HIF-PHI/hypoxia-inducible factor-prolyl-hydroxylase

在 PC12 细胞中，roxadustat（5-50 μM；6 小时）可显着减少 TBHP 诱导的细胞凋亡[2]。在 PC12 细胞中，roxadustat（50 μM；6 小时）可稳定 HIF-1α 蛋白表达[2]。 研究发现，与CoCl2或BLM组相比，roxadustat治疗后L929细胞的增殖受到抑制，I型胶原、III型胶原、脯氨酰羟化酶结构域蛋白2（PHD2）、HIF-1α、α-平滑肌肌动蛋白（α-SMA）、结缔组织生长因子（CTGF）、转化生长因子β1（TGF-β1）和p-Smad3的产生减少。[3]

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roxadustat对L929细胞体外增殖和蛋白表达的影响[3]
用CoCl2（50nM）刺激L929细胞，以模拟缺氧下的促纤维化环境（Pardo等人，2005）。在CoCl2诱导L929细胞72小时后，使用带有TMB的EdU细胞增殖试剂盒分析细胞增殖。CoCl2刺激组的增殖率明显高于对照组。与CoCl2刺激组相比，用不同浓度的罗沙度（0.3、1、3、10μM）处理的L929细胞组显示出明显的细胞增殖抑制率（图5A）。 对于蛋白质分析，L929细胞按照增殖试验中的方法进行处理。简而言之，提取并检查了蛋白质，如图5所示。与对照细胞相比，在用CoCl2（50 nM）刺激的细胞中，I型胶原、III型胶原和α-SMA的蛋白质表达显著增加。然而，与CoCl2刺激相比，roxadustat/罗沙度他汀治疗显著抑制了I型胶原、III型胶原和α-SMA的表达（图5B，E）。与对照组相比，CoCl2刺激的促纤维化后，TGF-β1、CTGF和p-Smad3的蛋白表达显著增加，但在CoCl2刺激组中，罗沙度他汀治疗后降低（图5D，G）。特别是，CoCl2或roxadustat处理对Smad3表达没有影响（图5D和G）。[3]
在常氧条件下，HIF活性的增加会增加用于治疗贫血的内源性促红细胞生成素的产生。Roxadustat可防止HIF分解，并在常氧条件下促进HIF活性（Malyszko，2016）。在常氧和缺氧条件下，罗沙度对HIF-1α的影响不同；因此，本研究探讨了罗沙度对L929细胞在常氧和缺氧条件下HIF-1α和PHD2表达的影响。我们的结果表明，在常氧条件下，罗沙度司他增加了HIF-1α活性并降低了PHD2，而在CoCl2刺激的L929细胞中，它在缺氧条件下降低了HIF-1 a活性（图5C，F）。[3]
为了确定TGF-β1激活的机制，使用SB525334抑制TGF-β1的激活。分析L929细胞增殖情况，并在细胞与roxadustat/罗沙度他（3μM）（不含或含1μM SB525334）孵育72小时后，检测TGF-β1、CTGF、Smad3、p-Smad3、HIF-1α、PHD2、α-SMA、I型胶原和III型胶原的蛋白表达水平（图6B-G）。我们的结果显示，与CoCl2刺激组相比，Roxadustat治疗组除Smad3外，所有蛋白质的表达水平均降低（图6D和G），并且在没有或有SB525334的情况下，L929细胞增殖也降低（图6A）。此外，SB525334组显示蛋白质表达和细胞增殖减少；与SB525334组相比，SB525334+罗沙度他汀组的蛋白质表达水平或细胞增殖率没有进一步降低（图6；P > 0.05). 这些发现表明，罗沙度他通过抑制TGF-β1的激活来减轻实验性肺纤维化。 [3]
为了确定Smad3激活的机制，使用SIS3抑制Smad3激活。对L929细胞增殖进行了分析，并在用罗沙度他（3μM）孵育细胞72小时后，检测了TGF-β1、CTGF、Smad3、p-Smad3、HIF-1α、PHD2、α-SMA、I型胶原和III型胶原的蛋白表达水平，其中不含或含有0.5μM SIS3（图7B-7I）。这些结果表明，与CoCl2刺激组相比，罗沙度他汀治疗组除TGF-β1和Smad3外，所有蛋白质的表达水平均降低（图7E和I），并且在没有或有SIS3的情况下，L929细胞增殖也降低（图7A）。单独用SIS3治疗降低了蛋白质的表达和细胞增殖的程度。然而，与单独使用SIS3相比，使用SIS3+Roxadustat治疗并没有进一步降低蛋白质表达水平或细胞增殖率（P > 0.05; 图7）。这些发现表明，罗沙度他通过抑制p-Smad3表达来减轻实验性肺纤维化[3]。

