Jing-Bo Peng

Association between well-characterized lung cancer lncRNA polymorphisms and platinum-based chemotherapy toxicity in Chinese patients with lung cancer.

Platinum-based chemotherapy is the standard first-line treatment for most lung cancer patients. However, the toxicity induced by platinum-based chemotherapy greatly impedes its clinical use. Previous studies showed that long non-coding RNAs (lncRNAs) with over 200 nucleotides in length affect drug response and toxicity. In the present study, we investigated the association of well-characterized lung cancer lncRNA polymorphisms with platinum-based chemotherapy toxicity in Chinese patients with lung cancer. A total of 467 lung cancer patients treated with platinum-based chemotherapy for at least two cycles were recruited. We primarily focused on gastrointestinal and hematological toxicities. A total of 14 potentially functional polymorphisms within 8 lncRNAs (HOTTIP, HOTAIT, H19, ANRIL, CCAT2, MALAT1, MEG3, and POLR2E) were genotyped. Unconditional logistical regression analysis was conducted to assess the associations. Gene-gene and gene-environment interactions were identified using the software generalized multifactor dimensionality reduction (GMDR). ANRIL rs1333049 was associated with severe overall toxicity in an additive model (adjusted OR=0.723, 95% CI=0.541–0.965, P=0.028). ANRIL rs1333049 was also associated with severe gastrointestinal toxicity in both the additive (adjusted OR=0.690, 95% CI=0.489–0.974, P=0.035) and dominant (adjusted OR=0.558, 95% CI=0.335–0.931, P=0.025) models. MEG3 rs116907618 was associated with severe gastrointestinal toxicity in an additive model (adjusted OR=1.717, 95% CI=1.007–2.927, P=0.047). GMDR identified the three-factor interaction model of POLR2E rs3787016-HOTTIP rs3807598-chemotherapy regimen as the best predictive model for hematological toxicity. In conclusion, ANRIL and MEG3 genetic polymorphisms are associated with severe platinum toxicity and could be considered as biomarkers for pretreatment evaluation in Chinese patients with lung cancer.

Searching the Cytochrome P450 Enzymes for the Metabolism of Meranzin Hydrate: A Prospective Antidepressant Originating from Chaihu-Shugan-San

Meranzin hydrate (MH), an absorbed bioactive compound from the Traditional Chinese Medicine (TCM) Chaihu-Shugan-San (CSS), was first isolated in our laboratory and was found to possess anti-depression activity. However, the role of cytochrome P450s (CYPs) in the metabolism of MH was unclear. In this study, we screened the CYPs for the metabolism of MH in vitro by human liver microsomes (HLMs) or human recombinant CYPs. MH inhibited the enzyme activities of CYP1A2 and CYP2C19 in a concentration-dependent manner in the HLMs. The Km and Vmax values of MH were 10.3±1.3 µM and 99.1±3.3 nmol/mg protein/min, respectively, for the HLMs; 8.0±1.6 µM and 112.4±5.7 nmol/nmol P450/min, respectively, for CYP1A2; and 25.9±6.6 µM and 134.3±12.4 nmol/nmol P450/min, respectively, for CYP2C19. Other human CYP isoforms including CYP2A6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 showed minimal or no effect on MH metabolism. The results suggested that MH was simultaneously a substrate and an inhibitor of CYP1A2 and CYP2C9, and MH had the potential to perpetrate drug-drug interactions with other CYP1A2 and CYP2C19 substrates.

The rs1142345 in TPMT Affects the Therapeutic Effect of Traditional Hypoglycemic Herbs in Prediabetes

Therapeutic interventions in prediabetes are important in the primary prevention of type 2 diabetes (T2D) and its chronic complications. However, little is known about the pharmacogenetic effect of traditional herbs on prediabetes treatment. A total of 194 impaired glucose tolerance (IGT) subjects were treated with traditional hypoglycemic herbs (Tianqi Jiangtang) for 12 months in this study. DNA samples were genotyped for 184 mutations in 34 genes involved in drug metabolism or transportation. Multinomial logistic regression analysis indicated that rs1142345 (A > G) in the thiopurine S-methyltransferase (TPMT) gene was significantly associated with the hypoglycemic effect of the drug (P = 0.001, FDR P = 0.043). The “G” allele frequencies of rs1142345 in the healthy (subjects reverted from IGT to normal glucose tolerance), maintenance (subjects still had IGT), and deterioration (subjects progressed from IGT to T2D) groups were 0.094, 0.214, and 0.542, respectively. Binary logistic regression analysis indicated that rs1142345 was also significantly associated with the hypoglycemic effect of the drug between the healthy and maintenance groups (P = 0.027, OR = 4.828) and between the healthy and deterioration groups (P = 0.001, OR = 7.811). Therefore, rs1142345 was associated with the clinical effect of traditional hypoglycemic herbs. Results also suggested that TPMT was probably involved in the pharmacological mechanisms of T2D.

Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The Km and Vmax values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC50 values were 16.00 μM and 9.83 μM, and Ki values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.

Validation of a Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry Method for Determination of All-Trans Retinoic Acid in Human Plasma and Its Application to a Bioequivalence Study

A sensitive, reliable and specific LC-MS-MS method was developed and validated for the identification and quantitation of all-trans retinoic acid (ATRA) in human plasma. Acitretin was used as the internal standard (IS). After liquid-liquid extraction of 500 μL plasma with methyl tert-butyl ether (MTBE), ATRA and the IS were chromatographed on a HyPURITY C18 column (150 mm × 2.1 mm, 5 μm) with the column temperature set at 40 °C. The mobile phase was consisted of 40% phase A (MTBE–methanol–acetic acid, 50:50:0.5, v/v) and 60% phase B (water–methanol–acetic acid, 50:50:0.5, v/v) with a flow rate of 0.3 mL/min. The API 4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) mode via the positive electrospray ionization interface using the transition m/z 301.4 → 123.1 for ATRA and m/z 326.9 → 177.1 for IS, respectively. The calibration curve was linear over the range of 0.45–217.00 ng/mL (r ≥ 0.999) with a lower limit of quantitation (LLOQ) of 0.45 ng/mL. The intra- and inter-day precisions values were below 8% relative standard deviation and the accuracy was from 98.98% to 106.19% in terms of relative error. The validated method was successfully applied in a bioequivalence study of ATRA in Chinese healthy volunteers.

Screening of drug metabolizing enzymes for fusidic acid and its interactions with isoform-selective substrates in vitro.

Abstract 1. Fusidic acid (FA) is widely used for the treatment of infections of sensitive osteomyelitis or skin and soft tissue caused by bacteria. However, the role of cytochrome P450s (CYPs) in the metabolism of FA is unclear. In the present study, we screened the main CYPs for the metabolism of FA and studied its interactions with isoform-selective substrates in vitro. 2. The main CYP450s were screened according to the inhibitory effect of specific inhibitors on the metabolism of FA in human liver microsomes (HLMs) or recombinant CYP isoforms. Enzyme kinetic parameters including Ki, Ki′, Vmax, and IC50 were calculated to determine the potential of FA to affect CYP-mediated metabolism of isoform-selective substrates. 3. FA metabolism rate was inhibited by 49.8% and 83.1% under CYP2D6, CYP3A4 selective inhibitors in HLMs. In recombinant experiment, the inhibitory effects on FA metabolism were 83.3% for CYP2D6 and 58.9% for CYP3A4, respectively. FA showed inhibition on CYP2D6 and CYP3A4 with Kis of 13.9 and 38.6 μM, respectively. Other CYP isoforms including CYP1A2, CYP2A6, CYP2C9, CYP2E1, and CYP2C19 showed minimal or no effect on the metabolism of FA. 4. FA was primarily metabolized by CYP2D6 and CYP3A4 and showed a noncompetitive inhibition on CYP2D6 and a mixed competitive inhibition on CYP3A4. Drug–drug interactions between FA and other chemicals, especially with substrates of CYP2D6 and CYP3A4, are phenomena that clinicians need to be aware of and cautious about.

Sodium tanshinone IIA sulfonate and its interactions with human CYP450s

Abstract 1.Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, a famous Chinese medicine used for many years to treat cardiovascular disorders. However, the role of cytochrome P450 (CYP) enzymes in the metabolism of STS was unclear. In this study, we screened the main CYPs for the metabolism of STS and studied their interactions in vitro. 2.Seven CYPs were screened for the metabolism of STS by human liver microsomes (HLMs) or recombinant CYP isoforms. To determine the potential of STS to affect CYP-mediated phase I metabolism in humans, phenacetin (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), metoprolol (CYP2D6), chlorzoxazone (CYP2E1), S-Mephenytoin (CYP2C19), and midazolam (CYP3A4) were used as the respective probe substrates. Enzyme kinetic studies were performed to investigate the mode of inhibition of the enzyme–substrate interactions. 3.STS inhibited the activity of CYP3A4 in a dose-dependent manner in the HLMs and CYP3A4 isoform. Other CYP isoforms, including CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on the metabolism of STS. 4.The results suggested that STS primarily inhibits the activities of CYP3A4 in vitro, and STS has the potential to perpetrate drug–drug interactions with other CYP3A4 substrates.

