BTK (Ki = 0.91 nM); BTK C481R (Ki = 1.3 nM); BTK C481S (Ki = 1.6 nM); BTK T474M (Ki = 3.4 nM); BTK T474I (Ki = 12.6 nM)

在一系列人类激酶生化检测中，当浓度为 1 μM 时，GDC-0853 仅抑制 286 种脱靶激酶中的 3 种。根据计算的 IC50 值，Btk 对这三种脱靶的选择性均 >100 倍：Bmx（153 倍）、Fgr（168 倍）和 Src（131 倍）。 GDC-0853 抑制单核细胞 FcγR 和 B 细胞 BCR 的信号传导。在体外生化 Btk 酶测定中，GDC-0853 与 Btk 的平均停留时间为 18.3 ± 2.8 小时。 GDC-0853 阻断 WT Btk 和 C481S 突变体的细胞自磷酸化[1]。在 BCR 刺激之前，GDC-0853 在体外对 CLL（慢性淋巴细胞白血病）细胞进行处理，导致 BTK 磷酸化降低，并降低 PLCγ2、AKT 和 ERK 等下游靶标的激活。 GDC-0853 减少激活、阻碍迁移并抑制 NF-κB 依赖性转录。 GDC-0853 不影响 T 细胞受体激活，也不抑制细胞系统中的 EGFR 或 ITK[3]。

GDC-0853 在通过腹腔注射 0.2 mg/kg 或口服 1 mg/kg 给药的大鼠中具有 27.4 mL/min/kg 的中等清除率和优异的生物利用度 (F=65%)。血浆半衰期（t_1/2）为2.2小时，分布容积（Vd）为5.42 L/kg，血浆清除率为27.4 mL/min/kg。 GDC-0853 在狗中也显示出积极的 PK 特征。在犬类毒理学研究中，3.8 小时的半衰期（Cl_p 10.9 mL/min/kg，V_d 2.96 L/kg）进一步实现了足够的暴露）和高口服生物利用度（85%）。大鼠和狗对 GDC-0853 的耐受性良好，并且总体上具有良好的安全性。 GDC-0853有助于治疗由B细胞或骨髓细胞介导的自身免疫性疾病，包括类风湿性关节炎。 GDC-0853 在两项研究中显示出优异的耐受性：单次递增剂量 (SAD) 试验（0.5 mg 至 600 mg）和持续 14 天的多次递增剂量 (MAD) 研究（250 mg BID 至 500 mg QD）。两项研究均表明没有剂量限制性毒性，也没有严重的不良事件。 GDC-0853 表现出剂量成比例的线性药代动力学并且吸收良好[1]。 GDC-0853 和其他结构多样的 BTK 抑制剂在 Sprague-Dawley (SD) 大鼠中给药 7 天或更长时间会导致胰腺病变，包括富含色素的巨噬细胞、炎症、纤维化和多灶性胰岛中心出血以及邻近的小叶外分泌腺泡细胞萎缩、变性和炎症。在高得多的暴露量下，在小鼠或狗中没有看到类似的结果[2]。

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类风湿关节炎和狼疮的临床前疗效[1]
为了评估GDC-0853 (29)的体内疗效，我们在B细胞和骨髓细胞依赖性炎症性关节炎模型中进行了测试。发生胶原诱导关节炎的雌性Lewis大鼠(30只)口服16天，其中29只每天服用一次(0.06、0.25、1、4和16 mg/kg QD)(图8A)或两次(0.125、0.5和2 mg/kg BID)(图8B)。29 . QD(图8A)和BID(图8B)给药方案后脚踝厚度的剂量依赖性降低。此外，29项研究显示，剂量反应对踝关节组织病理学参数(炎症、肿块、软骨损伤、骨吸收;数据未显示)。我们还评估了以曲线下面积(AUC)表示的踝关节直径作为疗效参数。与各自的疾病对照相比，QD组16 mg/kg(减少99%)、4 mg/kg(89%)、1 mg/kg(71%)和0.25 mg/kg 29(26%)或BID组2 mg/kg(96%)、0.5 mg/kg(79%)和0.125 mg/kg 29(50%)的大鼠脚踝直径AUC显著降低至正常水平。QD 16 mg/kg剂量和BID 2 mg/kg剂量29与地塞米松的疗效相当(略有改善)。在研究过程中，大鼠的踝关节直径没有变化，正常大鼠的踝关节直径明显小于CIA大鼠(P < 0.05)。

