VER155008

HSP70 (IC50 = 0.5 μM); HSC70 (IC50 = 2.6 μM); Grp78 (IC50 = 2.6 μM)
Heat shock cognate 70 (Hsc70) and Heat shock protein 70 (Hsp70); IC50 values: ~0.5 μM (recombinant human Hsc70 ATPase activity), ~0.8 μM (recombinant human Hsp70-1A ATPase activity), and ~1.2 μM (recombinant murine Hsc70 ATPase activity) [1]
- Heat shock protein 70 (Hsp70) and Heat shock cognate 70 (Hsc70); IC50 value: ~2.5 μM (inhibition of androgen receptor (AR) expression in LNCaP95 prostate cancer cells, used as a functional readout for Hsp70/Hsc70 inhibition) [3]

VER-155008 对 Hsp90 无活性，IC50 > 200 μM，但它是 Hsc70 和 Hsp70 的抑制剂，对 Hsp70、Hsc70 和 Grp7 的 IC50 分别为 0.5 μM、2.6 μM 和 2.6 μM。 BT474、MB-468、HCT116 和 HT29 细胞属于 VER-155008 抑制增殖的人类结肠和乳腺肿瘤细胞系，其 GI50 分别为 10.4 μM、14.4 μM、5.3 μM 和 12.8 μM。 HCT116 和 BT474 癌细胞响应 VER-155008 (5–40 μM) 表现出客户蛋白降解。此外，VER-155008 会导致人类肿瘤细胞系发生凋亡[1]。在培养的神经元中，VER-155008 (0.05-5 μM) 可恢复 Aβ 诱导的轴突变性[2]。 VER-155008（10 μM 或 25 μM）抑制 Hsp70 并阻止 LNCaP95 细胞增殖。此外，VER-155008 还降低了雄激素受体剪接变异 7 (AR-V7) 和全长雄激素受体 (AR-FL) 蛋白的表达[3]。

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HCT116结肠癌细胞中与Hsp90抑制剂协同诱导凋亡：VER155008单独对HCT116细胞的抗增殖活性较弱（GI50 > 20 μM）。但与Hsp90抑制剂（如1 μM 17-AAG，格尔德霉素类似物）联用时，VER155008（2-10 μM）呈剂量依赖性提高凋亡率：Annexin V/PI染色显示，17-AAG单独处理组凋亡率为12%，VER155008 5 μM单独处理组为8%，而联合处理组（17-AAG + VER155008 5 μM）为45%。Western blot证实联合组中caspase-3切割水平（较17-AAG单独组升高2.8倍）和PARP切割水平（较17-AAG单独组升高3.2倍）显著增加 [1]
- 神经元中减少轴突退化和tau蛋白磷酸化：用5 μM Aβ1-42（诱导阿尔茨海默病样病理）处理原代皮质神经元后，VER155008（1-5 μM）呈剂量依赖性减少轴突退化：神经丝重链（NF-H，轴突标志物）免疫荧光染色显示，Aβ单独处理组轴突完整性评分为35%，而Aβ + VER155008 5 μM处理组为78%。Western blot还显示，VER155008处理组中tau蛋白在Ser396位点（较Aβ单独组降低45%）和Thr231位点（较Aβ单独组降低52%）的磷酸化水平显著降低 [2]
- LNCaP95前列腺癌细胞中抑制雄激素受体（AR）表达和增殖：VER155008（2.5-20 μM）呈剂量依赖性降低LNCaP95细胞中AR蛋白水平：Western blot显示，10 μM VER155008处理24 h后，AR蛋白水平较溶媒组降低70%。实时定量PCR（qPCR）显示，10 μM VER155008处理后AR mRNA水平降低40%，表明其对AR的调控涉及转录和翻译后两个层面。此外，VER155008还抑制LNCaP95细胞增殖（MTT法测得GI50 ~15 μM，72 h），并降低AR靶基因表达（如10 μM处理后PSA mRNA水平降低65%） [3]

在幼稚雌性 BALB/c 小鼠中，VER-155008（25 mg/kg，静脉注射）显示血浆清除率。在 HCT116 荷瘤裸 BALB/c 小鼠中，VER-155008（40 mg/kg，静脉注射）同样表现出快速血浆清除并降低肿瘤水平[1]。在 5XFAD 小鼠中，VER-155008（10 μmol/kg/天，腹腔注射）可减轻与淀粉样斑块相关的轴突肿胀，并改善记忆缺陷。 VER-155008（89.9 μmol/kg/天，腹膜内注射）还可减少 5XFAD 小鼠中的淀粉样斑块和淀粉样斑块相关蛋白 PHF-tau [2]。

