CDK2; JAK2; FLT3

SB1317 (TG02) 是一种新型小分子强效 CDK/JAK2/FLT3 抑制剂。SB1317 可溶性好，在 Caco-2 细胞中渗透性高，与小鼠、狗和人类血浆的结合率 > 99%。与小鼠和大鼠相比，它在人类和狗肝微粒体中的代谢稳定。SB1317 在体外主要由 CYP3A4 和 CY1A2 代谢。SB1317 在体外不抑制除 CYP2D6（IC50=1 μM）以外的任何主要人类 CYP。SB1317 在体外不显著诱导人肝细胞中的 CYP1A 和 CYP3A4。临床前物种肝微粒体中的代谢特征与人类相似。

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一般来说，这两种化合物具有大致相似的药理特征，但Zotiraciclib在细胞中总是更具活性，特别是在前列腺癌症细胞系DU145中，两种化合物之间存在5倍的差异。Zotiraciclib更高的细胞效力可能是由于其对酶的效力通常更高，但也可能是由于更好的溶解性和渗透性。此外，26g的可溶性明显低于Zotiraciclib。综上所述，这些数据支持Zotiraciclib作为首选化合物，因此，它被选择用于高级分析。[1]
细胞内药效学标志物研究表明，Zotiraciclib能有效抑制HCT-116中的CDK2生物标志物pRb（磷酸化Rb，视网膜母细胞瘤肿瘤抑制蛋白）（图8）。在40 nM时可以检测到效果，在200 nM时蛋白质磷酸化完全被抑制。在白血病细胞系中进行的类似研究（36）表明，Zotiraciclib也对MV4-11细胞中的pRb有效（IC50=0.13μM），并且还抑制了同一细胞系中的pFLT3和pSTAT5。 [1]
图8。在变性裂解之前，HCT-116细胞分别用指定浓度的Zotiraciclib处理24小时。每次处理的30μg裂解物在10%SDS-PAGE上溶解，转移到PVDF膜上，并用抗磷酸Rb和β-actin的抗体进行检测。 [1]
其他地方报道了广泛的生物学特征，包括激酶谱、细胞内机制研究以及对各种白血病细胞系的抗增殖作用。这些数据表明，Zotiraciclib具有高度新颖的激酶抑制谱，可抑制63组中的17种激酶，其中11种是CDK/JAK/FLT家族成员。其他的Lck、Fyn、Fms、TYRO3、ERK5和p38δ与炎症和增殖过程有关，正在进行进一步的生物学研究，以更好地了解体内环境中的这些活动。

在药代动力学研究中，SB1317 表现出中等至高全身清除率（相对于肝血流量）、高分布容积（> 0.6 L/kg），小鼠、大鼠和狗的口服生物利用度分别为 24%、∼ 4% 和 37%；小鼠体内组织分布广泛。SB1317 良好的 ADME 支持其作为口服候选药物的临床前开发。

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基于其对广谱肿瘤细胞系的疗效和良好的口服生物利用度，我们选择了Zotiraciclib在人类肿瘤异种移植物小鼠模型中进行评估。根据其与癌症的相关性选择了两种模型：HCT-116结肠癌癌症和Ramos B细胞淋巴瘤。在进行这两个实验之前，探索了每个模型中的给药方案，并为每个模型选择了在实验期间可以耐受的最佳时间表。 在癌症结肠癌模型中，皮下注射HCT-116细胞，并建立平均组大小约为100 mm3的肿瘤。在细胞接种后8天开始使用Zotiraciclib治疗，剂量为50和75mg/kg po，每周3次，时间为周一、周三、周五，持续15天。以75 mg/kg po q.d 3×/周的剂量使用Zotiraciclib治疗显著抑制了肿瘤的生长，平均TGI为82%，而较低剂量的50 mg/kg po 3×/周则略有效果（图9）。[1]