BTK 激酶assay。[1]
Btk激酶的活性和抑制作用是根据先前发表的肽磷酸化测定和使用基因泰克表达和纯化的野生型和突变型Btk酶进行评估的。Btk蛋白使用纯化后的蛋白，未采取特殊的活化措施。抑制常数(Ki)由抑制剂滴定数据计算如下。Btk分数活性(vi /vo)与试验品浓度进行拟合，数据使用Genedata筛选软件(Genedata;巴塞尔，瑞士)到紧密结合抑制方程2来计算表观抑制常数，Ki app。

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激酶选择性。[1]
Btk抑制剂激酶的选择性在浓度为1µM时，在多达287个重组人激酶活性和结合试验中进行评估，如脂质激酶、丝氨酸/苏氨酸激酶、细胞质激酶和受体酪氨酸激酶。结合实验追踪ATP位点结合探针的位移，而激酶活性实验量化肽磷酸化或ADP的产生。对于每个激酶，活性测定中使用的ATP浓度通常在实验确定的表观米切里斯常数(Km^app)值的两倍以内，而结合测定中使用的竞争结合示踪剂浓度通常在实验确定的解离常数(Kd)值的三倍以内。对于每一种激酶，抑制剂都是重复测试的，并且报告了抑制的平均百分比。同样的测定法用于10点抑制剂滴定，以确定对在测试浓度下被抑制近80%或超过80%的激酶产生50%抑制(IC50)的抑制剂浓度。

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激酶试验[3]
将160 ng人重组野生型和C481S BTK与DMSO或1µM GDC-0853孵育30分钟。然后将重组蛋白与50µM三磷酸腺苷和5µg聚肽(4:1,Glu:Tyr)在1倍反应缓冲液中室温结合30分钟，使肽底物磷酸化。然后用ADP-glo激酶试剂和激酶检测试剂分别淬灭和定量反应。使用DTX880plate阅读器测量发光。

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肝微粒体代谢稳定性测定[1]
在人、小鼠和大鼠肝微粒体中评估了试验化合物的代谢稳定性。最终孵育液中含有:1µM试验化合物，1 mM NADPH, 0.5 mg/mL微粒体蛋白，0.1 M磷酸钾缓冲液(pH 7.4)。在5分钟的预孵育期后，将NADPH和测试化合物添加到磷酸盐缓冲盐水稀释的微粒体中，启动酶促反应。分别于37℃孵育0、20、40、60 min，采用LC-MS/MS测定所得化合物浓度。基于微粒体稳定性数据的内在清除率使用底物耗尽法确定，并使用充分搅拌模型缩放到肝脏清除率。

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肝细胞代谢稳定性测定[1]
实验用小鼠、大鼠、狗、食蟹猴和人肝细胞进行了代谢稳定性分析。台盼蓝法测定细胞的膜完整性。将测试化合物(1µM, 0.1% DMSO)与细胞(50万个细胞/mL)在37°C的95%空气/5% CO2气氛中孵育0,20,40或60分钟。通过LC-MS/MS测定肝细胞孵育中测试化合物的浓度。使用底物耗尽法确定内在清除率，并使用充分搅拌模型(见上)缩放到肝脏清除率。

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GDC-0853停留时间测定[1]
Btk酶(10 nM)与GDC-0853 (11 nM)或DMSO在50 mM HEPES缓冲液(pH 7.5)中室温孵育2 h。孵育后，将Btk样品稀释200倍至含有ATP和肽底物1的实验混合物中，大约每2.5分钟监测一次未反应的底物肽和磷酸化肽产物的水平，持续8.5 h。与GDC-0853预孵育的Btk形成的产物的进展曲线符合描述活性恢复的方程4:

细胞Btk磷酸化。[1]
GDC-0853和ibrutinib对细胞野生型Btk和Btk C481S突变体Y223磷酸化的影响在瞬时转染的HEK293T细胞中进行了评估。

