In vitro inhibitory effects of components from Salvia miltiorrhiza on catalytic activity of three human Arachidonic acid ω-hydroxylases

CYP4 enzymes are involved in the metabolism of xenobiotics and endogenous molecules. 20-Hydroxyeicosatetraenoic acid (20-HETE), the arachidonic acid (AA) ω-hydroxylation metabolite catalyzed by CYP4A/4F enzymes, is implicated in various biological functions. The goal of this investigation is to examine the inhibitory effects of components from Salvia miltiorrhiza(Danshen) on AA ω-hydroxylation using recombinant CYP4A11, CYP4F2, CYP4F3B, and microsomal systems. Tanshinone IIA had noncompetitive inhibition on CYP4F3B (K_i = 4.98 μM). Cryptotanshinone (K_i = 6.87 μM) and tanshinone I (K_i = 0.42 μM) had mixed-type inhibition on CYP4A11. Dihydrotanshinone I had mixed-type inhibition on CYP4A11 (K_i = 0.09 μM), and noncompetitive inhibition on CYP4F2 (K_i = 4.25 μM) and CYP4F3B (K_i = 3.08 μM). Salvianolic acid A had competitive inhibition on CYP4A11 (K_i = 19.37 μM), and noncompetitive inhibition on CYP4F2 (K_i = 15.28 μM) and CYP4F3B (K_i = 6.45 μM). Salvianolic acid C had noncompetitive inhibition on CYP4F2 (K_i = 5.70 μM) and CYP4F3B (K_i = 18.64 μM). In human kidney, human liver or rat heart microsomes, 20-HETE formation was significantly inhibited (P < 0.05) by dihydrotanshinone I (5 and 20 μM) and salvianolic acid A (20 and 50 μM). Given that low plasma concentrations of Danshen components after oral administration, Danshen preparations may not play pharmacological roles by inhibiting AA ω-hydroxylases; however, as Danshen components may reach high concentration in human intestine, drugs that have an important pre-systemic metabolism by these CYP4A/4F enzymes should avoid being co-administered with Danshen preparations.     

Synthesis and evaluation of tofacitinib analogs designed to mitigate metabolic activation

Tofacitinib (TFT), a JAK inhibitor used for the treatment of rheumatoid arthritis and other diseases, is associated with severe liver injury that is believed to be caused by its reactive aldehyde or epoxide metabolites. In this study, we synthesized six tofacitinib analogs designed to avoid the formation of reactive metabolites and evaluated their JAK3 inhibitory activity, metabolic stability, CYP3A time-dependent inhibition, and cytotoxicity. Our data indicated that purine analog 3, which showed little inhibition of CYP3A and cytotoxicity and inhibited JAK3 in the nanomolar range, could be a safer drug candidate than TFT. In addition, the results of the bioactivation study using TFT and its analogs suggest that the epoxide metabolite might contribute to TFT-induced CYP3A4 mechanism-based inhibition and hepatic toxicity.     

New insights of CYP1A in endogenous metabolism: a focus on single nucleotide polymorphisms and diseases

Cytochrome P450 1A (CYP1A), one of the major CYP subfamily in humans, not only metabolizes xenobiotics including clinical drugs and pollutants in the environment, but also mediates the biotransformation of important endogenous substances. In particular, some single nucleotide polymorphisms (SNPs) for CYP1A genes may affect the metabolic ability of endogenous substances, leading to some physiological or pathological changes in humans. This review first summarizes the metabolism of endogenous substances by CYP1A, and then introduces the research progress of CYP1A SNPs, especially the research related to human diseases. Finally, the relationship between SNPs and diseases is discussed. In addition, potential animal models for CYP1A gene editing are summarized. In conclusion, CYP1A plays an important role in maintaining the health in the body.     

