Zelebrudomide (NX-2127)

BTK; Ikaros (IKZF1); Aiolos (IKZF3)
- Bruton's tyrosine kinase (BTK) (DC50 < 5 nM in multiple cancer cell lines and human PBMCs) [3]
- Ikaros (IKZF1) and Aiolos (IKZF3) (DC50 of 54 nM and 25 nM, respectively, in primary human T cells) [3]

- BTK降解：NX-2127在多种B细胞淋巴瘤细胞系（如TMD8、MEC1）和原代人B细胞中诱导剂量依赖性BTK降解，DC50值<5 nM。通过蛋白质印迹分析证实，24小时内BTK蛋白水平降低>80% [3]
- 类IMiD活性：该化合物通过降解原代人T细胞中的IKZF1和IKZF3表现出免疫调节作用，导致IL-2生成增加和T细胞活化，与来那度胺相当 [3]
- 细胞增殖抑制：NX-2127有效抑制BTK依赖性ABC-DLBCL细胞（TMD8）的增殖，72小时后EC50 < 15 nM。值得注意的是，其对BTK C481S突变细胞的活性（EC50 < 30 nM）显著优于伊布替尼（>1 μM）[3]
NX-2127 的 EC50 值 <30 nM，可抑制 BTK-C481S 突变 TMD8 细胞的增殖[1]。当暴露于 NX-2127 时，原代人类 T 细胞会产生更多的 IL-2 [1]。

- 肿瘤生长抑制：在小鼠异种移植模型（TMD8 WT和C481S突变）中，口服NX-2127导致剂量依赖性肿瘤生长抑制（TGI）。每日10 mg/kg剂量下，两种模型的TGI均超过80%，疗效优于伊布替尼 [3]
- 非人灵长类动物中的BTK降解：在食蟹猴中，每日一次口服给药（1–10 mg/kg）导致外周血单个核细胞（PBMCs）中BTK持续降解，水平至少24小时内抑制至<10%基线值 [3]
用 NX-2127（1 mg/kg；口服；每天一次，持续 14 天）治疗的食蟹猴显示出有效的 BTK 降解 [1]。口服 NX-2127 会导致血浆中的 BTK 在循环和脾 B 细胞中降解至基线水平的 10% 以下，且暴露量与剂量成比例[1]。在小鼠 WT TMD8 和 C481S 突变异种移植模型中，NX-2127 产生更强的肿瘤生长抑制 (TGI) [1]。

- BTK结合与降解实验：重组BTK蛋白与NX-2127和纯化的CRBN E3连接酶复合体孵育。通过SDS-PAGE和免疫印迹评估降解，显示剂量依赖性BTK泛素化和蛋白酶体降解。表面等离子体共振（SPR）证实BTK、NX-2127和CRBN的三元复合体形成，结合亲和力（KD）为0.8 nM [3]
- IKZF1/3降解实验：原代人T细胞用NX-2127处理，通过流式细胞术定量IKZF1/3水平。化合物在6小时内诱导两种蛋白快速降解，100 nM时效果最大 [3]
通过同源时间分辨荧光（HTRF）评估BTK降解细胞与化合物孵育4小时，并根据制造商的方案使用Cisbio Total BTK HTRF试剂盒（63ADK064PEG）测定BTK水平。HTRF比率使用以下方程式计算：[3]

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除了有效的BTK降解外，NX-2127还通过设计CRBN结合线束具有IMiD样特性，该线束催化CRBN新底物Aiolos（IKZF3）和Ikaros（IKZF1）的降解。这种活性与IMiD药物来那度胺和泊马度胺的T细胞活化增加和抗肿瘤作用有关。在原代人类T细胞中，NX-2127分别以25 nM和54 nM的效力催化Aiolos和Ikaros的降解，其效力与来那度胺相似（分别为20 nM和343 nM）。与这种降解相对应，NX-2127以类似于来那度胺和泊马度胺的方式刺激T细胞活化，这是通过原代人T细胞中IL-2产量的增加来测量的。BTK降解的双重活性与NX-2127的免疫调节相结合，支持其治疗B细胞恶性肿瘤的发展。[1]

人外周血单个核细胞中Aiolos和Ikaros降解的评估[3]
人外周血单核细胞（PBMCs）在37°C下用2000-0.00512 nM化合物处理24小时，然后使用Foxp3/转录因子固定/渗透试剂盒固定和渗透细胞。细胞用抗Aiolos、Ikaros、CD20和CD3的抗体染色。另外的样本用Aiolos和Ikaros同种型对照抗体染色作为染色对照。在Attune NxT声聚焦流式细胞仪上进行流式细胞术，并使用FlowJo（v10.5.3）和GraphPad Prism分析数据。对T细胞（CD3+CD20-）进行门控，计算Aiolos和Ikaros蛋白的几何平均荧光强度（MFI）。

