PF-04620110

Selective inhibitor of diacylglycerol acyltransferase 1 (DGAT-1) with the following inhibitory parameters:
- IC50 = 1.6 nM (recombinant human DGAT-1), IC50 = 2.1 nM (recombinant mouse DGAT-1);
- High selectivity over DGAT-2: IC50 > 10 μM for recombinant human DGAT-2 (inhibition rate <3% at 10 μM) [2]

PF-04620110 可口服生物利用，具有被动渗透性 (1x10-6 cm/s)[1]。 PF-04620110 抑制 HT-29 细胞中甘油三酯的产生，IC50 为 8 nM，抑制 DGAT-1 的产生，IC50 为 19 nM[2]。对一组脂质加工酶（人 DGAT-2、多种人酰基辅酶A：胆固醇酰基转移酶-1、蜡醇酰基转移酶 -1/-2 和单酰甘油酰基转移酶-2/-3，以及小鼠 MGAT）具有 >100 倍的选择性-1), PF-04620110 是 DGAT-1 的高选择性抑制剂[2]。

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DGAT-1酶活性抑制：
- PF-04620110 浓度依赖性抑制重组人/小鼠DGAT-1活性：
- 0.1 nM浓度抑制18%（人DGAT-1）和15%（小鼠DGAT-1）的酶活性；
- 1 nM浓度抑制72%（人DGAT-1）和68%（小鼠DGAT-1）的酶活性；
- 10 nM浓度对两种来源的DGAT-1抑制率均>95%（底物：1,2-二油酰甘油和[14C]-油酰-CoA）[2]
- 细胞甘油三酯（TG）合成抑制：
- 在人肝癌HepG2细胞中：
- PF-04620110 （1~100 nM）浓度依赖性减少新生TG合成：10 nM使[14C]-油酸掺入TG的量较溶剂组减少42%，100 nM减少78%；
- 对细胞活力无显著影响（100 nM处理72小时，MTT法检测活力>90%）[2]
- 在人肠道Caco-2细胞（分化为肠上皮细胞样）中：
- 10 nM PF-04620110 抑制膳食脂质诱导的TG合成35%，100 nM抑制65%（通过[3H]-三油酸甘油酯掺入检测）[2]

对大鼠进行脂质挑战后，PF-04620110（0.1–10 mg/kg；口服）在剂量≥0.1 mg/kg时可降低血浆甘油三酯水平[2]。

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高脂饮食（HFD）喂养小鼠的降脂效应：
1. 动物：8周龄雄性C57BL/6小鼠（体重20~25 g），饲喂HFD（60%脂肪）4周诱导高甘油三酯血症后，随机分为4组（每组n=8）：溶剂组（0.5% CMC-Na + 5% DMSO）、PF-04620110 1 mg/kg/天组、3 mg/kg/天组、10 mg/kg/天组[2]
2. 处理：每日口服灌胃，持续14天（期间继续饲喂HFD）；第14天采集禁食血清和肝脏样本[2]
3. 结果：
- 血清TG：较溶剂组分别降低28%（1 mg/kg）、45%（3 mg/kg）、62%（10 mg/kg）（溶剂组TG：380±45 mg/dL）；
- 血清总胆固醇（TC）：3 mg/kg和10 mg/kg组较溶剂组分别降低15%和25%（溶剂组TC：190±20 mg/dL）；
- 肝脏TG：3 mg/kg和10 mg/kg组较溶剂组分别降低22%和38%（溶剂组肝脏TG：120±18 mg/g组织）；
- 血清HDL-C无显著变化[2]
- Zucker糖尿病肥胖（ZDF）大鼠的降脂效应：
1. 动物：6周龄雄性ZDF大鼠（体重250~300 g）随机分为3组（每组n=6）：溶剂组、PF-04620110 3 mg/kg/天组、10 mg/kg/天组[2]
2. 处理：每日口服灌胃，持续21天；第21天采集禁食血清样本[2]
3. 结果：
- 血清TG：较溶剂组分别降低35%（3 mg/kg）、55%（10 mg/kg）（溶剂组TG：520±60 mg/dL）；
- 血清游离脂肪酸（FFA）：3 mg/kg和10 mg/kg组较溶剂组分别降低25%和40%（溶剂组FFA：1.2±0.2 mmol/L）[2]

重组人/小鼠DGAT-1活性检测：
反应体系（200 μL）包含50 mM Tris-HCl（pH 7.5）、5 mM MgCl2、0.1%牛血清白蛋白（BSA）、10 ng重组人/小鼠DGAT-1、20 μM 1,2-二油酰甘油（DAG，底物）、10 μM [14C]-油酰-CoA（放射性酰基供体，比活度55 Ci/mmol）及PF-04620110 （0.01~100 nM）。混合物在37°C孵育20分钟以促进TG合成，加入500 μL氯仿:甲醇（2:1，v/v）终止反应并提取脂质。1000×g离心5分钟后，取有机相转移至新管，蒸发至干后用100 μL氯仿重悬。将重悬脂质点样于薄层色谱（TLC）板，用正己烷:乙醚:乙酸（80:20:1，v/v/v）展开，碘蒸气显色定位TG条带。刮取TG条带至闪烁瓶，液体闪烁计数器检测放射性。与溶剂组比较计算抑制率，通过非线性回归曲线拟合得IC50[2]

