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Identification of MT-45 Metabolites: In Silico Prediction, In Vitro Incubation with Rat Hepatocytes and In Vivo
Camilla Montesano1, Gabriele Vannutelli1, Federico Fanti1
1Department of Chemistry, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.
Abstract:
MT-45 is a synthetic opioid with a pharmacological activity comparable to morphine and it has been involved in intoxications and fatalities reported in Europe and in USA. It was recently subject to control measures, but to date the metabolic pathways of the substance are still unknown. Using rat hepatocytes and LC-HRMS, 14 novel Phase I and II MT-45 metabolites were identified, products of monohydroxylation, dihydroxylation and N-dealkylation; glucuronide conjugation of mono- and dihydroxylated metabolites also occurred. The detected metabolites were firstly predicted in silico, then incubation of the drug with rat hepatocytes was carried out and the obtained metabolites were identified by LC-HRMS, with retention times, mass shift between theoretical mass and observed mass (<5 ppm), peak abundance and fragmentation pattern. Hydroxylated MT-45 was found to be the major metabolite of MT-45 in vitro experiments. The presence of all metabolites was confirmed by in vivo experiments in urine samples of CD-1 male mice; in these samples hydroxy-MT-45-glucuronide and di-hydroxy-MT-45-glucuronide are the most abundant metabolites, while the parent drug is found at concentration <10 ng mL-1 after 300 min. The knowledge of Phase I and II MT-45 metabolite structure is then crucial to develop analytical methods to identify MT-45 consumption in clinical and forensic testing.
Insights
Researchers identified 14 new metabolites of the synthetic opioid MT-45 in rat liver cells and mouse urine. Understanding these metabolic pathways is crucial for detecting MT-45 use in forensic toxicology.
Area of Science:
- Forensic Toxicology
- Pharmacology
- Metabolism Studies
Background:
- MT-45 is a potent synthetic opioid implicated in intoxications and fatalities.
- Despite control measures, the metabolic fate of MT-45 remains largely unknown.
- Understanding metabolism is key for developing detection methods.
Purpose of the Study:
- To elucidate the metabolic pathways of MT-45 in vitro and in vivo.
- To identify novel Phase I and Phase II metabolites of MT-45.
- To provide structural information for forensic analysis.
Main Methods:
- In silico prediction of MT-45 metabolites.
- Incubation of MT-45 with rat hepatocytes.
- Identification of metabolites using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS).
- Confirmation of metabolites in mouse urine samples.
Main Results:
- Fourteen novel Phase I and II MT-45 metabolites were identified.
- Major metabolic pathways include monohydroxylation, dihydroxylation, and N-dealkylation.
- Hydroxylated metabolites and their glucuronide conjugates were predominant in vitro and in vivo.
- The parent drug concentration was very low in mouse urine after 300 minutes.
Conclusions:
- The study successfully identified key MT-45 metabolites, including hydroxylated and glucuronidated forms.
- Established metabolic pathways are essential for developing sensitive analytical methods for MT-45 detection.
- This research supports forensic and clinical toxicology in identifying MT-45 consumption.
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