Entrectinib (NMS-E 628; RXDX101; ROZLYTREK)   中文名称: 恩曲替尼

TrkA (IC50 = 1 nM); TrkB (IC50 = 3 nM); TrkC (IC50 = 5 nM); ROS1 (IC50 = 12 nM); ALK (IC50 = 7 nM)

体外活性：Entrectinib 选择性阻断 ALK 依赖性细胞系的增殖，并有效抑制 ALK 依赖性信号传导。 Entrectinib 还高度抑制带有 EML4-ALK 重排的 NSCLC 细胞系 NCI-H2228 的细胞生长。激酶测定：Entrectinib 是一种有效的口服 Trk、ROS1 和 ALK 抑制剂；抑制 TrkA、TrkB、TrkC、ROS1 和 ALK，IC50 值分别为 1、3、5、12 和 7 nM。细胞测定：将 NLF、NLF-TrkB、SY5Y 或 SY5Y-TrkB 细胞接种于 96 孔板中，并暴露于不同浓度的药物（1、5、10、20、30、50 和 100 nM 的 entrectinib，1.5 μM Irino 和 50 μM TMZ，分别）一小时，然后添加 100 ng/mL BDNF。添加药物后 24、48 和 72 小时收获平板。使用标准 SRB 测定方案对板进行处理并分析细胞活力。

在携带 Karpas-299 和 SR-786 异种移植物的小鼠中，Entrectinib (po) 诱导肿瘤完全消退。在 NPM-ALK 转基因小鼠中，Entrectinib 诱导胸腺和淋巴结中观察到的肿瘤块完全消退。在NB异种移植模型中，Entrectinib联合治疗增强了常规化疗的疗效。

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在体内，连续十天口服携带Karpas - 299或SR - 786异种移植物的SCID小鼠，NMS - E628诱导肿瘤完全消退，离体分析表明，单次治疗后，剂量依赖性靶标调节可维持长达18小时。NMS‐E628在靶向T细胞表达人类NPM‐ALK的转基因小鼠白血病模型中也非常有效。后一种模型真实再现了人类ALCL的病理特征，用NMS - E628连续治疗NPM - ALK转基因小鼠仅3天，就能诱导胸腺和淋巴结中肿瘤块的完全消退。[3]

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神经母细胞瘤（NB）是最常见和最致命的儿童实体瘤之一。这些肿瘤的特征是临床异质性，从自发消退到无情进展，神经营养因子受体Trk家族在这种异质性行为中起着重要作用。我们想确定恩曲替尼（NMS-E 628）（RXDX-101，Ignyta，股份有限公司），一种口服Pan-Trk、Alk和Ros1抑制剂，在我们的NB模型中是否有效。在稳定转染TrkB的SH-SY5Y细胞系上研究了entrectinib作为单一药物或与化疗药物伊立替康（Irino）和替莫唑胺（TMZ）联合使用的体外效果。在NB异种移植物中研究了体内生长抑制活性，无论是单独使用还是与Irino TMZ联合使用。Entrectinib (NMS-E 628)在体外显著抑制了表达TrkB的NB细胞的生长，联合使用时显著增强了Irino TMZ的生长抑制作用。与对照组动物相比，单药治疗导致用entrectinib治疗的动物的肿瘤生长受到显著抑制[无事件生存率（EFS）p<0.0001]。与赋形剂或Irino TMZ治疗的动物相比，在Irino TMZ中添加entrectinib也显著改善了动物的EFS[联合用药与对照组的p<0.0001，联合用药与Irino TMZ=0.0012]。我们发现，entrectinib在体外和体内抑制了表达TrkB的NB细胞的生长，并在体内模型中增强了常规化疗的疗效。我们的数据表明，entrectinib是一种强效的Trk抑制剂，应在NBs和其他表达Trk的肿瘤的临床试验中进行测试[5]。

Entrectinib 抑制 TrkA、TrkB、TrkC、ROS1 和 ALK，IC50 值分别为 1、3、5、12 和 7 nM。它是一种强效且易于使用的 Trk、ROS1 和 ALK 口服抑制剂。