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在淋巴瘤模型中，皮下注射Ramos细胞，建立平均组大小约为200mm3的肿瘤。在该模型中探索了两种不同的Zotiraciclib给药方案：在细胞接种15天后12天开始，按2天和5天的计划每天口服75mg/kg，按5天和5天后的计划每天腹腔注射15mg/kg。有两个载体对照组接受MC/Tween或DMA/CRE治疗（详见实验部分）。将治疗组与相应的溶媒对照组进行比较，以评估TGI百分比。使用任一方案的Zotiraciclib治疗显著抑制了肿瘤的生长，口服和ip给药方法的平均TGI分别为42%和63%（图10）。鉴于在这些具有挑战性的模型中，口服和ip给药方案都观察到令人鼓舞的TGI，因此选择了Zotiraciclib进行进一步的临床前开发[1]。

酶活分析[1]
使用重组酶（CDK2/细胞周期蛋白A、JAK2和FLT3）。所有测定均在384孔白色微量滴定板上使用Cambrex的PKLight测定系统进行。该测定平台是一种光度测定法，用于使用萤光素酶偶联反应检测反应中的ATP。在从10μM开始的3倍或4倍连续稀释制备的8个浓度下，对Zotiraciclib等化合物进行了测试。对于CDK2/细胞周期蛋白A测定，反应混合物由25μL测定缓冲液（50 mM Hepes，pH 7.5，10 mM MgCl2，5 mM MnCl2，5 mmol BGP，1 mM DTT，0.1 mM原钒酸钠）中的以下成分组成，1.4μg/mL CDK2/细胞循环蛋白A复合物，0.5μM RbING底物和0.5μM ATP。将混合物在室温下孵育2小时。然后加入13μL PKLight ATP检测试剂，将混合物孵育10分钟。在多标记平板阅读器上检测到发光信号。其他激酶测定类似，但试剂存在以下差异：对于FLT3测定，混合物含有2.0μg/mL FLT3酶、5μM聚（Glu，Tyr）底物和4μM ATP。对于JAK2测定，反应含有0.35μg/mL JAK2酶、10μM聚（Glu、Ala、Tyr）底物和0.15μM ATP。使用分析软件Prism 5.0从数据中生成IC50值。

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高通量溶解度测定[1]
该测定以高通量模式测量化合物在PBS中的溶解度。使用96孔V形底部半透明PP微孔板和96孔UV透明微孔板进行测定。在含有20%DMSO的10 mM磷酸盐缓冲液（pH 7.0）中制备化合物溶液（250μM），总体积为0.2 mL。将平板放置在转速为600 rpm的摇床上1.5小时，然后在室温下静置2小时。将平板在1500g下离心15分钟。将上清液转移到紫外透明微孔板上，在适当的最大吸收值下通过紫外分光光度法进行分析。使用校准曲线定量上清液中化合物的浓度。对于250±30μM的计算溶解度，溶解度报告为>250μM（>150μg/mL）。

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肝微粒体的代谢稳定性[1]
在含有50 mM磷酸钾缓冲液（pH 7.4）和NADPH再生系统的反应混合物中，在37°C下，在总反应体积为1 mL的条件下，将化合物（5μM）与MLM（小鼠肝微粒体）、RLM（大鼠肝微粒物）、DLM（狗肝微粒剂）和HLM（人肝微粒机）（最终微粒体浓度约为0.87 mg/mL）一起孵育。在乙腈和DMSO（80:20）的冷冻混合物中孵育0、15、30、45和60分钟后终止反应。将混合物涡旋5分钟，在4°C下以13 200 rpm离心15分钟，通过LC-MS/MS分析上清液。通过在对数线性标度上绘制母体化合物剩余百分比随时间的变化来评估稳定性，并使用一阶方程t1/2=0.693/k从对数线性曲线的线性部分估算半衰期，其中k是曲线的斜率（等于一阶消除速率常数）。

