Mimicking of Phase I Metabolism Reactions of Molindone by HLM and Photocatalytic Methods with the Use of UHPLC-MS/MS

Establishing the metabolism pathway of the drug undergoing the hepatic biotransformation pathway is one of the most important aspects in the preclinical discovery process since the presence of toxic or reactive metabolites may result in drug withdrawal from the market. In this study, we present the structural elucidation of six, not described yet, metabolites of an antipsychotic molecule: molindone. The elucidation of metabolites was supported with a novel photocatalytical approach with the use of WO3 and WS2 assisted photochemical reactions. An UHPLC-ESI-Q-TOF combined system was used for the registration of all obtained metabolite profiles as well as to record the high resolution fragmentation spectra of the observed transformation products. As a reference in the in vitro metabolism simulation method, the incubation with human liver microsomes was used. Chemometric comparison of the obtained profiles pointed out the use of the WO3 approach as being more convenient in the field of drug metabolism studies. Moreover, the photocatalysis was used in the direction of the main drug metabolite synthesis in order to further isolation and characterization.

Compound Characterization and Metabolic Profile Elucidation after In Vitro Gastrointestinal and Hepatic Biotransformation of an Herniaria hirsuta Extract Using Unbiased Dynamic Metabolomic Data Analysis

Herniaria hirsuta L. (Caryophyllaceae) is used for treatment of urinary stones and as a diuretic. Little is known about the active compounds and the mechanism of action. The phytochemical composition of H. hirsuta was comprehensively characterized using UHPLC-UV-HRMS (Ultrahigh-Performance Liquid Chromatography-Ultraviolet-High Resolution Mass Spectrometry) data. An in vitro gastrointestinal model was used to simulate biotransformation, which allowed the monitoring of the relative abundances of individual compounds over time. To analyze the longitudinal multiclass LC–MS data, XCMS, a platform that enables online metabolomics data processing and interpretation, and EDGE, a statistical method for time series data, were used to extract significant differential profiles from the raw data. An interactive Shiny app in R was used to rate the quality of the resulting features. These ratings were used to train a random forest model. The most abundant aglycone after gastrointestinal biotransformation was subjected to hepatic biotransformation using human S9 fractions. A diversity of compounds was detected, mainly saponins and flavonoids. Besides the known saponins, 15 new saponins were tentatively identified as glycosides of medicagenic acid, acetylated medicagenic acid and zanhic acid. It is suggested that metabolites of phytochemicals present in H. hirsuta, most likely saponins, are responsible for the pharmaceutical effects. It was observed that the relative abundance of saponin aglycones increased, indicating loss of sugar moieties during colonic biotransformation, with medicagenic acid as the most abundant aglycone. Hepatic biotransformation of this aglycone resulted in different metabolites formed by phase I and II reactions.

Acid Sphingomyelinase Inhibition Stabilizes Hepatic Ceramide Content and Improves Hepatic Biotransformation Capacity in a Murine Model of Polymicrobial Sepsis

Liver dysfunction during sepsis is an independent risk factor leading to increased mortality rates. Specifically, dysregulation of hepatic biotransformation capacity, especially of the cytochrome P450 (CYP) system, represents an important distress factor during host response. The activity of the conserved stress enzyme sphingomyelin phosphodiesterase 1 (SMPD1) has been shown to be elevated in sepsis patients, allowing for risk stratification. Therefore, the aim of the present study was to investigate whether SMPD1 activity has an impact on expression and activity of different hepatic CYP enzymes using an animal model of polymicrobial sepsis. Polymicrobial sepsis was induced in SMPD1 wild-type and heterozygous mice and hepatic ceramide content as well as CYP mRNA, protein expression and enzyme activities were assessed at two different time points, at 24 h, representing the acute phase, and at 28 days, representing the post-acute phase of host response. In the acute phase of sepsis, SMPD1+/+ mice showed an increased hepatic C16- as well as C18-ceramide content. In addition, a downregulation of CYP expression and activities was detected. In SMPD1+/− mice, however, no noticeable changes of ceramide content and CYP expression and activities during sepsis could be observed. After 28 days, CYP expression and activities were normalized again in all study groups, whereas mRNA expression remained downregulated in SMPD+/+ animals. In conclusion, partial genetic inhibition of SMPD1 stabilizes hepatic ceramide content and improves hepatic monooxygenase function in the acute phase of polymicrobial sepsis. Since we were also able to show that the functional inhibitor of SMPD1, desipramine, ameliorates downregulation of CYP mRNA expression and activities in the acute phase of sepsis in wild-type mice, SMPD1 might be an interesting pharmacological target, which should be further investigated.