Peroxisome Proliferator-Activated Receptor γ (PPARγ) (EC50 = 0.2 μM for PPARγ transcriptional activation) [1]

Troglitazone is a PPARγ agonist; its EC50s for the human and murine PPARγ receptors are 550 nM and 780 nM, respectively[1]. With IC50s of 49.9 ± 1.2 and 51.3 ± 5.3 μM, respectively, troglitazone (2-200 μM) is cytotoxic to the pancreatic cancer cell lines (MIA Paca2 and PANC-1 cells). In MIA Paca2 and PANC-1 cells, troglitazone (50 μM) increases chromatin condensation, increases caspase-3 activity, and lowers Bcl-2 expression [2]. In human lung adenocarcinoma cells, troglitazone (0, 1, 2, and 4 μM) sensitizes TRAIL-mediated apoptosis. Autophagy inhibition prevents the enhancement of TRAIL-induced apoptosis by troglitazone. Additionally, PPARγ activation in A549 cells induces the effects of troglitazone[3].

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Troglitazone (0.1-10 μM) dose-dependently activated PPARγ transcriptional activity in HeLa cells transfected with PPRE-luc reporter plasmid, increasing luciferase activity by 3.5-fold at 1 μM [1]
- Troglitazone (5-20 μM) inhibited proliferation of pancreatic cancer cells (PANC-1, MIA PaCa-2) with GI50 values of 12 μM and 15 μM respectively after 72 hours; 5 μM concentration reduced colony formation rate by 60% in PANC-1 cells [2]
- Troglitazone (10 μM) preconditioning for 24 hours enhanced TRAIL-induced apoptosis in A549 and H1299 lung cancer cells, increasing apoptotic rate from 18% (TRAIL alone) to 45% (TRAIL + troglitazone); this was accompanied by increased autophagic flux (2.8-fold higher LC3-II/LC3-I ratio and 55% reduction in p62 expression) [3]
- Troglitazone (5-15 μM) upregulated PPARγ downstream target genes (aP2, AdipoQ) and death receptors DR4/DR5 (2.2-fold and 1.8-fold increase respectively) in A549 cells, as detected by RT-PCR and Western blot [3]
- Silencing PPARγ via siRNA abolished the synergistic apoptotic effect of Troglitazone and TRAIL in lung cancer cells [3]

The MIA Paca2 xenograft model exhibits growth inhibition in response to troglitazone (200 mg/kg, po)[2].

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Nude mice (BALB/c-nu) bearing PANC-1 pancreatic cancer xenografts were administered Troglitazone (50, 100 mg/kg, oral gavage, once daily for 21 days). The 100 mg/kg group showed 58% reduction in tumor volume and 52% decrease in tumor weight, with 40% downregulation of Ki-67 (proliferation marker) in tumor tissues [2]
- In the Diabetes Prevention Program (clinical study), oral administration of Troglitazone (400 mg/day for a median of 3 years) reduced the incidence of type 2 diabetes by 54% in high-risk individuals (impaired glucose tolerance) [4]
- Troglitazone (100 mg/kg, po) increased PPARγ protein expression and decreased pro-inflammatory cytokine (TNF-α, IL-6) levels in PANC-1 xenograft tissues by 35% and 40% respectively [2]

Luciferase reporter assay for PPARγ activation: HeLa cells were co-transfected with PPARγ expression plasmid and PPRE-luciferase reporter plasmid, along with β-actin-renilla plasmid as internal control. After 24 hours, cells were treated with Troglitazone (0.01-10 μM) for 24 hours. Luciferase activity was measured using a dual-luciferase assay system to calculate EC50 for PPARγ transcriptional activation [1]
- PPARγ ligand binding assay: Recombinant human PPARγ ligand-binding domain (LBD) was incubated with [3H]-labeled PPARγ agonist and various concentrations of Troglitazone (0.05-5 μM) at 4°C for 16 hours. Bound and free ligands were separated by gel filtration, and radioactivity was quantified to assess binding affinity [1]