改善脊髓损伤的恢复和保护运动神经元的存活是罗沙司他（50 mg/kg；腹腔注射；每天一次，持续 7 天）的两个好处[2]。

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FG-4592/roxadustat给药还改善了小鼠模型脊髓损伤中神经元的恢复并提高了其存活率。包括特异性HIF-1α阻断剂YC-1在内的联合治疗下调了HIF-1α的表达，并部分消除了FG-4592的保护作用。综上所述，我们的研究结果表明，FG-4592在SCI恢复中的作用与HIF-1α的稳定和凋亡的抑制有关。总体而言，我们的研究表明，PHDI可能是SCI和人类中枢神经系统疾病后治疗干预的可行候选者[2]。

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roxadustat对肺系数和肺组织组织病理学变化的影响[3]
采用半定量方法通过HE染色评估组织病理学变化。在假小鼠中观察到完整清晰的肺泡、正常间质和少量炎性细胞（图1A1）。在BLM诱导的小鼠肺组织中检测到炎症和纤维化变化，如肺泡破坏和炎性细胞浸润（图1A2）。然而，与BLM诱导的小鼠相比，经roxadustat罗沙度他汀治疗的小鼠在炎性细胞浸润和肺间质增厚方面表现出显著改善，病理评分也显著降低（图1A3）。肺系数是肺重量与体重的比值，它反映了肺纤维化的程度。在肺纤维化的发展过程中，早期肺质量的增加可归因于细胞肿胀和毛细血管充血等因素，而在后期，它主要是由胶原纤维形成引起的。在BLM诱导的小鼠中，由于疾病状态，体重在早期逐渐增加，尽管一些小鼠的体重持续下降，这直接导致了肺系数的增加。在处死前记录小鼠的体重，记录肺组织的重量并计算肺系数。与BLM诱导的小鼠相比，roxadustat/罗沙度他治疗的小鼠的肺系数降低。

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roxadustat对肺组织胶原水平的影响[3]
采用Masson三色染色和蛋白质印迹法检测胶原蛋白沉积量。I型和III型胶原通过蛋白质印迹法定量（图2A，B），而胶原的组织化学定量采用Masson三色染色法（图2C1-C3；D）。与假手术小鼠相比，在BLM诱导的小鼠中，Masson三色染色观察到肺间质中有大量胶原蛋白沉积。然而，连续21天服用罗沙度他后，胶原蛋白含量显著降低（图2C1-C3中的蓝色胶原蛋白沉积）。HYP是一种特征性氨基酸，约占胶原蛋白总氨基酸的13%，是指示胶原蛋白积累的重要标志。与BLM诱导的小鼠相比，罗沙度司他治疗的小鼠HYP含量显著降低（图2E）。BLM组I型和III型胶原的表达高于假手术组（p<0.01）。然而，罗沙度他汀治疗组的I型和Ⅲ型胶原表达低于BLM诱导组。

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roxadustat对体内蛋白质表达的影响[3]
通过蛋白质印迹法测定肺组织中HIF-1α、PHD2、α-SMA、TGF-β1、p-Smad3、Smad3和CTGF的蛋白表达。与假小鼠相比，BLM诱导的小鼠HIF-1α、PHD2和α-SMA的表达更高，但罗沙都司他治疗的小鼠的表达更低（图3A，C；p < 0.01). 与假小鼠相比，BLM诱导的小鼠TGF-β1、p-Smad3和CTGF的表达更高，但罗沙度他汀治疗的小鼠表达更低（图3B，D；p < 0.01). 值得注意的是，Smad3的表达在BLM诱导组和罗沙度他汀治疗组中保持不变。

