Discovery that theonellasterol a marine sponge sterol is a highly selective FXR antagonist that protects against liver injury in cholestasis

Background

The farnesoid-x-receptor (FXR) is a bile acid sensor expressed in the liver and gastrointestinal tract. Despite FXR ligands are under investigation for treatment of cholestasis, a biochemical condition occurring in a number of liver diseases for which available therapies are poorly effective, mice harboring a disrupted FXR are protected against liver injury caused by bile acid overload in rodent models of cholestasis. Theonellasterol is a 4-methylene-24-ethylsteroid isolated from the marine sponge Theonella swinhoei. Here, we have characterized the activity of this theonellasterol on FXR-regulated genes and biological functions.

Principal Findings

Interrogation of HepG2 cells, a human hepatocyte cell line, by microarray analysis and transactivation assay shows that theonellasterol is a selective FXR antagonist, devoid of any agonistic or antagonistic activity on a number of human nuclear receptors including the vitamin D receptor, PPARs, PXR, LXRs, progesterone, estrogen, glucorticoid and thyroid receptors, among others. Exposure of HepG2 cells to theonellasterol antagonizes the effect of natural and synthetic FXR agonists on FXR-regulated genes, including SHP, OSTα, BSEP and MRP4. A proof-of-concept study carried out to investigate whether FXR antagonism rescues mice from liver injury caused by the ligation of the common bile duct, a model of obstructive cholestasis, demonstrated that theonellasterol attenuates injury caused by bile duct ligation as measured by assessing serum alanine aminostrasferase levels and extent of liver necrosis at histopathology. Analysis of genes involved in bile acid uptake and excretion by hepatocytes revealed that theonellasterol increases the liver expression of MRP4, a basolateral transporter that is negatively regulated by FXR. Administering bile duct ligated mice with an FXR agonist failed to rescue from liver injury and downregulated the expression of MRP4.

Conclusions

FXR antagonism in vivo results in a positive modulation of MRP4 expression in the liver and is a feasible strategy to target obstructive cholestasis.     

Hepatotoxicity in Mice of a Novel Anti-parasite Drug Candidate Hydroxymethylnitrofurazone: A Comparison with Benznidazole

Background

Treatment of Chagas disease, caused by Trypanosoma cruzi, relies on nifurtimox and benznidazole (BZL), which present side effects in adult patients, and natural resistance in some parasite strains. Hydroxymethylnitrofurazone (NFOH) is a new drug candidate with demonstrated trypanocidal activity; however, its safety is not known.

Methods

HepG2 cells dose response to NFOH and BZL (5–100 µM) was assessed by measurement of ROS, DNA damage and survival. Swiss mice were treated with NFOH or BZL for short-term (ST, 21 d) or long-term (LT, 60 d) periods. Sera levels of cellular injury markers, liver inflammatory and oxidative stress, and fibrotic remodeling were monitored.

Results

HepG2 cells exhibited mild stress, evidenced by increased ROS and DNA damage, in response to NFOH, while BZL at 100 µM concentration induced >33% cell death in 24 h. In mice, NFOH ST treatment resulted in mild-to-no increase in the liver injury biomarkers (GOT, GPT), and liver levels of inflammatory (myeloperoxidase, TNF-α), oxidative (lipid peroxides) and nitrosative (3-nitrotyrosine) stress. These stress responses in NFOH LT treated mice were normalized to control levels. BZL-treated mice exhibited a >5-fold increase in GOT, GPT and TNF-α (LT) and a 20–40% increase in liver levels of MPO activity (ST and LT) in comparison with NFOH-treated mice. The liver inflammatory infiltrate was noted in the order of BZL>vehicle≥NFOH and BZL>NFOH≥vehicle, respectively, after ST and LT treatments. Liver fibrotic remodeling, identified after ST treatment, was in the order of BZL>vehicle>NFOH; lipid deposits, indicative of mitochondrial dysfunction and in the order of NFOH>vehicle>BZL were evidenced after LT treatment.

Conclusions

NFOH induces mild ST hepatotoxicity that is normalized during LT treatment in mice. Our results suggest that additional studies to determine the efficacy and toxicity of NFOH are warranted.     