Inhibitory effects of sesamin on CYP2C9-dependent 7-hydroxylation of S-warfarin

A recent report demonstrated that sesamin strongly and non-competitively inhibits S-warfarin 7-hydroxylation activity in human liver microsomes with a K_i value of 0.2 μM. This finding suggests that sesamin predominantly binds to CYP2C9 at another site for which it has a higher affinity than its affinity for the active site, thereby inhibiting the activity of CYP2C9 non-competitively. In this study, we found that sesamin competitively inhibited the 7-hydroxylation activity of S-warfarin in human liver microsomes with a K_i value of 15.7 μM. In addition, the recombinant CYP2C9-dependent 7-hydroxylation activity of S-warfarin was competitively inhibited by sesamin with a Ki value of 13.1 μM. These results are consistent with the fact that sesamin is a good substrate of CYP2C9, and its activity follows Michaelis-Menten kinetics. As the plasma concentration of sesamin after its administration is usually lower than 0.01 μM, the inhibition of S-warfarin metabolism by sesamin does not appear to be severe.     

Cloning and tissue expression of cytochrome P450 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 in marmosets

Common marmoset (Callithrix jacchus) is an attractive animal model primate species for potential use in drug metabolism and pharmacokinetic studies. In this study, marmoset cytochrome P450 (P450) 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 cDNAs were isolated from marmoset tissues (brains, lungs, livers, kidneys, and jejunums). Deduced amino acid sequences (89–98% homologous) of the marmoset P450 gene suggested similarity of molecular characteristics of marmoset P450s to human counterparts, compared with those of pig, rabbit, and rodents. Phylogenetic analysis using amino acid sequences indicated 11 marmoset P450 forms clustered with those of human and other primate counterparts, suggesting marmoset P450s have an evolutionary close relationship to human and other primate counterparts. Tissue expression patterns of these P450 mRNAs except for P450 7B1 mRNA were generally similar to those of human P450s in the five tissue types analyzed. These results suggest similarity of molecular characteristics for P450 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 between marmosets and humans, in addition to the orthologs of human P450 1, 2, 3, and 4 families previously identified and characterized in marmosets.     

Sulfate conjugates are the major metabolites in rats administrated with sesamin

Sesamin is known to have various biological effects. Although several metabolites of sesamin have been identified, its metabolism by phase II enzymes remains unclear, because usually its sulfo- and glucurono-conjugates in plasma and urine are analyzed after sulfatase/β-glucuronidase treatment. In this study, the metabolites of sesamin in rats administrated with sesamin (100 mg/kg b.w.) were analyzed without sulfatase/β-glucuronidase treatment. Two sulfate conjugates of sesamin monocatechol (SC-1) were detected in the liver and plasma. Their C_max values were 5- and 10-times higher than that of sesamin itself. The V_max/K_m values for sulfate conjugation in the cytosol fraction of human liver were 1.7-times larger than that in the cytosol fraction of rat liver, suggesting that sulfate conjugation also occurs in human liver. The recombinant human proteins SULT1A1, 1A3, 1B1, and 1E1 expressed in Saccharomyces cerevisiae cells produced sulfate conjugates effectively. Our results could help revealing the mechanism of physiological effects of sesamin.     

In situ metabolomics in nephrotoxicity of aristolochic acids based on air flow-assisted desorption electrospray ionization mass spectrometry imaging

Understanding of the nephrotoxicity induced by drug candidates is vital to drug discovery and development. Herein, an in situ metabolomics method based on air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) was established for direct analysis of metabolites in renal tissue sections. This method was subsequently applied to investigate spatially resolved metabolic profile changes in rat kidney after the administration of aristolochic acid I, a known nephrotoxic drug, aimed to discover metabolites associated with nephrotoxicity. As a result, 38 metabolites related to the arginine–creatinine metabolic pathway, the urea cycle, the serine synthesis pathway, metabolism of lipids, choline, histamine, lysine, and adenosine triphosphate were significantly changed in the group treated with aristolochic acid I. These metabolites exhibited a unique distribution in rat kidney and a good spatial match with histopathological renal lesions. This study provides new insights into the mechanisms underlying aristolochic acids nephrotoxicity and demonstrates that AFADESI-MSI-based in situ metabolomics is a promising technique for investigation of the molecular mechanism of drug toxicity.     