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人体外CYP450抑制试验[1]
Zotiraciclib与人肝微粒体（CYP1A和CYP3A4为0.25mg/mL，CYP2C19和CYP2D6为0.5mg/mL，CYP2C9为1mg/mL）和NADPH（1mM）一起孵育（DMSO中浓度为0.05、0.25、0.5、2.5、5、25μM；DMSO终浓度为0.35%），在探针底物乙氧基间苯二酚（0.5μM）（CYP1A）、甲磺丁脲（120μM A（5μM）30分钟（CYP2D6）和咪达唑仑（2.5μM）5分钟（CYP3A4）在37°C下。选择性抑制剂α-萘黄酮、磺胺苯唑、反苯环丙胺、奎尼丁和酮康唑分别用作CYP1A、CYP2C9、CYP2C19、CYP2D6和CYP3A4抑制剂的阳性对照。对于CYP1A，通过加入甲醇终止反应，并通过荧光（激发波长为535nm，发射波长为595nm）监测代谢物间苯二酚的形成。对于CYP2C9、CYP2C19、CYP2D6和CYP3A4孵育，通过加入含有内标的甲醇终止反应。将样品离心，合并上清液，通过LC-MS/MS同时分析4-羟基甲苯磺丁脲、4-羟基美苯妥英、右美沙芬、1-羟基咪达唑仑和内标。在分析前，将去离子水中的甲酸（终浓度为0.1%）加入最终样品中。与载体对照相比，代谢物形成的减少用于计算IC50值（产生50%抑制的测试化合物浓度）。

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体外血浆蛋白结合 平衡透析在腔室体积为500μL的微型平衡透析器中进行（每个腔室的体积为250μL）。使用的半透膜用Milli-Q水冲洗，并在PBS中浸泡10分钟。将Zotiraciclib加入血浆（来自小鼠、狗和人类）中，使终浓度达到1000 ng/mL。将掺入的血浆涡旋，并将250μL等分到透析器细胞的一个腔室中。另一个腔室装有250μL PBS缓冲液。将组装好的细胞放入37°C的水浴中，透析4小时。透析后，将50μL含有游离Zotiraciclib的PBS透析样品转移到2 mL Eppendorf管中，一式三份进行提取。使用混合器，在电机速度设置为60并带有脉冲的情况下，用1500μL MTBE（甲基叔丁基醚）提取样品30分钟。30分钟后，在微量离心机中以13000 rpm的速度在4°C下离心样品管10分钟。将上清液（1400μL）转移到新鲜的2 mL Eppendorf管中，在43°C的SpeedVac中干燥35分钟。用100μL甲醇/Milli-Q H2O（60:40）复溶干燥的样品，并通过LC-MS/MS进行分析。

细胞增殖试验[1]
所有细胞系均按照推荐的指南进行培养。对于96孔板中的增殖测定，将20000个细胞接种在100μL培养基中，第二天用浓度高达10μM的化合物（一式三份）处理48小时。使用CellTiter-96水溶液细胞增殖试验监测细胞存活率。绘制剂量-反应曲线，使用XL拟合软件确定化合物的IC50值。

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细胞药效学测定[1]
在药物处理前16-24小时，将HCT-116细胞（2×105，在5 mL补充有2 mM l-谷氨酰胺和10%胎牛血清的McCoy培养基中）接种在60 mM培养皿中。在使用改良的放射免疫沉淀缓冲液（50 mM Tris-HCl、150 mM NaCl、1%脱氧胆酸钠、0.25 mM EDTA（pH 8.0）、1%Triton X-100、0.2%NaF和蛋白酶抑制剂混合物）裂解之前，用不同浓度的Zotiraciclib或DMSO分别处理每皿24小时。使用Bradford测定法测量蛋白质，每种处理的30μg裂解物在10%SDS-PAGE上溶解，并转移到PVDF膜上。使用供应商推荐的稀释液，使用抗磷酸Rb和β-actin抗体进行蛋白质印迹分析。使用Pierce ECL Western印迹底物进行放射自显影检测信号。