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涉及ALK酪氨酸激酶基因的染色体易位t(2;5)(p23;q35)导致NPM‐ALK融合蛋白的表达，该融合蛋白代表了间变性大细胞淋巴瘤亚群存活和增殖的驱动力。最近，在非小细胞肺癌患者中，ALK基因的染色体重排导致了一种新的融合变异EML4 - ALK，已被确定为一种低频事件，与EGFR和K - ras突变相互排斥。正如之前在NPM‐ALK中发现的那样，这种新的融合变体具有组成活性的ALK激酶，并被证明具有很强的致癌潜力。综上所述，这些发现支持了这样的假设，即ALK代表了肿瘤中含有易位ALK的ALCL和NSCLC患者癌症治疗的创新和有价值的靶点。[3]
在这里，我们进一步描述NMS - E628的临床前特征，NMS - E628是一种口服的ALK激酶活性小分子抑制剂。在广泛的人类肿瘤细胞系上的增殖分析表明，该化合物选择性地阻断ALK依赖性细胞系的增殖，并有效抑制ALK依赖性信号传导。[3]

将 NLF、NLF-TrkB、SY5Y 或 SY5Y-TrkB 细胞接种于 96 孔板中，并置于不同浓度的 entrectinib（1、5、10、20、30、50 和 100 nM、1.5 μM Irino 和 50 nM）中。 μM TMZ，分别）持续一小时。随后，添加 100 ng/mL 的 BDNF。添加药物后，在 24、48 和 72 小时后收获平板。准备好板，并使用 SRB 测定方案来分析细胞活力。

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体外实验及Western Blot分析[6]
为了确定肠替尼对TrkB磷酸化的抑制作用，在标准培养条件下，将细胞在10 cm3培养皿中培养到70-80%的合流度。细胞在2% FBS培养基中血清饥饿2小时，然后暴露于不同浓度的肠替尼(10 - 200 nM) 1小时。用100 ng/mL的TrkB配体，BDNF刺激细胞15分钟，然后收集总蛋白用于Western blots分析。用抗phospho Trk抗体(p-Trk, Tyr-490)或抗pan -Trk抗体确认Trk表达。使用抗phospho-Akt、抗phospho- erk1 /2抗体分析下游信号传导抑制作用，以总Akt、抗erk1 /2和肌动蛋白为负载对照。

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硫代胺B (SRB)测定[6]
采用Sulforhodamine B (SRB)法测定enterrectinib单用和与Irino-TMZ合用对表达trkb的NB细胞存活和生长的影响。将NLF、NLF- trkb、SY5Y或SY5Y- trkb细胞(5×103/孔)分别置于96孔板中，分别以不同浓度(1、5、10、20、30、50和100 nM的肠替尼、1.5 μM Irino和50 μM TMZ)暴露1小时，然后加入100 ng/mL的BDNF。在添加药物后24、48和72小时收获板。用标准SRB测定方案处理后分析细胞活力。所有体外实验一式三次，至少重复3次。

Male C57BL/6 mice (6-8 weeks old, 20-25 g; Bleomycin-induced pulmonary fibrosis model)[1].
20, 40, 60 mg/kg
Intragastric Administration; single daily for 7 days.

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Entrectinib (RXDX-101) is an orally available small molecule inhibitor of pan-Trk, Alk and Ros1 tyrosine kinases. It was dissolved in DMSO to obtain stocks for in vitro studies. For in vivo experiments, it was reconstituted in 0.5% methylcellulose (viscosity 400cP, 2% in H2O) containing 1% Tween 80 at a final dosing volume of 10 ml/kg (e.g., 0.2 ml for a 20 gm mouse). Entrectinib solution was stirred at RT for 30 min, and then sonicated in a water bath sonicator for 20 min. This formulation was made fresh every week. Animals were dosed BID, 7 days/week at 60 mg/kg.[6]

