Bioactivation of 3-n-Butylphthalide via Sulfation of Its Major Metabolite 3-Hydroxy-NBP: Mediated Mainly by Sulfotransferase 1A1

Abstract

3-n-Butylphthalide (NBP) [(6)-3-butyl-1(3H)-isobenzofuranone] is an anti-cerebral-ischemia drug. Moderate hepatotoxicity has been observed in clinical applications. One of the major metabolites, 3-N-acetylcysteine-NBP, has been detected in human urine, indicating the formation of a reactive metabolite. We elucidated the formation mechanism of the reactive metabolite and its association with the hepatotoxicity of NBP. The in vitro incubations revealed that 3-glutathione-NBP (3-GSH-NBP) was observed only in fresh rat liver homogenate rather than in liver microsomes, liver cytosol, or liver 9,000g supernatant supplemented with NADPH and GSH. We also detected 3-GSH-NBP when 39-phosphoadenosine-59-phosphosulfate was added in GSH-fortified human liver cytosol (HLC). The formation of 3-GSH-NBP was 39.3-fold higher using 3-hydroxy-NBP (3-OH-NBP) as the substrate than NBP. The sulfotransferase (SULT) inhibitors DCNP (2,6-dichloro-4-nitrophenol) and quercetin suppressed 3-GSH-NBP formation in HLC by 75 and 82%, respectively, suggesting that 3-OH-NBP sulfation was involved in 3-GSH-NBP formation. Further SULT phenotyping revealed that SULT1A1 is the major isoform responsible for the sulfation. Dose-dependent toxicity was observed in primary rat hepatocytes exposed to 3-OH-NBP, with an IC 50 of approximately 168 mM. Addition of DCNP and quercetin significantly increased cell viability, whereas L-buthionine-sulfoximine (a GSH depleter) decreased cell viability. Overall, our study revealed the underlying mechanism for the bioactivation of NBP is as follows. NBP is first oxidized to 3-OH-NBP and further undergoes sulfation to form 3-OH-NBP sulfate. The sulfate spontaneously cleaves off, generating highly reactive electrophilic cations, which can bind either to GSH to detoxify or to hepatocellular proteins to cause undesirable side effects.

A Review of Recent Advances in Neuroprotective Potential of 3-N-Butylphthalide and Its Derivatives

“…Additionally, it has been found that the likelihood of drug-to-drug interaction is minimized by the diversity of enzymes, such as P450, alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH), involved in its metabolism [6]. However, the participation of 3-hydroxy-NBP sulfate, one of the sulfated metabolites of dl-NBP in the process, is believed to be the main reason for dl-NBP's side effect (mild hepatotoxicity) [7]. Many researches involving stroke animal models have also concluded that NBP, through multiple mechanisms, significantly reduces the cerebral infarct size and oxidative damage, inhibits platelet aggregation, improves blood flow and mitochondrial functions, possesses antithrombotic and anti-inflammatory effects, and reduces neural apoptosis [8–10], hence the importance of understanding its mechanisms and the value it might hold for future management of ischemic stroke as well as neurodegenerative diseases.…”

In Vitro and In Vivo Human Metabolism of Synthetic Cannabinoids FDU-PB-22 and FUB-PB-22

“…Human hepatocytes were selected, rather than human liver microsomes (HLM), to better simulate a physiological liver environment (20,21). The hepatocyte incubation model was successful in our previous studies for identifying appropriate markers for AB-FUBINACA (19), AB-PINACA (22), and AH-7921 (23), among others.…”

High-Resolution Mass Spectrometry for Characterizing the Metabolism of Synthetic Cannabinoid THJ-018 and Its 5-Fluoro Analog THJ-2201 after Incubation in Human Hepatocytes

“…Human hepatocytes were chosen over human liver microsomes (HLMs) because hepatocytes contain all endogenous drug-metabolizing enzymes and essential cofactors (18 ). Metabolites identified with human hepatocytes can serve as useful markers for identifying novel SCs (19 ).…”