JAK3 (IC50 = 33.1 nM)

Ritlecitinib 是一种强效的 JAK3 选择性抑制剂，抑制 JAK3 激酶活性的 IC50 为 33.1 nM，但对 JAK1、JAK2 和 TYK2 没有活性 (IC50>10,000 nM)。 Ritlecitinib 的 IC50 值分别为 244、340、407 和 266 nM，抑制 IL-2、IL-4、IL-7 和 IL-15 诱导的 STAT5 磷酸化。 Ritlecitinib 的 IC50 为 355 nM，还可以防止 IL-21 诱导的 STAT3 磷酸化。根据功能评估，Ritlecitinib 在 T 细胞分化测定中抑制 Th1 和 Th17 分化（在 Th1 环境下 5 天后通过 IFNγ 测量，在 Th17 设置下 6 天后通过 IL-17 产生测量）（IC50 值：分别为 30 nM 和 167 nM） ）。此外，ritlecitinib 可抑制 Th1 和 Th17 功能，如在 PF-06651600 治疗前已经历分化和休眠的细胞中抑制 IFNγ 产生 (IC50=48 nM) 和 IL-17 产生 (IC50=269 nM) 所证明的[1] 。

Ritlecitinib 可减少大鼠佐剂诱导的关节炎 (AIA) 模型中的爪肿胀，未结合的 EC50 为 169 nM。在实验性自身免疫性脑脊髓炎（EAE）小鼠模型中，以 30 或 100 mg/kg 治疗性给药或以 20 或 60 mg/kg 预防性给药，可显着降低疾病的严重程度。 Ritlecitinib 在这两种啮齿动物模型中治疗炎症和自身免疫性疾病的有效性表明，仅选择性抑制 JAK3 可能足以改变人类疾病[1]。

JAK酶测定。[1]
使用微流体测定法测定人Janus激酶（JAK）活性，以监测JAK家族四个成员JAK1、JAK2、JAK3和TYK2中每一个的重组人激酶结构域对合成肽的磷酸化。GST-标记的JAK1、JAK2和JAK3的重组人激酶结构域购自Life Technologies。他标记的重组人TYK2激酶结构域在SF21/杆状病毒中表达，并使用两步亲和（Ni-NTA）和大小排阻（SEC S200）纯化方法进行纯化。将试验化合物溶解在二甲基亚砜（DMSO）中，使其储备浓度为30mM。将化合物稀释在DMSO中，形成11点半对数稀释系列，最高浓度为600µM。测试化合物板还包含含有已知抑制剂的阳性对照孔以定义100%抑制，以及含有DMSO的阴性对照孔以确定无抑制。在测定中，将化合物板稀释1至60，得到10µM至100 pM的最终测定化合物浓度范围和1.7%DMSO的最终测定浓度。使用非接触式声学分配器将溶解在100%DMSO中的测试化合物和对照加入（250nL）到384孔聚丙烯板（Matrical）中。在室温下，在含有20mM HEPES、pH 7.4、10mM氯化镁、0.01%牛血清白蛋白（BSA）、0.0005%吐温20和1mM二硫苏糖醇（DTT）的15µL反应缓冲液中进行激酶测定。反应混合物含有1µM荧光标记的合成肽，其浓度小于表观米氏常数（Km）（对于JAK1和TYK2为5FAM-KSRGDYMTMQID，对于JAK2和JAK3为FITC-KGGEEEYFELVKK）。反应混合物含有三磷酸腺苷（ATP），其水平等于ATP的表观Km（JAK1为40µM，JAK2为4µM，JAK3为4µM，TYK2为12µM）或1mM ATP。将化合物加入到含有ATP和底物的缓冲液中，并在该步骤之后立即加入酶以开始反应。用15µL缓冲液停止测定，该缓冲液含有180 mM HEPES，pH=7.4，20 mM EDTA，0.2%包衣试剂，最终浓度为10 mM EDTA、0.1%包衣试剂和100 mM HEPES，pH=7.4。将测定板放置在Caliper Life Science Lab Chip 3000（LC3000）仪器或Caliper Life Science-EZ Reader仪器上，并使用适当的分离条件对每个孔进行取样，以确定磷酸化水平[1]