Bile acid homeostasis in female mice deficient in Cyp7a1 and Cyp27a1

Bile acids (BAs) are amphipathic molecules important for metabolism of cholesterol, absorption of lipids and lipid soluble vitamins, bile flow, and regulation of gut microbiome. There are over 30 different BA species known to exist in humans and mice, which are endogenous modulators of at least 6 different membrane or nuclear receptors. This diversity of ligands and receptors play important roles in health and disease; however, the full functions of each individual BA in vivo remain unclear. We generated a mouse model lacking the initiating enzymes, CYP7A1 and CYP27A1, in the two main pathways of BA synthesis. Because females are more susceptible to BA related diseases, such as intrahepatic cholestasis of pregnancy, we expanded this model into female mice. The null mice of Cyp7a1 and Cyp27a1 were crossbred to create double knockout (DKO) mice. BA concentrations in female DKO mice had reductions in serum (63%), liver (83%), gallbladder (94%), and small intestine (85%), as compared to WT mice. Despite low BA levels, DKO mice had a similar expression pattern to that of WT mice for genes involved in BA regulation, synthesis, conjugation, and transport. Additionally, through treatment with a synthetic FXR agonist, GW4064, female DKO mice responded to FXR activation similarly to WT mice.     

Analysis of inhibition kinetics of three beverage ingredients,bergamottin, dihydroxybergamottin and resveratrol,on CYP2C9 activity

Some grapefruit juice (GFJ) ingredients and resveratrol, a fruit-derived phytoalexin, are known to inhibit cytochrome P450 (CYP) 2C9. However, their inhibition modes and detailed inhibition kinetics remain undetermined. This study aimed to investigate the inhibitory effects of two GFJ ingredients, bergamottin (BG) and dihydroxybergamottin (DHB), and resveratrol on CYP2C9 activity in vitro. DHB inhibited CYP2C9 activity, as assessed by warfarin 7-hydroxylation, in a preincubation time-dependent manner (i.e., mechanism-based inhibition; MBI), in the same manner as CYP2C19 and CYP3A4. The maximal inactivation rate (k_inact,max) was 0.0638 min⁻¹ and 0.12- and 0.26-fold of that for CYP2C19 and CYP3A4, respectively. BG showed both MBI and time-independent competitive inhibition. Resveratrol showed non-competitive inhibition with an inhibition constant (K_i) of 3.64 μM. Unlike the inhibition of CYP2C19 and CYP3A4, resveratrol did not induce MBI. These findings are important for estimating the risk of drug interactions between CYP2C9 substrates and some beverages. (146 words)     

Metabolomics study on the therapeutic effect of traditional Chinese medicine Xue-Fu-Zhu-Yu decoction in coronary heart disease based on LC-Q-TOF/MS and GC-MS analysis

The present study aims is to investigate the metabolic mechanism of Xue-Fu-Zhu-Yu decoction (XFZYD) in the treatment of blood-stasis syndrome in Coronary Heart Disease (CHD). To that end, 30 CHD patients with Blood-Stasis Syndrome (BSS) and 20 healthy subjects were enrolled. LC-Q-TOF/MS analysis determined that in comparison between CHD with BSS patients (Group A) and healthy subjects (Group C), 59 significantly differential metabolites in the positive mode and 18 significantly differential metabolites in the negative mode. The metabolite constituents in the plasma of 30 CHD with BSS patients before (group A) and after 30 days of treatment (Group B), and 20 healthy subjects (Group C) were analyzed using LC-Q-TOF/MS and GC-MS. Based on multivariate statistical analysis (PCA, PLS-DA and OPLS-DA), we determined 69 differential metabolites. The levels of hemorheology indexes were significantly down-regulated after treatment. Metabolic pathway attribution analysis showed that lipid metabolism, amino acid metabolism and bile acid metabolism pathways are involved. Our study identifies the metabolic networks of CHD and demonstrates the efficacy of this metabolomics approach to systematically study the therapeutic effect of XFZYC on CHD.     