Quercetin Significantly Inhibits the Metabolism of Caffeine, a Substrate of Cytochrome P450 1A2 Unrelated to CYP1A2*1C (−2964G>A) and *1F (734C>A) Gene Polymorphisms

Background. Quercetin is abundant in plants and human diets. Previous studies indicated that quercetin inhibited the activity of CYP1A2, and the combination of quercetin with the substrates of CYP1A2 might produce herb-drug interactions. This research aims to determine the effects of quercetin and the CYP1A2 gene polymorphisms, namely, CYP1A2*1C  (−2964G>A) and *1F (734C>A), on the metabolism of caffeine. Method. The experiment was designed into two treatment phases separated by a 2-week washout period. Six homozygous individuals for the CYP1A2*1C/*1F (GG/AA) genotype and 6 heterozygous individuals for the CYP1A2*1C/*1F (GA/CA) genotype were enrolled in the study. Quercetin capsules (500 mg) were given to each volunteer once daily for 13 consecutive days, and after that, each subject was coadministrated 100 mg caffeine capsules with 500 mg quercetin on the 14th day. Then a series of venous blood samples were collected for HPLC analysis. Correlation was determined between pharmacokinetics of caffeine and paraxanthine with caffeine metabolite ratio. Results. Quercetin significantly affected the pharmacokinetics of caffeine and its main metabolite paraxanthine, while no differences were found in the pharmacokinetics of caffeine and paraxanthine between GG/AA and GA/CA genotype groups. Conclusion. Quercetin significantly inhibits the caffeine metabolism, which is unrelated to CYP1A2*1C (−2964G>A) and *1F (734C>A) gene polymorphisms.

The potent mechanism-based inactivation of CYP2D6 and CYP3A4 with fusidic acid in in vivo bioaccumulation

Abstract 1. The accumulation of fusidic acid (FA) after multiple doses of FA has been reported on in previous studies but the related mechanisms have not been clarified fully. In the present study, we explain the mechanisms related to the mechanism-based inactivation of CYP2D6 and CYP3A4. 2. The irreversible inhibitory effects of FA on CYP2D6 and CYP3A4 were examined via a series of experiments, including: (a) time-, concentration- and NADPH-dependent inactivation, (b) substrate protection in enzyme inactivation and (c) partition ratio with recombinant human CYP enzymes. Metoprolol α-hydroxylation and midazolam 1′-hydroxylation were used as marker reactions for CYP2D6 and CYP3A4 activities, and HPLC-MS/MS measurement was also utilised. 3. FA caused to the time- and concentration-dependent inactivation of CYP2D6 and CYP3A4. About 55.8% of the activity of CYP2D6 and 75.8% of the activity of CYP3A4 were suppressed after incubation with 10 μM FA for 15 min. KI and kinact were found to be 2.87 μM and 0.033 min−1, respectively, for CYP2D6, while they were 1.95 μM and 0.029 min−1, respectively, for CYP3A4. Inhibition of CYP2D6 and CYP3A4 activity was found to require the presence of NADPH. Substrates of CYP2D6 and CYP3A4 showed that the enzymes were protected against the inactivation induced by FA. The estimated partition ratio for the inactivation was 7 for CYP2D6 and 12 for CYP3A4. 4. FA is a potent mechanism-based inhibitor of CYP2D6 and CYP3A4, which may explain the accumulation of FA in vivo.

1H NMR based pharmacometabolomics analysis of metabolic phenotype on predicting metabolism characteristics of losartan in healthy volunteers

Inter-individual variability in drug metabolism and disposition is common in both preclinical and clinical researches. Losartan and its active metabolite EXP3174 present a high degree of inter-individual differences in blood concentrations that affect drug efficacy and side effect. Pharmacometabolomics has been increasingly applied on predicting the drug responses by analyzing the differences in metabolic profile. A pre-dose metabolic phenotype was investigated to interpret inter-individual variations in the metabolism characteristics of losartan. 1H Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolic profiles were performed on 36 healthy Chinese male volunteers by measuring their pre-dose plasma samples. After oral administration of losartan, the concentrations of losartan and its bioactive metabolite EXP3174 were monitored by liquid chromatography-mass spectrometry (LC-MS). Orthogonal partial least-squares (O-PLS) model was conducted to select potential biomarkers that substantially contributed to the inter-individual variations in the metabolism features via analyzing the ratio of pharmacokinetics (PK) parameters of its metabolite to parent drug. Potential metabolites such as glycine, phosphorylcholine, choline, creatine, creatinine, lactate, citrate, α-glucose, and lipids showed strong correlations with metabolism features of losartan. In addition, the pathway analysis revealed that baseline lipid metabolism, the glycine, serine and threonine pathway, and glycolysis or gluconeogenesis metabolism pathway were significantly associated with the ratio of PK parameters of EXP3174 to losartan. Step-wise multiple linear regression (MLR) was constructed to investigate the potential roles of the selected biomarkers in predicting individualized metabolism characteristics of losartan. These results showed that the pre-dose individual metabolic traits may be a useful approach for characterizing individual differences in losartan metabolism characteristics and therefore for expediting personalized dose-setting in further clinical studies.