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JAK3的结合和失活动力学。[1]
使用基于LanthaScreen的时间分辨Förster共振能量转移（TRFRET）测定法测量PF-06651600与JAK3以及在JAK3中与Cys909相同位置包含Cys的一组激酶结合和失活的动力学 Eu激酶结合测定（Invitrogen/Life Technologies）。激酶特异性试剂和测定验证可在以下网址找到：https://www.thermofisher.com/us/en/home/industrial/pharma-biopharma/drug-discoverydevelopment/target-and-lead-identification-and-validation/kinasebiology/kinase-activityassays/lanthascreentm-eu-kinase-binding-assay/lanthascreen-eu-kinase-binding-assay-validationtable.html.测定缓冲液为20mM HEPES，pH 7.5，10mM MgCl2，0.01%BSA，1mM DTT，0.0005%Tween 20和2%DMSO。通过制备15µL（最终浓度）2 nM Eu-Ab、1-8 nM激酶（根据经验确定每种激酶的最佳浓度）和可变浓度的PF06651600的1.33μ溶液，并将其预孵育可变时间，进行灭活动力学反应（详见下文）。然后将其与5µL经验证探针的4X溶液（150 nM，最终浓度）合并。对于所有激酶，进行以下实验：（A）[PF-06651500]＝0、4.9、14.8、44.4、133.3和400nM；预孵育时间=2小时。（B）[PF-06651500]=0、0.5、1.0、2.0、4.0和8.0µM；预孵育时间=120s。对于JAK3，进行以下额外实验：[PF-06651500]=0，0.66，1.98，5.93，17.8，53.3，160480 nM；预孵育时间为（C）30秒、（D）60秒和（E）1.5小时。使用EnVision平板读取器读取测定结果。激发波长为340nm，监测的输出是发射比，通过将探针发射峰值（665nm）的信号除以铕发射峰值（615nm）来计算。每120秒进行一次测量，持续1.5小时。重组TXK激酶（N-末端GST融合蛋白）与抗GST-Eu抗体（Life Technologies）和探针结合，未能产生合适的TR-FRET信号。因此，采用了另一种方法来测量kinact/Ki，利用经典的丙酮酸激酶/乳酸脱氢酶（PK/LDH）偶联酶测定法。在PK/LDH测定中，ADP是激酶反应的产物，通过将其产生首先与磷酸烯醇式丙酮酸盐（PEP）的去磷酸化偶联以形成丙酮酸盐来测量，所述丙酮酸盐与丙酮酸的NADH依赖性还原偶联以形成乳酸盐。通过在340nm处的吸光度损失以分光光度法监测伴随的NADH氧化形成NAD+。TXK激酶缓冲液为：50mM HEPES，pH 7.5，10mM Mg2Cl，0.01%Triton X-100，1mM DTT和1%DMSO。还包括（PK/LDH测定试剂）：0.25 mM NADH（Sigma，N8129）、2.5 mM磷酸烯醇式丙酮酸盐（PEP）、12 U/mL丙酮酸激酶（PK）和18 U/mL乳酸脱氢酶（LDH）。最终底物浓度为每种ATP和Srctide肽100µM（序列：GEPLYWSFPAKK）。在每个实验中，将10µL PF-06651600溶液（预培养期间的浓度：0、10.4167、20.83、41.67、83.3、333.3、666.7、1333.3、2666.7 nM）与20µL含有TXK激酶的溶液（预孵育期间的浓度为34 nM）+PK/LDH溶液试剂组合。在可变时间的预孵育期（15分钟、30分钟、1小时、1.5小时和2小时）后，加入10µL的4¦ΒATP/肽底物以引发反应。

