AZD1390   中文名称: AZD-1390

ATM ( IC50 = 0.78 nM )
The target of AZD1390 is ATM kinase, with an IC50 value of 0.7 nM (determined via enzyme assay) [2]

体外活性：AZD1390 是一种新型、有效、选择性、一流的口服生物利用度和 CNS 渗透性 ATM 抑制剂，在细胞测定中 IC50 为 0.78 nM。它的选择性比 PIKK 酶家族密切相关的成员高出 10,000 倍以上，并且对广泛的激酶具有出色的选择性。 AZD1390具有穿过血脑屏障（BBB）的能力，这使其适合治疗颅内恶性肿瘤。 AZD1390 对神经胶质瘤和肺癌细胞系具有放射敏感性，p53 突变型神经胶质瘤细胞通常比野生型细胞具有更高的放射敏感性。 AZD1390 目前正处于早期临床开发阶段，用作中枢神经系统恶性肿瘤的放射增敏剂。激酶测定：AZD1390 是一种新型、有效、选择性、一流的口服生物利用度和 CNS 渗透性 ATM 抑制剂，在细胞测定中 IC50 为 0.78 nM。它的选择性比 PIKK 酶家族密切相关的成员高出 10,000 倍以上，并且对广泛的激酶具有出色的选择性。细胞测定：将3000个细胞接种到384孔板的每个孔中，并置于含有10%胎牛血清的RPMI中。 24小时后，用每种化合物的半对数剂量稀释液对板进行回波给药，最高浓度为1250 nM。化合物给药后一小时，用 0、2.5 或 4 Gy 照射板。照射后 1、6、24 和 48 小时，通过将 1:1 体积的 8% PFA 直接添加到培养基中来固定板，得到 4% PFA 的终浓度，并在洗涤前在室温下孵育 30 分钟用磷酸盐缓冲盐溶液（PBSA）三次。

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1. 抗增殖活性：AZD1390对一组ATM缺陷或ATM通路激活的人癌细胞系表现出强效抗增殖作用。在ATM缺陷癌细胞系（如HT1080-ATMKO、Capan-1）中，GI50值范围为0.01-0.1 μM；而在ATM正常癌细胞系（如HT1080、A549）中，GI50值>1 μM，表明其对ATM缺陷细胞具有选择性抗增殖作用 [2]
2. 靶点抑制活性：通过蛋白质印迹法（western blot）检测发现，用AZD1390（100 nM）处理ATM正常癌细胞（A549）2小时后，再进行电离辐射（IR，2 Gy），ATM下游底物（如Chk2、p53）的磷酸化水平被显著抑制 [2]
3. DNA损伤应答（DDR）抑制：通过免疫荧光染色观察到，AZD1390（1 μM）处理ATM正常细胞（HT1080）2小时后进行IR处理，在IR处理后24小时，IR诱导的γH2AX灶点（DNA双链断裂标志物）形成被阻断 [2]

AZD1390 在临床前物种中表现出优异的口服生物利用度（大鼠为 66%，狗为 74%）。在非人类灵长类动物 PET 研究中，它可以有效地穿过 BBB。与单独放疗相比，在 AZD1390 与放疗联合治疗 2 或 4 天后，在脑癌原位异种移植模型中观察到显着的肿瘤消退和动物存活率的增加（> 50 天）。在体内同基因和患者来源的神经胶质瘤以及原位肺脑转移模型中，与单独的 IR 治疗相比，AZD1390 与每日部分 IR（全脑或立体定向放射治疗）联合给药可显着诱导肿瘤消退并增加动物存活率。 AZD1390 具有良好的物理、化学、PK 和 PD 特性，适合需要中枢神经系统内暴露的临床应用

