞产生反应而增殖，并似乎保持了亲代Ba/F3细胞的特性[1]。

细胞在96孔板中以7.5×10个细胞/200μL的密度培养，并加入不同浓度的Butyzamide或重组细胞因子。将平板在37°C、含5%CO2的加湿室中孵育48小时，并在培养的最后4小时向每个孔中添加10μL WST-8试剂。使用680 型96孔微孔板读取器在450nm的波长下测量吸光度。[1]

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人骨髓源性CD34+细胞的液体培养[1]
人骨髓来源的CD34细胞在24孔板中以5.0×10细胞/mL的密度培养。作为无血清培养基，我们使用Iscove改良的Dulbecco培养基，添加20% BIT9500。细胞分别用1 nM rhTPO或3 μMButyzamide处理3次，37°C, 5% CO2加湿室处理10天。

Immunodeficient NOD/Shi-scid,IL-2Rγ (NOG) mice (8–10 weeks old) were irradiated with 2.4 Gy (125 kV, 10 mA, 0.45 Gy/min) by MBR-1520R-3 (Hitachi Medico, Tokyo, Japan), and on the next day, 6.7×10 human fetal liver (FL)-derived CD34 cells were transplanted intravenously. Two months after transplantation, the NOG mice were randomized into three groups, based on the number of human platelets and body weight. The vehicle or butyzamide at a dose of 10 or 50 mg/kg was administered orally for 21 days. The numbers of human platelets and megakaryocytes were calculated as described below [1].

[1]. The effect of a novel, small non-peptidyl molecule butyzamide on human thrombopoietin receptor and megakaryopoiesis. Haematologica. 2008 Oct;93(10):1495-504.

Background: Thrombocytopenia is a common problem in the management of patients with cancer and other conditions that affect hematopoietic cells. In previous clinical trials, the polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor increased platelet counts in patients with idiopathic thrombocytopenic purpura and solid tumors undergoing chemotherapy. However, antibodies to polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor develop in healthy volunteers and patients undergoing chemotherapy and cross-react with endogenous thrombopoietin. As a result, clinical development of polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor was discontinued in 1998. The aim of this study was to identify an orally bioavailable human Mpl activator that does not develop autoantibodies against endogenous thrombopoietin.[1]
Design and methods: We screened our chemical library and created a novel non-peptidyl thrombopoietin receptor, Mpl activator named butyzamide. We evaluated the effect of butyzamide on megakaryopoiesis in vitro using Ba/F3 cells expressing Mpl and human hematopoietic stem cells. For the evaluation of its in vivo effect, we administered butyzamide orally to immunodeficient NOD/Shi-scid,IL-2R gamma(null) (NOG) mice transplanted with human fetal liver-derived CD34(+) cells and investigated the production of human platelets.[1]
Results: Butyzamide specifically reacted with human Mpl and activated the same signal transduction pathway as thrombopoietin. However, unlike thrombopoietin, butyzamide did not react with murine Mpl and was shown to require the histidine residue in the transmembrane domain of Mpl for its agonistic activity. Butyzamide induced colony-forming unit-megakaryocytes and polyploid megakaryocytes from human CD34(+) hematopoietic progenitor cells, and its effects were comparable to those of thrombopoietin. When butyzamide was administered orally at the doses of 10 and 50 mg/kg for 20 days to NOG mice transplanted with human fetal liver-derived CD34(+) cells, the human platelet count increased by 6.2- and 22.9-fold, respectively.[1]
Conclusions: Butyzamide is an orally bioavailable human Mpl activator, and appears to have potential for clinical development as a therapeutic agent for patients with thrombocytopenia.[1]

C29H32CL2N2O5S

590.14

1110767-45-7

44602781

White to off-white solid

7.6

126Ų

2

7

11

39

857

0

CCCOC(C1=CC=CC(=C1OC)C2=CSC(=N2)NC(=O)C3=CC(=C(C(=C3)Cl)/C=C(\C)/C(=O)O)Cl)C(C)(C)C

BBEZGQPYGPBSLA-FOWTUZBSSA-N

InChI=1S/C29H32Cl2N2O5S/c1-7-11-38-25(29(3,4)5)19-10-8-9-18(24(19)37-6)23-15-39-28(32-23)33-26(34)17-13-21(30)20(22(31)14-17)12-16(2)27(35)36/h8-10,12-15,25H,7,11H2,1-6H3,(H,35,36)(H,32,33,34)/b16-12+

(E)-3-[2,6-dichloro-4-[[4-[3-(2,2-dimethyl-1-propoxypropyl)-2-methoxyphenyl]-1,3-thiazol-2-yl]carbamoyl]phenyl]-2-methylprop-2-enoic acid

Butyzamide; 1110767-45-7; (E)-3-(2,6-Dichloro-4-((4-(3-(2,2-dimethyl-1-propoxypropyl)-2-methoxyphenyl)thiazol-2-yl)carbamoyl)phenyl)-2-methylacrylic acid; SCHEMBL3059522; SCHEMBL3059524;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~169.05 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。