Zeneng Cheng

MiR-183/-96/-182 cluster is up-regulated in most breast cancers and increases cell proliferation and migration

IntroductionThe miR-183/-96/-182 cluster is a conserved polycistronic microRNA (miRNA) cluster which is highly expressed in most breast cancers. Although there are some sporadic reports which demonstrate the importance of each miRNA in this cluster in breast cancer, the biological roles of this cluster as a whole and its regulation mechanisms in breast cancer are still unclear. We compared the expression of this cluster in different cancer types, analyzed the regulation mechanism of this cluster, identified new target genes, and examined the impact of this cluster on breast cancer cells.MethodsThe miRNA level was detected by LNA-based northern blot and Real-time PCR, and was also analyzed from TCGA dataset. Bioinformatics research and luciferase assay were applied to find the promoter regions and transcription factors. To investigate the biological effects of the miR-183/-96 /-182 cluster in breast cancer, we generated miR-96, miR-182 and miR-183 overexpression stable cell lines to check the overdose effects; we also used miR-Down™ antagomir for each miRNA as well as miR-183/-96 /-182 cluster sponge lentivirus to check the knockdown effects. Growth, migration, cell cycle profile and survival of these cells was then monitored by colony formation assay, MTT assay, cell wound healing assay, flow cytometry and microscopy. The target gene was validated by Real-time PCR, luciferase assay, Western blot and Phalloidin/DAPI counterstaining.ResultsThe miR-183/-96/-182 cluster was highly expressed in most breast cancers, and its transcription is disordered in breast cancer. The miR-183/-96/-182 cluster was transcribed in the same pri-miRNA and its transcription was regulated by ZEB1 and HSF2. It increased breast cell growth by promoting more rapid completion of mitosis, promoted cell migration and was essential for cell survival. MiR-183 targeted the RAB21 mRNA directly in breast cancer.ConclusionThe miR-183/-96/-182 cluster is up-regulated in most breast cancer. It functions as an oncogene in breast cancer as it increases cell proliferation and migration.

In vitro dissolution similarity factor (f2) and in vivo bioequivalence criteria, how and when do they match? Using a BCS class II drug as a simulation example

The present study examined the agreement between in vitro dissolution f2 similarity and in vivo bioequivalence criteria for BCS class II drugs. Dissolution test profiles were generated using the First-order model with varied dissolution parameters around the standard values of a reference profile. The in vivo curves were derived from in vitro dissolution profiles with the drugs pharmacokinetics parameters by numerical convolution method. The Cmax, Tmax, AUC0-t and AUC0-∞ obtained from in vivo test and reference concentration-time curves were compared, and the CmaxR (Cmax ratio), TmaxDif (Tmax difference), AUC0-tR (AUC0-t ratio) and AUC0-∞R (AUC0-∞ ratio) were determined. The relationships between CmaxR, AUC0-tR, AUC0-∞R, f2 and the First-order model parameters demonstrated that the Similarity Region 1 enclosed by the f2 contour line labeled 50 was completely within the Bioequivalence Region enclosed by the contour lines labeled 0.80 and 1.20 of AUC0-tR, AUC0-∞R, and CmaxR, and the Similarity Region 2 enclosed by the f2 contour line labeled 35 was nearly overlapped with the Bioequivalence Region, but did not exactly match. The results indicate that the public standard for in vitro dissolution f2 similarity criterion (f2⩾50) is probably slightly conservative and may be widened to an appropriate lower critical value.

No effect of MDR1 C3435T polymorphism on oral pharmacokinetics of telmisartan in 19 healthy Chinese male subjects.

Abstract Background: Considerable interindividual differences in both drug response and pharmacokinetics of telmisartan have been identified. This study was designed to investigate the influence of MDR1 C3435T polymorphism on pharmacokinetics of telmisartan after a single oral dose in healthy Chinese volunteers. Methods: A total of 61 unrelated male volunteers were genotyped for MDR1 C3435T polymorphism by using the polymerase chain reaction-restriction fragment length polymorphism method. Six 3435CC homozygotes, eight 3435CT heterozygotes, and five 3435TT homozygotes were randomly selected and received a single oral dose of 40 mg telmisartan. Plasma concentrations of telmisartan were determined by the high performance liquid chromatography-mass spectrometry method up to 48 h after telmisartan administration. Results: Interindividual variation for tmax, Cmax, t1/2, AUC0–48, AUC0–∞, and clearance (CL) for telmisartan was approximately 6-, 33-, 16-, 17-, 20-, and 20-fold, respectively. Cmax (p=0.010), t1/2 (p=0.029) and CL (p=0.010) of telmisartan were not normally distributed. MDR1 3435TT homozygotes seemed to show increases in Cmax, tmax, t1/2, AUC0–48, and AUC0–∞. However, no significant differences in Cmax, tmax, t1/2, AUC0–48, AUC0–∞, and CL among MDR1 C3435T genotypes were observed. Conclusions: MDR1 C3435T polymorphism does not affect oral pharmacokinetics of telmisartan. Clin Chem Lab Med 2009;47:38–43.