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Caco-2双向渗透性测定[1]
Zotiraciclib在Hank's平衡盐溶液（HBSS）中以5μM的浓度放置在Transwell测定板上的21-28天融合单层细胞中，DMSO的最终浓度低于1%。心尖侧和基底外侧的pH值均维持在7.4。当在心尖侧给药时，评估A→B方向的渗透性，当在基底外侧给药时评估B→A方向。在2小时时对心尖侧和基底外侧侧进行取样。使用四点校准曲线通过LC/MS测定Zotiraciclib的浓度。单层批次的质量控制中使用了阿替洛尔（Papp<0.5×10-6cm/s）、普萘洛尔（15×10-6cm-s0.4×10-6cm2）和地高辛（流出比>3）。单层的完整性是通过测量实验前的TEER（在450至650Ωcm2之间）和使用路西法黄（流出≤0.5%）来确定的。外排率定义为Papp，B→A与Papp，A→B的比率。

Pharmacokinetics [1]
Male BALB/c mice (aged ∼10–12 weeks and weighing 17–22 g), male Beagle dogs (∼6–7 months of age, weighing 10–14 kg), and male Wistar rats (aged 6–8 weeks, weighing 239–249 g) were used in this study. All the animal studies were performed as per approved internal protocols for animal care and use. The oral doses for mice, dogs, and rats were 75, 10, and 10 mg/kg, respectively. The doses were administered by gavage as suspensions (0.5% methylcellulose and 0.1% Tween 80) to mice and rats, and as gelatin capsules (12 Torpac) to dogs. Following oral dosing serial blood samples were collected (cardiac puncture in mice, jugular vein in dogs, and superior vena cava in rats) at different time points (0–24 h) in tubes containing K3EDTA as anticoagulant, centrifuged, and plasma was separated and stored at −70 °C until analysis. Plasma samples were processed and analyzed by LC–MS/MS. Pharmacokinetic parameters were estimated by noncompartmental methods.

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Materials and Methods for HCT-116 and Ramos Studies [1]
1 Mice/Husbandry [1]
Female BALB/c nude mice (ARC, West Australia), 10–12 weeks of age, were fed with sterilized tap water (ad libitum water) and irradiated standard rodent diet consisting of 19% protein, 5% fat, and 5% fiber. Mice were housed in individual ventilated cages on a 12 h light cycle at 21–22 °C and 40–60% humidity. The use of animals is compliant with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care.

2 Tumor Implantation [1]
2.1 For HCT-116 Study [1]
Mice were implanted subcutaneously in the right flank with 5 × 106 cells of HCT-116 human colon carcinoma. Each tumor was monitored twice per week and subsequently daily as the neoplasms reached the desired size of approximately 100 mm3. At day 8, when the tumors attained a calculated tumor volume between 75 to 144 mm3, the animals were pair-matched and distributed randomly into various treatment groups (the mean tumor volume in each group was 105 mm3). Estimated tumor volume was calculated using the formula where w is the width and l is the length in mm of an HCT-116 carcinoma.

2.2 For Ramos Study [1]
Mice were implanted subcutaneously in the right flank with 7 × 106 cells of Ramos cells (100 μL). The tumor size was monitored twice per week and subsequently daily as the neoplasms reached the desired size, approximately 200 mm3. On day 12, when the tumors attained a volume of between 75 and 405 mm3, the animal were pair-matched and distributed randomly into various treatment groups (the mean tumor volume in each group was 216 mm3). Estimated tumor volume was calculated using the formula where w is the width and l is the length in mm of a Ramos tumor.

3 Drug [1]
Zotiraciclib hydrochloride was synthesized at S*BIO PTE LTD and dissolved in 0.5% methyl cellulose/0.1% Tween 80 (MC/Tween) for oral (po) dosing or in 10% dimethylacetamide (DMA) and 10% Cremophor (DMA/CRE) for ip dosing. Dosing solutions were prepared weekly in a feeding volume of 10 mL per kilogram body weight and stored at 4 °C.