Whole-cell dependent biosynthesis of N- and S-oxides using human flavin containing monooxygenases expressing budding yeast

Flavin containing monooxygenases (FMOs) represent one of the predominant types of phase I drug metabolizing enzymes (DMEs), and thus play an important role in the metabolism of xeno- and endobiotics for the generation of their corresponding oxides. These oxides often display biological activities, however they are difficult to study since their chemical or biological synthesis is generally challenging even though only small amounts are required to evaluate their efficacy and safety. Previously, we constructed a DME expression system for cytochrome P450, UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) using yeast cells, and successfully produced xenobiotic metabolites in a whole-cell dependent manner. In this study, we developed a heterologous expression system for human FMOs, including FMO1–FMO5, in Saccharomyces cerevisiae and examined its N- and S-oxide productivity. The recombinant yeast cells expressed each of the FMO successfully, and the FMO4 transformant produced N- and S-oxide metabolites at several milligrams per liter within 24 h. This whole-cell dependent biosynthesis enabled the production of N- and S-oxides without the use of the expensive cofactor NADPH. Such novel yeast expression system could be a powerful tool for the production of oxide metabolites.     

Bioengineered miR-328-3p modulates GLUT1-mediated glucose uptake and metabolism to exert synergistic antiproliferative effects with chemotherapeutics

MicroRNAs (miRNAs or miRs) are small noncoding RNAs derived from genome to control target gene expression. Recently we have developed a novel platform permitting high-yield production of bioengineered miRNA agents (BERA). This study is to produce and utilize novel fully-humanized BERA/miR-328-3p molecule (hBERA/miR-328) to delineate the role of miR-328-3p in controlling nutrient uptake essential for cell metabolism. We first demonstrated successful high-level expression of hBERA/miR-328 in bacteria and purification to high degree of homogeneity (>98%). Biologic miR-328-3p prodrug was selectively processed to miR-328-3p to suppress the growth of highly-proliferative human osteosarcoma (OS) cells. Besides glucose transporter protein type 1, gene symbol solute carrier family 2 member 1 (GLUT1/SLC2A1), we identified and verified large neutral amino acid transporter 1, gene symbol solute carrier family 7 member 5 (LAT1/SLC7A5) as a direct target for miR-328-3p. While reduction of LAT1 protein levels by miR-328-3p did not alter homeostasis of amino acids within OS cells, suppression of GLUT1 led to a significantly lower glucose uptake and decline in intracellular levels of glucose and glycolytic metabolite lactate. Moreover, combination treatment with hBERA/miR-328 and cisplatin or doxorubicin exerted a strong synergism in the inhibition of OS cell proliferation. These findings support the utility of novel bioengineered RNA molecules and establish an important role of miR-328-3p in the control of nutrient transport and homeostasis behind cancer metabolism.     

Identification of Major Esterase Involved in Hydrolysis of Soft Anticholinergic (2R3’R-SGM) Designed From Glycopyrrolate in Human and Rat Tissues

The glycopyrrolate soft analog, SGM, designed to be easily hydrolyzed into the significantly less active zwitterionic metabolite, SGa, typifies soft drug that reduces systemic side effects (a problem often seen with traditional anticholinergics) following local administration. In this study, hydrolysis of 2R3’R-SGM, the highest pharmacologically active stereoisomer of SGM, was investigated in human and rat tissues. In both species, 2R3’R-SGM was metabolized to 2R3’R-SGa in plasma but was stable in liver and intestine. The half-life of 2R3’R-SGM was found to be 16.9 min and 9.8 min in human and rat plasma, respectively. The enzyme inhibition and stimulation experiments showed that plasma paraoxonase 1 (PON1) is responsible for the hydrolysis of 2R3’R-SGM in humans and rats. The PON1-mediated hydrolysis of 2R3’R-SGM was confirmed in the lipoprotein-rich fractions of human plasma. As PON1 is naturally attached to high-density lipoprotein, it might be absent in topical tissues where 2R3’R-SGM is applied, supporting its local stability and efficacy. The metabolic behavior of 2R3’R-SGM indicates that it is an ideal soft drug to be detoxified as soon as it moves into systemic circulation. Furthermore, the similarity of 2R3’R-SGM metabolism in humans and rats showed that the rat is a suitable animal for preclinical study.