Lipase; fatty acid synthase (FASN)

Orlistat (40 μM; 2 days) invoiced repair proteins by 30-70% in human peripheral blood mononuclear cells, two leukocytes, and another Turkish line, but had no effect on MGMT levels in human melanoma cell lines.

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Tetrahydrolipstatin (orlistat), an inhibitor of lipases and fatty acid synthase, is used orally for long-term treatment of obesity. Although the drug possesses striking antitumor activities in vitro against human cancer cells and in vitro and in vivo against animal tumors, it also induces precancerous lesions in rat colon. Therefore, researchers tested the in vitro effect of orlistat on the expression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that plays an essential role in the control of mutagenesis and carcinogenesis. Western blot analysis demonstrated that 2-day continuous exposure to 40 µM orlistat did not affect MGMT levels in a human melanoma cell line, but downregulated the repair protein by 30-70% in human peripheral blood mononuclear cells, in two leukemia and two colon cancer cell lines. On the other hand, orlistat did not alter noticeably MGMT mRNA expression. Differently from lomeguatrib (a false substrate, strong inhibitor of MGMT) orlistat did not reduce substantially MGMT function after 2-h exposure of target cells to the agent, suggesting that this drug is not a competitive inhibitor of the repair protein. Combined treatment with orlistat and lomeguatrib showed additive reduction of MGMT levels. More importantly, orlistat-mediated downregulation of MGMT protein expression was markedly amplified when the drug was combined with a DNA methylating agent endowed with carcinogenic properties such as temozolomide. In conclusion, even if orlistat is scarcely absorbed by oral route, it is possible that this drug could reduce local MGMT-mediated protection against DNA damage provoked by DNA methylating compounds on gastrointestinal tract epithelial cells, thus favoring chemical carcinogenesis [1].

Orlistat (10 mg/kg/day) considerably improved blood lipid profile, upregulated antioxidant enzyme expression and anti-inflammatory marker expression, and downregulated pro-inflammatory marker expression as compared to the nutrition (OB) group [2].

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The aim of this study was to determine the anti-atherosclerotic effect of orlistat on obesity-induced vascular oxidative stress in obese male rats. Twenty-four male Sprague-Dawley rats were categorized into two groups: normal (Normal group, n = 6) and high-fat diet (HFD group, n = 12). After six weeks, obese rats in the HFD group were administered either with distilled water (OB group) or orlistat 10 mg/kg/day (OB/OR group) for another six weeks. The OB group had a significant increase in lipid profiles (total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL)) and decrease in high-density lipoprotein (HDL) level compared to the Normal group. The aortic antioxidants enzymes activities (superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), and catalase (CAT)) as well as total glutathione (GSH) and total antioxidant capacity (TAC) of the OB group were significantly decreased compared to the Normal group. Furthermore, pro-inflammatory atherosclerotic markers (tumour necrosis factor-alpha (TNF-ɑ), vascular cell adhesion molecule-1 (VCAM-1), and intercellular cell adhesion molecule-1 (ICAM-1)) expressions were increased significantly, and anti-inflammatory marker (interleukin-10 (IL-10)) was decreased significantly in the OB group compared to the Normal group. Treatment with orlistat significantly improved lipid profile, increased antioxidant enzymes and expression of anti-inflammatory markers, and decreased the expression of the pro-inflammatory marker compared to the OB group. These findings may suggest the therapeutic effect of orlistat in attenuating the progression of the atherosclerotic stage in obesity[2].

