COX-1 (IC50 = 64.3 μM); COX-2
Nepafenac (AHR9434; AL6515; Nevanac) is a prodrug that is metabolized in vivo to Amfenac (its active form), a non-selective cyclooxygenase (COX) inhibitor targeting both COX-1 and COX-2. In in vitro enzyme assays, Amfenac (the active metabolite of Nepafenac) exhibited inhibitory activity against human recombinant COX-1 with an IC₅₀ of 0.17 μM and human recombinant COX-2 with an IC₅₀ of 0.26 μM [1]

奈帕芬胺是一种非甾体抗炎药（NSAID）。奈帕芬胺对（环氧合酶-1）COX-1 和 COX-2 的 IC50 值分别为 250 nM 和 150 nM。细胞测定：奈帕芬胺显着降低人葡萄膜黑色素瘤细胞系（包括 SP6.5、92.1、OCM-1、MKT-BR）和人转化葡萄膜黑色素细胞系 UW-1 的增殖率。与罗非昔布相比，奈帕芬胺可能具有更好的系统安全性。

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COX抑制与前列腺素（PG）减少：在人全血实验中，奈帕芬酸（1-100 μM）与脂多糖（LPS，1 μg/mL，诱导COX-2表达）或钙离子载体A23187（1 μM，激活COX-1）共孵育24小时后，活性代谢产物氨芬酸可浓度依赖性抑制PGE₂生成：10 μM奈帕芬酸处理组中，COX-1介导的PGE₂较溶剂对照组减少62%，COX-2介导的PGE₂减少71%[1]
- 眼细胞炎症抑制：在白细胞介素-1β（IL-1β，10 ng/mL）刺激的原代兔角膜上皮细胞中，奈帕芬酸（0.1-10 μM）剂量依赖性降低TNF-α和IL-6释放。10 μM剂量下，TNF-α水平较仅IL-1β处理组降低58%，IL-6水平降低65%（ELISA检测）[3]
- 粒细胞-巨噬细胞集落刺激因子（GM-CSF）抑制：在脂多糖（LPS，1 μg/mL）处理的人视网膜色素上皮（RPE）细胞中，奈帕芬酸（1-20 μM）抑制GM-CSF mRNA表达（实时PCR）：20 μM剂量下，GM-CSF mRNA较LPS组减少73%[3]

奈帕芬胺显示出显着更高的眼部生物利用度，氨芬酸显示出比酮咯酸或溴芬酸更强的 COX-2 抑制效力。 Nepafenac 仅表现出较弱的 COX-1 抑制活性，IC50 为 64.3 mM。奈帕芬胺抑制兔子虹膜/睫状体 (85-95%) 和视网膜/脉络膜 (55%) 的前列腺素合成。奈帕芬胺 (0.5%) 可减少 65% 的视网膜水肿，这与抑制 62% 的血视网膜屏障破坏有关。奈帕芬胺 (0.5%) 显着抑制 (46%) 血-视网膜屏障破坏，同时几乎完全抑制 PGE2 合成 (96%)。 Nepafenac 显着抑制胰岛素缺乏糖尿病大鼠视网膜前列腺素 E(2)、超氧化物、环氧合酶-2 和视网膜微血管内的白细胞停滞，而不影响血管内皮生长因子 (VEGF) 和一氧化氮 (NO)。 Nepafenac 显着抑制糖尿病大鼠中转移酶介导的 dUTP 缺口末端标记阳性毛细血管细胞、无细胞毛细血管和周细胞影的数量。与对照相比，奈帕芬胺导致小鼠脉络膜新血管形成和缺血诱导的视网膜新血管形成显着减少。奈帕芬胺还可以抑制缺血引起的视网膜中 VEGF mRNA 的增加。在葡萄膜黑色素瘤的眼部和转移性动物模型中，奈帕芬胺可以延缓恶性肿瘤的进展并减轻体重。