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B细胞和单核细胞检测[1]
采用FicollPaque PLUS分离法从外周血单个核细胞(PBMCs)中分离人B细胞或单核细胞，并按照制造商说明进行磁性细胞分选负选择。用10或25µg/mL山羊抗igm - f (ab’)2或10µg/mL CD40L刺激人B细胞，用[3H]胸腺嘧啶掺入法检测细胞增殖。人单核细胞用40µg/mL固定的HSA/anti - HSA ic孵育。ELISA法检测fc - γ -活化后TNFα的生成。<人力资源> 用二甲基亚砜(DMSO)或GDC-0853处理的细胞同样成粒，然后在含有DMSO或GDC-0853的10%胎牛血清RPMI-1640培养基中重悬。在几天内进行的实验包括每天添加药物和更换培养基。[3]

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NK细胞介导的ADCC[3]
效应NK细胞从美国红十字会获得的白细胞中分离出来，与装载放射性Cr51的靶CLL细胞以效应靶比25:1孵育。用DMSO、1µM GDC-0853或1µM ibrutinib处理纯化NK细胞1小时后，CLL细胞与曲妥珠单抗、阿仑单抗、利妥昔单抗、ofatumumab或obinutuzumab以10µg/mL的浓度孵育，并与NK细胞共培养，使其溶解。共培养4小时后，收集上清并用PerkinElmer Wizard2 γ计数器测量辐射。β衰变测量根据无nk细胞共培养组与基线CLL裂解和洗涤剂处理CLL组完全裂解进行缩放。

Sprague-Dawley, Wistar-Han and Fischer-344 rats (6 to 12 weeks old)
5 or 10 mL/kg
p.o.

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Rat whole blood pBtk assay [1]
Sprague-Dawley rats were euthanized using CO2 asphyxiation. Blood was collected in heparin tubes by cardiac puncture. Rat whole blood was incubated with a titration of GDC-0853 (starting at 6 µM followed by 3-fold dilution for a 11-point dilution curve) for 4 h at 37 ºC. Blood was treated with an equal volume of MSD lysis buffer containing protease and phosphatase inhibitors. Thirty-five µL of lysate was added to MSD plates coated with 100 ng/well of total anti-BTK antibody and incubated for 2 h with shaking at room temperature. Wells were washed three times with TBST buffer and incubated with 12 µg/mL of anti-rabbit pBTK antibody detection antibody for 2 h at room temperature with constant shaking. Wells were washed and then incubated with 1 µg/mL of sulfo-tag anti-rabbit antibody for 45 min at room temperature with constant shaking. After incubation, wells were finally washed with S34 TBST and pBTK levels were detected by adding 150 µL of MSD Reading buffer in each well and read on a MSD Sector Imager 6000. IC50 values were calculated using Prism software.

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Bruton's tyrosine kinase (BTK) is a member of the Tec family of cytoplasmic tyrosine kinases involved in B-cell and myeloid cell signaling. Small molecule inhibitors of BTK are being investigated for treatment of several hematologic cancers and autoimmune diseases. GDC-0853 ((S)-2-(3'-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4'-bipyridin]-2'-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one) is a selective and reversible oral small-molecule BTK inhibitor in development for the treatment of rheumatoid arthritis and systemic lupus erythematosus. In Sprague-Dawley (SD) rats, administration of GDC-0853 and other structurally diverse BTK inhibitors for 7 days or longer caused pancreatic lesions consisting of multifocal islet-centered hemorrhage, inflammation, fibrosis, and pigment-laden macrophages with adjacent lobular exocrine acinar cell atrophy, degeneration, and inflammation. Similar findings were not observed in mice or dogs at much higher exposures. Hemorrhage in the peri-islet vasculature emerged between four and seven daily doses of GDC-0853 and was histologically similar to spontaneously occurring changes in aging SD rats. This suggests that GDC-0853 could exacerbate a background finding in younger animals. Glucose homeostasis was dysregulated following a glucose challenge; however, this occurred only after 28 days of administration and was not directly associated with onset or severity of pancreatic lesions. There were no changes in other common serum biomarkers assessing endocrine and exocrine pancreatic function. Additionally, these lesions were not readily detectable via Doppler ultrasound, computed tomography, or magnetic resonance imaging. Our results indicate that pancreatic lesions in rats are likely a class effect of BTK inhibitors, which may exacerbate an islet-centered pathology that is unlikely to be relevant to humans.[3]