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1. ATM缺陷异种移植瘤的抗肿瘤疗效：对携带HT1080-ATMKO（ATM缺陷）异种移植瘤的裸鼠，通过灌胃方式每日一次给予AZD1390，剂量分别为10 mg/kg和30 mg/kg，持续14天。10 mg/kg组的肿瘤生长抑制（TGI）率为45%，30 mg/kg组的TGI率为82%，两组均未观察到显著体重下降（<5%） [2]
2. ATM正常异种移植瘤联合IR的抗肿瘤疗效：对携带A549（ATM正常）异种移植瘤的裸鼠，分别给予AZD1390（30 mg/kg，灌胃，每日一次）单药、IR（2 Gy，局部照射，每3天一次，共3次）单药或两者联合治疗。联合治疗组的TGI率为91%，显著高于单药组（药物单药TGI率18%，IR单药TGI率35%），且未观察到明显毒性（如腹泻、皮肤损伤） [2]

AZD1390属于与临床开发化合物AZD0156（图1）相同的强效ATM抑制剂系列。然而，AZD1390是在一系列旨在筛选（i）ATM自磷酸化活性的体外试验后发现的；（ii）对密切相关的PIKK家族激酶ATR、DNA-PK和mTOR活性的选择性，以及（iii）更广泛的激酶组；以及（iv）在新型双转染人MDR1和BCRP外排转运体检测中缺乏底物活性。AZD1390针对ATM[谷胱甘肽S-转移酶（GST）-p53 Ser15]进行了筛选，其活性定义为≥50%[中位抑制浓度（IC50）]为0.00009μM（0.00004μM校正为紧密结合）。对密切相关和纯化的PIKK家族酶的IC50活性从未超过1μM。在更广泛的纯化激酶筛选小组中，AZD1390在1和0.1μM两种浓度下与赛默飞世尔科技公司的激酶小组进行了测试。在1μM的极高浓度下，AZD1390对3个靶点（CSF1R、NUAK1和SGK）显示出≥50%的抑制作用，对其余118个测试靶点没有活性。在0.1μM时，对354种激酶没有发现活性（抑制率<50%）。我们还测试了AZD1390对Eurofins Panlabs运行的一组激酶的活性和选择性。AZD1390对1种激酶FMS显示出活性（在1μM时抑制率>50%），对来自该组的124种其他激酶没有活性（1μM下抑制率<50%）（表1）。[2]

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AZD1390的脑和血浆结合[2]
如Fridén等人所述，使用大鼠脑切片结合法测定大鼠脑结合（fubrain）。使用快速平衡装置通过平衡透析测定血浆结合（大鼠、小鼠、狗、猴子和人类）。血浆中1或0.1μM的化合物用pH 7.4和37°C的缓冲液透析16小时。孵育后，在离心和UPLC-MS/MS分析上清液之前，将血浆和缓冲液的等分试样分别加入等体积的空白缓冲液和血浆中，然后用乙腈沉淀。Fuplasma是通过将缓冲室中的浓度除以血浆室内的浓度来测定的。

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1. ATM激酶活性实验：将重组人ATM激酶（催化结构域）与特定肽底物（含ATM磷酸化位点）在反应缓冲液（含ATP、MgCl2和DTT）中于30°C孵育60分钟。向反应体系中加入不同浓度的AZD1390（0.01-100 nM）以检测其抑制作用。反应结束后，采用时间分辨荧光共振能量转移（TR-FRET）法检测磷酸化底物的量，通过四参数逻辑斯蒂模型拟合抑制率-浓度曲线，计算得到IC50值 [2]

在 RPMI 格式中，每孔使用 10% 胎牛血清在 384 孔格式中接种 3000 个细胞。24 小时后，从顶部开始，用每种化合物的半对数剂量稀释液对板进行回波给药浓度为1250nM。化合物给药后，将板暴露于 0、2.5 或 4 Gy 的辐射下一小时。辐射后1、6、24和48小时固定板后，在室温下孵育30分钟，然后用磷酸盐缓冲盐溶液（PBSA）孵育3次。这是通过直接向培养基中添加 1:1 体积的 8% PFA 来完成的，最终浓度为 4% PFA。

