Selective inhibitory effects of HYIpro-3-1 on CYP1A2 in human liver microsomes.

CYP1A2 is one of the main Cytochrome P450 enzymes in the human liver associated with the metabolism of several xenobiotics. CYP1A2 is especially involved in the metabolic activation of different procarcinogens. Therefore, the development of cancer may be inhibited by inhibiting CYP1A2 activity. Here, the inhibitory effect of HYIpro-3-1 and its derivatives on CYP1A2 activity in human liver microsomes (HLM) was studied through LC-MS/MS using a cocktail assay. Among the four compounds, HYIpro-3-1 showed the most selective and strongest inhibitory effect on CYP1A2 at IC50 values of 0.1 µM in HLMs and inhibition was confirmed using purified human CYP1A2. It was determined that inhibition is reversible because the inhibitory effect of HYIpro-3-1 is not dependent on preincubation time. HYIpro-3-1 showed a typical pattern of competitive inhibition for CYP1A2-catalyzed phenacetin O-deethylation, based on the Lineweaver-Burk plot, with a Ki value of 0.05 µM in HLMs; the secondary plot also showed a linear pattern. In our study, HYIpro-3-1 was proposed as a novel inhibitor with the capacity to selectively inhibit CYP1A activity in HLMs.

Identification of suberosin metabolites in human liver microsomes by high-performance liquid chromatography combined with high-resolution quadrupole-orbitrap mass spectrometer.

Suberosin is a natural prenylated coumarin derivative isolated from Citropsis articulata. It has various pharmacological properties, especially as an anticoagulant, for which it has been used since antiquity. However, its metabolic pathway and metabolites have not yet been studied. Therefore, this study characterizes its metabolic pathway and metabolites in human liver microsomes (HLMs) using high-resolution quadrupole-orbitrap mass spectrometry (HRMS/MS). Eight metabolites (M1-M8) were found, including three monohydroxylated (M1-M3), one hydrated (M4), three dihydroxylated (M5-M7), and one glucuronide conjugate (M8). Furthermore, forms of cytochrome P450 (CYPs) responsible for suberosin metabolism in HLMs were characterized. CYP1A2 was identified as a major enzyme for the production of M1 and M5 metabolites. The M2, M3, and M7 metabolites were predominantly generated by CYP2B6. M8 was the only phase II metabolite, identified as a glucuronide conjugate from either M1 or M2. This glucuronide conjugate may be the only promising metabolite from phase II metabolism. Phase I metabolism, especially hydroxylation, was found to provide a predominant metabolic pathway of suberosin in HLMs. Further studies should be conducted to explore the metabolites, examining their efficacy and their toxicity in an in vivo system.

Characterization of hydrocoptisonine metabolites in human liver microsomes using a high-resolution quadrupole-orbitrap mass spectrometer.

Hydrocoptisonine is a new compound that has been isolated from the rhizomes of Coptis chinensis, which belongs to the Ranunculaceae family of Chinese medicines. Although studies on C. chinensis have been reported, the metabolic pathway of hydrocoptisonine in human liver microsomes (HLMs) remains unelucidated. We identified 13 metabolites in HLMs, including six Phase I metabolites and seven glucuronide conjugates, using a high-resolution quadrupole-orbitrap mass spectrometer. The major metabolic pathway was the O-demethylation and mono-hydroxylation of hydrocoptisonine in HLMs. Notably, M3 metabolite was O-demethylated in dioxolane structures (cyclohexa-3,5-diene-1,2-dione), which was mediated by cytochrome P450 1A2. The locations of hydroxylation and hydroxyl-glucuronidation were identified by analyzing the signature fragments generated as a result of tandem mass spectrometry, indicating hydroxylation at an aliphatic chain or aromatic ring. We determined whether the hydroxylation and glucuronidation occurred in an aromatic moiety (M5 and M12) or an aliphatic moiety (M6 and M13), respectively, based on signature fragments of the metabolites.

Characterization of red ginseng-drug interaction by CYP3A activity increased in high dose administration in mice.

