MDM-2/p53 (IC50 = 47-73 nM); HSP90
NF-κB pathway (IC50 = 10 nM for NF-κB activation inhibition in Jurkat cells) [1]
- HSP70 (Ki = 2.5 μM, determined by binding assay) [3]
- STAT3 (EC50 = 15 nM for STAT3 phosphorylation inhibition in MDA-MB-231 cells) [2]

Triptolide is a diterpene triepoxide with strong anti-inflammatory and immunosuppressive properties. The purine-box/nuclear factor and NF-κB mediated transcription activation of IL-2 in activated T cells is shown to be inhibited by triptolide. [1] At incredibly low concentrations (2–10 ng/mL), triptolide prevents the growth and colony formation of tumor cells. Triptolide inhibits the growth of HL-60 leukemia cell line, stomach, and breast cancer cells. By inhibiting NF-κB activation and making tumor cells more susceptible to TNF-&alpha-induced programmed cell death, triptolide causes apoptosis in tumor cells. [2]

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Inhibited NF-κB activation in Jurkat T cells and HeLa cells in a concentration-dependent manner, blocking TNF-α-induced NF-κB nuclear translocation by ~90% at 10 nM Triptolide (PG490) [1]
- Exhibited potent antiproliferative activity against various human cancer cell lines: IC50 values ranged from 5 to 50 nM (MDA-MB-231 breast cancer, PC-3 prostate cancer, HCT116 colon cancer) [2]
- Induced apoptosis in cancer cells via caspase-dependent pathway: 20 nM concentration increased caspase-3/9 activation by ~3-4 fold and upregulated Bax/Bcl-2 ratio by ~5 fold in HCT116 cells [2]
- Suppressed STAT3 phosphorylation and downstream target gene expression (Bcl-xL, cyclin D1) by ~70% at 15 nM in MDA-MB-231 cells [2]
- Bound to HSP70 and disrupted HSP70-client protein interactions, reducing HSP70-mediated protein folding activity by ~60% at 2.5 μM [3]
- Inhibited LPS-induced nitric oxide (NO) production and iNOS expression by ~80% at 50 nM in RAW 264.7 macrophages [4]
- Reduced cancer cell clone formation: 10 nM Triptolide (PG490) decreased colony number by ~90% compared to control in PC-3 cells [2]

In animal models, triptolide and cyclosporin A work together to promote graft survival and prevent graft versus host disease after allogeneic bone marrow transplants. Additionally, by suppressing the induction of c-IAP2 and c-IAP1, it causes tumor cells to undergo apoptosis and enhances tumor necrosis factor (TNF-α)-induced apoptosis. [1] [3] The growth of xenografts made of four different tumor cell lines (B16 melanoma, MDA-435 breast cancer, TSU bladder cancer, and MGC80-3 gastric carcinoma) is inhibited by triptolide treatment for two to three weeks, demonstrating that TPL has a wide range of activity against tumors that contain both wild-type and mutant forms of p53.

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In nude mouse MDA-MB-231 breast cancer xenograft model, intraperitoneal administration of Triptolide (PG490) (0.5 mg/kg, twice weekly for 4 weeks) inhibited tumor growth by ~75% and reduced tumor weight by ~70% compared to vehicle control [2]
- In C57BL/6 mouse LPS-induced sepsis model, intravenous injection of Triptolide (PG490) (0.2 mg/kg) 1 hour post-LPS challenge reduced serum TNF-α and IL-6 levels by ~65% and ~70%, respectively, and improved survival rate from 30% to 75% [4]
- In rat adjuvant-induced arthritis model, subcutaneous administration of Triptolide (PG490) (0.1 mg/kg, once daily for 14 days) reduced paw edema volume by ~60% and inhibited synovial inflammation [4]
- Did not cause significant weight loss in tumor-bearing mice at therapeutic doses (≤0.5 mg/kg), but higher doses (≥1 mg/kg) led to mild body weight reduction (~10%) [2]

NF-κB activation assay: Jurkat cells were transfected with a NF-κB-dependent luciferase reporter plasmid and a Renilla luciferase internal control. After 24 hours, cells were treated with Triptolide (PG490) (0.1-100 nM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 6 hours. Cells were lysed, and luciferase activity was measured to calculate NF-κB inhibition rate; IC50 was determined based on dose-response curves [1]
- HSP70 binding assay: Recombinant human HSP70 protein was incubated with fluorescently labeled client peptide and various concentrations of Triptolide (PG490) (0.1-10 μM) in binding buffer. The mixture was incubated at 37°C for 1 hour, and fluorescence polarization was measured. Ki value was calculated by fitting the competition data to a binding model [3]
- STAT3 phosphorylation assay: MDA-MB-231 cells were serum-starved for 24 hours, treated with Triptolide (PG490) (0.1-50 nM) for 2 hours, then stimulated with EGF (20 ng/mL) for 30 minutes. Cell lysates were analyzed by western blot with anti-phospho-STAT3 and anti-total STAT3 antibodies; EC50 was determined by densitometric quantification [2]

The survival of differentiated PC12 cells given various doses of triptolide. Differentiated PC12 cells are cultured on 96-well plates with RPMI 1640 medium for stabilization before being exposed to Triptolide at various concentrations (0.01, 0.1, and 1 nM) for 24 hours. The study's concentrations have been selected. Next, the MTT assay is used to determine cell viability. Three times each condition and experiment are performed[3].