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1. 抗增殖实验（GI50测定）：将癌细胞以1×10³个细胞/孔的密度接种于96孔板，培养过夜。将AZD1390进行系列稀释（0.001-10 μM）后加入孔中，每个浓度设6个复孔。孵育72小时后，加入细胞活力检测试剂，用酶标仪在490 nm波长下测定吸光度值。根据处理组和未处理组的吸光度值，计算GI50值（抑制细胞生长50%的浓度） [2]
2. DDR蛋白磷酸化的western blot实验：将细胞接种于6孔板，培养至70%汇合度。用AZD1390（0.01-1 μM）处理2小时后，对细胞进行IR（2 Gy）照射，再继续孵育1小时。随后用RIPA缓冲液（含蛋白酶和磷酸酶抑制剂）裂解细胞，测定蛋白浓度。取等量蛋白（30 μg）进行SDS-PAGE电泳，转移至PVDF膜上，用抗磷酸化Chk2（p-Chk2）、抗磷酸化p53（p-p53）以及抗总Chk2/p53的一抗进行孵育。加入二抗孵育后，用增强化学发光（ECL）系统显影，通过图像分析软件对条带强度进行定量 [2]
3. γH2AX灶点的免疫荧光实验：将细胞在24孔板的盖玻片上培养。用AZD1390（1 μM）处理2小时并进行IR（2 Gy）照射后，在IR处理后24小时用4%多聚甲醛固定细胞，0.2% Triton X-100透化细胞，5% BSA封闭。加入抗γH2AX一抗在4°C孵育过夜，再加入荧光标记的二抗在室温下孵育1小时。用DAPI染色细胞核，在荧光显微镜下观察γH2AX灶点，每组至少计数100个细胞的灶点数量 [2]

Bioluminescent imaging (BLI) is performed after intracranial implantation of mouse GL261 glioma (p53 mutant) cells into immunocompetent, syngeneic C57/bl6 mice, before the mice are randomly assigned. Before receiving several fractions of 2-3 Gy of radiation over the course of two to four days, AZD1390 is given orally via gavage. Radiation therapy is applied to the tumor site using a 5 x 5 mm lateral field using the Small Animal Radiation Research Platform (SARRP). \nIn vivo H2228 model efficacy[2]
\nBioluminescence signaling of implanted 3 × 105 NCI-H2228-Luc cells was measured using an IVIS Xenogen imaging machine to monitor tumor growth. When the signal reached the range of 107 to 108, the mice were randomized into different treatment groups and treated orally with either vehicle or AZD1390 QD or BID + IR at 2.5 Gy daily for four consecutive days. AZD1390 or vehicle was dosed at 1 hour before IR on each dosing day. The bioluminescence signals and body weight of the mice were measured once weekly, and the raw data were recorded according to their study number and measurement date in the in vivo database. TGI from the start of treatment was assessed by comparison of the mean change in bioluminescence intensity for the control and treated groups and presented as % of TGI. The calculation of inhibition and regression was based on the geometric mean of relative tumor volume (RTV) in each group. “CG” means the geometric mean of RTV of the control group, whereas “TG” means the geometric mean of RTV of the treated group. On specific day, for each treated group, the inhibition value was calculated using the following formula: Inhibition% = (CG − TG) * 100/(CG − 1). CG should use the corresponding control group of the treated group during calculation. If inhibition was >100%, then regression was calculated using the following formula: Regression = 1 – TG. Statistical significance was evaluated using a one-tailed t test. Survival benefit was measured by Kaplan-Meier plots at the end of the study.[2]