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In Vivo Experiments[6]
For the xenograft studies, animals were injected subcutaneously in the flank with 1 × 107 SY5Y-TrkB cells in 0.1 ml of Matrigel (BD Bioscience, Palo Alto, CA). Tumors were measured 2 times per week in 3 dimensions, and the volume calculated as follows: [(0.523xLxWxW)/1000]. Body weights were measured at least twice a week, and the dose of compound was adjusted accordingly. Treatment with entrectinib, Irino and TMZ started about 15–17 days after tumor inoculation when the average tumor size was 0.2 cm3. Mice were sacrificed when tumor volume reached 3 cm3. Tumors were harvested and flash frozen on dry ice for analysis of protein expression using Western blot. Tumor lysates were obtained using Fast Prep 24 System in the presence of a protease inhibitor cocktail and phosphatase inhibitor cocktail. The following antibodies were used for the Western blot: anti-TrkB (Abcam), anti-phospho- TrkB (Tyr816); anti-Trk (pan-Trk); anti-phospho-Akt (Ser473); anti-Akt; anti-phosphop44/42 Erk (Thr202/Tyr204); anti-p44/42 Erk; anti-Phospho-PLCγ1 (Tyr783) and anti- PLCγ1. Plasma was obtained at different times points after dosing for PK/PD studies.[6]

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Pharmacokinetic studies[6]
Entrectinib was dosed at 60 mg/kg BID, for the entire duration of the study. After the final dose was given, the blood samples were drawn from 4 mice per time point via retro-orbital bleeding and collected in heparinized tubes on wet ice. The plasma was then separated by centrifugation at 1200 g for 10 minutes at 4°C. The concentration of entrectinib (free base) was measured by LC-MS-MS. The pharmacokinetic analysis was performed using the Watson system, and plotted using GraphPad Prism (mean ±SD).

Absorption, Distribution and Excretion
Entrectinib has a Tmax of 4-5 h after administration of a single 600 mg dose. Food does not produce a significant effect on the extent of absorption.
After a single radio-labeled dose of entrectinib, 83% of radioactivity was present in the feces and 3% in the urine. Of the dose in the feces, 36% was present as entrectinib and 22% as M5.
Entrectinib has an apparent volume of distribution of 551 L. The active metabolite, M5, has an apparent volume of distribution of 81.1 L. Entrectinib is known to cross the blood-brain barrier.
The apparent clearance of entrectinib is 19.6 L/h while the apparent clearance of the active metabolite M5 is 52.4 L/h.

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Metabolism / Metabolites
CYP3A4 is responsible for 76% of entrectinib metabolism in humans including metabolism to the active metabolite, M5. M5 has similar pharmacological activity to entrectinib and exists at approximately 40% of the steady state concentration of the parent drug. In rats, six in vivo metabolites have been identified including N-dealkylated, N-oxide, hydroxylated, and glucuronide conjugated metabolites.

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Biological Half-Life
Entrectinib has a half-life of elimination of 20 h. The active metabolite, M5, has a half-life of 40 h.

Hepatotoxicity
In the prelicensure clinical trials of entrectinib in patients with NTRK fusion gene positive solid tumors and ROS1 fusion gene positive non-small cell lung cancer, liver test abnormalities were frequent although usually mild. Some degree of ALT elevation arose in 38% of entrectinib treated patients, but were above 5 times the upper limit of normal (ULN) in only 2% to 3% (although the incidence may have been underestimated as 4.5% of patients had no post-treatment liver function tests). In these trials that enrolled approximately 355 patients, entrectinib was discontinued early due to increased AST or ALT in 0.8% of patients. Thus, in preregistration trials of entrectinib there were no instances of clinically apparent liver injury with jaundice, but therapy was associated with a high rate of serum ALT elevations and the total clinical experience with its use has been limited. The product label for entrectinib recommends monitoring for routine liver tests before, at 2 week intervals during the first month of therapy, and monthly thereafter as clinically indicated.
Likelihood score: E* (unproven but suspect rare cause of clinically apparent liver injury).

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Protein Binding
Entrectinib is over 99% bound to plasma proteins.

[1]. Expert Opin Investig Drugs. 2015;24(11):1493-500.