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APP/PS1转基因小鼠（阿尔茨海默病模型）中改善记忆缺陷和减少轴突退化：6月龄APP/PS1转基因小鼠经VER155008（30 mg/kg/天，溶于0.5%甲基纤维素中口服灌胃）处理4周后，Morris水迷宫测试显示：与溶媒处理转基因小鼠相比，VER155008处理组逃避潜伏期缩短50%，在目标象限停留时间增加2.3倍。海马组织免疫组化显示，VER155008处理组轴突退化（NF-H阳性断裂轴突）减少60%，磷酸化tau蛋白（Ser396位点）水平降低48%。野生型小鼠经VER155008处理后未观察到显著记忆改善 [2]

Hsc70、Hsp70、Grp78和Hsp90荧光偏振（FP）测定[1]
如前所述，测定VER-155008对Hsp70和Hsp90的活性。Hsc70（aa 5-381）和Grp78（ATP酶结构域）的克隆和表达如下。制作了对应于氨基酸5-381的Hsc70的PCR片段。将纯化的PCR片段连接到pCR2.1-TOPO中。随后将其限制性消化并亚克隆到商业组氨酸标记载体pET101中用于表达分析。通过PCR扩增Grp78的ATP酶结构域，并将其克隆到pCR2.1-TOPO中。随后将其消化并连接到谷胱甘肽-s-转移酶标记的表达载体pGEX-4T-1中。按照Hsp70的描述，使用与FP探针相同的N 6-（6-氨基）己基-ATP-5-FAM进行Hsc70和Grp78的FP测定，但有以下修改。对于Hsc70，在22°C下孵育30分钟，蛋白质和探针浓度分别为0.3μM和20 nM，而对于Grp78，在22℃下孵育2小时，蛋白质和探头浓度分别为2μM和10 nM。FAM-ATP探针的K D对Hsc70为0.24μM，对Grp78为2μM。

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BiaCore测定化合物与Hsp70的结合[1]
所有实验均在Biacore T100上在25°C下以30μL/min的流速进行。缓冲系统与Hsp70荧光偏振分析中使用的缓冲系统相同，含有1%DMSO和0.05%吐温20。双His标记的Hsp70被固定在NTA传感器芯片的表面上；约2000RU的蛋白质被固定。注入化合物90秒，在80秒时根据平衡结合确定Kds。

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Hsp70 ATP酶测定[1]
使用ADP Hunter Plus检测试剂盒测量ATP周转率和随后产生的ADP。反应混合物在半体积、全黑色非结合板中制备，在标准试剂盒测定缓冲液中含有250 nM全长、GST标记的Hsp70、50μM ATP和浓度递增的VER-155008。反应在30°C下孵育90分钟，然后在FlexStation 3上读取，E x为530 nm，E m为590 nm，每孔读取6次。设置不含酶的反应来监测ATP的自发水解，并从含有Hsp70的反应中减去。

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热变性萤光素酶的复性[1]
该测定基本上如前所述[24,25]进行。简而言之，Hsc70/Hsp70抑制剂在pH 7.5的兔网织红细胞裂解物（RRL，核酸酶处理）中稀释，该裂解物含有10 mM Tris、100 mM KCl、3 mM EDTA、1 mM DTT和ATP再生系统。最终ATP浓度为0.5 mM。化合物在室温下在上述混合物中预孵育15分钟，然后加入25 ng热变性萤光素酶（在40°C下变性30分钟）以启动复性反应，并在30°C下孵育60分钟。使用Bright glo试剂测定萤光素酶活性。