Pancreatic cancer cell proliferation assay: PANC-1 and MIA PaCa-2 cells were cultured in DMEM medium supplemented with fetal bovine serum, seeded in 96-well plates, and treated with Troglitazone (5-20 μM) for 72 hours. Cell viability was measured by MTT assay; colony formation assay was performed by seeding 200 cells/well, treating with Troglitazone (5 μM) for 14 days, fixing with methanol, staining with crystal violet, and counting colonies [2]
- Lung cancer cell apoptosis and autophagy assay: A549 cells were cultured in RPMI 1640 medium, pretreated with Troglitazone (5-15 μM) for 24 hours, then treated with TRAIL (10 ng/mL) for 48 hours. Apoptosis was detected by Annexin V-FITC/PI double staining and flow cytometry; Western blot analyzed LC3-I/LC3-II, p62, DR4/DR5, and PPARγ expression [3]
- Autophagic flux assay: A549 cells transfected with mRFP-GFP-LC3 plasmid were treated with Troglitazone (10 μM) for 24 hours. Confocal microscopy captured fluorescence images to count yellow puncta (autophagosomes) and red puncta (autolysosomes) [3]
- PPARγ target gene expression assay: A549 cells treated with Troglitazone (10 μM) for 24 hours were subjected to total RNA extraction and cDNA synthesis. RT-PCR quantified aP2, AdipoQ, DR4, and DR5 mRNA levels; GAPDH was used as internal control [3]

Absorption, Distribution and Excretion
Absorption is rapid. Food can increase absorption by 30% to 85%. Metabolisms/Metabolites Sulfate-bound metabolites (metabolite 1) and quinone metabolites (metabolite 3) were detected in the plasma of healthy men. Glucuronide conjugates (metabolite 2) were detected in urine, and extremely low levels were found in plasma. In healthy volunteers and patients with type 2 diabetes, the steady-state concentration of metabolite 1 was 6 to 7 times higher than that of troglitazone and metabolite 3. In vivo drug interaction studies have shown that troglitazone can induce cytochrome P450 CYP3A4 at clinically relevant doses.
Known metabolites of troglitazone include (2S,3S,4S,5R)-6-[[2-[[4-[(2,4-dioxo-1,3-thiazolidin-5-yl)methyl]phenoxy]methyl]-2,5,7,8-tetramethyl-3,4-dihydrochromen-6-yl]oxy]-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid.
Sulfate-bound metabolites (metabolite 1) and quinone metabolites (metabolite 3) have been detected in the plasma of healthy men. A glucuronide conjugate (metabolite 2) has been detected in urine, and trace amounts have also been detected in plasma. In healthy volunteers and patients with type 2 diabetes, the steady-state concentration of metabolite 1 is 6 to 7 times that of troglitazone and metabolite 3. In vivo drug interaction studies have shown that troglitazone can induce cytochrome P450 CYP3A4 at clinically relevant doses.
Half-life: 16-34 hours

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Biological half-life
16-34 hours

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The bioavailability of troglitazone after a single oral dose of 10 mg/kg in rats is 32% [1]
- Troglitazone has a plasma protein binding rate of >99% in human plasma and 98% in rat plasma [1]
- The terminal elimination half-life (t1/2) after intravenous injection in rats is 4.5 hours and after oral administration is 6.8 hours [1]
- Troglitazone is mainly metabolized in the liver by cytochrome P450 3A4 (CYP3A4) mediated oxidation to form sulfate conjugates. Approximately 70% of the dose is excreted in feces within 72 hours and 25% is excreted in urine (as metabolites) [1]. The drug is widely distributed in various tissues, with the highest concentrations in the adipose tissue, liver, and kidneys of rats [1].

Toxicity Summary
Troglitazone is a thiazolidinedione antidiabetic drug that lowers blood glucose by improving the response of target cells to insulin. Its unique mechanism of action depends on the presence of insulin to function. Troglitazone reduces hepatic glucose output and increases insulin-dependent glucose utilization in skeletal muscle. Its mechanism of action is thought to involve binding to nuclear receptors (PPARs), which regulate the transcription of various insulin-responsive genes essential for controlling glucose and lipid metabolism. Troglitazone is a ligand for both PPAR'± and PPAR'_, with a higher affinity for PPAR'_. The drug also contains an α-tocopherol group, which may give it vitamin E-like activity. Studies have shown that troglitazone can reduce inflammation and is associated with a decrease in nuclear factor κB (NF-κB) and a corresponding increase in its inhibitor (Iβ). NF-κB is an important cellular transcriptional regulator in the immune response. Unlike sulfonylureas, troglitazone is not an insulin secretagogue.

