Ferroptosis; DPP-4 (IC50 = 1 nM)

体外活性：利格列汀在体外显示出对 DPP-4 的有效抑制作用，并且对 hERG 通道和 M1 受体具有低亲和力 (IC50 295 nM)。 Linagliptin 是一种竞争性抑制剂，Ki 为 1 nM，对 DPP-4 的选择性比 DPP-8、DPP-9、氨基肽酶 N 和 P、脯氨酰寡肽酶、胰蛋白酶、纤溶酶和凝血酶高 10,000 倍。体外选择性比成纤维细胞激活蛋白高 90 倍。激酶测定：EDTA 血浆 (20 μL) 用 30 μL DPP-4 测定缓冲液（100 mM Tris 和 100 mM NaCl，用 HCl 调节至 pH 7.8）稀释，并与 50 μL H-Ala-Pro-7- 混合。酰胺基-4-三氟甲基香豆素。将 200 mM 二甲基甲酰胺储备液用水按 1:1000 稀释，得到 100 μM 的终浓度。将板在室温下孵育 10 分钟，并使用 Victor 1420 Multilabel Counter 在 405 nm 的激发波长和 535 nm 的发射波长下测定孔中的荧光。为了检测伤口裂解物中的 DPP-4 活性，使用来自各个伤口裂解物的 100 μg 蛋白质代替 20 μL 血浆。还从 100 μg 各自的伤口组织样本中检测到活性 GLP-1，并使用小鼠/大鼠总活性 GLP-1 检测试剂盒进行分析。细胞测定：将每孔总共4.0×107个角质形成细胞接种到24孔板中。达到 50% 汇合后，用 DMEM 将细胞饥饿 24 小时。在存在 10% 胎牛血清和增加利格列汀浓度（3、30、300 或 600 nM）的 DMEM 中使用 1 μCi/mL [3H]甲基胸苷 24 小时来评估细胞增殖。然后用磷酸盐缓冲盐水洗涤细胞两次，并在 5% 三氯乙酸中于 4°C 孵育 30 分钟，并将 DNA 在 0.5mol/LNaOH 中于 37°C 溶解 30 分钟。最后，确定[3H]胸苷掺入。

在雄性 Wistar 大鼠、Beagle 犬和恒河猴中，利格列汀对 DPP-4 表现出高效、持久和强效的抑制活性，口服 1 mg/kg 后，所有三个物种的抑制率均超过 70%。在口服葡萄糖耐量试验前 45 分钟，给 db/db 小鼠口服利格列汀，以剂量依赖性方式将血浆葡萄糖偏移从 0.1 mg/kg（15% 抑制）降低至 1 mg/kg（66% 抑制）。通过抑制 DPP-4 活性，利格列汀可降低促炎标记物环氧合酶-2 和巨噬细胞炎症蛋白-2 的表达，并增强 ob/ob 小鼠伤口愈合过程中肌成纤维细胞的形成。

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在HanWistar大鼠中，BI 1356给药后24小时的DPP-4抑制作用比任何其他DPP-4抑制剂都更为显著[2]。 在C57BL/6J小鼠和Zucker fatty（fa/fa）大鼠中，对葡萄糖耐量的作用持续时间按BI 1356>（西格列汀/沙格列汀）>维格列汀的顺序减少。这些作用是通过控制胰高血糖素样肽-1和胰岛素介导的。总之，BI 1356比维达列汀、西格列汀、沙格列汀和阿格列汀更有效地抑制DPP-4，有可能成为第一个真正每天一次治疗2型糖尿病的DPP-4抑制剂。[2]

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近年来，用于血糖控制的新型有效治疗剂已被添加到标准糖尿病治疗中：二肽基肽酶-4（DPP-4）抑制剂，可延长内源性分泌的肠促胰岛素激素胰高血糖素样肽-1（GLP-1）的生物利用度。在野生型（C57BL/6J）和糖尿病[C57BL/6J肥胖/肥胖（ob/ob）]小鼠中进行全层切除损伤。在愈合过程中口服利格列汀可抑制DPP-4活性。使用组织学、分子和生化技术分析伤口组织。在健康小鼠中，DPP-4在无损伤皮肤的角质形成细胞中组成型表达。皮肤损伤后，DPP-4表达下降，在组织重组最活跃的阶段最低。相比之下，在ob/ob小鼠中，我们观察到DPP-4水平在晚期增加，此时延迟的组织修复仍然发生。口服DPP-4抑制剂利格列汀显著降低了DPP-4活性，稳定了慢性伤口中的活性GLP-1，并改善了ob/ob小鼠的愈合。在受伤后第10天，利格列汀治疗的ob/ob小鼠显示出大量上皮化的伤口，其特征是缺乏中性粒细胞。此外，DPP-4抑制降低了促炎标志物环氧化酶-2和巨噬细胞炎性蛋白-2的表达，但增强了ob/ob小鼠愈合伤口中肌成纤维细胞的形成。我们的数据表明，DPP-4抑制在糖尿病影响的伤口愈合中具有潜在的有益作用[3]。