Oral dosing with GDC-0853 (29) at 1, 4, and 16 mg/kg QD maintained plasma concentrations above the rat whole blood pBtk potency, as determined by inhibition of Btk Y223 autophosphorylation (IC50 = 9 nM, IC70 = 27 nM, IC90 = 135 nM), for a minimum of 12 h of the 24-h dosing period. BID dosing at 0.125, 0.5, and 2 mg/kg (Figure 9B) also maintained plasma concentrations above the IC50, IC70, and IC90 levels for a minimum of 6 h of the 12-h dosing period. Doses of 1 mg/kg QD and 0.5 mg/kg BID were associated with plasma concentrations that, at a minimum, exceeded the whole blood pBtk IC70 (27 nM) for approximately 12 h in a 24-h period. This exposure-efficacy relationship suggests that plasma concentrations of 29 in excess of the IC70 for 12 h were required to achieve efficacy, defined as a reduction of ∼75% of ankle swelling. Increased target coverage, as seen at the 4 mg/kg QD dose covered the IC90 for 24 h and offered further efficacy improvement. The efficacy of Btk inhibition by noncovalent inhibitors has also been assessed in mouse models of SLE. (14) Taken together, these impressive in vivo efficacy results with our noncovalent Btk inhibitors, combined with the excellent preclinical pharmacologic, pharmacokinetic and in vitro safety profile of 29, gave us confidence to progress this molecule into tolerability studies.[1]

In studies designed to assess the safety of the molecule, GDC-0853 (29) was well tolerated in both rats and dogs and displayed an overall favorable safety profile. The no observed adverse effect level (NOAEL) in dogs, the most sensitive preclinical species, was >80-fold higher than the targeted efficacious exposure, i.e., exceeding for 12 h the IC70 concentration (from the human whole blood CD69 assay). In Sprague–Dawley rats,GDC-0853 (29) and other structurally distinct Btk inhibitors have been shown to be associated with islet-centric pancreatic lesions at clinically relevant doses. After a thorough investigation involving evaluation of strain and species sensitivity differences, Btk knockout (KO) mice, and literature reports of humans with XLA mutations, we concluded that the GDC-0853-related pancreas findings in the Sprague–Dawley strain were the result of a rat-specific, strain-variable, on-target effect of Btk inhibition that is not relevant for humans. These conclusions have been supported by a histologic evaluation of the pancreas of untreated Btk KO Sprague–Dawley rats, that demonstrated the presence of identical pancreatic pathology. With a favorable safety profile and evidence that the observed pancreatic toxicity was a rat-specific phenomenon, we selected GDC-0853 (29) as our lead candidate for clinical development.[1]

[1]. J Med Chem . 2018 Mar 22;61(6):2227-2245.

[2]. J Pharmacol Exp Ther . 2017 Jan;360(1):226-238.

[3]. Blood . 2018 Sep 6;132(10):1039-1049.

Fenebrutinib is under investigation in clinical trial NCT03174041 (A Drug-Drug Interaction Study Between GDC-0853 and Midazolam, Itraconazole, Rosuvastatin, and Simvastatin).
Fenebrutinib is an orally available inhibitor of Bruton's tyrosine kinase (BTK) with potential antineoplastic activity. Upon administration, fenebrutinib inhibits the activity of BTK and prevents the activation of the B-cell antigen receptor (BCR) signaling pathway. This prevents both B-cell activation and BTK-mediated activation of downstream survival pathways, which leads to the inhibition of the growth of malignant B-cells that overexpress BTK. BTK, a member of the Src-related BTK/Tec family of cytoplasmic tyrosine kinases, is overexpressed in B-cell malignancies; it plays an important role in B-lymphocyte development, activation, signaling, proliferation and survival.

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Bruton's tyrosine kinase (Btk) is a nonreceptor cytoplasmic tyrosine kinase involved in B-cell and myeloid cell activation, downstream of B-cell and Fcγ receptors, respectively. Preclinical studies have indicated that inhibition of Btk activity might offer a potential therapy in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Here we disclose the discovery and preclinical characterization of a potent, selective, and noncovalent Btk inhibitor currently in clinical development. GDC-0853 (29) suppresses B cell- and myeloid cell-mediated components of disease and demonstrates dose-dependent activity in an in vivo rat model of inflammatory arthritis. It demonstrates highly favorable safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles in preclinical and Phase 2 studies ongoing in patients with rheumatoid arthritis, lupus, and chronic spontaneous urticaria. On the basis of its potency, selectivity, long target residence time, and noncovalent mode of inhibition, 29 has the potential to be a best-in-class Btk inhibitor for a wide range of immunological indications.[1]