[2]. Cancer Res (2015) 75 (15_Supplement): 5390.

[2]. Mol Cancer Ther (2009) 8 (12_Supplement): A244.

[4]. Thorac Cancer. 2022 Nov;13(21):3032-3041.

[5]. Clin Cancer Res. 2021 Feb 15;27(4):1184-1194.

[6]. Cancer Lett. 2016 Mar 28;372(2):179-86.

Pharmacodynamics
Entrectinib and its active metabolite suppress several pathways which contribute to cell survival and proliferation. This suppression shifts the balance in favor of apoptosis thereby preventing cancer cell growth and shrinking tumors.

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Introduction: Receptor tyrosine kinases (RTKs) and their signaling pathways, control normal cellular processes; however, their deregulation play important roles in malignant transformation. In advanced non-small cell lung cancer (NSCLC), the recognition of oncogenic activation of specific RTKs, has led to the development of molecularly targeted agents that only benefit roughly 20% of patients. Entrectinib is a pan-TRK, ROS1 and ALK inhibitor that has shown potent anti-neoplastic activity and tolerability in various neoplastic conditions, particularly NSCLC. Areas covered: This review outlines the pharmacokinetics, pharmacodynamics, mechanism of action, safety, tolerability, pre-clinical studies and clinical trials of entrectinib, a promising novel agent for the treatment of advanced solid tumors with molecular alterations of Trk-A, B and C, ROS1 or ALK. Expert opinion: Among the several experimental drugs under clinical development, entrectinib is emerging as an innovative and promising targeted agent. The encouraging antitumor activity reported in the Phase 1 studies, together with the acceptable toxicity profile, suggest that entrectinib, thanks to its peculiar mechanism of action, could play an important role in the treatment-strategies of multiple TRK-A, B, C, ROS1, and ALK- dependent solid tumors, including NSCLC and colorectal cancer. That being said, further evidence for its clinical use is still needed. [1]

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Purpose: Neuroblastoma (NB) is one of the most common and deadly solid tumors of childhood. The Trk family of neurotrophin receptors plays an important role in clinical behavior of NBs. Overexpression of TrkB and its ligand, BDNF, is associated with poor prognosis. We wanted to determine if RXDX-101, an oral pan-TRK, ROS1 and ALK inhibitor, would be effective in our NB xenograft model, either alone or in combination with conventional chemotherapy. Experimental Design: We tested the in vitro effects of RXDX-101 as a single agent, or in combination with the chemotherapeutic agents irinotecan and temozolomide (Irino-TMZ), using a subclone of the SH-SY5Y NB cell line transfected with TrkB. We also examined in vivo growth inhibition of TrkB-expressing NB xenografts with RXDX-101 alone or in combination with Irino-TMZ. Results: RXDX-101 significantly inhibited growth of TrkB-expressing NB cells in vitro. Enhanced in vitro inhibition was observed when RXDX-101 was used in combination with Irino-TMZ. Single agent therapy with RXDX-101 resulted in significant tumor growth inhibition compared to control animals [p<0.0001 for event-free survival (EFS)]. The addition of RXDX-101 to Irino-TMZ also significantly improved the EFS of animals compared to vehicle or Irino-TMZ treated animals (p<0.0001 for combination vs. control, p = 0.0012 for combination vs. Irino-TMZ). Conclusions: We show that RXDX-101 inhibits growth of TrkB expressing NB cells in vitro and in vivo. Furthermore, RXDX-101 cotreatment enhanced the efficacy of conventional chemotherapy in our NB xenograft model. Our data suggest that RXDX-101 has potential for incorporation in clinical trials for NB and other Trk expressing tumors. [2]