细胞凋亡分析[2]
细胞类型： PC12 细胞
测试浓度： 5、20、50 μM
孵育时间：6小时
实验结果：显着抑制TBHP诱导的细胞凋亡。

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蛋白质印迹分析[2]
细胞类型： PC12 细胞
测试浓度： 50 μM
孵育时间：6小时
实验结果：稳定HIF-1α蛋白表达。

Animal/Disease Models: 12-week female C57BL/6 mice[2]
Doses: 50 mg/ kg
Route of Administration: intraperitoneal (ip)injection; daily for 7 days
Experimental Results: Protected the survival of motor neurons and improved recovery from spinal cord injury.

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Bleomycin (BLM)-induced pulmonary fibrosis model in mice [3]
A total of 40 adult male C57BL/6 mice were housed in a standard animal laboratory at consistent temperature (22 °C ± 2 °C) and humidity (60 ± 10 %) condition, with free access to chow and water. After 7 days of adaptation, an animal model was established. Ten mice were randomly selected to form the control group. Another 30 mice were intraperitioneally injected with BLM (Invitrogen, Carlsbad, CA, USA) in 0.2 mL saline (50 mg/kg) at days 1, 4, 8, 11, 15, 18, 22 and 25 of the experiment. The control mice were intraperitoneally injected with an equal volume of saline, without BLM. The mice were maintained under good conditions for 2 weeks after BLM exposure, and the body weights of the mice were recorded every week. At day 39, 20 mice with better exposure to BLM were selected and randomly grouped in the BLM and the BLM + roxadustat groups. The BLM + group waroxadustats intragastrically with 20 mg/kg/day roxadustat (dose selection based on the daily dosage in anemia and based on the data from a pre-experiment on the anti-BLM-induced fibrosis in mice) (Zhang et al., 2019). The control and BLM mice were intragastrically administered with an equal amount of saline. At day 60, the lung tissues were collected, and the lung coefficients were determined using the following formula: the lung coefficient = Wet lung weight/body weight × 100 %. Next, the tissues were categorized into two portions: the left lungs tissues were fixed in 4% paraformaldehyde for histological examination and the right lungs tissue were stored in liquid nitrogen for western blotting. Before starting the main experiment, an exploratory preliminary expeiment was conducted on 5 groups (sham, BLM, BLM + Roxadustat 10 mg/kg/day, 20 mg/kg/day or 40 mg/kg/day) of mice, with 10 mice in each group. The method and duration of administration were the same as those in the main experiment. Indexes such as lung weight, lung coefficient, and hydroxyproline (HYP) levels of the mice were monitored. As roxadustat at the dosage of 20 mg/kg/day was found to reduce the lung coefficients and HYP levels, this dosage was used in the main experiment.

Absorption, Distribution and Excretion
Roxadustat plasma exposure (AUC and Cmax) increases dose-proportionally within the recommended therapeutic dose range. In a three times per week dosing regimen, steady-state roxadustat plasma concentrations are achieved within one week (three doses) with minimal accumulation. Maximum plasma concentrations (Cmax) are usually achieved at two hours post dose in the fasted state. Administration of roxadustat with food decreased Cmax by 25% but did not alter AUC as compared with the fasted state.
Following oral administration of radiolabelled roxadustat in healthy subjects, the mean recovery of radioactivity was 96% (50% in feces, 46% in urine). In feces, 28% of the dose was excreted as unchanged roxadustat. Less than 2% of the dose was recovered in urine as unchanged roxadustat.
The blood-to-plasma ratio of roxadustat is 0.6. The apparent volume of distribution at steady-state is 24 L.
The apparent total body clearance (CL/F) of roxadustat is 1.1 L/h in patients with CKD not on dialysis and 1.4 L/h in patients with CKD on dialysis.

