Toxic metabolites and metabolic soft spots of celastrol based on glutathione metabolic capture and high-resolution mass spectrometry

ABSTRACT

Background

Celastrol is known as one of the most medicinally valuable compounds. However, the pharmaceutical application of celastrol is significantly limited due to high toxicity, while there are few reports on the mechanism of toxicity.

Methods

This study searched for possible toxic metabolites through phase I in vitro metabolism and glutathione capture experiments. Then in vivo metabolism experiments in mice and rats were conducted to look for metabolites in vivo. Finally, mice in vivo toxicity experiment was conducted to verify the toxicity of different doses of celastrol to mice.

Results

In the in vivo and in vitro metabolism experiments, we found 7 phase I metabolites in vitro, 9 glutathione conjugation metabolites in vitro, and 20 metabolites in vivo. The metabolic soft points of celastrol could be the quinone methyl structure at C3-OH and C6. In vivo toxicity experiments show that celastrol causes weight loss, diarrhea, gastrointestinal tract and liver inflammation in mice.

Conclusions

This study analyzed the metabolites and possible metabolic soft spots of celastrol, and its hepatotoxicity and gastrointestinal toxicity were demonstrated through in vivo studies for the first time. The results might provide an important basis for potential structural modification to increase the druggability of celastrol.

KEYWORDS:

• Celastrol
• toxic metabolite
• metabolic soft spot
• hepatotoxicity
• gastrointestinal toxicity

CRediT author statement

Y Lin, Q Xie, C Wu, X Jiang, C Yuan and R Ding: writing - original draft, conceptualization, methodology, funding acquisition. C Wu: resources; X Jiang, C Yuan, D Lu, X Peng and Z Dong: data curation, writing - review & editing; X Jiang, C Yuan and C Zhu: formal analysis; X Jiang and R Ding: Validation. All authors approved the final version of the article.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

One reviewer has been recruited by Research Square. Reviewers with declared or apparent competing interests are not utilized for these reviews. This reviewer was paid a small honorarium for completing the review within a specified timeframe. Honoraria for reviews such as this are paid regardless of the reviewer recommendation. The remaining reviewers have no other relevant financial relationships or otherwise to disclose.

Ethics

All animal care and experimental procedures were approved by the Animal Ethics Committee of Xiamen University (XMULAC20220126), and the animals were self-fed for one week before experiment.

Additional information

Funding

This research was funded by National Natural Science Foundation of China [grant numbers 82070423, 82222068, 82141215, and 82173779]; Fujian Province Science and Technology Project [grant numbers 2021J02058 and 2020Y0013]; Fundamental Research Funds for the Chinese Central Universities (20720230070).