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The chromosomal translocation t(2;5)(p23;q35) involving the ALK tyrosine kinase gene results in expression of the NPM‐ALK fusion protein which represents the driving force for survival and proliferation of a subset of Anaplastic Large Cell Lymphoma. More recently, a distinct chromosomal rearrangement of the ALK gene leading to a new fusion variant EML4‐ALK, has been identified as a low frequency event, mutually exclusive with respect to EGFR and K‐ras mutation, in Non Small Cell Lung cancer patients. As previously found for NPM‐ALK, this new fusion variant has constitutively active ALK kinase and was demonstrated to have strong oncogenic potential. Taken together these findings support the hypothesis that ALK represents an innovative and valuable target for cancer therapy both in ALCL and NSCLC patients whose tumors harbor translocated ALK. Here we further describe the preclinical characterization of NMS‐E628, an orally available small‐molecule inhibitor of ALK kinase activity. Proliferation profiling on a wide panel of human tumor cell lines demonstrated that the compound selectively blocks proliferation of ALK‐dependent cell lines and potently inhibits ALK‐dependent signaling. In vivo, NMS‐E628 induced complete tumor regression when administered orally for ten consecutive days to SCID mice bearing Karpas‐299 or SR‐786 xenografts, with ex vivo analyses demonstrating dose‐dependent target modulation that was maintained for up to 18 hours after single treatment. NMS‐E628 was also highly efficacious in a transgenic mouse leukemia model in which human NPM‐ALK expression was targeted to T cells. In this latter model, which faithfully recapitulates pathological features of human ALCL, treatment of NPM‐ALK transgenic mice with NMS‐E628 for as little as 3 consecutive days induced complete regression of tumor masses observed in the thymus and in lymph nodes. NMS‐E628 was also highly efficacious in inhibiting the in vitro and in vivo growth of the NSCLC cell line NCI‐H2228, which bears the EML4‐ALK rearrangement. Complete regressions were also achieved in this model, and prolonged inhibition of ALK phosphorylation and downstream effector activation were observed at active doses. NMS‐E628 has favorable pharmacokinetic and toxicological properties and biodistribution analysis revealed that it is able to cross the blood‐brain barrier in different animal species. To confirm that therapeutic doses are reached in the brain, NCI‐H2228 cells were injected intracranially in nude mice and NMS‐E628 was administered orally with different schedules. Dose‐dependent increase in survival, together with inhibition of tumor growth as assessed by MRI, confirmed that NMS‐E628 does indeed possess antitumor activity in this setting, an important finding considering that a significant proportion of NSCLC patients develop brain metastases. [3]

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Background: ROS1 tyrosine kinase inhibitors (TKIs) have demonstrated significant clinical benefit for ROS1+ NSCLC patients. However, TKI resistance inevitably develops through ROS1 kinase domain (KD) modification or another kinase driving bypass signaling. While multiple TKIs have been designed to target ROS1 KD mutations, less is known about bypass signaling in TKI-resistant ROS1+ lung cancers. Methods: Utilizing a primary, patient-derived TPM3-ROS1 cell line (CUTO28), we derived an entrectinib-resistant line (CUTO28-ER). We evaluated proliferation and signaling responses to TKIs, and utilized RNA sequencing, whole exome sequencing, and fluorescence in situ hybridization to detect transcriptional, mutational, and copy number alterations, respectively. We substantiated in vitro findings using a CD74-ROS1 NSCLC patient's tumor samples. Last, we analyzed circulating tumor DNA (ctDNA) from ROS1+ NSCLC patients in the STARTRK-2 entrectinib trial to determine the prevalence of MET amplification. Results: CUTO28-ER cells did not exhibit ROS1 KD mutations. MET TKIs inhibited proliferation and downstream signaling and MET transcription was elevated in CUTO28-ER cells. CUTO28-ER cells displayed extrachromosomal (ecDNA) MET amplification without MET activating mutations, exon 14 skipping, or fusions. The CD74-ROS1 patient samples illustrated MET amplification while receiving ROS1 TKI. Finally, two of 105 (1.9%) entrectinib-resistant ROS1+ NSCLC STARTRK-2 patients with ctDNA analysis at enrollment and disease progression displayed MET amplification. Conclusions: Treatment with ROS1-selective inhibitors may lead to MET-mediated resistance. The discovery of ecDNA MET amplification is noteworthy, as ecDNA is associated with more aggressive cancers. Following progression on ROS1-selective inhibitors, MET gene testing and treatments targeting MET should be explored to overcome MET-driven resistance. [4]

