Orlistat (Tetrahydrolipstatin)   中文名称: 奥利司他

Lipase; fatty acid synthase (FASN)

奥利司他（40 μM；2 天）使人外周血单核细胞、两种白细胞和另一种土耳其细胞系中的修复蛋白增加 30-70%，但对人黑色素瘤细胞系中的 MGMT 水平没有影响。

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四氢前列腺素（奥利司他）是一种脂肪酶和脂肪酸合酶抑制剂，长期口服用于治疗肥胖症。尽管该药物在体外对人癌症细胞以及在体外和体内对动物肿瘤具有显著的抗肿瘤活性，但它也会诱导大鼠结肠癌前病变。因此，研究人员测试了奥利司他对O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）表达的体外影响，MGMT是一种在控制突变和致癌中起重要作用的DNA修复酶。蛋白质印迹分析表明，连续暴露于40µM奥利司他2天不会影响人黑色素瘤细胞系中的MGMT水平，但会下调人外周血单核细胞、两种白血病和两种癌症细胞系中修复蛋白30-70%。另一方面，奥利司他没有明显改变MGMT mRNA的表达。与洛米加trib（一种假底物，MGMT的强抑制剂）不同，奥利司他在靶细胞暴露于该药物2小时后没有显著降低MGMT功能，这表明该药物不是修复蛋白的竞争性抑制剂。奥利司他和洛美加治疗显示MGMT水平呈加性降低。更重要的是，当奥利司他与具有致癌特性的DNA甲基化剂（如替莫唑胺）联合使用时，奥利司他介导的MGMT蛋白表达下调显著增强。总之，即使奥利司他几乎不被口服吸收，该药物也可能降低局部MGMT介导的对胃肠道上皮细胞DNA甲基化化合物引起的DNA损伤的保护作用，从而有利于化学致癌[1]。

与营养（OB）组相比，奥利司他（10 mg/kg/天）显着改善血脂状况，上调抗氧化酶表达和抗炎标志物表达，并下调促炎标志物表达[2]。

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本研究旨在确定奥利司他对肥胖雄性大鼠肥胖诱导的血管氧化应激的抗动脉粥样硬化作用。24只雄性Sprague-Dawley大鼠分为两组：正常组（正常组，n=6）和高脂饮食组（HFD组，n=12）。六周后，HFD组的肥胖大鼠被给予蒸馏水（OB组）或奥利司他10mg/kg/天（OB/or组），持续六周。与正常组相比，OB组的脂质谱（总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白（LDL））显著增加，高密度脂蛋白水平降低。与正常组相比，OB组的主动脉抗氧化酶活性（超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GPx）、谷胱甘肽还原酶（GR）、谷胱甘肽S-转移酶（GST）和过氧化氢酶（CAT））以及总谷胱甘肽（GSH）和总抗氧化能力（TAC）显著降低。此外，与正常组相比，OB组的促炎动脉粥样硬化标志物（肿瘤坏死因子-α（TNF-α）、血管细胞粘附分子-1（VCAM-1）和细胞间粘附分子-1）表达显著增加，抗炎标志物（白细胞介素-10（IL-10））显著降低。与OB组相比，奥利司他治疗显著改善了脂质状况，增加了抗氧化酶和抗炎标志物的表达，并降低了促炎标志物的表达式。这些发现可能表明奥利司他在减轻肥胖动脉粥样硬化阶段进展方面的治疗作用[2]。

MGMT活性测定[1]
通过测量3H-甲基从DNA底物转移到MGMT蛋白来测定MGMT活性。简而言之，将细胞颗粒（1×106个细胞）重新悬浮在1 ml补充有蛋白酶抑制剂混合物的裂解缓冲液（0.5%CHAPS、50 mM Tris-HCl pH 8.0、1 mM EDTA、3 mM二硫苏糖醇、100 mM NaCl、10%甘油）中，并在4°C下孵育30分钟。然后将细胞裂解物在4°C下以18000×g离心10分钟。然后将上清液的等分试样稀释在pH 8.3的50 mM Tris-HCl缓冲液中，该缓冲液含有1 mM EDTA和3 mM二硫苏糖醇，并在37°C下与10μg 3H甲基化DNA一起孵育1小时。然后在1 N高氯酸的存在下，在75°C下加热样品45分钟，使DNA水解，并使用1 mg牛血清白蛋白作为载体沉淀蛋白质。用1N高氯酸洗涤颗粒，重新悬浮在0.01N NaOH中，并在加入闪烁液后在液体闪烁计数器中测量放射性。根据Bradford的方法，使用Bio-Rad蛋白质测定染料试剂和牛血清白蛋白作为标准，评估上清液中的蛋白质浓度。MGMT活性以细胞提取物中每毫克蛋白质转移的3H-甲基的fmole表示。