EDTA 血浆 (20 μL) 用 30 μL DPP-4 测定缓冲液（100 mM Tris 和 100 mM NaCl，校正 pH 7.8）稀释后与 50 μL H-Ala-Pro-7-amido-4-三氟甲基香豆素混合。与 HCl）。为了获得 100 μM 的终浓度，将二甲基甲酰胺中的 200 mM 储备液用水按 1:1000 稀释。室温孵育 10 分钟后，使用 Victor 1420 Multilabel Counter 测量孔中的荧光，激发波长设置为 405 nm，发射波长设置为 535 nm。使用来自相应伤口裂解物的 100 μg 蛋白质代替 20 μL 血浆来检测裂解物中的 DPP-4 活性。利用小鼠/大鼠总活性 GLP-1 检测试剂盒，还可在 100 μg 相应伤口组织样本中鉴定出活性 GLP-1。

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BI 1356[拟议商品名ONDERO；（R）-8-（3-氨基哌啶-1-基）-7-丁-2-炔基-3-甲基-1-（4-甲基-喹唑啉-2-基甲基）-3,7-二氢-嘌呤-2,6-二酮]是一种新型二肽基肽酶（DPP）-4抑制剂，正在临床开发中，用于治疗2型糖尿病。在这项研究中，我们研究了BI 1356的体外和体内效力、选择性、机制和作用持续时间，并将其与其他DPP-4抑制剂进行了比较。BI 1356在体外抑制DPP-4活性，IC（50）约为1 nM，与西格列汀（19 nM）、阿格列汀（24 nM），沙格列汀（50 nM）和维达列汀（62 nM）相比。BI 1356是一种竞争性抑制剂，K（i）为1nM。BI 1356的计算k（关闭）速率为3.0 x 10（-5）/s（而维达列汀为2.1 x 10（-4）/s）。BI 1356对DPP-4的选择性是DPP-8、DPP-9、氨基肽酶N和P、脯氨酰肽酶、胰蛋白酶、纤溶酶和凝血酶的10000倍以上，在体外对成纤维细胞活化蛋白的选择性是其90倍以上[2]。

在 24 孔板中，每孔接种 4.0×10⁷ 角质形成细胞。 50% 汇合后，细胞用 DMEM 饥饿一整天。在含有 10% 胎牛血清和不同浓度利格列汀（3、30、300 或 600 nM）的 DMEM 中使用 1 μCi/mL [³H]甲基胸苷 24 小时，增殖测量细胞数。用磷酸盐缓冲盐水洗涤两轮后，将细胞在 5% 三氯乙酸中于 4°C 下孵育 30 分钟。随后，将 DNA 在 0.5mol/LNaOH 中于 37°C 溶解 30 分钟。最后发现[³H]胸苷的掺入。

There are ten separate ob/ob mice (n=10) in each experimental group (car or linagliptin treatment). Animals are given oral treatment once a day (8:00 AM) either with linagliptin (3 mg/kg body weight in 1% methylcellulose) or vehicle (1% methylcellulose) starting two days (day−2) prior to wounding. Animals that have been wounded are then given treatment once a day for ten days.