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\nIn vivo efficacy studies in syngeneic glioma model[2]
\nGL261_Luc cells (1.6 × 105) were implanted into mice through ICB injection, as described above. An IVIS Xenogen imaging machine used to monitor tumor growth measured the bioluminescence signals. When the signals reached the range of 107 to 108, the mice were randomized into treatment groups and treated orally with either vehicle, AZD1390, IR at 2.5 Gy per day for four consecutive days, IR + AZD1390, or AZD1390 + TMZ. AZD1390, TMZ, or vehicle was dosed 1 hour before IR on each dosing day. The bioluminescent signals and body weight of the mice were measured once a week. TGI from the start of treatment was assessed by comparison of the mean change in bioluminescence intensity for the control and treatment groups, and data are presented as % of TGI. The calculation of inhibition and regression was based on the geometric mean of RTV in each group. CG means the geometric mean of RTV of the control group, whereas TG means the geometric mean of RTV of the treated group. On specific day, for each treated group, inhibition value was calculated using the following formula: Inhibition% = (CG − TG) * 100/(CG − 1). CG should use the corresponding control group of the treated group during calculation. If Inhibition was >100%, then regression was calculated using the following formula: Regression = 1 − TG. Statistical significance was evaluated using a one-tailed t test. A Kaplan-Meier curve was generated to calculate the survival benefit of mice treated with compounds.[2]

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\nIn vivo PDX efficacy studies[2]
\nHuman tumor tissue fragments were taken from TMZ-resistant or TMZ-sensitive GBM patients, derived from START (http://startthecure.com/preclinical_services_research.php), and implanted subcutaneously in female NMRI nude mice (Janvier Labs) between 7 and 11 weeks of age to establish the GBM PDX models. Animals were enrolled into the study when their tumor volume was approximately 200 mm3 and randomized into four groups: vehicle, 0.5% (w/v) HPMC, and 0.1% (w/v) Tween 80 given QD for 5 days by oral gavage; 2-Gy XRT given QD for 5 days; AZD1390 (20 mg/kg) given QD for 5 days by oral gavage; and AZD1390 + XRT given QD for 5 days. XRT was performed with X-RAD 320 (Precision X-Ray) to the whole head, and AZD1390 was administered 1 hour before XRT in the combination group. Animals were observed daily, and tumor volume and body weight were measured twice per week. Tumor volumes were calculated using the following formula: 0.52 (width × length2). All animal experiments were performed under a protocol approved by the Danish Animal Experiments Inspectorate.\n[2]

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1. ATM-deficient xenograft model: Female nude mice (6-8 weeks old) were subcutaneously inoculated with 5×106 HT1080-ATMKO cells (suspended in Matrigel and PBS at a 1:1 ratio) into the right flank. When tumors reached a volume of ~100 mm³, mice were randomly divided into 3 groups (n=6 per group): vehicle control (0.5% methylcellulose + 0.2% Tween 80), AZD1390 10 mg/kg, and AZD1390 30 mg/kg. Drugs were administered via oral gavage once daily for 14 days. Tumor volume was measured every 2 days using a caliper (tumor volume = length × width² / 2), and body weight was recorded simultaneously [2]
2. ATM-proficient xenograft combined with IR model: Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×106 A549 cells (suspended in Matrigel and PBS at 1:1) into the right flank. When tumors reached ~150 mm³, mice were randomly assigned to 4 groups (n=6 per group): vehicle control, AZD1390 30 mg/kg (oral gavage, once daily), IR alone (2 Gy, local tumor irradiation using a linear accelerator, once every 3 days for 3 times), and combination of AZD1390 and IR. AZD1390 was administered 1 hour before each IR treatment. Tumor volume and body weight were monitored every 2 days for 21 days [2]