Western Blot分析[1]
细胞类型：人黑色素瘤细胞系M10、外周血单核细胞、人Jurkat CD4+ T细胞白血病细胞系、人早幼粒细胞白血病细胞系HL-60。会显着改变 MGMT mRNA 表达 [1]。 、上皮结肠癌 HCT116 细胞、非贴壁单核细胞 (NAMNC) [1]
测试浓度：Jurkat 细胞为 2.5、5、10、20、40 μM； HCT116 细胞为 20 和 40 μM；正常 NAMNC、M10 黑色素瘤，40 μM 用于 HL-60 早幼粒细胞白血病和 HT-29 结肠癌细胞。
孵育时间：Jurkat 细胞为 2 天； HCT116 细胞 2 或 4 天； NAMNC、M10 黑色素瘤、HL-60 早幼粒细胞白血病、HT-29 结肠癌 2 天
实验结果：对于 Jurkat 细胞，MGMT 水平在浓度为 40 μM 时降低 > 50% ，而在较低浓度下，几乎没有看到任何影响。用过的。 HCT116 细胞中的 MGMT 表达在 40 μM 时下调。在 40 μM 浓度下，正常 NAMNC、HL-60 早幼粒细胞白血病、

Animal/Disease Models: Eighteen male SD (SD (Sprague-Dawley)) strain rats, aged 8-10 weeks, weighing 200-250 g[2]
Doses: 10 mg/kg/day
Doses: po (po (oral gavage)) Six-week
Experimental Results: Treatment There was a sustained recovery of the gained weight, which was observed Dramatically from the ninth week until the end of the experimental period.

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After a one-week acclimatization period, the rats were divided into two groups and fed either with normal pellet (Normal group, n = 6/group) or high-fat diet (HFD) (n = 12/group) for six weeks to induce obesity. Obese rats were subjected to treatment either with distilled water (1 mL/day) as positive control (OB group, n = 6/group) or orlistat at 10 mg/kg/day (OB/OR group, n = 6/group) for another six weeks. The dose of orlistat was selected based on a previous study. The rats in the Normal group continued to receive distilled water until the end of experimental period. [2]

Absorption, Distribution and Excretion
The systemic absorption and exposure of orlistat is low, however, systemic absorption of the drug is not required for orlistat activity. After an oral dose with 360 mg of radiolabeled orlistat, plasma radioactivity achieved a peak at about 8 hours. Plasma concentrations of unchanged parent drug were close to the lower end of detection limits (<5 ng/mL). In plasma samples of patients taking orlistat, the detection of unchanged drug was sporadic and very low concentrations were detected (<10 ng/mL or 0.02 μM) with no evidence suggesting drug accumulation.
After single oral dose of radiolabled orlistat in both normal weight and obese volunteers fecal excretion of the unabsorbed drug was found to be the major route of elimination with <2% urinary excretion. Fecal elimination of orlistat is estimated between 95-97%. Complete excretion by both routes occurs within in 3 to 5 days.
Volume of distribution cannot be obtained because the absorption of orlistat is minimal. Orlistat is minimally distributed to erythrocytes and is primarily bound to proteins.
Orlistat works locally within the GI tract, and therefore systemic absorption of the drug is not required for activity. In fact, systemic absorption of orlistat is minimal, and effects on systemic lipases are unlikely. Fecal excretion of unabsorbed drug is the major route of elimination.
Systemic exposure to orlistat is minimal. Following oral dosing with 360 mg 14C-orlistat, plasma radioactivity peaked at approximately 8 hours; plasma concentrations of intact orlistat were near the limits of detection (<5 ng/mL). In therapeutic studies involving monitoring of plasma samples, detection of intact orlistat in plasma was sporadic and concentrations were low (<10 ng/mL or 0.02 uM), without evidence of accumulation, and consistent with minimal absorption.
The average absolute bioavailability of intact orlistat was assessed in studies with male rats at oral doses of 150 and 1000 mg/kg/day and in male dogs at oral doses of 100 and 1000 mg/kg/day and found to be 0.12%, 0.59% in rats and 0.7%, 1.9% in dogs, respectively.
In vitro orlistat was >99% bound to plasma proteins (lipoproteins and albumin were major binding proteins). Orlistat minimally partitioned into erythrocytes.
For more Absorption, Distribution and Excretion (Complete) data for ORLISTAT (6 total), please visit the HSDB record page.