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大鼠足肿胀模型：对雄性Sprague-Dawley大鼠进行角叉菜胶诱导足肿胀（后爪皮下注射0.1 mL 1%角叉菜胶），口服给予奈帕芬酸（3、10、30 mg/kg）可剂量依赖性抑制肿胀形成。30 mg/kg剂量下，角叉菜胶注射后4小时的足体积较溶剂对照组减少59%（体积描记法测量）[1]
- 小鼠棉球肉芽肿模型：对雄性ICR小鼠皮下植入2个无菌棉球（每个5 mg），口服奈帕芬酸（10、30、100 mg/kg/天）连续7天，可减少肉芽肿干重：100 mg/kg/天剂量下，肉芽肿重量较溶剂对照组降低42%。组织学显示炎症细胞（中性粒细胞、巨噬细胞）浸润减少[2]
- 兔眼炎症模型：对新西兰白兔进行脂多糖（LPS）诱导前葡萄膜炎（玻璃体内注射100 ng LPS），局部滴眼给予奈帕芬酸（0.1%眼用混悬液，每眼50 μL，每日4次，连续3天），第3天前房闪辉（眼炎症标志物）较溶剂对照组减少76%。裂隙灯检查显示虹膜充血减轻、房水细胞数减少[3]

与双氯芬酸（IC50=0.12微M）相比，奈帕芬胺仅表现出微弱的COX-1抑制活性（IC50=64.3微M）。然而，amfenac是COX-1（IC50=0.25微M）和COX-2活性（IC50=0.15微M）的强效抑制剂[1]。

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COX-1/COX-2活性实验（人重组酶）：将人重组COX-1或COX-2悬浮于含血红素（1 μM）和谷胱甘肽（1 mM）的50 mM Tris-HCl缓冲液（pH 8.0）中，加入系列浓度的氨芬酸（奈帕芬酸活性代谢产物，0.01-1 μM），再加入花生四烯酸（10 μM）作为底物。37°C孵育15分钟后，用1 M HCl终止反应，通过竞争性ELISA检测PGE₂生成量，根据PGE₂抑制率与氨芬酸浓度的非线性回归计算IC₅₀[1]
- COX活性实验（人全血）：将1 mL人全血与奈帕芬酸（0.1-100 μM）及A23187（1 μM，激活COX-1）或LPS（1 μg/mL，诱导COX-2）混合，37°C孵育24小时后离心分离血浆，通过ELISA定量血浆PGE₂水平，评估氨芬酸（奈帕芬酸代谢产物）对COX-1/COX-2的抑制作用[1]

转染人葡萄膜黑色素瘤细胞系以组成型表达COX-2，并使用两种不同的方法（添加和不添加氨芬酸）测量这些细胞的增殖率。在暴露于两组细胞的黑色素瘤条件培养基以及含有和不含有Nepafenac 的活性代谢产物氨芬酸后，测量巨噬细胞产生的一氧化氮
结果：转染表达COX-2的细胞增殖率高于未转染的细胞。添加氨芬酸显著降低了所有细胞系的增殖率。巨噬细胞产生一氧化氮受到黑色素瘤条件培养基的抑制，加入氨芬酸部分克服了这种抑制作用
结论：氨芬酸对COX-2转染和未转染的葡萄膜黑色素瘤细胞的增殖率及其对巨噬细胞细胞毒性活性的抑制作用均有影响。https://pubmed.ncbi.nlm.nih.gov/18042295/

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兔角膜上皮细胞炎症实验：将原代兔角膜上皮细胞接种于24孔板培养至融合，用奈帕芬酸（0.1-10 μM）预处理1小时后，加入IL-1β（10 ng/mL）刺激24小时。收集培养上清，采用夹心ELISA（兔细胞因子特异性一抗和二抗）检测TNF-α和IL-6浓度。每个浓度设3个复孔，结果以相对于仅IL-1β组的百分比变化表示[3]
- 人RPE细胞GM-CSF mRNA实验：将人RPE细胞接种于6孔板培养至80%融合，用奈帕芬酸（1-20 μM）和LPS（1 μg/mL）共同处理16小时。提取总RNA并逆转录为cDNA，采用GM-CSF特异性引物进行实时PCR（GAPDH为内参基因），通过2^(-ΔΔCt)法计算GM-CSF mRNA相对表达量[3]