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Cancer cell antiproliferation assay: MDA-MB-231, PC-3, and HCT116 cells were seeded in 96-well plates and treated with Triptolide (PG490) (0.1-100 nM) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were calculated [2]
- Apoptosis assay: HCT116 cells were treated with Triptolide (PG490) (5-50 nM) for 24 hours. Apoptotic cells were detected by Annexin V-FITC/PI staining and flow cytometry. Caspase-3/9 activity was measured using colorimetric kits, and Bax/Bcl-2 expression was analyzed by western blot [2]
- Macrophage NO production assay: RAW 264.7 macrophages were seeded in 24-well plates and treated with Triptolide (PG490) (0.1-100 nM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected, and NO concentration was measured by Griess reagent. iNOS expression was detected by western blot [4]
- Clone formation assay: PC-3 cells were seeded in 6-well plates at low density and treated with Triptolide (PG490) (0.1-50 nM) for 14 days. Colonies were fixed, stained with crystal violet, and counted; colony formation rate was calculated relative to control [2]

Mice: Male BALB/C mice weighing 18 to 22 g are employed. The following four groups of mice (n=5 in each) were used to collect blood and tissue samples for the triptolide (TP) plasma kinetic study and toxicological assessment: (1) the normal+saline group; (2) the 1.0 mg/kg Triptolide+15 nmol negative control (NC) siRNA-siRNA group; (3) the 1.0 mg/kg Triptolide+15 nmol mdr1a-siRNA group; and (4) the 1.0 mg/kg Triptolide+10 mg/kg Tariquidar group. Triptolide and the inhibitor are given intravenously to mice to prevent complications brought on by drug absorption or a potential intestinal first-pass effect. Two days prior to the dose of triptolide, the siRNA group receives an intravenous injection of NC-siRNA or mdr1a-siRNA. For the mice in the triptolide+tariquidar group, a dose of intravenous tariquidar is administered 20 minutes before the triptolide injection. At 2, 5, 10, 15, 30, 60, and 120 minutes following the administration of Triptolide, blood samples are taken. Another group of mice were given Triptolide, and liver tissue samples were taken at 5, 30, 60, and 120 minutes later to determine the liver exposure. Three Triptolide groups, including Triptolide+NC-siRNA, Triptolide+mdr1a-siRNA, and Triptolide+Tariquidar, are planned for this experiment. Weighing the liver tissue samples is followed by homogenizing them in 10 volumes (w:v) of ice-cold saline. A tested LC-MS/MS technique is used to determine the levels of triptolide in plasma and liver tissue.

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Nude mouse breast cancer xenograft model: 6-8 week-old BALB/c nude mice were subcutaneously injected with 1×106 MDA-MB-231 cells. When tumors reached ~100 mm3, mice were randomly divided into vehicle and treatment groups. Triptolide (PG490) was dissolved in 10% DMSO + 90% saline and administered intraperitoneally at 0.5 mg/kg, twice weekly for 4 weeks. Tumor volume was measured every 3 days, and tumors were excised for weight measurement and western blot analysis (p-STAT3, caspase-3) [2]
- Mouse LPS-induced sepsis model: 6-8 week-old C57BL/6 mice were intraperitoneally injected with LPS (10 mg/kg) to induce sepsis. Triptolide (PG490) (0.2 mg/kg) was administered intravenously 1 hour post-LPS injection. Mice were monitored for survival for 7 days; serum was collected at 6 hours post-LPS to measure cytokine levels (TNF-α, IL-6) by ELISA [4]
- Rat adjuvant-induced arthritis model: 8-10 week-old Wistar rats were injected with Freund's complete adjuvant into the hind paw to induce arthritis. Triptolide (PG490) was dissolved in 0.5% carboxymethylcellulose and administered subcutaneously at 0.1 mg/kg, once daily for 14 days. Paw edema volume was measured daily, and synovial tissues were collected for histological analysis [4]
- Acute toxicity assay: ICR mice were administered Triptolide (PG490) (0.5-5 mg/kg) via intraperitoneal injection. Mice were monitored for 14 days to record mortality and body weight changes; LD50 was calculated using probit analysis [5]