Association between Common CYP1A2 Polymorphisms and Theophylline Metabolism in Non-smoking Healthy Volunteers

This study was designed to investigate the impact of cytochrome P450 (CYP) 1A2 polymorphisms on theophylline metabolism in a non‐smoking healthy male Chinese population. Four polymorphisms CYP1A2*1C (G‐3860A), G‐3113A, CYP1A2*1F (C‐163A) and CYP1A2*1B (C‐5347T) were screened in 238 unrelated male volunteers. Then, a single oral 200‐mg dose of theophylline was administered to 37 volunteers, who were selected from 238 volunteers based on the CYP1A2 genotype. CYP1A2 activities were evaluated by plasma 1,7‐dimethylxanthine/caffeine ratios (17X/137X) after administration of 100‐mg caffeine. The plasma concentrations of theophylline, 17X and 137X were determined by high‐performance liquid chromatography. The activity of CYP1A2 was lower in volunteers with the ‐3113 AA genotype compared with those with the ‐3113 AG genotype (0.35 ± 0.04 versus 0.48 ± 0.07, p = 0.016) or the ‐3113 GG genotype (0.35 ± 0.04 versus 0.58 ± 0.22, p = 0.037). CYP1A2*1F polymorphisms were associated with increased CYP1A2 activity in volunteers with ‐3860G/‐3113G/5347C homozygosity (0.66 ± 0.24 versus 0.46 ± 0.05, p = 0.034). However, theophylline metabolism showed no difference among volunteers carrying different haplotype pairs. CYP1A2 genetic polymorphisms influenced CYP1A2 enzyme activity as measured by caffeine, but CYP1A2 gene polymorphisms appeared to have limited influence on theophylline metabolism in our study.

Evaluation of CYP3A activity in humans using three different parameters based on endogenous cortisol metabolism

AbstractAim:Currently, there is considerable debate as to which method is more accurate for measuring the activity of CYP3A in vivo: cortisol 6β-hydroxylation clearance (Clm(6β)) or the urinary ratio of 6β-OHF to F (6β-OHF/F). Furthermore, the value of measuring endogenous levels of cortisol over a 24 h period (AUCF) needs to be confirmed. The aim of the present study was to determine which method was most effective at measuring changes in the in vivo activity of CYP3A: AUCF, Clm(6β), or 6β-OHF/F.Methods:A two phase, cross-over design was adopted in this study. A total of 24 subjects (12 males and 12 females) were randomly assigned to one of two groups: the test group subjects were given 250 mg clarithromycin tablets twice a day for a period of 4 d, whereas the control group received a placebo twice daily for a similar period. On d 5 of the study, the last dose of either clarithromycin or placebo was supplemented with an oral dose of 7.5 mg midazolam (MDZ); blood and urine samples were then collected at various times. All samples collected at the same sampling times on d 4 were used to evaluate the effects of MDZ administration on cortisol levels and metabolism. The ratio of 1-hydroxymidazolam (1-OHMDZ) concentration to MDZ concentration at 1 h (MR) was taken as a measure of the in vivo CYP3A activity. AUCF, Clm(6β), and 6β-OHF/F were also used as biomarkers for CYP3A activity.Results:No correlations were found (either before or after inhibition) between CYP3A activity and any of the following measures: AUCF, Clm(6β), or 6β-OHF/F (r<0.4, P>0.05). After 4 d of clarithromycin administration, CYP3A activity (MR) decreased by 75% (P=0.000), whereas AUCF increased by 19% (P=0.040), and Clm(6β) and 6β-OHF/F decreased by 54.2% (P=0.000) and 50% (P=0.003), respectively. No significant changes in AUCF (P=0.178), or in the amount of urinary 6β-OHF (P=0.169), or in F (P=0.391) were found over a 24 h time period, either with or without MDZ administration.Conclusion:Although Clm(6β) and 6β-OHF/F can reflect the decline in CYP3A activity, the impression they provide is neither accurate nor complete. AUCF is completely ineffective for evaluating variations in CYP3A activity. MDZ administration had no evident effects on either cortisol metabolism or excretion over a period of 24 h.

Investigation of chiral inversion and pharmacokinetics of laevo-ornidazole by high-performance liquid chromatography.

What is known and Objective:  Laevo‐ornidazole is an enantiomer of ornidazole, a 5‐nitroimidazole antimicrobial agent. It is not known whether chiral inversion of laevo‐ornidazole occurs in humans. The objective of this study was to investigate the possible chiral inversion and pharmacokinetics of the drug in vivo.

Simultaneous determination of bendamustine and its active metabolite, gamma-hydroxy-bendamustine in human plasma and urine using HPLC-fluorescence detector: Application to a pharmacokinetic study in Chinese cancer patients

A simple, sensitive and cost-effective assay based on reversed phase high performance liquid chromatography (RP-HPLC) with isocratic mode for simultaneous determination of bendamustine (BM) and its active metabolite, gamma-hydroxy-bendamustine (γ-OH-BM) in human plasma and urine was developed and validated. Sample preparation involved protein precipitation by 10% perchloric acid-methanol solution. The peaks were recorded by using fluorescence detector (excitation wavelength 328 nm and emission wavelength 420 nm). The calibration curves were linear over concentration ranges of 8.192-10,000 ng mL(-1) and 5-1,000 ng mL(-1) for BM in human plasma and urine as well as 10-1,000 ng mL(-1) and 5-1,000 ng mL(-1) for γ-OH-BM in human plasma and urine, respectively. Intra- and inter-run precisions of BM and γ-OH-BM were less than 15% and the bias were within ± 15% for both plasma and urine. This validated method was successfully applied to a pharmacokinetic study enrolling 10 Chinese patients with indolent B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia administered a single intravenous infusion of 100 mg m(2) bendamustine hydrochloride.