Ginseng (Panax ginseng Meyer) is a popular traditional herbal medicine used worldwide. Patients often take ginseng preparations with other medicines where the ginseng dose could exceed the recommended dose during long-term administration. However, ginseng-drug interactions at high doses of ginseng are poorly understood. This study showed the possibility of herb-drug interactions between the Korean red ginseng (KRG) extract and cytochrome P450 (CYP) substrates in higher administration in mice. The CYP activities were determined in vivo after oral administration of KRG extract doses of 0.5, 1.0, and 2.0 g/kg for 2 or 4 weeks by monitoring the concentration of five CYP substrates/metabolites in the blood. The area under the curve for OH-midazolam/midazolam catalysed by CYP3A was increased significantly by the administration of 2.0 g/kg KRG extract for 2 and 4 weeks. CYP3A-catalysed midazolam 1'-hydroxylation also increased significantly in a dose- and time-dependent manner in the S9 fraction of mouse liver which was not related to induction by transcription. Whereas CYP2D-catalysed dextromethorphan O-deethylation decreased in a dose- and time-dependent manner in vivo. In conclusion, interactions were observed between KRG extract and CYP2D and CYP3A substrates at subchronic-high doses of KRG administration in mice.

In vitro characterization of glycyrol metabolites in human liver microsomes using HR-resolution MS spectrometer coupled with tandem mass spectrometry.

1. Glycyrol is a coumestan derivative that is isolated from roots of Glycyrrhiza uralensis. Glycyrol exhibits several biological effects, including anti-oxidative and anti-inflammatory effects.2. Herein, we characterized glycyrol metabolism by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM), human liver cytosol, human intestinal microsomes, or human recombinant cDNA-expressed CYPs and UGTs. The analysis was conducted using high resolution mass spectroscopy (HR-MS) on a Q ExactiveTM HF Hybride Quadrupole-Orbitrap mass spectrometer.3. NADPH-supplemented HLM generated six glycyrol metabolites (M1-M6) via hydroxylation, oxidation, and hydration; both NADPH- and UDPGA-supplemented liver microsomes generated three glucuronides (M7-M9). Reaction phenotyping revealed that CYP1A2 is the primary enzyme responsible for phase I metabolism, with minor involvement of the CYP3A4/5, CYP2D6, and CYP2E1 enzymes. Glucuronidation of glycyrol was primarily mediated by UGT1A1, UGT1A3, UGT1A9, and UGT2B7.4. In conclusion, glycyrol undergoes the efficient metabolic hydroxylation and glucuronidation reactions in human liver microsomes, which are predominantly catalyzed by CYP1A2, UGT1A1/3/9, and UGT2B7.

Characterization of osthenol metabolism in vivo and its pharmacokinetics.

Osthenol, a prenylated coumarin, is a C8-prenylated derivative of umbelliferone isolated from the root of Angelica koreana and Angelica dahurica, an intermediate and is known as a major metabolite of desmethyl-osthole.The various pharmacological effects of osthenol have been reported. In previous studies, we investigated five hydroxylated metabolites by cytochromes P450 (CYP) and glucuronide conjugates of osthenol by uridine diphosphate-glucuronosyltransferases (UGTs). However, osthenol have very few studies have been reported on its pharmacokinetic (PK) profiling, we reported the PK parameters in mouse of osthenol through this study.After oral (5 and 20 mg/kg) and intravenous (5 mg/kg) administration, the concentration of osthenol in plasma was determined by LC-MS/MS. The quantitative method was validated in terms of linearity, accuracy, and precision. When 5 and 20 mg/kg of osthenol were orally administered, the bioavailability (BA) was found to be very low at 0.43 and 0.02%, respectively.In fact, osthenol was mostly metabolized to a two-Phase II conjugates, a sulfonyl and glucuronyl-osthenol, in the blood, which was determined by LC-HR/MS analysis of the blood sample. Because osthenol is rapidly metabolized to two conjugates by first-pass effect the BA of osthenol is low after oral administration.

Assessing Drug Interaction and Pharmacokinetics of Loxoprofen in Mice Treated with CYP3A Modulators.

Loxoprofen (LOX) is a non-selective cyclooxygenase inhibitor that is widely used for the treatment of pain and inflammation caused by chronic and transitory conditions. Its alcoholic metabolites are formed by carbonyl reductase (CR) and they consist of trans-LOX, which is active, and cis-LOX, which is inactive. In addition, LOX can also be converted into an inactive hydroxylated metabolite (OH-LOXs) by cytochrome P450 (CYP). In a previous study, we reported that CYP3A4 is primarily responsible for the formation of OH-LOX in human liver microsomes. Although metabolism by CYP3A4 does not produce active metabolites, it can affect the conversion of LOX into trans-/cis-LOX, since CYP3A4 activity modulates the substrate LOX concentration. Although the pharmacokinetics (PK) and metabolism of LOX have been well defined, its CYP-related interactions have not been fully characterized. Therefore, we investigated the metabolism of LOX after pretreatment with dexamethasone (DEX) and ketoconazole (KTC), which induce and inhibit the activities of CYP3A, respectively. We monitored their effects on the PK parameters of LOX, cis-LOX, and trans-LOX in mice, and demonstrated that their PK parameters significantly changed in the presence of DEX or KTC pretreatment. Specifically, DEX significantly decreased the concentration of the LOX active metabolite formed by CR, which corresponded to an increased concentration of OH-LOX formed by CYP3A4. The opposite result occurred with KTC (a CYP3A inhibitor) pretreatment. Thus, we conclude that concomitant use of LOX with CYP3A modulators may lead to drug-drug interactions and result in minor to severe toxicity even though there is no direct change in the metabolic pathway that forms the LOX active metabolite.