Absorption, Distribution and Excretion
Oral bioavailability of linagliptin is 30%.
84.7% of linagliptin is eliminated in the feces and 5.4% is eliminated in the urine.
A single intravenous dose of 5mg results in a volume of distribution of 1110L. However an intravenous infusion of 0.5-10mg results in a volume of distribution of 380-1540L.
Total clearance of linagliptin is 374mL/min.
Available animal data have shown excretion of linagliptin in milk at a milk-to-plasma ratio of 4:1.
After oral administration of a single 5-mg dose to healthy subjects, peak plasma concentrations of linagliptin occurred at approximately 1.5 hours post dose (Tmax); the mean plasma area under the curve (AUC) was 139 nmol*h/L and maximum concentration (Cmax) was 8.9 nmol/L.
The absolute bioavailability of linagliptin is approximately 30%. High-fat meal reduced Cmax by 15% and increased AUC by 4%; this effect is not clinically relevant. Tradjenta may be administered with or without food.
Following administration of an oral (14C)-linagliptin dose to healthy subjects, approximately 85% of the administered radioactivity was eliminated via the enterohepatic system (80%) or urine (5%) within 4 days of dosing. Renal clearance at steady state was approximately 70 mL/min.
For more Absorption, Distribution and Excretion (Complete) data for Linagliptin (6 total), please visit the HSDB record page.

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Metabolism / Metabolites
An oral dose of linagliptin is excreted primarily in the feces. 90% of an oral dose is excreted unchanged in the urine and feces. The predominant metabolite in the plasma is CD1790 and the predominant metabolite recovered after excretion was M489(1). Other metabolites are produced through oxidation, oxidative degradation, N-acetylation, glucuronidation, and cysteine adduct formation. Other metabolites have been identified through mass spectrometry though no structures were determined. Metabolism of linagliptin is mediated by cytochrome P450 3A4, aldo-keto reductases, and carbonyl reductases.
Following oral administration, the majority (about 90%) of linagliptin is excreted unchanged, indicating that metabolism represents a minor elimination pathway. A small fraction of absorbed linagliptin is metabolized to a pharmacologically inactive metabolite, which shows a steady-state exposure of 13.3% relative to linagliptin.

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Biological Half-Life
The terminal half life of linagliptin is 155 hours.
The effective half-life for accumulation of linagliptin, as determined from oral administration of multiple doses of linagliptin 5 mg, is approximately 12 hours.
Plasma concentrations of linagliptin decline in at least a biphasic manner with a long terminal half-life (>100 hours), related to the saturable binding of linagliptin to DPP-4.

Toxicity Summary
IDENTIFICATION AND USE: Linagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus, but should not be used in patients with type 1 diabetes or for the treatment of diabetic ketoacidosis. HUMAN EXPOSURE AND TOXICITY: In a pooled dataset of 14 placebo-controlled clinical trials, adverse reactions that occurred in > or = 2% of patients receiving Tradjenta (Linagliptin) (n = 3625) were nasopharyngitis (7.0%), diarrhea (3.3%), and cough (2.1%). Other adverse reactions reported in clinical studies with treatment of Tradjenta (Linagliptin) were hypersensitivity (e.g., urticaria, angioedema, localized skin exfoliation, or bronchial hyperreactivity), and myalgia. Additional adverse reactions have been identified during postapproval use of Tradjenta (Linagliptin); acute pancreatitis, including fatal pancreatitis, hypersensitivity reactions including anaphylaxis, angioedema, exfoliative skin conditions, and rash. ANIMAL STUDIES: Linagliptin did not increase the incidence of tumors in male and female rats in a 2-year study at doses of 6, 18, and 60 mg/kg. Linagliptin did not increase the incidence of tumors in mice in a 2-year study at doses up to 80 mg/kg (males) and 25 mg/kg (females). Higher doses of Linagliptin in female mice (80 mg/kg) increased the incidence of lymphoma. In fertility studies in rats, Linagliptin had no adverse effects on early embryonic development, mating, fertility, or bearing live young up to the highest dose of 240 mg/kg. Linagliptin crossed the placenta into the fetus following oral dosing in pregnant rats and rabbits and available animal data have shown excretion of linagliptin in milk at a milk-to-plasma ratio of 4:1. Linagliptin was not mutagenic or clastogenic with or without metabolic activation in the Ames bacterial mutagenicity assay, a chromosomal aberration test in human lymphocytes, and an in vivo micronucleus assay.

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Hepatotoxicity
In large clinical trials, rates of serum enzyme elevations were similar with linagliptin therapy (
Likelihood score: D (possible rare cause of clinically apparent acute liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of linagliptin during breastfeeding. Linagliptin's plasma protein binding ranges from 80% to over 99%, so it is unlikely to pass into breastmilk in clinically important amounts and might be a better choice among drugs in this class for nursing mothers. However, an alternate drug may be preferred, especially while nursing a newborn or preterm infant. Monitor breastfed infants for signs of hypoglycemia such as jitteriness, excessive sleepiness, poor feeding, seizures cyanosis, apnea, or hypothermia. If there is concern, monitoring of the breastfed infant's blood glucose is advisable during maternal therapy with linagliptin.[1]
◉ 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
Linagliptin is 99% protein bound at a concentration of 1nmol/L and 75-89% protein bound at a concentration of >30nmol/L.