Ketoconazole Inhibits the Cellular Uptake of Anandamide via Inhibition of FAAH at Pharmacologically Relevant Concentrations

Background

The antifungal compound ketoconazole has, in addition to its ability to interfere with fungal ergosterol synthesis, effects upon other enzymes including human CYP3A4, CYP17, lipoxygenase and thromboxane synthetase. In the present study, we have investigated whether ketoconazole affects the cellular uptake and hydrolysis of the endogenous cannabinoid receptor ligand anandamide (AEA).

Methodology/Principal Findings

The effects of ketoconazole upon endocannabinoid uptake were investigated using HepG2, CaCo2, PC-3 and C6 cell lines. Fatty acid amide hydrolase (FAAH) activity was measured in HepG2 cell lysates and in intact C6 cells. Ketoconazole inhibited the uptake of AEA by HepG2 cells and CaCo2 cells with IC₅₀ values of 17 and 18 µM, respectively. In contrast, it had modest effects upon AEA uptake in PC-3 cells, which have a low expression of FAAH. In cell-free HepG2 lysates, ketoconazole inhibited FAAH activity with an IC₅₀ value (for the inhibitable component) of 34 µM.

Conclusions/Significance

The present study indicates that ketoconazole can inhibit the cellular uptake of AEA at pharmacologically relevant concentrations, primarily due to its effects upon FAAH. Ketoconazole may be useful as a template for the design of dual-action FAAH/CYP17 inhibitors as a novel strategy for the treatment of prostate cancer.     

Mushroom Ganoderma lucidum Prevents Colitis-Associated Carcinogenesis in Mice

Background

Epidemiological studies suggest that mushroom intake is inversely correlated with gastric, gastrointestinal and breast cancers. We have recently demonstrated anticancer and anti-inflammatory activity of triterpene extract isolated from mushroom Ganoderma lucidum (GLT). The aim of the present study was to evaluate whether GLT prevents colitis-associated carcinogenesis in mice.

Methods/Principal Findings

Colon carcinogenesis was induced by the food-borne carcinogen (2-Amino-1-methyl-6-phenylimidazol[4,5-b]pyridine [PhIP]) and inflammation (dextran sodium sulfate [DSS]) in mice. Mice were treated with 0, 100, 300 and 500 mg GLT/kg of body weight 3 times per week for 4 months. Cell proliferation, expression of cyclin D1 and COX-2 and macrophage infiltration was assessed by immunohistochemistry. The effect of GLT on XRE/AhR, PXR and rPXR was evaluated by the reporter gene assays. Expression of metabolizing enzymes CYP1A2, CYP3A1 and CYP3A4 in colon tissue was determined by immunohistochemistry. GLT treatment significantly suppressed focal hyperplasia, aberrant crypt foci (ACF) formation and tumor formation in mice exposed to PhIP/DSS. The anti-proliferative effects of GLT were further confirmed by the decreased staining with Ki-67 in colon tissues. PhIP/DSS-induced colon inflammation was demonstrated by the significant shortening of the large intestine and macrophage infiltrations, whereas GLT treatment prevented the shortening of colon lengths, and reduced infiltration of macrophages in colon tissue. GLT treatment also significantly down-regulated PhIP/DSS-dependent expression of cyclin D1, COX-2, CYP1A2 and CYP3A4 in colon tissue.

Conclusions

Our data suggest that GLT could be considered as an alternative dietary approach for the prevention of colitis-associated cancer.     

Regulation of Multidrug Resistance Proteins by Genistein in a Hepatocarcinoma Cell Line: Impact on Sorafenib Cytotoxicity

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of these transporters. Genistein (GNT) is a phytoestrogen abundant in soybean that exerts its genomic effects through Estrogen-Receptors and Pregnane-X-Receptor (PXR), which are involved in the regulation of the above-mentioned transporters. We evaluated the effect of GNT on the expression and activity of P-gp, MRP2, MRP3 and BCRP in HCC-derived HepG2 cells. GNT (at 1.0 and 10 μM) increased P-gp and MRP2 protein expression and activity, correlating well with an increased resistance to sorafenib cytotoxicity as detected by the methylthiazole tetrazolium (MTT) assay. GNT induced P-gp and MRP2 mRNA expression at 10 but not at 1.0 μM concentration suggesting a different pattern of regulation depending on the concentration. Induction of both transporters by 1.0 μM GNT was prevented by cycloheximide, suggesting translational regulation. Downregulation of expression of the miR-379 by GNT could be associated with translational regulation of MRP2. Silencing of PXR abolished P-gp induction by GNT (at 1.0 and 10 μM) and MRP2 induction by GNT (only at 10 μM), suggesting partial mediation of GNT effects by PXR. Taken together, the data suggest the possibility of nutrient-drug interactions leading to enhanced chemoresistance in HCC when GNT is ingested with soy rich diets or dietary supplements.     