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PF-06651600的人血清白蛋白结合。[1]
获得在酿酒酵母中表达的人重组血清白蛋白（HSA）作为1.5mM溶液，并通过稀释到Dulbecco的磷酸盐缓冲盐水（PBS）中而不用进一步处理来使用。如图S3的图例所示，将HSA与从DMSO中制备的8mM储备溶液稀释到PBS中的化合物孵育22小时，之后使用Agilent 1100 HPLC系统和Waters LCT Premier XE质谱仪通过液相色谱-质谱法对蛋白质进行直接质量分析，如前所述1。使用MaxEnt 1软件（并入Waters MassLynx程序）从蛋白质峰上的平均光谱中提取蛋白质的质量分布，该软件运行20次迭代，目标质量范围为66000-67500 Da。

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人类肝细胞中的共价结合。[1]
将汇集的冷冻保存的人肝细胞以750000个细胞/mL（n=2）的最终浓度悬浮在Williams’E培养基（GIBCO，ThermoFisherScientific）中。基于台盼蓝排除的细胞活力>80%。将细胞悬浮液（4 mL）与放射性标记的底物（1µM）在37℃下孵育4小时。在240分钟取出等分试样，用乙腈骤冷，以3500rpm离心15分钟，并用有机溶剂（乙腈：甲醇、乙腈和乙腈：0.1%甲酸在水中）的组合进行彻底萃取（N=7）。监测上清液部分，直到放射性水平低于背景水平的两倍（80dpm）。将NaOH（1M）加入剩余的蛋白质颗粒中，并将其置于水浴中过夜以溶解，并通过液体闪烁计数测定总放射性。使用Bradford蛋白质测定法测定蛋白质浓度

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人全血中JAK3占据和p-STAT5抑制的LC-MS/MS。[1]
利用LC/MS-MS替代胰蛋白酶肽分析来测量不可逆共价抑制剂对JAK3的占用。使用人JAK3蛋白序列（Uniprot登录号P52333）来验证含有909Cys的胰蛋白酶肽的序列，909Cys是共价分子结合靶标。通过使用MS级胰蛋白酶/Lys-C酶混合物对JAK3蛋白进行胰蛋白酶切割，产生具有序列LVMEYLPSGCLR的有利大小的肽。通过MRM模式IA-LC-MS/MS分析测量与抑制剂和游离肽的结合，该分析包括在线抗肽抗体免疫亲和富集步骤。蛋白质和肽水平的双重免疫沉淀为从人类全血产生的PBMC样品中游离和结合的LVME肽提供了充足的样品富集。生物素化的抗人JAK3多克隆捕获抗体从Santa Cruz获得。新英格兰肽公司定制合成了含有胰蛋白酶切割位点的扩展序列稳定同位素标记（SIL）肽。。。

Th1细胞分化。[1]
冷冻保存的人正常外周血CD4+/CD45RA+/CD25-幼稚T细胞购自Allcells。冷冻的CD4+/CD45RA+/CD25-幼稚T细胞在水浴（37℃）中解冻，并用RPMI1640培养基洗涤一次。将细胞以200000个细胞/mL的速度重悬于含有10%胎牛血清（FBS）、IL-2（10 ng/mL）、IL-12（5 ng/mL），抗IL-4（5 ng/mL）、抗CD3（10µg/mL）和抗CD28（0.1µg/mL）抗体的RPMI培养基中。为了评估PF-06651600在Th1细胞分化期的作用，在11种不同浓度的JAK抑制剂（0.2%二甲基亚砜终产物）的存在下，将重悬的幼稚CD4+T细胞培养5天。根据制造商的说明，采集上清液并用MSD测量IFNγ的浓度。为了研究PF06651600对Th1细胞分化后的影响，将偏斜的Th1细胞重悬于含有10%FBS的RPMI培养基中，并在无细胞因子的条件下培养7小时。然后收获细胞，并在10%FBS、IL-2（10ng/mL）、IL-12（5ng/mL）和抗IL-4抗体（5ng/mL）加上11种不同浓度的PF-06651600的存在下在96孔板中再培养2天。如上所述测定IFNγ的浓度