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Interactions
Trajenta is not indicated in combination with insulin due to an increase in cardiovascular risk, which cannot be excluded.
Insulin secretagogues and insulin are known to cause hypoglycemia. The use of Tradjenta in combination with an insulin secretagogue (e.g., sulfonylurea) was associated with a higher rate of hypoglycemia compared with placebo in a clinical trial. The use of Tradjenta in combination with insulin in subjects with severe renal impairment was associated with a higher rate of hypoglycemia. Therefore, a lower dose of the insulin secretagogue or insulin may be required to reduce the risk of hypoglycemia when used in combination with Tradjenta.
Rifampin decreased linagliptin exposure, suggesting that the efficacy of Tradjenta may be reduced when administered in combination with a strong P-gp or CYP3A4 inducer. Therefore, use of alternative treatments is strongly recommended when linagliptin is to be administered with a strong P-gp or CYP3A4 inducer.
Sulfonylureas and insulin are known to cause hypoglycemia. Therefore, caution is advised when linagliptin is used in combination with a sulfonylurea and/or insulin. A dose reduction of the sulfonylurea or insulin, may be considered.
Linagliptin is a weak to moderate inhibitor of cytochrome P-450 (CYP) isoenzyme 3A4; however, it does not inhibit or induce CYP isoenzymes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, or 4A11 in vitro. In vivo studies indicate that drug interactions are unlikely with substrates of CYP isoenzymes 3A4, 2C9, or 2C8. No adjustment of linagliptin dosage is recommended based on results of pharmacokinetic studies. Inducers of CYP3A4 (e.g., rifampin) decrease exposure to linagliptin, resulting in subtherapeutic and likely ineffective concentrations. The manufacturer states that alternatives to linagliptin are strongly recommended in patients who require therapy with potent CYP3A4 inducers.

[1]. J Med Chem . 2007 Dec 27;50(26):6450-3.

[2]. J Pharmacol Exp Ther . 2008 Apr;325(1):175-82.

[3]. J Pharmacol Exp Ther . 2012 Jul;342(1):71-80.

[4]. Cell Rep . 2017 Aug 15;20(7):1692-1704.

Therapeutic Uses
Hypoglycemic Agents
Tradjenta tablets are indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. /Included in US product label/