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The clinical success of ibrutinib validates Bruton tyrosine kinase (BTK) inhibition as an effective strategy for treating hematologic malignancies, including chronic lymphocytic leukemia (CLL). Despite ibrutinib's ability to produce durable remissions in patients, acquired resistance can develop, mostly commonly by mutation of C481 of BTK in the ibrutinib binding site. Here, we characterize a novel BTK inhibitor, GDC-0853, to evaluate its preclinical efficacy in ibrutinib-naive and ibrutinib-resistant CLL. GDC-0853 is unique among reported BTK inhibitors in that it does not rely upon covalent reaction with C481 to stabilize its occupancy within BTK's adenosine triphosphate binding site. As with ibrutinib, GDC-0853 potently reduces B-cell receptor signaling, viability, NF-κB-dependent transcription, activation, and migration in treatment naïve CLL cells. We found that GDC-0853 also inhibits the most commonly reported ibrutinib-resistant BTK mutant (C481S) both in a biochemical enzyme activity assay and in a stably transfected 293T cell line and maintains cytotoxicity against patient CLL cells harboring C481S BTK mutations. Additionally, GDC-0853 does not inhibit endothelial growth factor receptor or ITK, 2 alternative targets of ibrutinib that are likely responsible for some adverse events and may reduce the efficacy of ibrutinib-antibody combinations, respectively. Our results using GDC-0853 indicate that noncovalent, selective BTK inhibition may be effective in CLL either as monotherapy or in combination with therapeutic antibodies, especially among the emerging population of patients with acquired resistance to ibrutinib therapy.[3]

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In summary, we have discovered a potential best-in-class Btk inhibitor, GDC-0853 (29), that is currently in clinical investigation for several immune disorders. It is highly potent and is the most selective Btk inhibitor reported to date. Its preclinical pharmacokinetic characteristics are favorable, indicating the potential for QD oral dosing. In addition, the supporting efficacy data reported here suggest that it could have utility in treating rheumatoid arthritis and other B-cell or myeloid cell mediated autoimmune diseases. These findings, combined with a very desirable tolerability and safety profile in multiple species, encouraged us to progress 29 into clinical studies in autoimmune diseases. In a single ascending dose (SAD) study (0.5 mg to 600 mg) and multiple ascending dose (MAD) study for 14 days (250 mg of BID to 500 mg of QD) in healthy volunteers, GDC-0853 was very well tolerated with no severe adverse events, no safety signals, and no dose limiting toxicities. Additionally, 29 was well absorbed and had linear, dose-proportional PK. Target engagement (CD63, CD69, pBTK) was assessed and complete suppression of PD markers was maintained over 24 h. With favorable Phase 1 results, 29 entered Phase 2 clinical studies in rheumatoid arthritis, lupus, and chronic urticaria. Further details and clinical results will be reported in due course.[1]

C37H45CLN8O4

701.26

700.325

2128304-54-9

1434048-34-6;2128304-54-9 (HCl);2128304-53-8 (mesylate);2128304-55-0 (sulfate);

154723936

Typically exists as solid at room temperature

1.59

119Ų

3

9

7

50

1340

1

C[C@H]1CN(CCN1C2=CN=C(C=C2)NC3=CC(=CN(C3=O)C)C4=C(C(=NC=C4)N5CCN6C7=C(CC(C7)(C)C)C=C6C5=O)CO)C8COC8.Cl

UFNQEETXQCTBNF-BQAIUKQQSA-N

InChI=1S/C37H44N8O4.ClH/c1-23-18-42(27-21-49-22-27)9-10-43(23)26-5-6-33(39-17-26)40-30-13-25(19-41(4)35(30)47)28-7-8-38-34(29(28)20-46)45-12-11-44-31(36(45)48)14-24-15-37(2,3)16-32(24)44;/h5-8,13-14,17,19,23,27,46H,9-12,15-16,18,20-22H2,1-4H3,(H,39,40);1H/t23-;/m0./s1

(S)-2-(3'-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4'-bipyridin]-2'-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one hydrochloride

RG-7845 HCl; GDC-0853 HCl; RG7845 HCl; GDC 0853; RG 7845 hydrochloride; GDC0853 HCl

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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。