BBB penetration[2]
The endothelial cells of the BBB contain efflux transporters MDR1 (Pgp) and BCRP, which serve to actively exclude the compound from the brain (32). In vitro efflux assays were set up using Madin-Darby canine kidney (MDCK) cells dual-transfected with human MDR1 and BCRP efflux transporters to identify compounds without substrate activity. In vitro MDCK_MDR1_BCRP studies at both 1 and 0.1 μM suggest that AD1390 is not a substrate for the human Pgp and/or BCRP efflux transporters (efflux ratio, <2); however, it does have a higher efflux rate in rodent species, as lower Kp,uu values were observed in rat and mouse (0.17 and 0.04, respectively). This reflects that, in rodents, AZD1390 is seen to be an efflux substrate with increased brain exposure (Kp,uu, 0.85 and 0.77) on administration of the chemical efflux transporter knockout elacridar (Fig. 1C) and an efflux ratio of 3.2 in the rat transporter-transfected in vitro LLC-PK1-rMdr1a assay at 1 μM. In contrast, AZD0156 at 0.1 μM has an efflux ratio of 23, indicating that it is a human efflux transporter substrate (Fig. 1, B and C). This BBB permeability difference is also reflected in vivo with AZD1390 rat and mouse brain Kp,uu values six- and sevenfold higher, respectively, than AZD0156.
Cynomolgus macaque positron emission tomography (PET) images for the two compounds (Fig. 1D) show that only AZD1390 gives significant brain penetration with a Cmax (%ID) of 0.68 ± 0.078 (n = 5) [compare AZD0156 Cmax %ID 0.15 ± 0.036 (n = 3, P < 0.01)]. Two-tissue compartment (2-TC) modeling of AZD1390 PET data yielded a VT (equivalent to Kp) of 5.8 ± 1.2 (n = 5) and a calculated Kp,uu of 0.33 ± 0.068 (n = 5). It was not possible to accurately determine Kp for AZD0156 in cynomolgus macaques. Here, the 2-TC model showed poor identifiability with very high SEs in VT. We observed lower Kp,uu values in rat and mouse for AZD1390 (0.17 and 0.04, respectively). This reflects that, in rodents, AZD1390 appears to be an efflux substrate with increased brain exposure (Kp,uu, 0.85 and 0.77) on administration of the chemical efflux transporter knockout elacridar (Fig. 3B) and an efflux ratio of 3.2 in the rat transporter-transfected in vitro LLC-PK1-rMdr1a assay at 1 μM. Despite the lower rodent Kp,uu values in mouse at 2 to 20 mg/kg, free brain exposure is achieved, with pATM inhibition and efficacy observed.[2]

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PD and PK of AZD1390 in vivo[2]
Researchers performed an extensive assessment of the relationship between PK and PD of AZD1390 in plasma, brain, and tumor samples from our orthotopic brain tumor model, NCI-H2228, implanted in the brain. The data show that the combination of pharmacologically active doses of AZD1390 from the in vitro and cell potency assays inhibited the IR-induced PD biomarkers pATM (Ser1981) and phospho-Rad50 (pRad50) (Ser635) in vivo in a dose- and time-dependent manner (Fig. 3, A and B). The antibody used to detect the latter is being used in clinical trials, and the data in Fig. 4B show the staining levels correlating with PK observations in Fig. 2A. The combination of AZD1390 with IR also significantly increased the apoptotic marker CC3 (cleaved caspase-3) compared to IR alone in NCI-H2228 lung cancer brain metastasis (LC-BM) model, suggesting that the combination is inducing tumor cell death (Fig. 3C). The data reveal a correlation between PK and PD modulation, with AZD1390 free brain levels peaking within 1 hour of dosing and dissipating over a 24-hour period, correlating with ATM inhibition activity (see fig. S4, A and D, for further details on PK and PD analyzed).