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Drug Warnings
/BOXED WARNING/ WARNING: RISK OF LACTIC ACIDOSIS. Lactic acidosis is a rare, but serious, complication that can occur due to metformin accumulation. The risk increases with conditions such as renal impairment, sepsis, dehydration, excess alcohol intake, hepatic impairment, and acute congestive heart failure. The onset is often subtle, accompanied only by nonspecific symptoms such as malaise, myalgias, respiratory distress, increasing somnolence, and nonspecific abdominal distress. Laboratory abnormalities include low pH, increased anion gap, and elevated blood lactate. If acidosis is suspected, Jentadueto should be discontinued and the patient hospitalized immediately. /Linagliptin and metformin hydrochloride combination product/
FDA is evaluating unpublished new findings by a group of academic researchers that suggest an increased risk of pancreatitis and pre-cancerous cellular changes called pancreatic duct metaplasia in patients with type 2 diabetes treated with a class of drugs called incretin mimetics. These findings were based on examination of a small number of pancreatic tissue specimens taken from patients after they died from unspecified causes. FDA has asked the researchers to provide the methodology used to collect and study these specimens and to provide the tissue samples so the Agency can further investigate potential pancreatic toxicity associated with the incretin mimetics. Drugs in the incretin mimetic class include exenatide (Byetta, Bydureon), liraglutide (Victoza), sitagliptin (Januvia, Janumet, Janumet XR, Juvisync), saxagliptin (Onglyza, Kombiglyze XR), alogliptin (Nesina, Kazano, Oseni), and linagliptin (Tradjenta, Jentadueto). These drugs work by mimicking the incretin hormones that the body usually produces naturally to stimulate the release of insulin in response to a meal. They are used along with diet and exercise to lower blood sugar in adults with type 2 diabetes. FDA has not reached any new conclusions about safety risks with incretin mimetic drugs. This early communication is intended only to inform the public and health care professionals that the Agency intends to obtain and evaluate this new information. ... FDA will communicate its final conclusions and recommendations when its review is complete or when the Agency has additional information to report. The Warnings and Precautions section of drug labels and patient Medication Guides for incretin mimetics contain warnings about the risk of acute pancreatitis. FDA has not previously communicated about the potential risk of pre-cancerous findings of the pancreas with incretin mimetics. FDA has not concluded these drugs may cause or contribute to the development of pancreatic cancer. At this time, patients should continue to take their medicine as directed until they talk to their health care professional, and health care professionals should continue to follow the prescribing recommendations in the drug labels. ...
There have been postmarketing reports of acute pancreatitis, including fatal pancreatitis, in patients taking Tradjenta. Take careful notice of potential signs and symptoms of pancreatitis. If pancreatitis is suspected, promptly discontinue Tradjenta and initiate appropriate management. It is unknown whether patients with a history of pancreatitis are at increased risk for the development of pancreatitis while using Tradjenta.
There have been postmarketing reports of serious hypersensitivity reactions in patients treated with Tradjenta. These reactions include anaphylaxis, angioedema, and exfoliative skin conditions. Onset of these reactions occurred within the first 3 months after initiation of treatment with Tradjenta, with some reports occurring after the first dose. If a serious hypersensitivity reaction is suspected, discontinue Tradjenta, assess for other potential causes for the event, and institute alternative treatment for diabetes. Angioedema has also been reported with other dipeptidyl peptidase-4 (DPP-4) inhibitors. Use caution in a patient with a history of angioedema to another DPP-4 inhibitor because it is unknown whether such patients will be predisposed to angioedema with Tradjenta.
For more Drug Warnings (Complete) data for Linagliptin (20 total), please visit the HSDB record page.

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Pharmacodynamics
A 5mg oral dose of linagliptin results in >80% inhibition of dipeptidyl peptidase 4 (DPP-4) for ≥24 hours. Inhibition of DPP-4 increases the concentration of glucagon-like peptide 1 (GLP-1), leading to decreased glycosylated hemoglobin and fasting plasma glucose.

C25H28N8O2

472.54

472.233

C, 63.54; H, 5.97; N, 23.71; O, 6.77

668270-12-0

Linagliptin-d4;2140263-92-7;Linagliptin-13C,d3;1398044-43-3

10096344

White to yellow solid; also reported as a crystalline solid

1.4±0.1 g/cm3

661.2±65.0 °C at 760 mmHg

202ºC

353.7±34.3 °C

0.0±2.0 mmHg at 25°C

1.717

1.99

116.86

1

7

4

35

885

1

O=C1C2=C(N(C([H])([H])[H])C(N1C([H])([H])C1N=C(C([H])([H])[H])C3=C([H])C([H])=C([H])C([H])=C3N=1)=O)N=C(N2C([H])([H])C#CC([H])([H])[H])N1C([H])([H])C([H])([H])C([H])([H])[C@]([H])(C1([H])[H])N([H])[H]

LTXREWYXXSTFRX-QGZVFWFLSA-N

InChI=1S/C25H28N8O2/c1-4-5-13-32-21-22(29-24(32)31-12-8-9-17(26)14-31)30(3)25(35)33(23(21)34)15-20-27-16(2)18-10-6-7-11-19(18)28-20/h6-7,10-11,17H,8-9,12-15,26H2,1-3H3/t17-/m1/s1

8-[(3R)-3-aminopiperidin-1-yl]-7-but-2-ynyl-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]purine-2,6-dione

Linagliptin; BI-1356; BI1356; 668270-12-0; Tradjenta; Ondero; BI-1356; BI 1356; Trajenta; Trazenta; BI 1356; trade names: Tradjenta, Trajenta

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: ≥ 25 mg/mL (52.91 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 250.0 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 2 中的溶解度: 2.5 mg/mL (5.29 mM) 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; 超声助溶.
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 3 中的溶解度: ≥ 2.5 mg/mL (5.29 mM) (饱和度未知) 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), 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900 μL 20% SBE-β-CD生理盐水溶液中，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 4 中的溶解度: 0.5% hydroxyethyl cellulose: 30 mg/mL

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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。