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Purpose: Desmoplastic small round cell tumor (DSRCT) is a highly lethal intra-abdominal sarcoma of adolescents and young adults. DSRCT harbors a t(11;22)(p13:q12) that generates the EWSR1-WT1 chimeric transcription factor, the key oncogenic driver of DSRCT. EWSR1-WT1 rewires global gene expression networks and activates aberrant expression of targets that together mediate oncogenesis. EWSR1-WT1 also activates a neural gene expression program. Experimental design: Among these neural markers, we found prominent expression of neurotrophic tyrosine kinase receptor 3 (NTRK3), a druggable receptor tyrosine kinase. We investigated the regulation of NTRK3 by EWSR1-WT1 and its potential as a therapeutic target in vitro and in vivo, the latter using novel patient-derived models of DSRCT. Results: We found that EWSR1-WT1 binds upstream of NTRK3 and activates its transcription. NTRK3 mRNA is highly expressed in DSRCT compared with other major chimeric transcription factor-driven sarcomas and most DSRCTs are strongly immunoreactive for NTRK3 protein. Remarkably, expression of NTRK3 kinase domain mRNA in DSRCT is also higher than in cancers with NTRK3 fusions. Abrogation of NTRK3 expression by RNAi silencing reduces growth of DSRCT cells and pharmacologic targeting of NTRK3 with entrectinib is effective in both in vitro and in vivo models of DSRCT. Conclusions: Our results indicate that EWSR1-WT1 directly activates NTRK3 expression in DSRCT cells, which are dependent on its expression and activity for growth. Pharmacologic inhibition of NTRK3 by entrectinib significantly reduces growth of DSRCT cells both in vitro and in vivo, providing a rationale for clinical evaluation of NTRK3 as a therapeutic target in DSRCT. [5]

C31H34F2N6O2

560.64

560.271

C, 66.41; H, 6.11; F, 6.78; N, 14.99; O, 5.71

1108743-60-7

25141092

Off-white to light yellow solid powder

1.3±0.1 g/cm3

717.5±60.0 °C at 760 mmHg

387.7±32.9 °C

0.0±2.3 mmHg at 25°C

1.672

5.66

85.52

3

8

7

41

847

0

FC1C([H])=C(C([H])=C(C=1[H])C([H])([H])C1C([H])=C([H])C2=C(C=1[H])C(=NN2[H])N([H])C(C1C([H])=C([H])C(=C([H])C=1N([H])C1([H])C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H])=O)F

HAYYBYPASCDWEQ-UHFFFAOYSA-N

InChI=1S/C31H34F2N6O2/c1-38-8-10-39(11-9-38)25-3-4-26(29(19-25)34-24-6-12-41-13-7-24)31(40)35-30-27-17-20(2-5-28(27)36-37-30)14-21-15-22(32)18-23(33)16-21/h2-5,15-19,24,34H,6-14H2,1H3,(H2,35,36,37,40)

N-[5-[(3,5-difluorophenyl)methyl]-1H-indazol-3-yl]-4-(4-methylpiperazin-1-yl)-2-(oxan-4-ylamino)benzamide

Entrectinib, RXDX-101, NMS-E628; RXDX101; RXDX 101; Rozlytrek; RXDX-101; NMS-E628; Entrectinib (RXDX-101); entrectinibum; Entrectinib(rxdx-101); RXDX-101; NMS E628; NMS-E-628; trade name: ROZLYTREK

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: ≥ 2.5 mg/mL (4.46 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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 (4.46 mM) (饱和度未知) in 5% DMSO + 95% (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。
*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 (3.71 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澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 4 中的溶解度: 2.08 mg/mL (3.71 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将100μL 20.8mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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 5 中的溶解度: ≥ 2.08 mg/mL (3.71 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900 μL 玉米油中，混合均匀。

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配方 6 中的溶解度: 5 mg/mL (8.92 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。