LC-MS/MS method for the quantification of cortisol of hepatocellular carcinoma.

The imbalance of steroid hormones is closely related to the occurrence and development of hepatocellular carcinoma (HCC). However, most research has focused on steroid hormone receptors, and reports about the relationship between the serum concentration of cortisol and the development of HCC are rare. The aim of this research was to establish a simple, specific, sensitive and reliable liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for the quantitation of cortisol in human serum and to compare the level of cortisol in serum between 221 HCC patients and 183 healthy volunteers. The results showed that the correlation coefficients of the linear regression with a weighing factor of 1/x2 ranged from 0.9933 to 0.9984 over the range of 2-1,000 ng/ml. The inter- and intra-day precision and accuracy were <10%. The matrix effect and recovery of cortisol were 94.9-102.5% and 96.3-99.8%, respectively. The concentration of cortisol in HCC patients was significantly higher than that in healthy volunteers (p < 0.05) and was not affected by sex, age, menopause or α-fetoprotein (AFP) level. The present study reveals that elevated cortisol might promote the progression of HCC.

Interaction between 3,4­dichlorophenyl­propenoyl­sec.­butylamine (3,4­DCPB), an antiepileptic drug, and cytochrome P450 in rat liver microsomes and recombinant human enzymes in vitro.

The compound 3,4­dichlorophenyl­propenoyl­sec.­butylamine (3,4­DCPB) is an antiepileptic drug. The purpose of the present research was to identify cytochrome P450 (CYP450) responsible for the metabolism of 3,4­DCPB and evaluate the effects of 3,4­DCPB on the activities of CYP450 enzymes. 3,4­DCPB was incubated with rat liver microsomes (RLMs) plus six CYP450 enzyme-specific inhibitors, or six recombinant human CYP450 enzymes (rhCYP450s). The concentrations of 3,4­DCPB and six CYP450 enzyme-activities probe drugs were detected by high-performance liquid chromatographic (HPLC). The results showed that the prototype of 3,4­DCPB was metabolized by multiple CYP450 enzymes into three metabolites, and the predominant isoforms were CYP2D6 (metabolite M1), CYP1A2 (M2), CYP2C19 and CYP3A4 (M3), respectively., in the presence of ß-NADPH (1 mM) in RLMs or rhCYP450s. Compared with the control (PB-), phenobarbital pre-treatment (PB+) significantly enhanced levels (all of p < 0.01) of hydroxylmethytobutamide (CYP2C9), 4­hydroxy­mephenytoin (CYP2C19), acetaminophen (CYP1A2), 6­hydroxychlorzoxazone (CYP2E1) and oxidized nifedipine (CYP3A4), respectively, in spite of dextrophan (CYP2D6) was not markedly enhanced in RLMs. Conversely, the inhibitory ratios of 3,4­DCPB (16 µg/mL, 59 µM) on the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6 were 97.6%, 59.0%, 53.5% and 36.5%, respectively. However, CYP2E1 (both of PB- and PB+) and CYP3A4 (PB+) were not inhibited by 3,4­DCPB in RLMs. In conclusion, the present study showed that 3,4­DCPB was metabolized by multiple CYP450 enzymes. 3,4­DCPB inhibited the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6, rather than that CYP2E1 and CYP3A4 enzymes, suggesting that the different effects of 3,4­DCPB on the CYP450 enzymes might influence metabolic drug-drug interaction in antiepileptics therapy.

Identification of cytochrome P450s involved in the metabolism of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1) using human recombinant enzymes and rat liver microsomes in vitro.

1. The aim of this study was to identify the hepatic metabolic enzymes, which involved in the biotransformation of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) in rat and human in vitro. 2. The parent drug of W-1 was incubated with rat liver microsomes (RLMs) or recombinant CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, respectively) in the presence or absence of nicotinamide adeninedinucleotide phosphate (NADPH)-regenerating system. The metabolites of W-1 were analyzed with liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). 3. The parent drug of W-1 was metabolized in a NADPH-dependent manner in RLMs. The kinetic parameters of prototype W-1 including Km, Vmax, and CLint were 2.3 µM, 3.3 nmol/min/mg protein, and 1.4 mL/min/mg protein, respectively. Two metabolites M1 and M2 were observed in shorter retention times (2.988 and 3.188 min) with a higher molecular ion at m/z 463.0160 (both M1 and M2) than that of the W-1 parent drug (6.158 min with m/z 447.0218). The CYP selective inhibition and recombinant enzymes also showed that two hydroxyl metabolites M1 and M2 are mainly mediated by CYP2C19 and CYP3A4. 4. The identification of CYPs involved in W-1 biotransformation is important to understand and minimize, if possible, the potential of drug-drug interactions.