4 Treatment Plan [1]
4.1 For HCT-116 Study [1]
On day 1, HCT-116-bearing nude mice were pair-matched and placed into 3 groups of 9–10 animals each. Treatment with all drugs was initiated on day 1. The test compound, Zotiraciclib, was administered po at the following dosing schedules: 50 and 75 mg/kg × 3 times a week (Monday, Wednesday, and Friday; 3/w). There was a vehicle control group that received vehicle (MC/Tween) on the same schedule. The study was terminated on day 15. 4.2 For Ramos Study [1]
Zotiraciclib was administered once daily at doses of 75 mg/kg po q.d. 2d_on–5d_off or 15 mg/kg ip q.d. 5d_on–5d_off. There were two vehicle control groups that received either MC/Tween or DMA/CRE. The treatment groups were compared with the corresponding vehicle control group for the percentage of the tumor growth inhibition (% TGI). The treatment was terminated after 14 days of dosing.

Extensive ADME Profiling of Zotiraciclib/26h [1]
In the Caco-2 bidirectional permeability assays, the permeability (Papp) of 26h in the apical to basolateral (Papp,A→B) direction and in the basolateral to apical (Papp,B→A) direction was 28.0 × 10–6 and 27.4 x10–6 cm/s, respectively. The efflux ratio, defined as the ratio of Papp, B→A to Papp,A→B, was less than 3 (1.0), indicating that 26h was not a substrate for efflux by intestinal P-gp transporters, suggestive of high intestinal absorption in humans (Table 6). In human liver microsomes (HLM) 26h was found to be stable with a half-life of 45 min, was moderately stable in DLM (t1/2 = 33 min), and was quite rapidly cleared in MLM (t1/2 = 12 min) and in RLM (t1/2 = 11 min). Human CYP1A2, 3A4, 2C9, and 2C19 isoforms were not inhibited by 26h at the highest tested concentration of 25 μM, but the compound inhibited CYP2D6 with IC50 = 0.95 μM, approximately at the plasma Cmax observed at the maximum tolerated dose. Compound 26h was highly bound to plasma proteins in human, mouse, and dog plasma with PPB ranging between 99.4% to 99.9%.

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Pharmacokinetics of Zotiraciclib/26h in Mice [1]
The PK properties of 26h in mice are summarized in Table 7. 26h showed high systemic clearance relative to liver blood flow and high volume of distribution at steady state, with a terminal half-life of ∼5.0 h. It showed rapid absorption (tmax = 0.5 h) and a mean Cmax and AUC of 1029 ng/mL and 2523 ng·h/mL, respectively, with a mean terminal half-life of 6.1 h following a single oral dose of 75 mg/kg. It showed an acceptable oral bioavailability of 24%. The exposures achieved in mice at the 75 mg/kg dose far exceeded the enzyme inhibiton (CDK2 IC50 = 0.013 μM, JAK2 IC50 = 0.073 μM, and FLT3 IC50 = 0.056 μM) and cell proliferation concentrations in HCT-116 (IC50 = 0.079 μM) and HL-60 (IC50 = 0.059 μM), correlating with the observed efficacy of 26h in preclinical pharmacology models at similar doses.

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Zotiraciclib/SB1317 (TG02) is a novel small molecule potent CDK/JAK2/FLT3 inhibitor. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-cancer agent. SB1317 was soluble, highly permeable in Caco-2 cells, and showed > 99% binding to plasma from mice, dog and humans. It was metabolically stable in human and dog liver microsomes relative to mouse and rat. SB1317 was mainly metabolized by CYP3A4 and CY1A2 in vitro. SB1317 did not inhibit any of the major human CYPs in vitro except CYP2D6 (IC50=1 μM). SB1317 did not significantly induce CYP1A and CYP3A4 in human hepatocytes in vitro. The metabolic profiles in liver microsomes from preclinical species were qualitatively similar to humans. In pharmacokinetic studies SB1317 showed moderate to high systemic clearance (relative to liver blood flow), high volume of distribution ( > 0.6 L/kg), oral bioavailability of 24%, ∼ 4 and 37% in mice, rats and dogs, respectively; and extensive tissue distribution in mice. The favorable ADME of SB1317 supported its preclinical development as an oral drug candidate.[2]

[1]. Discovery of kinase spectrum selective macrocycle (16E)-14-methyl-20-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene (SB1317/TG02), a potent inhibitor of cyclin dependent kinases (CDKs), Janus kinase 2 (JAK2), and fms-like tyrosine kinase-3 (FLT3) for the treatment of cancer. J Med Chem . 2012 Jan 12;55(1):169-96.