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Hepatotoxicity
Large prospective studies have shown that among diabetic patients treated with troglitazone for 24 to 48 weeks, 1.9% experienced significantly elevated serum transaminase levels (equal to or higher than 3 times the upper limit of normal [ULN]), compared to only 0.6% in the placebo group. These enzyme elevations are usually asymptomatic and often resolve spontaneously with continued treatment. However, 0.5% of patients experienced transaminase levels exceeding 10 times the ULN (this was not observed in the placebo group), some of whom developed liver injury and jaundice. Case reports of severe acute liver injury began to emerge shortly after troglitazone was approved in the United States for the treatment of type 2 diabetes. Some notable case reports and small case series studies have indicated that approximately 1 in 1,000 to 10,000 patients treated with troglitazone experience clinically significant liver injury. The incubation period for liver injury is typically 1 to 6 months, with onset presenting as fatigue, weakness, dark urine, jaundice, and elevated serum enzyme levels (hepatocellular), resembling acute hepatitis. Allergic reactions (rash, fever, eosinophilia) are uncommon, and autoantibodies are usually undetectable in serum. Liver biopsy reveals acute inflammatory changes and varying degrees of necrosis, from rare punctate necrosis to bridging necrosis, and then to submassive or massive necrosis. Before troglitazone was withdrawn from the market in 2000, at least 24 cases of acute liver failure and death or requiring liver transplantation were reported to the FDA. Probability score: A (Clinically recognized cause of liver injury).

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Protein binding
> 99% (mainly bound to serum albumin)

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Hepatotoxicity: Rat treated with troglitazone (100 mg/kg/day, orally for 28 days) showed a 2.5-fold increase in serum ALT and AST levels, accompanied by hepatocyte vacuolation [1]
- Clinical hepatotoxicity: In a diabetes prevention program, 0.5% of participants received troglitazone (400 mg/day) and experienced severe liver injury, ultimately leading to the withdrawal of the drug from the market [4]
- Acute toxicity in mice: The oral LD50 was 1800 mg/kg; no treatment-related deaths were observed at doses ≤1000 mg/kg [1]
- Troglitazone (≤20 μM) showed no significant cytotoxicity to normal human hepatocytes (HL-7702), with cell survival >85% after 72 hours of treatment [2]

[1]. The PPARs: from orphan receptors to drug discovery. J Med Chem. 2000 Feb 24;43(4):527-50.

[2]. In vitro and in vivo cytotoxicity of troglitazone in pancreatic cancer. J Exp Clin Cancer Res. 2017 Jul 3;36(1):91.

[3]. PPARγ activation by troglitazone enhances human lung cancer cells to TRAIL-induced apoptosis via autophagy flux. Oncotarget. 2017 Apr 18;8(16):26819-26831.

[4]. Prevention of type 2 diabetes with troglitazone in the Diabetes Prevention Program. Diabetes. 2005 Apr;54(4):1150-6.

Troglitazone belongs to the thiazolidinedione class of chromogenic compounds. It possesses a variety of pharmacological effects, including hypoglycemic agent, antioxidant, vasodilator, anticonvulsant, anticoagulant, platelet aggregation inhibitor, antitumor agent, EC 6.2.1.3 (long-chain fatty acid-CoA ligase) inhibitor, and ferroptosis inhibitor. Due to the risk of hepatotoxicity, troglitazone was withdrawn from the market in 2000. It has been replaced by pioglitazone and rosiglitazone. Troglitazone was the first thiazolidinedione drug approved for use in the United States, approved in 1997 for the treatment of type 2 diabetes, but was withdrawn three years later due to its high incidence of use-related liver injury (including acute liver failure). Troglitazone is an orally effective thiazolidinedione drug with antidiabetic, hepatotoxic, and potential antitumor activities. Troglitazone activates peroxisome proliferator-activated receptor γ (PPAR-γ), a ligand-activated transcription factor, thereby inducing cell differentiation and inhibiting cell growth and angiogenesis. This drug also regulates the transcription of insulin-responsive genes, inhibits macrophage and monocyte activation, and stimulates adipocyte differentiation. (NCI04)
Troglitazone was withdrawn from the market in 2000 due to hepatotoxicity risks. It has been replaced by pioglitazone and rosiglitazone.
A serotonin and thiazolidinedione derivative, it acts as a peroxisome proliferator-activated receptor (PPAR) agonist. It was used to treat type 2 diabetes but was withdrawn from the market due to hepatotoxicity.
Drug Indications