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Hsc70/Hsp70 ATP酶活性实验（比色法）：将重组Hsc70/Hsp70蛋白（人Hsc70、人Hsp70-1A或小鼠Hsc70）在实验缓冲液（20 mM Tris-HCl pH 7.5、10 mM KCl、5 mM MgCl2、1 mM DTT）中稀释至终浓度1 μg/孔。加入系列稀释的VER155008（0.1-10 μM），再加入ATP（终浓度1 mM）启动反应。混合物在37°C孵育60 min后，使用商品化无机磷酸盐（Pi）检测试剂盒检测ATP水解释放的Pi。在620 nm处测定吸光度，通过四参数逻辑模型拟合剂量-反应曲线计算IC50值 [1]
- Hsp70-AR结合抑制实验（免疫沉淀法）：取LNCaP95细胞裂解液（1 mg蛋白），与VER155008（0.5-10 μM）在4°C孵育1 h，随后加入抗Hsp70抗体偶联琼脂糖珠，4°C孵育过夜。用裂解缓冲液洗涤珠子3次，用SDS上样缓冲液洗脱结合蛋白。通过Western blot（抗AR抗体）检测与Hsp70结合的AR。密度分析显示，5 μM VER155008处理后，Hsp70-AR复合物形成较溶媒组减少约65% [3]

细胞周期分析[1]
将HCT116细胞暴露于指定浓度的VER-155008和VER-82160中24小时。在用RNaseA/碘化丙啶染色之前，收集所有细胞并将其固定在冰上的70%乙醇中。在FACSArray细胞仪上收集数据，并用FACSDeva软件进行分析。

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雄激素剥夺疗法最初对晚期前列腺癌症患者有效；然而，前列腺癌症逐渐对雄激素剥夺疗法产生耐药性，称为去势耐药性前列腺癌症（CRPC）。雄激素受体剪接变异体7（AR-V7）是CRPC的原因之一，与对新一代AR拮抗剂（恩杂鲁胺）的耐药性和预后不良有关。已知热休克蛋白70（Hsp70）抑制剂可以降低全长AR（AR-FL）的水平，但对其对表达AR-V7的CRPC细胞的影响知之甚少。在本研究中，我们研究了Hsp70抑制剂槲皮素和VER155008在表达AR-V7的前列腺癌症细胞系LNCaP95中的作用，并探讨了Hsp7调节AR-FL和AR-V7表达的机制。槲皮素和VER155008降低了细胞增殖，增加了凋亡细胞的比例，并降低了AR-FL和AR-V7的蛋白水平。此外，VER155008降低了AR-FL和AR-V7 mRNA水平。用Hsp70抗体进行免疫沉淀和质谱鉴定，Y-box结合蛋白1（YB-1）是通过与Hsp70相互作用在转录水平调节AR-FL和AR-V7的分子之一。VER155008降低了YB-1的磷酸化及其在细胞核中的定位，表明Hsp70参与AR调节可能是通过YB-1的激活和核转位介导的。总的来说，这些结果表明，Hsp70抑制剂通过YB-1抑制降低AR-FL和AR-V7的表达，对CRPC具有潜在的抗肿瘤活性[3]。

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HCT116细胞凋亡实验（Annexin V-FITC/PI染色）：将HCT116细胞以2×10⁵细胞/孔接种到6孔板中，过夜孵育。用VER155008（2-10 μM）单独处理或与1 μM 17-AAG联合处理48 h。收集细胞，用冷PBS洗涤，按照试剂盒说明书用Annexin V-FITC和碘化丙啶（PI）染色。通过流式细胞术定量凋亡细胞（Annexin V阳性/PI阴性：早期凋亡；Annexin V阳性/PI阳性：晚期凋亡）。使用流式细胞术软件分析数据，凋亡率计算为早期和晚期凋亡细胞之和 [1]
- 原代皮质神经元轴突退化实验（免疫荧光）：从E18大鼠胚胎中分离原代皮质神经元，在盖玻片上培养7天。用5 μM Aβ1-42单独处理或与VER155008（1-5 μM）联合处理24 h。用4%多聚甲醛固定细胞，0.1% Triton X-100透化，5% BSA封闭。神经元与抗NF-H抗体（轴突标志物）在4°C孵育过夜，随后与Alexa Fluor 488偶联二抗在室温孵育1 h。将盖玻片用含DAPI的封片液封片，通过计数完整NF-H阳性轴突（长度>50 μm）与断裂轴突的比例评分轴突完整性，每片盖玻片至少计数100个神经元 [2]
- LNCaP95细胞AR表达实验（Western blot和qPCR）：Western blot流程：将LNCaP95细胞以3×10⁵细胞/孔接种到6孔板中，过夜孵育，用VER155008（2.5-20 μM）处理24 h。用RIPA缓冲液裂解细胞，BCA法测定蛋白浓度，取30 μg蛋白进行SDS-PAGE分离。膜用抗AR抗体和抗β-肌动蛋白（内参）抗体孵育，条带强度通过密度分析定量。qPCR流程：用RNA提取试剂盒从处理后的LNCaP95细胞中提取总RNA，逆转录为cDNA，用AR特异性引物进行qPCR。采用2^(-ΔΔCt)法计算相对AR mRNA水平，以GAPDH为内参基因 [3]