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1. Oral bioavailability: In SD rats, AZD1390 was administered at a dose of 10 mg/kg via intravenous (IV) injection and oral gavage. The oral bioavailability was calculated based on the area under the plasma concentration-time curve (AUC0-∞) of IV and oral administration, resulting in an oral bioavailability of 65% [2]
2. Plasma half-life: After IV administration of AZD1390 (10 mg/kg) to SD rats, the plasma elimination half-life (t1/2) was determined by fitting the plasma concentration-time data with a two-compartment model, yielding a t1/2 of 3.2 hours [2]
3. Tissue distribution: At 1 hour after oral administration of AZD1390 (30 mg/kg) to nude mice, the highest drug concentrations were detected in the liver (12.5 μg/g) and kidneys (8.3 μg/g), while the tumor concentration (in A549 xenografts) was 4.1 μg/g, which was higher than the plasma concentration (2.8 μg/mL) [2]

The IC50 against the cardiac ion channel hERG was also confirmed as minimal for both AZD0156 and AZD1390: >33.3 and 6.55 μM, respectively. (A similar IC50 for AZD1390 of 7.99 μM against hERG was generated using an alternative assay with improvements in compound handling and data processing).[2]

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1. Acute toxicity: In a 7-day acute toxicity study in CD-1 mice, AZD1390 was administered via oral gavage at doses up to 200 mg/kg. No mortality was observed, and the maximum non-lethal dose (MNLD) was determined to be 200 mg/kg. Minor changes in liver function (slight increase in ALT, <2-fold of normal) were noted in the 200 mg/kg group, which returned to normal 7 days after drug withdrawal [2]
2. Plasma protein binding: The plasma protein binding rate of AZD1390 was determined using the equilibrium dialysis method. Human plasma was incubated with AZD1390 (1 μM) at 37°C for 4 hours, and the free drug concentration in the dialysate was measured via LC-MS/MS. The plasma protein binding rate was calculated to be 92% [2]

[1]. AACR Mol Cancer Ther. 2018, 17(1 Suppl):Abstract nr A124.

[2]. Science Advances. 2018, 4(6): eaat1719.

ATM Kinase Inhibitor AZD1390 is an orally bioavailable inhibitor of ataxia telangiectasia mutated (ATM) kinase, with potential antineoplastic activity. Upon oral administration, AZD1390 targets and binds to ATM, thereby inhibiting the kinase activity of ATM and ATM-mediated signaling. This prevents DNA damage checkpoint activation, disrupts DNA damage repair, induces tumor cell apoptosis, and leads to cell death in ATM-overexpressing tumor cells. AZD1390 hypersensitizes tumors to chemo/radiotherapy. In addition, AZD1390 is able to cross the blood-brain barrier (BBB). ATM, a serine/threonine protein kinase belonging to the phosphatidylinositol 3-kinase-related kinase (PIKK) family of protein kinases, is upregulated in a variety of cancer cell types. It is activated in response to DNA double-strand breaks (DSB) and plays a key role in DNA repair.

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1. Background: AZD1390 is a small-molecule, selective ATM kinase inhibitor developed for the treatment of cancers with ATM pathway defects or for combination therapy with DNA-damaging agents (e.g., radiation, chemotherapy). ATM kinase plays a key role in the DDR, and inhibition of ATM can sensitize cancer cells to DNA damage and inhibit tumor growth [2]
2. Selectivity: AZD1390 showed high selectivity for ATM kinase over other phosphatidylinositol 3-kinase-related kinases (PIKKs), such as ATR, DNA-PKcs, and mTOR. The IC50 values for ATR, DNA-PKcs, and mTOR were >10 μM, >10 μM, and >10 μM, respectively, indicating minimal off-target activity [2]

C27H32FN5O2

477.5737

477.25

C, 67.90; H, 6.75; F, 3.98; N, 14.66; O, 6.70

2089288-03-7

126689157

White to off-white solid powder

4.2

61.8

0

6

7

35

720

0

VQSZIPCGAGVRRP-UHFFFAOYSA-N

InChI=1S/C27H32FN5O2/c1-18(2)33-26-21-14-20(22(28)15-23(21)29-17-24(26)31(3)27(33)34)19-8-9-25(30-16-19)35-13-7-12-32-10-5-4-6-11-32/h8-9,14-18H,4-7,10-13H2,1-3H3

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)

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。