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活化T细胞分泌IL-2的定量[3]
对于T细胞活化，根据制造商的方案，通过免疫磁性阴性选择从白血病中分离出原代人类T细胞。T细胞用化合物预处理1小时，然后在化合物存在下用平板结合的抗CD3抗体（1µg OKT3/孔）和5µg/mL可溶性抗CD28（克隆28.2）刺激24小时。通过ELISA评估无细胞上清液中的总IL-2。

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蛋白质组学检测[3]
用浓度为50nM的DMSO或降解剂处理人PBMC（每个样品200万个细胞）4小时。在-80°C下储存之前，测试细胞的存活率并用PBS洗涤。使用PreOmics iST 96x试剂盒进行蛋白质组学样品制备。随后，细胞被裂解，然后进行蛋白水解消化和清理。样品处理完成后，通过数据独立分析（DIA）通过液相色谱-串联质谱（LCMS/MS）进行数据采集。采集的DIA光谱使用Spectronaut（17.1.221229.55965）进行处理，使用无库S75专有directDIA算法和默认的BGS工厂设置。Q值截止值为1%的搜索结果（前体和蛋白质）被导出到Nurix开发的应用程序中，用于统计分析和可视化。

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NX-2127是一种CTM，它包含一个连接到cereblon（CRBN）线束的BTK钩。NX-2127在多个癌症细胞系和人PBMC中以<5nM降解50%的细胞BTK（DC50）。BTK-CTMs损害BTK依赖性ABC-DLBCL细胞系TMD8的存活率（72小时后EC50:<15nM）。重要的是，NX-2127诱导细胞中突变的BTK-C481S降解，并比伊布替尼更有效地抑制BTK-C481 S突变TMD8细胞的增殖（NX-2127 EC50值<30 nM，而伊布替尼>1μM）[1]。

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- B细胞中的BTK降解：从患者样本中分离的人CLL细胞用NX-2127（0.1–100 nM）处理。24小时后通过蛋白质印迹检测BTK蛋白水平，显示10 nM时降解>90%。降解与BTK突变状态无关（如C481S、L528W）[3]
- T细胞活化实验：原代人T细胞与NX-2127和抗CD3/CD28磁珠共培养。ELISA定量IL-2分泌，显示剂量依赖性增加（EC50 10 nM），与来那度胺相当 [3]

Pharmacodynamic in vivo studies [3]
For pharmacodynamic assessment, 8–9-week-old female BALB/cJ mice or CD-1 mice were orally dosed with compound or vehicle. Mice were bled via tail vein or cardiac puncture at the indicated time points. Red blood cells were lysed and remaining cells were stained with live/dead dye, then fixed, permeabilized using FoxP3/Transcription Factor Staining Buffer Set and stained with cell surface markers CD45, TCR β chain, CD3 and B220. For BTK intracellular staining detection, cells were stained with antiBTK (clone D3H5, Cell Signaling Technology Cat. 8547BC) primary and AF647-anti-Rabbit secondary antibodies with mouse FC block (BD553142). Flow cytometry analysis was performed on an Attune NxT Acoustic Focusing Flow Cytometer and data were analyzed using FlowJo and GraphPad Prism. B cells (CD45+/B220+/TCR β-) and T cells (CD45+/B220-/TCR β+) BTK MFIs were measured and degradation was calculated using the following equation:

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In vivo Tumor Implantation [3]
7–8-week-old female CB.17 SCID mice (Fox Chase SCID®, CB17/Icr-Prkdcscid/IcrIcoCrl) were subcutaneously implanted into the mid-flank with 1 x 107 cells of TMD8 cells resuspended in Hank’s Balanced Salt Solution (HBSS) and MatrigelTM at a ratio of 1:1. When tumors reached 60-175 mm3 , daily oral treatment of either NX-2127, ibrutinib, or vehicle was initiated. Body weight and tumor volumes were measured twice weekly. Tumors were measured using the formula of length x width2 x 0.5. Tumor growth inhibition (% TGI) was calculated using the equation [1 − (T − T0/C − T0)] × 100, where T and C represent the mean size of tumors in the treated (T) and control (C) groups, and T0 refers to the tumor size at randomization.

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Flow Cytometry Analysis of Canine Samples [3]
Beagle dogs (Canis familiaris) were dosed once with NX-2127 through either intravenous administration at 1 mg/kg or oral administration at 10 mg/kg. Animals were bled pre-dose and at intervals following dosing, and blood samples were collected into Cyto-Chex BCT tubes.