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Metabolism / Metabolites
Orlistat is hydrolyzed in the intestinal wall. In a radiolabeled orlistat mass balance study in obese patients, two metabolites were identified. The first metabolite, M1, was the hydrolyzed β-lactone ring product of orlistat. The second metabolite, M3, was produced from M1’s cleavage of the N-formyl leucine side-chain. Both metabolites accounted for about 42% of total plasma radioactivity. Both M1 and M3 are considered pharmacologically inactive.
Based on animal data, it is likely that the metabolism of orlistat occurs mainly within the gastrointestinal wall. Based on an oral 14C-orlistat mass balance study in obese patients, two metabolites, M1 (4-member lactone ring hydrolyzed) and M3 (M1 with N-formyl leucine moiety cleaved), accounted for approximately 42% of total radioactivity in plasma. M1 and M3 have an open beta-lactone ring and extremely weak lipase inhibitory activity (1000- and 2500-fold less than orlistat, respectively). In view of this low inhibitory activity and the low plasma levels at the therapeutic dose (average of 26 ng/mL and 108 ng/mL for M1 and M3, respectively, 2 to 4 hours after a dose), these metabolites are considered pharmacologically inconsequential. The primary metabolite M1 had a short half-life (approximately 3 hours) whereas the secondary metabolite M3 disappeared at a slower rate (half-life approximately 13.5 hours). In obese patients, steady-state plasma levels of M1, but not M3, increased in proportion to orlistat doses.

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Biological Half-Life
The half-life of orlistat of the small amount of absorbed orlistat ranges between 1-2 hours.
Based on limited data, the half-life of the absorbed orlistat is in the range of 1 to 2 hours.
Based on an oral 14C-orlistat mass balance study in obese patients, ... the primary metabolite M1 had a short half-life (approximately 3 hours) whereas the secondary metabolite M3 disappeared at a slower rate (half-life approximately 13.5 hours). In obese patients, steady-state plasma levels of M1, but not M3, increased in proportion to orlistat doses.

Hepatotoxicity
Orlistat acts my binding pancreatic and gastric lipase in the intestinal tract. Systemic absorption is not needed for its effect. Indeed, little of orally administered orlistat is absorbed (1% to 3%) and plasma levels are usually undetectable or less than 4 ng/mL (too little to inhibit serum lipase activities). Thus, systemic side effects of orlistat were not expected. In large clinical trials, serum liver test abnormalities were no more common with orlistat than with placebo therapy. Nevertheless, there have been several case reports of clinically apparent acute liver injury attributed to orlistat and in 2010 the FDA announced safety concerns regarding hepatotoxicity. The onset of injury in published cases was between 2 to 12 weeks of starting orlistat. The usual pattern of serum enzyme elevations was hepatocellular and some cases were severe with signs of hepatic failure and progression to death or need for liver transplantation. Features of hypersensitivity were not prominent and autoimmune markers were absent. None of the published cases included results of rechallenge. Thus, despite the number of published case reports, the hepatotoxicity of orlistat remains controversial and far from proven.
Likelihood score: C (probable rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Orlistat is poorly absorbed orally, but a small amount has been detected in the milk of one woman. It is unlikely that orlistat will be absorbed by the infant in amounts that would adversely affect the breastfed infant. Because it inhibits the absorption of fat-soluble vitamins, mothers using it should take a multivitamin supplement at bedtime.
◉ 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
Orlistat is >99% bound to plasma proteins (mainly lipoproteins and albumin).

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Interactions
In a multiple-dose study in 30 normal-weight subjects, coadministration of orlistat and 40 grams of alcohol (eg, approximately 3 glasses of wine) did not result in alteration of alcohol pharmacokinetics, orlistat pharmacodynamics (fecal fat excretion), or systemic exposure to orlistat.
Preliminary data from a orlistat and cyclosporine drug interaction study indicate a reduction in cyclosporine plasma levels when orlistat was coadministered with cyclosporine.
A pharmacokinetic interaction study showed a 30% reduction in beta-carotene supplement absorption when concomitantly administered with orlistat. Orlistat inhibited absorption of a vitamin E acetate supplement by approximately 60%. The effect of orlistat on the absorption of supplemental vitamin D, vitamin A, and nutritionally-derived vitamin K is not known at this time.
In 12 normal-weight subjects receiving orlistat 80 mg three times a day for 5 days, orlistat did not alter the pharmacokinetics or pharmacodynamics (blood glucose-lowering) of glyburide.
For more Interactions (Complete) data for ORLISTAT (6 total), please visit the HSDB record page.