MGMT activity assay [1]
MGMT activity was determined by measuring the transfer of 3H-methyl groups from a DNA substrate to the MGMT protein. Briefly, cell pellets (1×106 cells) were re-suspended in 1 ml of lysis buffer (0.5% CHAPS, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 3 mM dithiothreitol, 100 mM NaCl, 10% glycerol) supplemented with a cocktail of protease inhibitors and incubated for 30 min at 4°C. Cell lysates were then centrifuged at 18,000 × g for 10 min at 4°C. Aliquots of supernatants were then diluted in 50 mM Tris-HCl buffer, pH 8.3, containing 1 mM EDTA, and 3 mM dithiothreitol, and incubated with 10 μg of 3H-methylated-DNA at 37°C for 1 h. DNA was then hydrolyzed by heating samples at 75°C for 45 min, in the presence of 1 N perchloric acid, and protein precipitated using 1 mg bovine serum albumin as carrier. Pellets were washed with 1 N perchloric acid, re-suspended in 0.01 N NaOH, and radioactivity measured in a liquid scintillation counter, after addition of scintillation liquid. Protein concentration in supernatants was evaluated according to the method of Bradford using the Bio-Rad Protein Assay Dye reagent and bovine serum albumin as standard. MGMT activity was expressed in terms of fmoles of 3H-methyl groups transferred per mg of protein in cell extract.

Western Blot analysis [1]
Cell Types: human melanoma cell line M10, peripheral blood mononuclear cells, human Jurkat CD4+ T cell leukemia cell line, human promyelocytic leukemia cell line HL-60. Will Dramatically change MGMT mRNA expression [1]. , epithelial colon cancer HCT116 cells, non-adherent mononuclear cells (NAMNC) [1]
Tested Concentrations: 2.5, 5, 10, 20, 40 μM for Jurkat cells; 20 and 40 μM for HCT116 cells; normal NAMNC, M10 melanoma , 40 μM for HL-60 promyelocytic leukemia and HT-29 colon cancer cells.
Incubation Duration: 2 days for Jurkat cells; 2 or 4 days for HCT116 cells; NAMNC, M10 melanoma, HL-60 promyelocytic leukemia, HT-29 colon cancer 2-day
Experimental Results: For Jurkat cells, MGMT levels were diminished by >50% at a concentration of 40 μM, whereas at lower concentrations, almost no effect was seen. used. MGMT expression in HCT116 cells was downregulated at 40 μM. At a concentration of 40 μM, MGMT levels were diminished by approximately 50% in normal NAMNC, HL-60 promyelocytic leukemia, a