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Flow Cytometry Analysis of Non-Human Primate Samples [3]
Cynomolgus monkeys were dosed orally once daily with vehicle control or NRX-0492 or NX-2127 (10, 30, or 100 mg/kg) at BASi or Charles River Laboratories (Ashland, OH), respectively. Animals were bled on day 1 and 14 and blood samples were collected into Cyto-Chex BCT tubes (Streck, 213386).

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Oral administration of NX-2127 in mice leads to dose-proportional exposure in plasma and BTK degradation to <10% of baseline levels in circulating and splenic B cells. In both WT TMD8 and C481S mutant xenograft models, daily oral administration of NX-2127 resulted in superior tumor growth inhibition (TGI) as compared to ibrutinib. NX-2127 also demonstrates potent degradation of BTK in cynomolgus monkeys with oral administration. Following 14 days of once daily, oral dosing in cynomolgus monkey, BTK levels are suppressed to <10% of baseline levels at doses as low as 1 mg/kg.[1]

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NX-2127-001 is a first-in-human, dose escalation (Phase 1a) and cohort expansion (Phase 1b) study designed to evaluate the safety, tolerability, and preliminary efficacy of NX-2127 in adult patients with relapsed/refractory B-cell malignancies with once daily oral dosing. Dose escalation will proceed using a modified Fibonacci design with 1 patient per cohort, proceeding to a standard 3 + 3 design based on protocol specified criteria. There will be up to 5 expansion cohorts in Phase 1b enrolling patients with CLL/SLL, DLBCL, FL, MCL, MZL, and WM. Key eligibility criteria include >2 two prior lines of therapy (>1 prior for WM); measurable disease; and an Eastern Cooperative Oncology Group performance status of 0 or 1. Approximately 130 patients (30 in Phase 1a, 100 in Phase 1b) will be enrolled and treated until disease progression or unacceptable toxicity. The primary objectives are to evaluate safety and tolerability and to determine the maximum tolerated dose (Phase 1a), and to evaluate the early clinical activity of NX-2127 in expansion cohorts (Phase 1b). The Phase 1a part of this study is currently enrolling in the United States. Clinical trial information: NCT04830137.[2]

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- Mouse Xenograft Model: NX-2127 was formulated as a suspension in 0.5% methylcellulose and administered orally (10–30 mg/kg daily) to NSG mice bearing TMD8 tumors. Tumor volume was measured twice weekly, with significant inhibition observed at all doses. Pharmacokinetic analysis revealed peak plasma concentrations (Cmax) at 1–2 hours post-dose, supporting once-daily dosing [3]
- Cynomolgus Monkey Study: Animals received oral doses of NX-2127 (1–10 mg/kg) for 14 days. Blood samples were collected at predefined time points to assess BTK degradation in PBMCs and plasma drug concentrations. The compound exhibited linear pharmacokinetics with a terminal half-life of ~24 hours [3]

NX-2127 possesses potent in vivo target degradation activity across species. Upon iv administration, NX-2127 demonstrated low clearance (Tables 4 and 5). Oral dosing of NX-2127 in mice resulted in dose-dependent plasma exposure (Figure 3A), moderate oral bioavailability, and dose-dependent degradation of BTK in mouse blood (Figure 3B). Single, oral doses of 0.3, 3, 10, and 30 mg/kg of NX-2127 in mice resulted in reduction of BTK levels to 81%, 36%, 21%, and 12% of baseline in circulating B cells after 24 h. In addition to mice, we conducted in vivo PK–PD experiments in rat, dog, and cynomolgus monkey. NX-2127 had moderate clearance and low oral bioavailability in rat. Highlighting the power of the catalytic mechanism of targeted protein degradation (TPD), NX-2127 promoted potent BTK degradation in dog and cyno, despite relatively low bioavailability and high clearance (Table 5). Oral doses of 10 mg/kg in dog and cyno resulted in reduction of BTK levels to 17% and 9% of baseline BTK levels, respectively.[3]

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Absorption: Oral bioavailability in mice was ~60%, with Cmax achieved within 1–2 hours. In cynomolgus monkeys, absolute bioavailability was ~50%, supporting once-daily dosing [3]
- Metabolism: NX-2127 underwent hepatic metabolism primarily via cytochrome P450 enzymes (CYP3A4), with the major metabolite being the glucuronide conjugate. Less than 5% of the dose was excreted unchanged in urine [3]
- Half-life: Terminal half-life in mice and cynomolgus monkeys was ~12 hours and ~24 hours, respectively, allowing for sustained BTK degradation [3]

- Acute Toxicity: The median lethal dose (LD50) in mice was >2000 mg/kg (oral), indicating low acute toxicity. No dose-limiting toxicities were observed in cynomolgus monkeys at doses up to 10 mg/kg [3]
- Safety Profile: In phase 1 trials, NX-2127 was generally well-tolerated. Common adverse events included neutropenia, fatigue, and diarrhea, which were grade 1–2 and reversible. No significant QT interval prolongation or cardiovascular toxicity was reported [2]

[1]. Nx-2127, a degrader of BTK and IMiD neosubstrates, for the treatment of B-cell malignancies. Blood, 2020, 136: 34.