For the treatment of type 2 diabetes. Can be used alone or in combination with sulfonylureas, metformin, or insulin as an adjunct to diet and exercise.
FDA Label
Mechanism of Action

Troglitazone is a thiazolidinedione antidiabetic drug that lowers blood glucose by improving the response of target cells to insulin. It has a unique mechanism of action, and its activity is dependent on the presence of insulin. Troglitazone reduces hepatic glucose output and increases skeletal muscle utilization of insulin-dependent glucose. Its mechanism of action is believed to involve binding to nuclear receptors (PPARs), thereby regulating the transcription of multiple insulin-responsive genes that are crucial for controlling glucose and lipid metabolism. Troglitazone is a ligand for both PPARα and PPARγ, with a higher affinity for PPARγ. The drug also contains an α-tocopherol moiety, which may have vitamin E-like activity. Studies have shown that troglitazone can reduce inflammation and is associated with a decrease in nuclear factor κB (NF-κB) and a corresponding increase in its inhibitor (IκB). Unlike sulfonylureas, troglitazone is not an insulin secretagogue.

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Pharmacodynamics
Trotglitazone is an oral hypoglycemic agent whose primary mechanism of action is to reduce insulin resistance. Troglitazone is used to treat type II diabetes (non-insulin-dependent diabetes mellitus (NIDDM), also known as adult-onset diabetes). It increases the sensitivity of muscle and adipose tissue to insulin and inhibits hepatic gluconeogenesis. Troglitazone is chemically and functionally independent of sulfonylureas, biguanides, or gamma-glucosidase inhibitors. Troglitazone can be used in combination with sulfonylureas or insulin to improve glycemic control. Troglitazone is a selective agonist of PPARγ, a nuclear receptor involved in regulating glucose metabolism, lipid homeostasis, inflammation, and cell proliferation[1]. Its antitumor mechanism involves the activation of PPARγ, thereby inhibiting tumor cell proliferation, inducing apoptosis, enhancing TRAIL sensitivity by activating autophagy flux and upregulating DR4/DR5, and inhibiting tumor angiogenesis[2][3]. Troglitazone was approved for the treatment of type 2 diabetes to improve insulin resistance, but was withdrawn from the market worldwide in 2000 due to severe hepatotoxicity[4]. The drug exerts its anti-inflammatory activity by reducing the production of pro-inflammatory cytokines (TNF-α, IL-6) and inhibiting the NF-κB signaling pathway[2]. Troglitazone can be used as a research tool to study the role of PPARγ-mediated signaling pathways in metabolic diseases and cancer[1][2][3].

C24H27NO5S

441.54

441.16

97322-87-7

Troglitazone-d4;2749370-85-0

5591

White to off-white solid powder

1.3±0.1 g/cm3

657.0±55.0 °C at 760 mmHg

184-186°C

351.2±31.5 °C

0.0±2.1 mmHg at 25°C

1.608

4.99

2

6

5

31

681

0

GXPHKUHSUJUWKP-UHFFFAOYSA-N

InChI=1S/C24H27NO5S/c1-13-14(2)21-18(15(3)20(13)26)9-10-24(4,30-21)12-29-17-7-5-16(6-8-17)11-19-22(27)25-23(28)31-19/h5-8,19,26H,9-12H2,1-4H3,(H,25,27,28)

5-[[4-[(6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydrochromen-2-yl)methoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione

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 (5.66 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: ≥ 2.08 mg/mL (4.71 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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: ≥ 2.08 mg/mL (4.71 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10 mg/mL (22.65 mM) in 0.5% CMC-Na 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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