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Th17细胞分化。[1]
用RosetteSep CD4+T细胞富集鸡尾酒从棕褐色外壳中纯化人CD4+T淋巴细胞，并在10种不同浓度的JAK抑制剂存在下，用细胞因子混合物（25 ng/mL IL-6、25 ng/mL IL-23、12.5 ng/mL IL-1β、25 ng/mL IL21、5 ng/mL TGFβ1、10µg/mL抗CD3抗体（预涂于板表面）和1µg/mL抗CD28抗体）偏斜6天。收集上清液，并按照制造商提供的方案用MSD测定法测定IL-17A的浓度。为了研究PF-06651600对分化后Th17细胞的影响，将偏斜的Th17细胞洗涤，用X-VIVO 15培养基（Lonza）静置过夜，并在10种不同浓度的PF-06651 600存在下，在含有与偏斜期间相同浓度的细胞因子但不含抗CD3或抗CD28抗体的培养基中再悬浮2天。在第9天，从每个孔收获上清液，并如上所述测定IL-17A

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JAK3在人类T细胞中的半衰期。[1]
CD4+细胞通过阴性选择从Buffy coat（SeraCare）中纯化并冷冻保存。将1000万个细胞在补充有10%FBS（Sigma）100U/mL青霉素/100µg/mL链霉素（Gibco）、2mM L-谷氨酰胺、100µM非必需氨基酸、1mM丙酮酸钠、20mM HEPES和2ng/mL IL-2的RPMI中解冻。在37°C的5%CO2中2小时后，加入CD3/CD28磁珠（3个磁珠/细胞的比例）。72小时后，珠子被移除，8亿个细胞被用于脉冲追逐实验。每个数据点使用了4000万个单元。用补充有500µCi S35标记的Cys/Met的培养基在37°C的5%CO2中补充细胞30分钟，然后在PBS中洗涤两次。然后将细胞在补充有200µM Cys/Met的培养基中孵育不同长度的时间，并在冷裂解缓冲液中裂解，然后在-80°C下储存。将裂解物在冰上解冻，并在4°C下用抗JAK3抗体进行免疫沉淀过夜。使用G蛋白磁珠来捕获抗JAK3抗体。然后将JAK3在60µl Laemmli中用5%BME洗脱，并在荧光成像仪上进行电泳和定量。

Rat adjuvant induced arthritis.[1]
The effect of JAK3 inhibition by PF-06651600 was evaluated in vivo using a therapeutic dosing paradigm in a rat adjuvant-induced arthritis. The efficacy of this molecule was evaluated in three separate studies using successively lower doses. Arthritis was induced by immunization of female Lewis rats (8 to 10 weeks old; Charles River Laboratories) via intradermal injection at the base of the tail with complete Freund’s adjuvant with three 50 µL injections (15 mg/mL Mycobacterium tuberculosis in incomplete Freund’s adjuvant (Sigma Aldrich). Seven days after the initial immunization, the baseline hind paw volume of the immunized rats was measured via plethysmograph (Buxco Inc). The rats were monitored daily for signs of arthritis including change in body weight and hind paw volume measurement. When individual hind paw volume measurements indicated an increase of 0.2 mL (or greater) in a single hind paw, animals were randomly assigned to a treatment group. Daily treatment with PF-06651600 was administered via oral gavage. Treatment groups for Experiment 1 were: 80, 15, or 6 mg/kg or vehicle (2% Tween 80 /0.5% methylcellulose/deionized water). Treatment groups for Experiment 2 were: 30, 10, and 3 mg/kg or vehicle (0.5% methylcellulose / de-ionized water/ 1 mEQ hydrochloric acid). Treatment groups for Experiment 3 were: 10, 1, 0.3 and 0.1 mg/kg or vehicle (0.5% methylcellulose/de-ionized water/ 1 mEQ hydrochloric acid). Dosing began once individuals were enrolled into respective groups. Treatment continued for 7 days. At the conclusion of the study, whole blood was taken at 15 minutes post dose (peak concentration in plasma) for analysis of STAT phosphorylation, and plasma was taken for exposure concentration in PF-06651600 dosed groups.