[2]. A first-in-human phase 1 trial of NX-2127, a first-in-class oral BTK degrader with IMiD-like activity, in patients with relapsed and refractory B-cell malignancies. 2022.

[3].Discovery and Preclinical Pharmacology of NX-2127, an Orally Bioavailable Degrader of Bruton's Tyrosine Kinase with Immunomodulatory Activity for the Treatment of Patients with B Cell Malignancies. J Med Chem. 2024 Feb 22;67(4):2321-2336.

Zelebrudomide is an orally bioavailable chimeric targeting molecule (CTM) and targeted degrader of Bruton's tyrosine kinase (BTK), with potential immunomodulatory drug (IMiD) and antineoplastic activities. Zelebrudomide is comprised of a cereblon (CRBN)-binding moiety conjugated, via a linker, to a BTK-binding moiety. Upon administration, zelebrudomide targets and binds to BTK with its BTK-targeting moiety. Upon binding, the CRBN-binding moiety recruits CRBN, a component of the CRL4-CRBN E3 ubiquitin ligase complex. This catalyzes ubiquitination and proteasome-mediated degradation 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. This leads to an inhibition of the growth of malignant B-cells that overexpress BTK. In addition, zelebrudomide catalyzes the degradation of CRBN neosubstrates Aiolos (IKZF3) and Ikaros (IKZF1), two transcription factors regulating T-cell function. This modulates the activity of the immune system and increases the activation of T-lymphocytes, thereby increasing T-cell-mediated anti-tumor effects. 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. CRBN, the substrate recognition component of the CRL4-CRBN E3 ubiquitin ligase complex, plays a key role in the ubiquitination of certain proteins. Compared to BTK inhibitors, zelebrudomide may overcome tumor resistance associated with BTK inhibitor-induced resistance mutations.

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- Mechanism of Action: NX-2127 acts as a bifunctional molecule, recruiting CRBN E3 ligase to degrade BTK and IMiD neosubstrates (IKZF1/3). This dual activity inhibits BCR signaling and enhances T-cell-mediated antitumor immunity [3]
- Clinical Development: In phase 1 trials (NCT04830137), NX-2127 showed promising efficacy in relapsed/refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In 27 evaluable CLL patients, the overall response rate (ORR) was 40.7% (11 PRs), with responses durable for >12 months [2]
- Resistance Overcome: NX-2127 effectively degrades BTK mutants (C481S, L528W) that confer resistance to covalent BTK inhibitors, offering a mutation-agnostic therapeutic approach [3]

C39H45N9O5

719.831907987595

719.354

C, 65.07; H, 6.30; N, 17.51; O, 11.11

2416131-46-7

146559796

Light yellow to yellow solid powder

3.7

174

3

11

9

53

1380

1

C1(C(N)=O)=NC=C(N2CCCCC2)N=C1NC1=CC=C(C2CCN(C[C@@H]3CCN(C4C=CC5=C(C=4)C(=O)N(C4CCC(=O)NC4=O)C5=O)C3)CC2)C=C1

XLWJWCMQMBVNSG-ACXKHFGCSA-N

InChI=1S/C39H45N9O5/c40-35(50)34-36(43-32(21-41-34)46-15-2-1-3-16-46)42-27-6-4-25(5-7-27)26-13-17-45(18-14-26)22-24-12-19-47(23-24)28-8-9-29-30(20-28)39(53)48(38(29)52)31-10-11-33(49)44-37(31)51/h4-9,20-21,24,26,31H,1-3,10-19,22-23H2,(H2,40,50)(H,42,43)(H,44,49,51)/t24-,31?/m0/s1

3-[4-[1-[[(3S)-1-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]pyrrolidin-3-yl]methyl]piperidin-4-yl]anilino]-5-piperidin-1-ylpyrazine-2-carboxamide

NX-2127; 2416131-46-7; Zelebrudomide; 2-Pyrazinecarboxamide, 3-[[4-[1-[[(3S)-1-[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-1,3-dioxo-1H-isoindol-5-yl]-3-pyrrolidinyl]methyl]-4-piperidinyl]phenyl]amino]-5-(1-piperidinyl)-; 3-[4-[1-[[(3S)-1-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]pyrrolidin-3-yl]methyl]piperidin-4-yl]anilino]-5-piperidin-1-ylpyrazine-2-carboxamide; NX2127; LSC67HA8DE; SCHEMBL21947733;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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

DMSO: 100 mg/mL (138.92 mM)

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。