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
Orlistat has low systemic absorption and exposure, but systemic absorption is not essential for its activity. Following oral administration of 360 mg of radiolabeled orlistat, peak plasma radioactivity is reached in approximately 8 hours. Unmetabolized plasma concentrations are close to the lower limit of detection (<5 ng/mL). In plasma samples from patients taking orlistat, unmetabolized drug is detected sporadically at extremely low concentrations (<10 ng/mL or 0.02 μM), with no evidence of drug accumulation. In normal-weight and obese volunteers, unabsorbed orlistat is primarily excreted via feces after a single oral dose, with urinary excretion <2%. The fecal excretion rate of orlistat is estimated to be between 95-97%. Both routes of excretion are complete within 3 to 5 days. Due to minimal absorption, its volume of distribution cannot be determined. Orlistat is hardly distributed to erythrocytes and is primarily bound to proteins. Orlistat acts locally in the gastrointestinal tract, thus requiring no systemic absorption to be active. In fact, systemic absorption of orlistat is minimal and unlikely to affect systemic lipases. Unabsorbed drug is primarily excreted in feces. Systemic exposure to orlistat is extremely low. Following oral administration of 360 mg of 14C-orlistat, plasma radioactivity peaks at approximately 8 hours; plasma concentrations of intact orlistat are close to the limit of detection (<5 ng/mL). In treatment studies involving plasma sample monitoring, the detection rate and concentration of intact orlistat in plasma were low (<10 ng/mL or 0.02 μM), with no signs of accumulation, consistent with minimal absorption.
In studies in male rats (oral doses of 150 and 1000 mg/kg/day, respectively) and male dogs (oral doses of 100 and 1000 mg/kg/day, respectively), the mean absolute bioavailability of intact orlistat was evaluated, showing 0.12% and 0.59% in rats and 0.7% and 1.9% in dogs, respectively.
In vitro studies showed that orlistat is bound to plasma proteins >99% (primarily lipoproteins and albumin). Orlistat is minimally distributed in erythrocytes.
For more complete data on absorption, distribution, and excretion of orlistat (6 items), 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, is a β-lactone ring product of orlistat hydrolysis. The second metabolite, M3, is produced by the cleavage of the N-formylleucine side chain of M1. These two metabolites account for approximately 42% of the total plasma radioactivity. Both M1 and M3 are considered pharmacologically inactive. Based on animal data, orlistat metabolism likely occurs primarily within the gastrointestinal wall. Based on a mass balance study of oral 14C-orlistat in obese patients, the two metabolites, M1 (a hydrolysis product of the four-membered lactone ring) and M3 (a partial cleavage product of the N-formylleucine side chain of M1), accounted for approximately 42% of the total plasma radioactivity. M1 and M3 have open β-lactone rings and exhibit extremely weak lipase inhibitory activity (1000-fold and 2500-fold lower than orlistat, respectively). Given their low inhibitory activity and low plasma concentrations at therapeutic doses (mean concentrations of M1 and M3 were 26 ng/mL and 108 ng/mL, respectively, 2 to 4 hours after administration), these metabolites are considered pharmacologically insignificant. The major metabolite M1 has a short half-life (approximately 3 hours), while the minor metabolite M3 disappears more slowly (half-life approximately 13.5 hours). In obese patients, the steady-state plasma concentration of M1 (but not M3) increases with increasing orlistat dose.
Biological half-life
The half-life of minimally absorbed orlistat is 1–2 hours.
Based on limited data, the half-life of absorbed orlistat is between 1 and 2 hours.
Based on a mass balance study of oral 14C-orlistat in obese patients, ... the major metabolite M1 has a short half-life (approximately 3 hours), while the minor metabolite M3 disappears more slowly (half-life approximately 13.5 hours). In obese patients, the steady-state plasma concentration of M1 (but not M3) increases with increasing orlistat dose.

Hepatotoxicity
Orlistat exerts its effect by binding to pancreatic and gastric lipases in the intestines. Its therapeutic effect does not require systemic absorption. In fact, the absorption rate of orlistat orally is very low (1% to 3%), and plasma concentrations are often undetectable or below 4 ng/mL (too low to inhibit serum lipase activity). Therefore, systemic side effects from orlistat are not expected. In large clinical trials, the incidence of serum liver function abnormalities was not higher in the orlistat group than in the placebo group. However, several cases of clinically significant acute liver injury have been reported, all related to orlistat. In 2010, the U.S. Food and Drug Administration (FDA) announced a safety concern regarding orlistat hepatotoxicity. In published cases, liver injury occurred 2 to 12 weeks after starting orlistat. The common pattern of elevated serum enzymes was hepatocellular; some cases were severe, showing signs of liver failure, even progressing to death or requiring liver transplantation. Hypersensitivity reactions were not prominent, and no autoimmune markers were observed. None of the published cases included results from re-administration of the drug. Therefore, despite the large number of published case reports, the hepatotoxicity of orlistat remains controversial and far from confirmed. Probability Score: C (Possibly a rare cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Orlistat is poorly absorbed orally, but trace amounts have been detected in the breast milk of one woman. It is unlikely that an infant will absorb a dose sufficient to adversely affect a breastfed infant. Because orlistat inhibits the absorption of fat-soluble vitamins, mothers taking this medication should take a multivitamin supplement at bedtime. ◉ Effects on Breastfed Infants No relevant published information was found as of the revision date. ◉ Effects on Lactation and Breast Milk No relevant published information was found as of the revision date. Protein Binding Orlistat binds to plasma proteins (primarily lipoproteins and albumin) at a rate >99%.