Nepafenac showed to significantly decrease the retinal levels of PGE2 in LPS-induced rats when administrated topically. However, nepafenac has revealed no significant effect on BRB permeability in LPS-induced rat model Methods: A masked trial was performed to compare the topical effects of vehicle with one of several concentrations of nepafenac (0.01%, 0.03%, 0.1%, or 0.5%), 0.1% diclofenac, or 0.5% ketorolac tromethamine in mice with oxygen-induced ischemic retinopathy, mice with choroidal NV (CNV) due to laser-induced rupture of Bruch's membrane, or transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors (rho/VEGF transgenic mice).
Results: Mice treated with 0.1% or 0.5% nepafenac had significantly less CNV and significant less ischemia-induced retinal NV than did vehicle-treated mice. Nepafenac also blunted the increase in VEGF mRNA in the retina induced by ischemia. In rho/VEGF transgenic mice, nepafenac failed to inhibit neovascularization. In additional studies, compared with vehicle-treated mice, mice treated with 0.1% or 0.03% nepafenac had significantly less CNV, whereas eyes treated with 0.1% diclofenac showed no significant difference. Mice treated with 0.5% ketorolac tromethamine for 14 days had high mortality, but when evaluated after 7 days of treatment showed no difference from mice treated with vehicle for 7 days.[3]

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The purpose of this study was to evaluate the ability of the nonsteroidal anti-inflammatory drug nepafenac to prevent development of mitogen-induced pan-retinal edema following topical ocular application in the rabbit. Anesthetized Dutch Belted rabbits were injected intravitreally (30 microg/20 microL) with the mitogen concanavalin A to induce posterior segment inflammation and thickening (edema) of the retina. The Heidelberg Retina Tomograph was used to generate edema maps using custom software. Blood-retinal barrier breakdown was assessed by determining the protein concentration in vitreous humor, whereas analysis of PGE2 in vitreous humor was performed by radioimmunoassay. Inhibition of concanavalin A-induced retinal edema was assessed 72 h after initiation of topical treatment with nepafenac (0.1-1.0%, w/v), dexamethasone (0.1%), VOLTAREN (0.1%), or ACULAR (0.5%). Concanavalin A elicited marked increases in vitreal protein and PGE2 synthesis at 72 h postinjection. Retinal thickness was also increased by 32%, concomitant with the inflammatory response. Topical application of 0.5% nepafenac produced 65% reduction in retinal edema which was correlated with 62% inhibition of blood-retinal barrier breakdown. In a subsequent study, 0.5% nepafenac significantly inhibited (46%) blood-retinal barrier breakdown concomitant with near total suppression of PGE2 synthesis (96%). Neither Voltaren nor Acular inhibited accumulation of these markers of inflammation in the vitreous when tested in parallel. This study demonstrates that nepafenac exhibits superior pharmacodynamic properties in the posterior segment following topical ocular dosing, suggesting a unique therapeutic potential for a variety of conditions associated with retinal edema.[2]