Effect of amlodipine on the pharmacokinetics of tacrolimus in rats.

Abstract 1. The immunosuppressant tacrolimus (TAC) is a substrate of cytochrome P450 3A2 (CYP3A2) and P-glycoprotein (P-gp) in rats. Amlodipine (AML) is an inhibitor of CYP3A2 in rats. We investigated the effect of AML on the pharmacokinetics of TAC in rats. 2. When co-administered with TAC orally or intravenously, AML decreased the oral clearance and raised the blood concentration of TAC in rats, but the T1/2 of TAC was not significantly affected by AML. Upon oral administration of TAC, the effect of 15 mg/kg of AML on the AUC of TAC was lower than that seen with 5 or 10 mg/kg. However, upon intravenous TAC administration, the effect of 15 mg/kg of AML on the AUC of TAC was higher than that seen with 5 mg/kg. 3. AML is an inhibitor of P-gp and CYP3A2 in rats. If AML and TAC are co-administered orally, AML elicits greater inhibition in P-gp than CYP3A2 during first-pass metabolism. If AML is given orally and TAC given intravenously concurrently, AML mainly inhibits CYP3A2 activity and increases the blood concentration of TAC. There are significant pharmacokinetic interactions between TAC and AML. AML raises the blood concentration of TAC in rats probably by inhibiting P-gp and CYP3A2.

Prediction of tacrolimus metabolism and dosage requirements based on CYP3A4 phenotype and CYP3A5＊3 genotype in Chinese renal transplant recipients

Aim:To examine how the endogenous CYP3A4 phenotype and CYP3A5*3 genotype of Chinese renal transplant recipients influenced the dose-corrected trough concentration (C0/D) and weight-corrected daily dose (D/W) of tacrolimus.Methods:A total of 101 medically stable kidney transplant recipients were enrolled, and their blood and urine samples were gathered. The endogenous CYP3A4 phenotype was assessed by the ratio of 6β-hydroxycortisol and 6β-hydroxycortisone to cortisol and cortisone in urine. CYP3A5*3 genotype was determined using PCR-RELP.Results:In overall renal transplant recipients, a multiple regression analysis including the endogenous CYP3A4 phenotype, CYP3A5*3 genotype and post-operative period accounted for 60.1% of the variability in C0/D ratio; a regression equation consisting of the endogenous CYP3A4 phenotype, post-operative period, body mass index, CYP3A5*3 genotype, gender, total bilirubin and age explained 61.0% of the variability in D/W ratio. In CYP3A5*3/*3 subjects, a combination of the endogenous CYP3A4 phenotype, post-operative period and age was responsible for 65.3% of the variability in C0/D ratio; a predictive equation including the endogenous CYP3A4 phenotype, post-operative period, body mass index, gender and age explained 61.2% of the variability in the D/W ratio. Base on desired target range of tacrolimus trough concentrations, individual daily dosage regimen was calculated, and all the observed daily doses were within the predicted range.Conclusion:This study provides the equations to predict tacrolimus metabolism and dosage requirements based on the endogenous CYP3A4 phenotype, CYP3A5*3 genotype and other non-genetic variables.

Pharmacokinetic study of benfotiamine and the bioavailability assessment compared to thiamine hydrochloride

Benfotiamine is a lipid‐soluble thiamine precursor which can transform to thiamine in vivo and subsequently be metabolized to thiamine monophosphate (TMP) and thiamine diphosphate (TDP). This study investigated the pharmacokinetic profiles of thiamine and its phosphorylated metabolites after single‐ and multiple‐dose administration of benfotiamine in healthy Chinese volunteers, and assessed the bioavailability of orally benfotiamine administration compared to thiamine hydrochloride. In addition, concentration of hippuric acid in urine which is produced in the transformation process of benfotiamine was determined. The results showed that thiamine and its phosphorylated metabolites exhibited different pharmacokinetic characteristics in plasma, blood and erythrocyte, and one‐compartment model provided the best fit for pharmacokinetic profiles of thiamine. The transformation process of benfotiamine to thiamine produced large amount of hippuric acid. No accumulation of hippuric acid was observed after multiple‐dose of benfotiamine. Compared to thiamine hydrochloride, the bioavailability of thiamine in plasma and TDP in erythrocyte after oral administration of benfotiamine were 1147.3 ± 490.3% and 195.8 ± 33.8%, respectively. The absorption rate and extent of benfotiamine systemic availability of thiamine were significantly increased indicating higher bioavailability of thiamine from oral dose of benfotiamine compared to oral dose of thiamine hydrochloride.