A secretome profile indicative of oleate-induced proliferation of HepG2 hepatocellular carcinoma cells.

Increased fatty acid (FA) is often observed in highly proliferative tumors. FAs have been shown to modulate the secretion of proteins from tumor cells, contributing to tumor survival. However, the secreted factors affected by FA have not been systematically explored. Here, we found that treatment of oleate, a monounsaturated omega-9 FA, promoted the proliferation of HepG2 cells. To examine the secreted factors associated with oleate-induced cell proliferation, we performed a comprehensive secretome profiling of oleate-treated and untreated HepG2 cells. A comparison of the secretomes identified 349 differentially secreted proteins (DSPs; 145 upregulated and 192 downregulated) in oleate-treated samples, compared to untreated samples. The functional enrichment and network analyses of the DSPs revealed that the 145 upregulated secreted proteins by oleate treatment were mainly associated with cell proliferation-related processes, such as lipid metabolism, inflammatory response, and ER stress. Based on the network models of the DSPs, we selected six DSPs (MIF, THBS1, PDIA3, APOA1, FASN, and EEF2) that can represent such processes related to cell proliferation. Thus, our results provided a secretome profile indicative of an oleate-induced proliferation of HepG2 cells.

Quantitative Proteomic Analysis of Changes Related to Age and Calorie Restriction in Rat Liver Tissue.

Calorie restriction (CR) is the most frequently studied mechanism for increasing longevity, protecting against stress, and delaying age-associated diseases. Most studies have initiated CR in young animals to determine the protective effects against aging. Although aging phenomena are well-documented, the molecular mechanisms of aging and CR remain unclear. In this study, we observe changes in hepatic proteins upon age-related and diet-restricted changes in the rat liver using quantitative proteomics. Quantitative proteomes are measured using tandem mass tag labeling followed by LC-MS/MS. We compare protein levels in livers from young (6 months old) and old (25 months old) rats with 40% calorie-restricted (YCR and OCR, respectively) or ad libitum diets. In total, 44 279 peptides and 3134 proteins are identified and 260 differentially expressed proteins are found. Functional enrichment analysis show that these proteins are mainly involved in glucose and fatty acid metabolism-related processes, consistent with the theory that energy metabolism regulation is dependent on age-related and calorie-restricted changes in liver tissue. In addition, proteins mediating inflammation and gluconeogenesis are increased in OCR livers, but not YCR livers. These results show that CR in old rats might not have antiaging benefits because liver inflammation is increased.

Characterization of developmental defects in the forebrain resulting from hyperactivated mTOR signaling by integrative analysis of transcriptomic and proteomic data.

Hyperactivated mTOR signaling in the developing brain has been implicated in multiple forms of pathology including tuberous sclerosis complex (TSC). To date, various phenotypic defects such as cortical lamination irregularity, subependymal nodule formation, dysmorphic astrocyte differentiation and dendritic malformation have been described for patients and animal models. However, downstream networks affected in the developing brain by hyperactivated mTOR signaling have yet to be characterized. Here, we present an integrated analysis of transcriptomes and proteomes generated from wild-type and Tsc1/Emx1-Cre forebrains. This led to comprehensive lists of genes and proteins whose expression levels were altered by hyperactivated mTOR signaling. Further incorporation of TSC patient data followed by functional enrichment and network analyses pointed to changes in molecular components and cellular processes associated with neuronal differentiation and morphogenesis as the key downstream events underlying developmental and morphological defects in TSC. Our results provide novel and fundamental molecular bases for understanding hyperactivated mTOR signaling-induced brain defects which can in turn facilitate identification of potential diagnostic markers and therapeutic targets for mTOR signaling-related neurological disorders.