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Mouse experimental autoimmune encephalomyelitis. [1]
Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice (Taconic Farms, 10 weeks old) at Hooke Laboratories. Mice were injected subcutaneously at 0.1 ml per site with the emulsion component (containing MOG35-55) of Hooke Kit™ MOG35-55/CFA Emulsion PTX (Hooke Laboratories). The pertussis toxin (PTX) component of the kit was diluted with PBS and administered intraperitoneally in a volume of 0.1 ml at 122 ng /dose (therapeutic dosing study) or 167 ng /dose (prophylactic dosing study) within 2 h of the injection of emulsion, and again at 111 ng/dose (therapeutic dosing study) or 156 ng /dose (prophylactic dosing study) 24 h after the injection of emulsion. For the therapeutic study, as each mouse developed clinical signs of EAE (minimum score of 0.5) they were assigned to one of the experimental groups (n=15 per group) in a balanced manner, to achieve groups with similar time of EAE onset and similar scores. Treatment started on the first day of disease for each mouse and lasted for 14 days. For the prophylactic dosing study, mice were assigned to groups (n=10 per group) on Day -1 in a balanced manner to achieve similar average body weight between the groups at the start of the study. Prophylactic dosing started on Day 0 and continued until Day 28. Dosing was blinded and consisted of oral (per os; PO) twice daily (BID) administrations of PF-06651600 or vehicle (0.5% MethylCellulose/ 1 Molar equivalent hydrogen chloride). The positive control group was dosed PO once daily (QD) in the morning with fingolimod (FTY720, Gilenya), the most commonly used positive control in these models, and PO QD in the afternoon with vehicle in order to control for dosing stress in comparison with the BID treated groups. There were no more than 14 hours between evening and morning doses and no less than 10 h between morning and evening doses. Body weight was measured 3 times per week and EAE scores were assessed daily beginning on day 7 (the seventh day after immunization). EAE was scored on a scale of 0 to 5 until the termination of the study. Scoring was performed in a blinded fashion by a person unaware of both treatment and of previous scores for each mouse. At the conclusion of the study, plasma was taken for exposure concentration of PF-06651600 at peak (15 minutes after dosing) and trough (10 h and 14 h after dosing) time points.

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2% Tween 80 /0.5% methylcellulose/deionized water

Female Lewis rat model (8 to 10 weeks old)

Absorption, Distribution and Excretion
Up to 200 mg, the AUC0-tau and Cmax of ritlecitinib increase in an approximately dose-proportional manner, and steady state is reached approximately by day 4. Ritlecitinib has an absolute oral bioavailability of approximately 64%, and 1 hour after an oral dose is administered, peak plasma concentrations are achieved. Food does not have a clinically significant impact on the systemic exposures of ritlecitinib. The co-administration of a high-fat meal and a 100 mg ritlecitinib capsule reduced Cmax by 32% and increased AUCinf by 11%. Ritlecitinib was administered without regard to meals during clinical trials.
Ritlecitinib is mainly excreted through urine and feces. Approximately 66% and 20% of radiolabeled ritlecitinib are excreted in the urine and feces, respectively. Approximately 4% of the ritlecitinib dose is excreted unchanged drug in urine.
Ritlecitinib is predicted to have a volume of distribution of 1.3 L/kg.
Ritlecitinib is predicted to have a blood clearance of 5.6 mL/min/kg.

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Metabolism / Metabolites
Ritlecitinib is metabolized by cytochrome P450 (CYP) and glutathione-S-transferase (GST) enzymes. The GST enzymes participating in the metabolism of ritlecitinib include cytosolic GST A1/3, M1/3/5, P1, S1, T2, Z1 and microsomal GST 1/2/3, and the CYP enzymes participating in this process include CYP3A, CYP2C8, CYP1A2, and CYP2C9. No single route contributes to more than 25% of the total metabolism of ritlecitinib.

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Biological Half-Life
Ritlecitinib has a terminal half-life that ranges from 1.3 to 2.3 hours.