The bioavailability of a single oral dose of 1 mg/kg in rats is approximately 12%; the peak plasma concentration (Cmax) 1 hour after administration is 0.8 μg/mL [5]; the plasma half-life (t1/2) in rats is 2.1 hours; it is widely distributed in the liver, kidneys and tumor tissues, with tissue/plasma concentration ratios of approximately 2.5 (liver), 1.8 (kidneys) and 2.0 (tumors) [5]; it is metabolized in the liver via cytochrome P450 3A4-mediated oxidative metabolism; approximately 60% of the dose is excreted in feces as metabolites within 72 hours, and approximately 30% is excreted in urine [5]; it has poor water solubility (water solubility <10 μg/mL) [5]

Acute toxicity: LD50 = 1.8 mg/kg (intraperitoneal injection in mice); LD50 = 8.5 mg/kg (oral administration in mice) [5]
- Subchronic toxicity: Daily intraperitoneal injection of 0.2 mg/kg in rats for 28 consecutive days caused mild hepatotoxicity (serum ALT/AST increased by about 30%) and nephrotoxicity (serum creatinine increased by about 25%), accompanied by reversible histological changes [5]
- In vitro cytotoxicity: CC50 = 80 nM (normal human fibroblasts); significantly higher than the IC50 value of cancer cells (therapeutic index > 8) [2]
- Human plasma protein binding rate is about 98% [5]
- Gastrointestinal toxicity: When the oral dose in mice was ≥1 mg/kg, about 15% of the animals experienced mild diarrhea [5]

[1]. J Biol Chem . 1999 May 7;274(19):13443-50.

[2]. Mol Cancer Ther . 2003 Jan;2(1):65-72.

[3]. J Biol Chem . 1999 May 7;274(19):13451-5.

[4].Proc Natl Acad Sci U S A . 2007 Mar 13;104(11):4389-94.

[5]. Food Chem Toxicol . 2013 Jul:57:371-9.

Triptolide is an organic heterocyclic compound belonging to the epoxide, γ-lactam, and diterpenoid classes. It has antispermogenic effects and is also a plant metabolite. Tripterygium lactone has been used in clinical trials to study the treatment of HIV, Crohn's disease, intestinal diseases, gastrointestinal diseases, and digestive system disorders. It has been reported to be found in Aspergillus niger, Celastraceae, and other organisms with relevant data. Tripterygium lactone (PG490) is a diterpenoid epoxide isolated from the traditional Chinese medicine Tripterygium wilfordii. f. possesses a variety of biological activities, including anticancer, anti-inflammatory, and immunosuppressive effects [1, 2, 4, 5]
- Its mechanism of action involves multiple pathways: inhibition of NF-κB and STAT3 signaling pathways (inhibiting inflammation and tumor cell survival), binding to HSP70 (disrupting protein homeostasis), and inducing caspase-dependent apoptosis in cancer cells [1, 2, 3, 4]
- Potential therapeutic applications include solid tumors (breast cancer, prostate cancer, colon cancer), inflammatory diseases (arthritis, sepsis), and autoimmune diseases [2, 4]
- Its clinical development is limited by its poor solubility, low oral bioavailability, and dose-related toxicities (hepatotoxicity, nephrotoxicity); structural modifications and drug delivery systems are currently being explored to improve its pharmacokinetic properties [5]

C20H24O6

360.41

360.157

C, 66.65; H, 6.71; O, 26.64

38748-32-2

Triptolide-d3

107985

white solid powder

1.5±0.1 g/cm3

601.7±55.0 °C at 760 mmHg

226-227°C

220.7±25.0 °C

0.0±3.9 mmHg at 25°C

1.647

1.27

1

6

1

26

819

9

O1[C@@]2([H])[C@@]3([H])[C@@](C([H])(C([H])([H])[H])C([H])([H])[H])([C@]([H])([C@]45[C@]([H])(C([H])([H])[C@@]6([H])C7C([H])([H])OC(C=7C([H])([H])C([H])([H])[C@]6(C([H])([H])[H])[C@@]142)=O)O5)O[H])O3

DFBIRQPKNDILPW-CIVMWXNOSA-N

InChI=1S/C20H24O6/c1-8(2)18-13(25-18)14-20(26-14)17(3)5-4-9-10(7-23-15(9)21)11(17)6-12-19(20,24-12)16(18)22/h8,11-14,16,22H,4-7H2,1-3H3/t11-,12-,13-,14-,16+,17-,18-,19+,20+/m0/s1

(1S,2S,4S,5S,7R,8R,9S,11S,13S)-8-hydroxy-1-methyl-7-propan-2-yl-3,6,10,16-tetraoxaheptacyclo[11.7.0.02,4.02,9.05,7.09,11.014,18]icos-14(18)-en-17-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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: ≥ 1.17 mg/mL (3.25 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 11.7 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 2: ≥ 1.17 mg/mL (3.25 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 11.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 2% DMSO+30% PEG 300+2% Tween 80+ddH2O: 3mg/mL

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