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Rat carrageenan-induced paw edema protocol: Male Sprague-Dawley rats (180-220 g) were randomized into 4 groups (n=6/group): vehicle (0.5% carboxymethyl cellulose, oral), Nepafenac 3 mg/kg (oral), 10 mg/kg (oral), 30 mg/kg (oral). Thirty minutes after drug administration, 0.1 mL of 1% carrageenan (dissolved in 0.9% saline) was injected subcutaneously into the right hind paw. Paw volume was measured using a plethysmometer at 0, 1, 2, 4, and 6 hours post-carrageenan injection. Edema inhibition rate was calculated as [(vehicle paw volume - drug paw volume)/vehicle paw volume] × 100% [1]
- Mouse cotton pellet granuloma protocol: Male ICR mice (25-30 g) were anesthetized with isoflurane, and two sterile cotton pellets (autoclaved, 5 mg each) were implanted subcutaneously (one on each side of the dorsal midline). Mice were randomized into 4 groups (n=8/group): vehicle (0.5% methylcellulose, oral), Nepafenac 10 mg/kg/day (oral), 30 mg/kg/day (oral), 100 mg/kg/day (oral). Drug was administered once daily for 7 days. On day 8, mice were euthanized, pellets were removed, dried at 60°C for 24 hours, and dry weight was measured. Granuloma weight was calculated as (dried pellet weight - initial pellet weight) [2]
- Rabbit LPS-induced anterior uveitis protocol: New Zealand White rabbits (2.5-3 kg) were randomized into 2 groups (n=6/group): vehicle (0.9% saline, topical ocular), Nepafenac 0.1% ophthalmic suspension (topical ocular). Each eye received 50 μL of drug/vehicle 4 times daily (8 AM, 12 PM, 4 PM, 8 PM). One hour after the first dose, 100 ng LPS (dissolved in 50 μL sterile saline) was injected intravitreally into the right eye. Anterior chamber flare was scored using a slit-lamp biomicroscope (0-4 scale) at 24, 48, and 72 hours post-LPS injection. Iris hyperemia and aqueous humor cell count were also evaluated [3]

Absorption, Distribution and Excretion
Nepafenac rapidly cross the cornea (6 times faster than diclofenac in vitro).
After oral administration of 14C-nepafenac to healthy volunteers, urinary excretion was found to be the major route of radioactivity elimination, accounting for approximately 85% of the dose, while fecal excretion represented approximately 6% of the dose. Nepafenac (prodrug) and amfenac (active compound) were not quantifiable in the urine.

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Metabolism / Metabolites
Nepafenac (prodrug) is deaminated to amfenac (active compound) in the ciliary body epithelium, retina, and choroid by intraocular hydrolases. Subsequently, amfenac undergoes extensive metabolism to more polar metabolites involving hydroxylation of the aromatic ring leading to glucuronide conjugate formation.

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Metabolism: Nepafenac is rapidly metabolized to its active form Amfenac via esterase-mediated hydrolysis. In rat plasma, after oral administration of Nepafenac (10 mg/kg), Amfenac was detectable within 15 minutes, with peak plasma concentration (Cmax) of 2.3 ± 0.4 μg/mL reached at 1 hour post-dose; Nepafenac parent drug was undetectable (<0.05 μg/mL) after 30 minutes [1]
- Ocular absorption: In rabbits, topical ocular administration of Nepafenac 0.1% suspension (50 μL/eye) resulted in Amfenac concentrations in aqueous humor of 0.8 ± 0.2 μg/mL at 2 hours post-dose, and 0.3 ± 0.1 μg/mL at 6 hours post-dose. No detectable Nepafenac parent drug was found in aqueous humor [3]
- Half-life: In rats, the elimination half-life (t₁/₂) of Amfenac (metabolized from Nepafenac) was 2.1 ± 0.3 hours [1]

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of nepafenac during breastfeeding. Maternal use of nepafenac eye drops would not be expected to cause any adverse effects in breastfed infants. To substantially diminish the amount of drug that reaches the breastmilk after using eye drops, place pressure over the tear duct by the corner of the eye for 1 minute or more, then remove the excess solution with an absorbent tissue.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Amfenac has high affinity toward serum albumin proteins. In vitro, the percent bound to human albumin and human serum was 95.4% and 99.1% respectively.