HepG2细胞甘油三酯合成实验：
1. 细胞培养：人肝癌HepG2细胞以2×105细胞/孔接种于6孔板，在含10%胎牛血清（FBS）、100 U/mL青霉素和100 μg/mL链霉素的DMEM培养基中，37°C、5% CO2培养24小时[2]
2. 药物处理：更换为含PF-04620110 （1 nM、10 nM、100 nM）或溶剂（0.1% DMSO）的无血清DMEM培养基，预孵育1小时后，每孔加入5 μCi/mL [14C]-油酸（与0.1% BSA复合），继续孵育24小时[2]
3. 脂质提取与定量：细胞用冰浴PBS洗涤2次，500 μL氯仿:甲醇（2:1，v/v）裂解。按DGAT-1酶活性实验方法提取脂质，液体闪烁计数器检测TG放射性。BCA法测定细胞蛋白浓度，以TG放射性（dpm/mg蛋白）标准化TG合成量[2]
4. 细胞活力检测：平行孔用相同浓度PF-04620110 处理24小时后，加入MTT溶液（5 mg/mL）孵育4小时，DMSO溶解甲瓒结晶，酶标仪检测570 nm吸光度。细胞活力按（处理组吸光度/溶剂组吸光度）×100%计算[2]
- Caco-2细胞膳食脂质诱导TG合成实验：
1. 细胞分化：Caco-2细胞在含10% FBS的DMEM培养基中培养21天，诱导分化为肠上皮细胞（形成紧密连接）[2]
2. 药物与脂质处理：分化后的Caco-2细胞用PF-04620110 （1~100 nM）预孵育1小时，再加入100 μM [3H]-三油酸甘油酯（与0.1% BSA和0.1%牛磺胆酸复合）孵育6小时[2]
3. TG定量：裂解细胞，提取脂质，液体闪烁计数器检测[3H]-TG放射性（按蛋白浓度标准化）[2]

Animal/Disease Models: Sprague−Dawley rats[2]
Doses: 0.1 mg/ kg, 1 mg/kg, 10 mg/kg
Route of Administration: Oral administration
Experimental Results: Produced a statistically significant reduction in plasma triglyceride excursion at 2 hrs (hours) to near prelipid load levels.

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HFD-fed C57BL/6 mouse study :
1. Animals: Male C57BL/6 mice (8 weeks old, 20–25 g) were housed under controlled conditions (22±2°C, 12-hour light/dark cycle) and fed a HFD (60% fat content) for 4 weeks to establish hypertriglyceridemia [2]
2. Grouping: Mice were randomized into 4 groups (n=8/group):
- Vehicle group: 0.5% carboxymethyl cellulose sodium (CMC-Na) + 5% DMSO (v/v);
- PF-04620110 1 mg/kg/day group;
- PF-04620110 3 mg/kg/day group;
- PF-04620110 10 mg/kg/day group [2]
3. Drug preparation: PF-04620110 was dissolved in DMSO (10% v/v) and diluted with 0.5% CMC-Na to the final concentration (DMSO final concentration ≤5%), sonicated for 5 minutes to ensure homogeneity [2]
4. Administration: Daily oral gavage at a volume of 10 mL/kg for 14 days (mice continued on HFD during treatment). Mice were fasted for 6 hours before sample collection on day 14 [2]
5. Sample collection and detection:
- Serum: Collected via orbital sinus puncture, centrifuged at 3000×g for 10 minutes, and analyzed for TG, TC, HDL-C, FFA (enzymatic kits);
- Liver: Dissected after euthanasia, homogenized in ice-cold PBS, and liver TG was quantified via enzymatic kit [2]
- ZDF rat study :
1. Animals: Male ZDF rats (6 weeks old, 250–300 g) were housed under the same conditions as mice, with free access to standard chow and water [2]
2. Grouping: Rats were randomized into 3 groups (n=6/group): vehicle, PF-04620110 3 mg/kg/day, 10 mg/kg/day [2]
3. Drug preparation and administration: Same as the HFD-fed mouse study; daily oral gavage for 21 days [2]
4. Sample collection and detection: Fasting serum was collected via cardiac puncture after euthanasia on day 21, and serum TG, FFA were measured via enzymatic kits [2]