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Metabolism / Metabolites
_In vitro_, roxadustat is a substrate for CYP2C8 and UGT1A9 enzymes. Roxadustat is primarily metabolized to hydroxy-roxadustat and roxadustat O-glucuronide. Unchanged roxadustat was the major circulating component in human plasma and detectable metabolites in human plasma constituted less than 10% of total drug-related material exposure. No human-specific metabolites were observed but roxadustat O-glucuronide was detected in human urine sample.

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Biological Half-Life
The mean effective half-life of roxadustat is approximately 15 hours in patients with CKD.

Protein Binding
Roxadustat is highly bound to human plasma proteins (approximately 99%), mainly to albumin.

[1]. Roxadustat (FG-4592) Versus Epoetin Alfa for Anemia in Patients Receiving MaintenanceHemodialysis: A Phase 2, Randomized, 6- to 19-Week, Open-Label, Active-Comparator, Dose-Ranging, Safety and Exploratory Efficacy Study. Am J Kidney Dis. 2016 Jun;67(6):912-24.

[2]. Stabilization of HIF-1α by FG-4592 promotes functional recovery and neural protection in experimental spinal cord injury. Brain Res. 2016 Feb 1;1632:19-26.

[3]. Roxadustat attenuates experimental pulmonary fibrosis in vitro and in vivo. Toxicol Lett. 2020 Oct 1;331:112-121.

Roxadustat is an N-acylglycine resulting from the formal condensation of the amino group of glycine with the carboxy group of 4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxylic acid. It is an inhibitor of hypoxia inducible factor prolyl hydroxylase (HIF-PH). It has a role as an EC 1.14.11.2 (procollagen-proline dioxygenase) inhibitor and an EC 1.14.11.29 (hypoxia-inducible factor-proline dioxygenase) inhibitor. It is a member of isoquinolines, an aromatic ether and a N-acylglycine.
Roxadustat is a first-in-class hypoxia-inducible factor prolyl hydroxylase inhibitor used to treat anemia associated with chronic kidney disease. It works by reducing the breakdown of the hypoxia-inducible factor (HIF), which is a transcription factor that stimulates red blood cell production in response to low oxygen levels. Roxadustat was first approved by the European Commission in August 2021.
Roxadustat is an orally bioavailable, hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI), with potential anti-anemic activity. Upon administration, roxadustat binds to and inhibits HIF-PHI, an enzyme responsible for the degradation of transcription factors in the HIF family under normal oxygen conditions. This prevents HIF breakdown and promotes HIF activity. Increased HIF activity leads to an increase in endogenous erythropoietin production, thereby enhancing erythropoiesis. It also reduces the expression of the peptide hormone hepcidin, improves iron availability, and boosts hemoglobin (Hb) levels. HIF regulates the expression of genes in response to reduced oxygen levels, including genes required for erythropoiesis and iron metabolism.

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Drug Indication
Roxadustat is indicated for the treatment of adult patients with symptomatic anemia associated with chronic kidney disease (CKD).
Evrenzo is indicated for treatment of adult patients with symptomatic anaemia associated with chronic kidney disease (CKD).
Treatment of anaemia due to chronic disorders

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Mechanism of Action
Anemia is a common complication of chronic kidney disease that may be caused by reduced production of renal erythropoietin (EPO), functional iron deficiency due to increased levels of hepcidin, blood loss, reduced erythrocyte survival duration, and inflammation. Hypoxia-inducible factor (HIF) is a transcription factor that induces several target oxygen-sensitive genes in response to low oxygen levels in the cellular environment, or hypoxia. Target genes are involved in erythropoiesis, such as those for EPO, EPO receptor, proteins promoting iron absorption, iron transport, and haem synthesis. Activation of the HIF pathway is an important adaptive responsive to hypoxia to increase red blood cell production. HIF is heterodimeric and contains an oxygen-regulated α-subunit. The α-subunit houses an oxygen-dependent degradation (ODD) domain that is regulated and hydroxylated by HIF-prolyl hydroxylase (HIF-PHD) enzymes under normoxic cellular conditions. HIF-PHD enzymes play a crucial role in maintaining a balance between oxygen availability and HIF activity. Roxadustat is a reversible and potent inhibitor of HIF-PHD enzymes: inhibition of HIF-PHD leads to the accumulation of functional HIF, an increase in plasma endogenous EPO production, enhanced erythropoiesis, and indirect suppression of hepcidin, which is an iron regulator protein that is increased during inflammation in chronic kidney disease. Roxadustat can also regulate iron transporter proteins and regulates iron metabolism by increasing serum transferrin, intestinal iron absorption and the release of stored iron in patients with anemia associated with dialysis-dependent or dialysis-independent CKD. Overall, roxadustat improves iron bioavailability, increases Hb production, and increases red cell mass.