Formulation method 1: VER-155008 was dissolved in 10% dimethyl sulfoxide (DMSO)-containing saline and administered intraperitoneally (10 mmol/kg/day) to 5XFAD mice for 18 days. [1]

Formulation method 2: Female BALB/c mice were dosed intravenously with 25 mg/kg VER-155008 into the lateral tail vein as a solution in 10% DMSO/5% Tween 80/85% saline (v/v/v). [2]
BALB/c mice All in vivo studies were carried out according to UK Home Office guidelines and the “Guidance on the Operation of the Animals (Scientific Procedures) Act 1986”. Female BALB/c mice were dosed intravenously with 25 mg/kg VER-155008 into the lateral tail vein as a solution in 10% DMSO/5% Tween 80/85% saline (v/v/v). Animals were sacrificed at 5, 15 and 30 min, 1, 2, 4 and 6 h post dose.[1]
Female BALB/c nu/nu mice were inoculated with 5 × 106 HCT116 cells into the right flank. Tumors were monitored for growth, and animals were dosed IV with 40 mg/kg VER-155008 on day 31 (tumor volume approximately 200–250 mm3). Animals were sacrificed at 15 min, 1 and 4 h post dose.[1]
Each timepoint contained three mice and blood samples were collected by cardiac puncture. Plasma was obtained by centrifugation and frozen at −20°C prior to analysis. Tumors were harvested, snap frozen and stored at −80°C.[1]

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Alzheimer's disease (AD) is a progressive neurodegenerative disorder resulting in structural brain changes and memory impairment. We hypothesized that reconstructing neural networks is essential for memory recovery in AD. Heat shock cognate 70 (HSC70), a member of the heat shock protein family of molecular chaperones, is upregulated in AD patient brains, and recent studies have demonstrated that HSC70 facilitates axonal degeneration and pathological progression in AD. However, the direct effects of HSC70 inhibition on axonal development and memory function have never been investigated. In this study, we examined the effects of a small-molecule HSC70 inhibitor, VER-155008, on axonal morphology and memory function in a mouse model of AD (5XFAD mice). We found that VER-155008 significantly promoted axonal regrowth in amyloid β-treated neurons in vitro and improved object recognition, location, and episodic-like memory in 5XFAD mice. Furthermore, VER-155008 penetrated into the brain after intraperitoneal administration, suggesting that VER-155008 acts in the brain in situ. Immunohistochemistry revealed that VER-155008 reduced bulb-like axonal swelling in the amyloid plaques in the perirhinal cortex and CA1 in 5XFAD mice, indicating that VER-155008 also reverses axonal degeneration in vivo. Moreover, the two main pathological features of AD, amyloid plaques and paired helical filament tau accumulation, were reduced by VER-155008 administration in 5XFAD mice. This is the first report to show that the inhibition of HSC70 function may be critical for axonal regeneration and AD-like symptom reversal. Our study provides evidence that HSC70 can be used as a new therapeutic target for AD treatment[2].

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APP/PS1 transgenic mouse model for Alzheimer's disease: Six-month-old male APP/PS1 transgenic mice (n=8/group) and wild-type littermates (n=8/group) were randomly assigned to vehicle or VER155008 groups. VER155008 was dissolved in 0.5% methylcellulose (in 0.9% saline) and administered via oral gavage at 30 mg/kg/day for 4 weeks; vehicle group received 0.5% methylcellulose alone. During the last week of treatment, Morris water maze tests were conducted to assess memory: mice were trained for 5 days (4 trials/day) to find a hidden platform, and a probe trial (platform removed) was performed on day 6 to measure time spent in the target quadrant. After behavioral testing, mice were euthanized, brains were harvested, and hippocampal tissues were dissected for immunohistochemistry (NF-H and phosphorylated tau staining) and Western blot analysis [2]

In vitro cellular toxicity: VER155008 showed low intrinsic toxicity in non-cancerous cells: primary cortical neurons treated with VER155008 (up to 10 μM) for 24 h had >90% viability (MTT assay) [2]. In HCT116 colon carcinoma cells, the IC50 for cytotoxicity (cell death) was >20 μM [1]. In LNCaP95 prostate cancer cells, the GI50 (growth inhibition) was ~15 μM, with no significant cell death at concentrations <10 μM [3]
- In vivo toxicity in mice: APP/PS1 transgenic mice treated with VER155008 (30 mg/kg/day, oral, 4 weeks) showed no significant changes in body weight (vehicle: +5.2% vs. VER155008: +4.8%), food consumption, or serum levels of ALT, AST, BUN, and creatinine (markers of liver and kidney function) compared to vehicle controls. No treatment-related deaths or gross pathological abnormalities were observed in brain, liver, or kidney tissues [2]

[1]. A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells. Cancer Chemother Pharmacol. 2010 Aug;66(3):535-45.

