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. 2025 Jan;43(1):130-141.
doi: 10.1007/s11419-024-00705-0. Epub 2024 Nov 22.

Metabolic profiling of the synthetic cannabinoid APP-CHMINACA (PX-3) as studied by in vitro and in vivo models

Cristian Camuto  1 , Fabio De-Giorgio  2   3 , Giorgia Corli  4 , Sabrine Bilel  4 , Monica Mazzarino  1   5 , Matteo Marti  4   6 , Francesco Botrè  7   8   9
Affiliations

Metabolic profiling of the synthetic cannabinoid APP-CHMINACA (PX-3) as studied by in vitro and in vivo models

Cristian Camuto et al. Forensic Toxicol. 2025 Jan.

Abstract

Purpose: The metabolic pathways of APP-CHMINACA were characterized to select the markers of intake for implementation into analytical assays used by the clinical and forensic communities. We have combined the evidences obtained by both in vitro experiments and administration studies on mice.

Methods: APP-CHMINACA was incubated with either human or mouse liver microsomes. Urine and blood samples were collected at different time points from mice after injection of a 3 mg/kg dose of the test compound. Samples were analyzed using liquid chromatography-tandem mass spectrometry.

Results: The in vitro studies allowed to isolate eight different metabolic reactions, formed by two metabolic routes, with no differences between human and mouse liver microsomes. The main biotransformation route involved the hydrolysis of the distal amide group and the subsequent hydroxylation on the cyclohexyl-methyl ring. The second route involved multiple hydroxylation of the parent compound, followed by reduction to generate minor metabolites. In blood samples, the most abundant substances identified were APP-CHMINACA unchanged and the metabolites formed by the hydrolysis of the distal amide together with its hydroxylated products. In urine samples, four metabolites formed following the hydroxylation of the distal amide hydrolysis metabolite were detected as the most abundant and long-term metabolites.

Conclusions: The outcomes of our study showed that the most suitable markers to detect the intake of APP-CHMINACA in blood and urine samples in the framework of toxicological, clinical and forensic investigations were the metabolite formed by the hydrolysis of the distal amide and its hydroxylated products.

Keywords: APP-CHMINACA; ICR-CD1 mice; Metabolite identification; PX-3; Synthetic cannabinoids.

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Conflict of interest statement

Declarations. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose. Ethical approval: All experimental protocols were in accordance with the U.K. Animals (Scientific Procedures) Act of 1986 and associated guidelines and the new European Communities Council Directive of September 2010 (2010/63/EU), and approved by the Italian Ministry of Health (license n. 223/2021-PR, CBCC2.46.EXT.21) and the Animal Welfare Body of the University of Ferrara. According to the ARRIVE guidelines, all possible efforts were made to minimise the animals’ pain and discomfort and to reduce the number of experimental subjects.

Figures

Fig. 1
Fig. 1
Characteristic fragmentation pathway of APP-CHMINACA and proposed fragmentation pattern
Fig. 2
Fig. 2
Metabolic reactions identified for APP-CHMINACA by two different metabolic routes (R1 and R2). R1: Distal amide hydrolysis (M1), distal amide hydrolysis and ketone formation on CHM (M2), distal amide hydrolysis and mono-hydroxylation on CHM (M4). R2: mono-hydroxylation on CHM (M3), mono-hydroxylation and ketone formation on CHM (M5), di-hydroxylation on CHM (M6), di-hydroxylation and ketone formation on CHM (M7), tri-hydroxylation on CHM (M8)
Fig. 3
Fig. 3
Extracted chromatogram of APP-CHMINACA and the principal metabolites identified after 1 h by the incubation with MLM a and HLM b. APP-CHMINACA distal amide hydrolysis (M1), distal amide hydrolysis and mono-oxidation (CHM) (M4.1–5)
Fig. 4
Fig. 4
Levels of APP-CHMINACA in blood samples over the range of 30–300 min by the intraperitoneal administration of a 3 mg/kg dose (A). Blood profile of APP-CHMINACA and its main metabolites over the range of 30–300 min by the intraperitoneal administration of a 3 mg/kg dose of APP-CHMINACA (B)
Fig. 5
Fig. 5
Excretion profile of the main metabolites found in urine after the intraperitoneal administration of a 3 mg/kg dose of APP-CHMINACA
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