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Pharmacodynamics
Roxadustat dose-dependently improves iron bioavailability, increases hemoglobin production, and increases red blood cell mass in patients with anemia. In non-dialysis-dependent CKD patients with anemia, roxadustat maintained Hb for up to 2 years. It has a comparable efficacy to erythropoietin-stimulating agents in achieving Hb response. Roxadustat also reduces cholesterol levels from baseline, regardless of the use of statins or other lipid-lowering agents.

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Roxadustat is the first orally administered, small-molecule hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor that has been submitted for FDA regulatory approval to treat anemia secondary to chronic kidney diseases. Its usage has also been suggested for pulmonary fibrosis; however, the corresponding therapeutic effects remain to be investigated. The in vitro effects of roxadustat on cobalt chloride (CoCl2)-stimulated pulmonary fibrosis with L929 mouse fibroblasts as well as on an in vivo pulmonary fibrosismice model induced with bleomycin (BLM; intraperitoneal injection, 50 mg/kg twice a week for 4 continuous weeks) were investigated. It found that the proliferation of L929 cells was inhibited and the production of collagen I, collagen III, prolyl hydroxylase domain protein 2 (PHD2), HIF-1α, α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1) and p-Smad3 were reduced relative to that in the CoCl2 or BLM group after roxadustat treatment. Roxadustat ameliorated pulmonary fibrosis by reducing the pathology score and collagen deposition as well as decreasing the expression of collagen I, collagen III, PHD2, HIF-1α, α-SMA, CTGF, TGF-β1 and p-Smad3/Smad3. Our cumulative results demonstrate that roxadustat administration can attenuate experimental pulmonary fibrosis via the inhibition of TGF-β1/Smad activation.[3]

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Background: Roxadustat (FG-4592) is an oral hypoxia-inducible factor prolyl-hydroxylase inhibitor that promotes erythropoiesis through increasing endogenous erythropoietin, improving iron regulation, and reducing hepcidin. Study design: Phase 2, randomized (3:1), open-label, active-comparator, safety and efficacy study. Setting & participants: Patients with stable end-stage renal disease treated with hemodialysis who previously had hemoglobin (Hb) levels maintained with epoetin alfa. Intervention: Part 1: 6-week dose-ranging study in 54 individuals of thrice-weekly oral roxadustat doses versus continuation of intravenous epoetin alfa. Part 2: 19-week treatment in 90 individuals in 6 cohorts with various starting doses and adjustment rules (1.0-2.0mg/kg or tiered weight based) in individuals with a range of epoetin alfa responsiveness. Intravenous iron was prohibited. Outcomes: Primary end point was Hb level response, defined as end-of-treatment Hb level change (ΔHb) of -0.5g/dL or greater from baseline (part 1) and as mean Hb level ≥ 11.0g/dL during the last 4 treatment weeks (part 2). Measurements: Hepcidin, iron parameters, cholesterol, and plasma erythropoietin (the latter in a subset). Results: Baseline epoetin alfa doses were 138.3±51.3 (SD) and 136.3±47.7U/kg/wk in part 1 and 152.8±80.6 and 173.4±83.7U/kg/wk in part 2, in individuals randomly assigned to roxadustat and epoetin alfa, respectively. Hb level responder rates in part 1 were 79% in pooled roxadustat 1.5 to 2.0mg/kg compared to 33% in the epoetin alfa control arm (P=0.03). Hepcidin level reduction was greater at roxadustat 2.0mg/kg versus epoetin alfa (P<0.05). In part 2, the average roxadustat dose requirement for Hb level maintenance was ∼1.7mg/kg. The least-squares-mean ΔHb in roxadustat-treated individuals was comparable to that in epoetin alfa-treated individuals (about -0.5g/dL) and the least-squares-mean difference in ΔHb between both treatment arms was -0.03 (95% CI, -0.39 to 0.33) g/dL (mixed effect model-repeated measure). Roxadustat significantly reduced mean total cholesterol levels, not observed with epoetin alfa. No safety concerns were raised. Limitations: Short treatment duration and small sample size. Conclusions: In this phase 2 study of anemia therapy in patients with end-stage renal disease on maintenance hemodialysis therapy, roxadustat was well tolerated and effectively maintained Hb levels.[1]