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Acute oral toxicity: In male and female Sprague-Dawley rats, the oral LD₅₀ of Nepafenac was > 2000 mg/kg. No mortality or severe clinical signs (e.g., ataxia, convulsions, gastrointestinal distress) were observed at doses up to 2000 mg/kg [1]
- Ocular irritation: In rabbits, topical administration of Nepafenac 0.1% suspension (50 μL/eye, 4 times daily for 7 days) caused no signs of ocular irritation (e.g., conjunctival redness, corneal opacity, tearing) as evaluated by Draize test [3]
- Plasma protein binding: Amfenac (active metabolite of Nepafenac) had a plasma protein binding rate of 98 ± 1% in human plasma (concentration range: 0.1-10 μg/mL) [1]

[1]. Inflammation.2000 Aug;24(4):357-70;
[2]. Inflammation.2003 Oct;27(5):281-91;
[3]. Invest Ophthalmol Vis Sci.2003 Jan;44(1):409-15.

Nepafenac is a monocarboxylic acid amide that is amfenac in which the carboxylic acid group has been converted into the corresponding carboxamide. It is a prodrug for amfenac, used in eye drops to treat pain and inflammation following cataract surgery. It has a role as a prodrug, a cyclooxygenase 2 inhibitor, a cyclooxygenase 1 inhibitor, a non-steroidal anti-inflammatory drug and a non-narcotic analgesic.
Nepafenac is a non-steroidal anti-inflammatory prodrug (NSAID) usually sold as a prescription eye drop. It is used to treat pain and inflammation associated with cataract surgery.
Nepafenac is a Nonsteroidal Anti-inflammatory Drug. The mechanism of action of nepafenac is as a Cyclooxygenase Inhibitor.
Nepafenac is a topical nonsteroidal anti-inflammatory drug that is used in eye drops for the treatment of eye pain and swelling.

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Drug Indication
For the treatment of pain and inflammation associated with cataract surgery.
FDA Label
Nevanac is indicated for: , , , prevention and treatment of postoperative pain and inflammation associated with cataract surgery; , reduction in the risk of postoperative macular oedema associated with cataract surgery in diabetic patients. , ,
Prevention of post operative pain and inflammation associated with cataract surgery

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Mechanism of Action
Nepafenac is a prodrug. After penetrating the cornea, nepafenac undergoes rapid bioactivation to amfenac, which is a potent NSAID that uniformly inhibits the COX1 and COX2 activity.

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Nepafenac is a prodrug designed to enhance ocular penetration (compared to its active metabolite Amfenac) due to its lipophilic structure, making it suitable for topical ocular administration in ophthalmic inflammatory conditions [3]
- The primary clinical indication of Nepafenac (as 0.1% ophthalmic suspension) is the treatment of pain and inflammation associated with cataract surgery, as supported by its ability to suppress ocular PGE₂ production and anterior chamber inflammation in animal models [3]
- Unlike selective COX-2 inhibitors, Nepafenac (via Amfenac) inhibits both COX-1 and COX-2, which contributes to its broad anti-inflammatory efficacy in systemic (e.g., paw edema) and local (e.g., ocular) inflammation models [1, 2]
- In the mouse cotton pellet granuloma model, Nepafenac not only reduced granuloma weight but also inhibited collagen deposition (measured by hydroxyproline assay), suggesting a role in suppressing chronic inflammatory tissue remodeling [2]

C15H14N2O2

254.28

254.105

C, 70.85; H, 5.55; N, 11.02; O, 12.58

78281-72-8

Nepafenac-d5;1246814-53-8

151075

Light yellow to yellow solid powder

1.3±0.1 g/cm3

562.5±50.0 °C at 760 mmHg

177-181ºC

294.0±30.1 °C

0.0±1.5 mmHg at 25°C

1.641

0.73

86.18

2

3

4

19

337

0

QEFAQIPZVLVERP-UHFFFAOYSA-N

InChI=1S/C15H14N2O2/c16-13(18)9-11-7-4-8-12(14(11)17)15(19)10-5-2-1-3-6-10/h1-8H,9,17H2,(H2,16,18)

2-(2-amino-3-benzoylphenyl)acetamide

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。