Zotiraciclib is under investigation in clinical trial NCT02942264 (Zotiraciclib (TG02) Plus Dose-dense or Metronomic Temozolomide Followed by Randomized Phase II Trial of Zotiraciclib (TG02) Plus Temozolomide Versus Temozolomide Alone in Adults With Recurrent Anaplastic Astrocytoma and Glioblastoma).
ZOTIRACICLIB is a small molecule drug with a maximum clinical trial phase of II (across all indications) and has 7 investigational indications.

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Herein, we describe the design, synthesis, and SAR of a series of unique small molecule macrocycles that show spectrum selective kinase inhibition of CDKs, JAK2, and FLT3. The most promising leads were assessed in vitro for their inhibition of cancer cell proliferation, solubility, CYP450 inhibition, and microsomal stability. This screening cascade revealed 26 h as a preferred compound with target IC(50) of 13, 73, and 56 nM for CDK2, JAK2 and FLT3, respectively. Pharmacokinetic (PK) studies of 26 h in preclinical species showed good oral exposures. Oral efficacy was observed in colon (HCT-116) and lymphoma (Ramos) xenograft studies, in line with the observed PK/PD correlation. 26h (SB1317/TG02) was progressed into development in 2010 and is currently undergoing phase 1 clinical trials in advanced leukemias and multiple myeloma.[1]

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We have described the discovery of a series of small molecule macrocycles as potent inhibitors of CDKs, JAK2, and FLT3, a spectrum selective profile not previously reported. Application of a hypothesis of conformational constraint generated macrocycles that were synthesized using a RCM strategy. Screening of initial compounds in functional biochemical assays against CDK2, JAK2, and FLT3 kinases allowed selection of a preferred linker moiety containing a phenolic ether, trans double bond, and allylic/benzylic N-methyl group. SAR and broader in vitro profiling, particularly cellular assays, identified 26h, a small molecule kinase inhibitor with a distinct kinase inhibitory spectrum, as the preferred lead candidate. Further evaluation revealed excellent pharmacokinetic properties of 26h and dose-dependent efficacy in mouse models of cancer including a HCT-116 model of colon cancer and a Ramos model of lymphoma. On the basis of its favorable pharmaceutical and pharmacological profile, 26h (SB1317/TG02) was advanced into development and is currently being evaluated in phase 1 clinical trials in leukemia patients. [1]

C23H25CLN4O

408.92

408.171

C, 62.03; H, 5.88; Cl, 15.92; N, 12.58; O, 3.59

1321626-25-8

(E/Z)-Zotiraciclib;937270-47-8; 1354567-82-0 (HCl);1204918-73-9 (citrate);1204918-72-8;937270-47-8;(E/Z)-Zotiraciclib

141730164

White to light yellow solid

50.3

2

5

0

29

499

0

CN1C/C=C/CCOC2=CC=CC(=C2)C3=NC(=NC=C3)NC4=CC=CC(=C4)C1.Cl

YPVRSANPCYTWDF-SQQVDAMQSA-N

InChI=1S/C23H24N4O.ClH/c1-27-13-3-2-4-14-28-21-10-6-8-19(16-21)22-11-12-24-23(26-22)25-20-9-5-7-18(15-20)17-27;/h2-3,5-12,15-16H,4,13-14,17H2,1H3,(H,24,25,26);1H/b3-2+;

(16E)-14-methyl-20-oxa-5,7,14,27-tetrazatetracyclo[19.3.1.12,6.18,12]heptacosa-1(25),2(27),3,5,8,10,12(26),16,21,23-decaene;hydrochloride

(E/Z)-Zotiraciclib HCl; TG02 HCl

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，避免吸湿/受潮。

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

DMSO: ~33.3 mg/mL (~81.5 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.11 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 (6.11 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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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。