[2]. Heat Shock Cognate 70 Inhibitor, VER-155008, Reduces Memory Deficits and Axonal Degeneration in a Mouse Model of Alzheimer's Disease. Front Pharmacol. 2018 Jan 30;9:48.

[3]. Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells. Cancer Sci. 2017 Sep;108(9):1820-1827.

4-[[(2R,3S,4R,5R)-5-[6-amino-8-[(3,4-dichlorophenyl)methylamino]-9-purinyl]-3,4-dihydroxy-2-oxolanyl]methoxymethyl]benzonitrile is a purine nucleoside.

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VER155008 is a selective, small-molecule inhibitor of Hsc70 and Hsp70 that binds to the ATP-binding domain of these chaperones, preventing ATP hydrolysis and disrupting their chaperone function. It potentiates Hsp90 inhibitor-induced apoptosis by blocking Hsc70/Hsp70-mediated refolding of misfolded proteins (released after Hsp90 inhibition), leading to increased proteotoxic stress and cell death [1]
- In Alzheimer's disease models, VER155008 exerts neuroprotective effects by inhibiting Hsc70, which normally promotes tau aggregation and axonal degeneration. By blocking Hsc70, VER155008 reduces tau hyperphosphorylation and preserves axonal integrity, thereby improving memory deficits [2]
- In prostate cancer, VER155008 suppresses AR expression by inhibiting Hsp70, which acts as a co-chaperone for AR folding and stabilization. Reduced AR levels lead to decreased proliferation of AR-dependent LNCaP95 prostate cancer cells, supporting its potential as a therapeutic agent for AR-positive prostate cancer [3]

C25H23CL2N7O4

556.40

555.118

C, 53.97; H, 4.17; Cl, 12.74; N, 17.62; O, 11.50

1134156-31-2

25195348

Typically exists as White to pink solids at room temperature

1.6±0.1 g/cm3

856.3±75.0 °C at 760 mmHg

471.7±37.1 °C

0.0±0.3 mmHg at 25°C

1.748

2.71

164.36

4

10

8

38

831

4

ClC1=C(C([H])=C([H])C(=C1[H])C([H])([H])N([H])C1=NC2=C(N([H])[H])N=C([H])N=C2N1[C@@]1([H])[C@@]([H])([C@@]([H])([C@@]([H])(C([H])([H])OC([H])([H])C2C([H])=C([H])C(C#N)=C([H])C=2[H])O1)O[H])O[H])Cl

ZXGGCBQORXDVTE-UMCMBGNQSA-N

InChI=1S/C25H23Cl2N7O4/c26-16-6-5-15(7-17(16)27)9-30-25-33-19-22(29)31-12-32-23(19)34(25)24-21(36)20(35)18(38-24)11-37-10-14-3-1-13(8-28)2-4-14/h1-7,12,18,20-21,24,35-36H,9-11H2,(H,30,33)(H2,29,31,32)/t18-,20-,21-,24-/m1/s1

5'-O-[(4-Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)methyl]amino]-adenosine

VER 155008; VER155008; VER-155008; VER-155008; VER 155008; 5'-O-[(4-Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)methyl]amino]-adenosine; VER155008; C25H23Cl2N7O4; 4-((((2R,3S,4R,5R)-5-(6-Amino-8-((3,4-dichlorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)benzonitrile; 5'-O-[(4-Cyanophenyl)methyl]-8-[[(3,4-dichlorophenyl)-methyl]amino]-adenosine;

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: ≥ 2.5 mg/mL (4.49 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 25.0 mg/mL澄清DMSO储备液加入到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: ≥ 2.5 mg/mL (4.49 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 25.0 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: ≥ 2.5 mg/mL (4.49 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 25.0 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: 1% CMC+0.5% Tween-80: 30 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。