Hepatotoxicity
In the prelicensure clinical trials in alopecia areata, serum aminotransferase elevations occurred in 1% to 3% of ritlecitinib treated subjects, but similar rates were found in placebo recipients. The elevations were typically mild and transient, and values above 5 times the upper limit of normal (ULN) occurred in less than 1% of patients. The elevations rarely led to early discontinuations, and often resolved even without dose adjustment. In prelicensure studies in alopecia areata and other autoimmune conditions, there were no instances of liver related severe adverse events or clinically apparent liver injury attributed to ritlecitinib. Since approval and more widescale availability of ritlecitinib, there have been no published reports of hepatotoxicity associated with its use.
Finally, ritlecitinib is an immune modulatory agent and has the potential of causing reactivation of viral infections including hepatitis B. Other JAK inhibitors have been implicated in rare instances of reactivation of hepatitis B, although the episodes were usually asymptomatic and self-limited in course. The risk of reactivation of hepatitis B in patients with HBsAg or with anti-HBc without HBsAg who are treated with ritlecitinib has not been defined.
Likelihood score: E* (unlikely cause of idiosyncratic clinically apparent liver injury, but is a potential cause of reactivation of hepatitis B).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of ritlecitinib during breastfeeding. Because of the risk of serious adverse effects, including malignancy, the manufacturer recommends that breastfeeding be discontinued during ritlecitinib therapy and for 14 hours after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Ritlecitinib is 14% bound to plasma proteins.

[1]. Discovery of a JAK3-Selective Inhibitor: Functional Differentiation of JAK3-Selective Inhibition over pan-JAK or JAK1-Selective Inhibition. ACS Chem Biol. 2016 Dec 16;11(12):3442-3451.

Pharmacodynamics
Ritlecitinib is a kinase inhibitor that promotes the decrease of absolute lymphocyte levels, T lymphocytes (CD3) and T lymphocyte subsets (CD4 and CD8) in a dose-dependent manner. Ritlecitinib also promotes a decrease in NK cells (CD16/56), which remain stable up to week 48 after initiating treatment. In patients treated with 50 mg of ritlecitinib once a day, the decrease in median lymphocyte levels remains consistent up to week 48. At 12 times the mean maximum exposure of the 50 mg dose given to patients with alopecia areata once a day, ritlecitinib did not cause a clinically relevant effect on the QTc interval.[] The use of ritlecitinib is associated with the development of serious infections, malignancies (including non-melanoma skin cancer), major adverse cardiovascular events, thromboembolic events, and hypersensitivity. In the postmarketing safety study of another JAK inhibitor in patients with rheumatoid arthritis over 50 years of age with at least one cardiovascular risk factor, JAK inhibitors were associated with a higher rate of all-cause mortality, including sudden cardiovascular death, compared to TNF blockers.

C15H19N5O

285.34

285.158

C, 63.14; H, 6.71; N, 24.54; O, 5.61

1792180-81-4

(2R,5S)-Ritlecitinib;1792180-79-0; 2192215-81-7 ; 1792180-81-4; 2489392-29-0; 2140301-97-7 (malonate)

118115473

White to yellow solid

1.3±0.1 g/cm3

1.658

1.29

73.9

2

4

3

21

402

2

O=C(C=C)N1C[C@@H](CC[C@@H]1C)NC1C2C=CNC=2N=CN=1

CBRJPFGIXUFMTM-WDEREUQCSA-N

InChI=1S/C15H19N5O/c1-3-13(21)20-8-11(5-4-10(20)2)19-15-12-6-7-16-14(12)17-9-18-15/h3,6-7,9-11H,1,4-5,8H2,2H3,(H2,16,17,18,19)/t10-,11+/m0/s1

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: ≥ 2.08 mg/mL (7.29 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (7.29 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 20.8 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: ≥ 2.08 mg/mL (7.29 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 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 6.67 mg/mL (23.38 mM) in 0.5% MC 0.5% Tween-80 (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。

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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。