[1]. Influence of fatty acid synthase inhibitor orlistat on the DNA repair enzyme O6-methylguanine-DNA methyltransferase in human normal or malignant cells in vitro. Int J Oncol. 2015 Aug;47(2):764-72.
[2]. Anti-Atherogenic Effects of Orlistat on Obesity-Induced Vascular Oxidative Stress Rat Model. Antioxidants (Basel). 2021 Feb 6;10(2):251.

Therapeutic Uses
Anti-Obesity Agents
The Food and Drug Administration (FDA) today-(February 07, 2007) approved orlistat capsules as an over-the-counter (OTC) weight loss aid for overweight adults. Orlistat was initially approved in 1999 as a prescription drug to treat obesity, and remains a prescription drug for obesity at a higher dose than the OTC version. OTC orlistat will be manufactured by GlaxoSmithKline under the name Alli and is indicated for use in adults ages 18 years and older along with a reduced-calorie, low-fat diet, and exercise program.
Orlistat is indicated for the management of obesity in persons with an initial body mass index (BMI) greater than or equal to 30 kg per square meter of body surface area (kg/sq m), or a BMI greater than or equal to 27 kg/sq m when other risk factors (such as hypertension, diabetes, or dyslipidemia are present. Orlistat should be use in conjunction with a reduced calorie diet for management of obesity, including weight loss, weight maintenance, and reduction of the risk of weight gain following previous weight loss. Weight loss has been observed within 2 week of initiation or orlistat therapy. /Included in US product label/

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Drug Warnings
Chronic malabsorption syndrome or cholestasis /are contraindications for orlistat therapy/
/Orlistat is contraindicated in patients with/ known hypersensitivity to orlistat or any ingredient in the formulation.
Caution /is advised/ in patients with a history of hyperoxaluria or calcium oxalate nephrolithiasis.
/Clinician should/ rule out organic causes of obesity (e.g., hypothyroidism) /before initiating treatment/.
For more Drug Warnings (Complete) data for ORLISTAT (12 total), please visit the HSDB record page.

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Pharmacodynamics
Orlistat helps with weight reduction and maintenance by inhibiting the absorption of dietary fats via the inhibition of lipase enzymes.

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In conclusion, the preliminary data illustrated in this report appear to disclose an unexpected source of concern about the clinical use of orlistat. Actually, the administration modality of the drug in severely overweight subjects implies that daily treatment with high doses of this agent is carried out for a period of one or two years. At the present time, we are not able to evaluate the impact that a chronic downregulation of a DNA repair enzyme such as MGMT could have on host’s surveillance against chemical carcinogenesis targeting digestive tract mucosa locally exposed to high concentrations of orlistat. Therefore, the studies illustrated in this report appear to provide the ground for casting a note of caution in the long-term clinical use of orlistat suggesting that appropriate control of the gastrointestinal apparatus appears to be unreservedly advisable. [1]

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In conclusion, orlistat treatment at 10 mg/kg/day for six weeks significantly mitigated the increased aortic oxidative stress and inflammation, partly via its effect in improving lipid profile, which in turn may attenuate the progression of atherosclerosis in obese rats. Therefore, orlistat may possess a therapeutic effect in attenuating the progression of atherosclerosis in obesity. However, future study is recommended to investigate its exact molecular mechanism. In addition, it is also suggested to study the effect of orlistat on oxidative stress and inflammation in adipose tissue to further support this conclusion. [2]

C29H53NO5

495.73

495.392

C, 70.26; H, 10.78; N, 2.83; O, 16.14

96829-58-2

Orlistat (Standard);96829-58-2

3034010

White to off-white solid powder

1.0±0.1 g/cm3

615.9±30.0 °C at 760 mmHg

<50ºC

326.3±24.6 °C

0.0±1.8 mmHg at 25°C

1.470

8.94

81.7

1

5

23

35

579

4

CCCCCCCCCCC[C@@H](C[C@H]1[C@@H](C(=O)O1)CCCCCC)OC(=O)[C@H](CC(C)C)NC=O

AHLBNYSZXLDEJQ-FWEHEUNISA-N

InChI=1S/C29H53NO5/c1-5-7-9-11-12-13-14-15-16-18-24(34-29(33)26(30-22-31)20-23(3)4)21-27-25(28(32)35-27)19-17-10-8-6-2/h22-27H,5-21H2,1-4H3,(H,30,31)/t24-,25-,26-,27-/m0/s1

[(2S)-1-[(2S,3S)-3-hexyl-4-oxooxetan-2-yl]tridecan-2-yl] (2S)-2-formamido-4-methylpentanoate

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 (5.04 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 (5.04 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 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 3 中的溶解度: ≥ 2.5 mg/mL (5.04 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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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。