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Drug Interactions
In a multidose study of 30 normal-weight subjects, concomitant administration of orlistat and 40 grams of alcohol (e.g., approximately 3 glasses of wine) did not alter alcohol pharmacokinetics, orlistat pharmacodynamics (fecal fat excretion), or systemic orlistat exposure.
Preliminary data from a study of orlistat-cyclosporine drug interactions showed that concomitant use of orlistat with cyclosporine resulted in a decrease in cyclosporine plasma concentrations.
A pharmacokinetic interaction study showed that concomitant use of orlistat with β-carotene supplements reduced the absorption of β-carotene supplements by 30%. Orlistat inhibited the absorption of vitamin E acetate supplements by approximately 60%. The effects of orlistat on the absorption of supplemented vitamin D, vitamin A, and dietary vitamin K are currently unknown.
In 12 normal-weight subjects, orlistat was administered three times daily at 80 mg for 5 days, and the results showed that orlistat did not alter the pharmacokinetics or pharmacodynamics (hypoglycemic effect) of glibenclamide. For more complete data on interactions of orlistat (6 in total), please visit the HSDB records 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 Drug The U.S. Food and Drug Administration (FDA) today (February 7, 2007) approved orlistat capsules as an over-the-counter (OTC) weight-loss adjunct to help overweight adults. Orlistat was initially approved as a prescription drug for the treatment of obesity in 1999 and remains approved for use in this condition, although at higher doses than the OTC version. OTC orlistat is manufactured by GlaxoSmithKline under the brand name Alli and is indicated for adults 18 years and older, in conjunction with a low-calorie, low-fat diet and exercise program. Orlistat is indicated for obese patients with an initial body mass index (BMI) ≥30 kg/m², or a BMI ≥27 kg/m² with other risk factors such as hypertension, diabetes, or dyslipidemia. Orlistat should be used in conjunction with a low-calorie diet to treat obesity, including weight loss, weight maintenance, and reducing the risk of weight regain after previous weight loss. Weight loss is typically observed within two weeks of starting orlistat treatment. /US Product Label Contains/

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Drug Warnings
Chronic malabsorption syndrome or cholestasis is a contraindication for orlistat. Orlistat Treatment/
/Orlistat is contraindicated in patients with hypersensitivity to orlistat or any component of its preparations.
Patients with a history of hyperoxaluria or calcium oxalate kidney stones should use with caution.
/Clinicians should rule out organic causes of obesity (e.g., hypothyroidism) before initiating treatment.
For more complete data on drug warnings for orlistat (12 in total), please visit the HSDB record page.

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Pharmacodynamics
Orlistat helps reduce and maintain weight by inhibiting the absorption of dietary fat through the inhibition of lipase.

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In summary, the preliminary data presented in this report appear to reveal an unexpected concern regarding the clinical use of orlistat. Indeed, for severely overweight patients, the route of administration implies the need for high daily doses for one to two weeks. For many years, we have been unable to assess the effects of chronic downregulation of DNA repair enzymes such as MGMT on host resistance to chemical carcinogenesis, particularly local exposure to high concentrations of orlistat in the gastrointestinal mucosa. Therefore, the findings in this report appear to offer a cautious recommendation for long-term clinical use of orlistat, suggesting that proper control of the gastrointestinal tract is absolutely necessary. [1]

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In summary, treatment with orlistat at a dose of 10 mg/kg/day for six consecutive weeks significantly reduced the increase in aortic oxidative stress and inflammation, partly due to its improved lipid profile, which may have slowed the progression of atherosclerosis in obese rats. Therefore, orlistat may have a therapeutic effect in slowing the progression of obesity-related atherosclerosis. However, future studies are recommended to explore its exact molecular mechanisms. Furthermore, studies on the effects of orlistat on… further support this conclusion regarding oxidative stress and inflammation in adipose tissue. [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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.04 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.04 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.04 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)