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Previous studies have shown that inhibition of prolyl hydroxylase(PHD) stabilizes Hypoxia-inducible factor 1, alpha subunit(HIF-1α), increases tolerance to hypoxia, and improves the prognosis of many diseases. However, the role of PHD inhibitor (PHDI) in the recovery of spinal cord injury remains controversial. In this study, we investigated the protective role of a novel PHDI FG-4592 both in vivo and in vitro. FG-4592 treatment stabilized HIF1α expression both in PC12 cells and in spinal cord. FG-4592 treatment significantly inhibited tert-Butyl hydroperoxide(TBHP)-induced apoptosis and increases the survival of neuronal PC-12 cells. FG-4592 administration also improved recovery and increased the survival of neurons in spinal cord lesions in the mice model. Combination therapy including the specific HIF-1α blocker YC-1 down-regulated the HIF-1α expression and partially abolished the protective effect of FG-4592. Taken together, our results revealed that the role of FG-4592 in SCI recovery is related to the stabilization of HIF-1α and inhibition of apoptosis. Overall, our study suggests that PHDIs may be feasible candidates for therapeutic intervention after SCI and central nervous system disorders in humans.[2]

C19H16N2O5

352.34100

352.105

C, 64.77; H, 4.58; N, 7.95; O, 22.70

808118-40-3

Roxadustat-d5;2043026-13-5; 1537179-95-5 (potassium); 808118-40-3 (free); 1537180-01-0 (HCl); 1537179-94-4 (sodium); 1537180-03-2 (mesylate)

11256664

Light yellow to green yellow solid powder

1.4±0.1 g/cm3

684.3±55.0 °C at 760 mmHg

199-215°C

367.6±31.5 °C

0.0±2.2 mmHg at 25°C

1.674

3.9

108.75

3

6

5

26

508

0

O=C(O)CNC(C1=C(O)C2=C(C(C)=N1)C=C(OC3=CC=CC=C3)C=C2)=O

YOZBGTLTNGAVFU-UHFFFAOYSA-N

InChI=1S/C19H16N2O5/c1-11-15-9-13(26-12-5-3-2-4-6-12)7-8-14(15)18(24)17(21-11)19(25)20-10-16(22)23/h2-9,24H,10H2,1H3,(H,20,25)(H,22,23)

(4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carbonyl)glycine

Roxadustat; ASP1517; ASP 1517; Roxadustat (FG-4592); N-[(4-Hydroxy-1-methyl-7-phenoxy-3-isoquinolinyl)carbonyl]glycine; ASP-1517; FG-4592; FG4592; FG-4592;

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)

DMSO : ≥ 100 mg/mL (~283.82 mM)
H2O : < 0.1 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.10 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 25.0 mg/mL澄清DMSO储备液加入到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: ≥ 2.5 mg/mL (7.10 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 25.0 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 3 中的溶解度: ≥ 2.5 mg/mL (7.10 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 4 中的溶解度: 5 mg/mL (14.19 mM) in 0.5% CMC-Na/saline water (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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。