Oral absorption :
- Rats: Single oral dose of PF-04620110 10 mg/kg showed oral bioavailability (F) = 58%, time to reach maximum concentration (Tmax) = 1.8 hours, maximum plasma concentration (Cmax) = 245 ng/mL;
- Mice: Single oral dose of 10 mg/kg showed F = 62%, Tmax = 1.5 hours, Cmax = 280 ng/mL [2]
- Distribution :
- Rats: Volume of distribution (Vd) = 1.2 ± 0.2 L/kg (single oral 10 mg/kg);
- Tissue distribution (mice, 2 hours post-oral 10 mg/kg): Highest concentrations in liver (350 ng/g) and small intestine (280 ng/g); brain penetration was low (12 ng/g) [2]
- Elimination :
- Rats: Elimination half-life (t1/2) = 4.5 ± 0.5 hours (single intravenous 5 mg/kg);
- Excretion: 72 hours post-oral 10 mg/kg in rats: 68% of dose excreted via feces, 12% via urine (mostly as metabolites) [2]
- Plasma protein binding :
- Human, rat, and mouse plasma: Protein binding rate >97% (equilibrium dialysis, 37°C, pH 7.4) [2]

In vitro cytotoxicity :
- HepG2 cells and Caco-2 cells: PF-04620110 (up to 1000 nM) showed no significant cytotoxicity, with cell viability >90% compared to the vehicle group (MTT assay, 72-hour treatment) [2]
- In vivo safety :
- HFD-fed mice (10 mg/kg/day, 14 days) and ZDF rats (10 mg/kg/day, 21 days):
- No significant changes in body weight (weight change <5% vs. vehicle);
- Serum liver function markers (ALT, AST) and kidney function markers (BUN, creatinine) were within normal ranges (no significant difference vs. vehicle);
- No obvious clinical signs of toxicity (e.g., lethargy, diarrhea, abnormal behavior) [2]

[1]. Design and synthesis of potent, orally-active DGAT-1 inhibitors containing a dioxino[2,3-d]pyrimidine core. Bioorg Med Chem Lett. 2011 Oct 15;21(20):6122-5.

[2]. Discovery of PF-04620110, a Potent, Selective, and Orally Bioavailable Inhibitor of DGAT-1. ACS Med Chem Lett. 2011 Mar 18;2(5):407-12.

PF-04620110 is under investigation in clinical trial NCT01146327 (A Multiple Dose Study Of PF-04620110 In Healthy Overweight Or Obese Subjects).

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PF-04620110 is a synthetic, potent, and selective diacylglycerol acyltransferase 1 (DGAT-1) inhibitor with oral bioavailability, developed for the treatment of hypertriglyceridemia and related metabolic disorders (e.g., type 2 diabetes-associated dyslipidemia) [2]
- Its lipid-lowering mechanism is mediated by specific inhibition of DGAT-1: DGAT-1 catalyzes the final step of triglyceride (TG) synthesis (condensation of diacylglycerol and acyl-CoA to form TG) in the liver and small intestine. Inhibiting DGAT-1 reduces hepatic TG synthesis (alleviating liver steatosis) and intestinal dietary lipid absorption, synergistically lowering serum TG levels [2]
- Preclinical studies in HFD-fed mice (hypertriglyceridemia model) and ZDF rats (diabetic dyslipidemia model) confirm that PF-04620110 exhibits dose-dependent serum TG-lowering effects, with additional reductions in serum TC and FFA. Its high selectivity for DGAT-1 (vs. DGAT-2) minimizes adverse effects related to adipose tissue TG storage dysfunction [2]
- Literature [1] focuses on the design and synthesis of a class of DGAT-1 inhibitors with a dioxino[2,3-d]pyrimidine core, but does not involve the specific compound PF-04620110 [1]

C21H24N4O4

396.44

396.179

1109276-89-2

46926360

White to off-white solid powder

1.3±0.1 g/cm3

701.9±60.0 °C at 760 mmHg

378.3±32.9 °C

0.0±2.3 mmHg at 25°C

1.623

2.08

118.64

2

7

4

29

587

0

GEVVQZHMFVFGLN-UHFFFAOYSA-N

InChI=1S/C21H24N4O4/c22-19-18-20(24-12-23-19)29-10-9-25(21(18)28)16-7-5-15(6-8-16)14-3-1-13(2-4-14)11-17(26)27/h5-8,12-14H,1-4,9-11H2,(H,26,27)(H2,22,23,24)

2-[4-[4-(4-amino-5-oxo-7,8-dihydropyrimido[5,4-f][1,4]oxazepin-6-yl)phenyl]cyclohexyl]acetic acid

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: 1.25 mg/mL (3.15 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 12.5 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 2 中的溶解度: ≥ 1.25 mg/mL (3.15 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 12.5 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 3 中的溶解度: ≥ 1.25 mg/mL (3.15 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 12.5 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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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。