Metabolic activity and mRNA levels of human cardiac CYP450s involved in drug metabolism

Background

Tissue-specific expression of CYP450s can regulate the intracellular concentration of drugs and explain inter-subject variability in drug action. The overall objective of our study was to determine in a large cohort of samples, mRNA levels and CYP450 activity expressed in the human heart.

Methodology

CYP450 mRNA levels were determined by RTPCR in left ventricular samples (n = 68) of explanted hearts from patients with end-stage heart failure. Samples were obtained from ischemic and non-ischemic hearts. In some instances (n = 7), samples were available from both the left and right ventricles. A technique for the preparation of microsomes from human heart tissue was developed and CYP450-dependent activity was determined using verapamil enantiomers as probe-drug substrates.

Principal Findings

Our results show that CYP2J2 mRNA was the most abundant isoform in all human heart left ventricular samples tested. Other CYP450 mRNAs of importance were CYP4A11, CYP2E1, CYP1A1 and CYP2C8 mRNAs while CYP2B6 and CYP2C9 mRNAs were present at low levels in only some of the hearts analyzed. CYP450 mRNAs did not differ between ischemic and non-ischemic hearts and appeared to be present at similar levels in the left and right ventricles. Incubation of verapamil with heart microsomes led to the formation of nine CYP450-dependent metabolites: a major finding was the observation that stereoselectivity was reversed compared to human liver microsomes, in which the R-enantiomer is metabolized to a greater extent.

Conclusions

This study determined cardiac mRNA levels of various CYP450 isozymes involved in drug metabolism and demonstrated the prevalent expression of CYP2J2 mRNA. It revealed that cardiomyocytes can efficiently metabolize drugs and that cardiac CYP450s are highly relevant with regard to clearance of drugs in the heart. Our results support the claim that drug metabolism in the vicinity of a drug effector site can modulate drug effects.     

Myeloid related protein induces muscle derived inflammatory mediators in juvenile dermatomyositis

Introduction

The aetiopathogenesis of juvenile dermatomyositis (JDM) remains poorly understood. In particular the contribution of monocytes or macrophages, which are frequently observed to be an infiltrate within muscle tissue very early in the disease process, is unknown. We hypothesised that these cells secrete the pro-inflammatory myeloid related protein (MRP) 8/14 which may then contribute to muscle pathology in JDM.

Methods

In this study of 56 JDM patients, serum MRP8/14 levels were compared with clinical measures of disease activity. Muscle biopsies taken early in disease were assessed by immunohistochemistry to determine the frequency and identity of MRP-expressing cells. The effects of MRP stimulation and endoplasmic reticulum (ER) stress on muscle were tested in vitro. Serum or supernatant levels of cytokines were analyzed by multiplex immunoassay.

Results

Serum MRP8/14 correlated with physician’s global assessment of disease activity in JDM (R = 0.65, p = 0.0003) and muscle strength/endurance, childhood myositis assessment score (CMAS, R = −0.55, p = 0.004). MRP8/14 was widely expressed by CD68+ macrophages in JDM muscle tissue. When cultured with human myoblasts, MRP8 led to the secretion of MCP-1 and IL-6, which was enhanced by ER stress. Both inflammatory mediators were detected in significantly higher levels in the serum of JDM patients compared to healthy controls.

Conclusions

This study is the first to identify serum MRP8/14 as a potential biomarker for disease activity in JDM. We propose that tissue infiltrating macrophages secreting MRP8/14 may contribute to myositis, by driving the local production of cytokines directly from muscle.     

Nuclear Multidrug-Resistance Related Protein 1 Contributes to Multidrug-Resistance of Mucoepidermoid Carcinoma Mainly via Regulating Multidrug-Resistance Protein 1: A Human Mucoepidermoid Carcinoma Cells Model and Spearman's Rank Correlation Analysis

Background

Multidrug resistance-related protein 1 (MRP1/ABCC1) and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1) are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH) and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR) of mucoepidermoid carcinoma (MEC). The present study investigated how MRP1 contributes to MDR in the nuclei of MEC cells.

Methods

Western blot and RT-PCR was carried out to investigate the change of multidrug-resistance protein 1 (MDR1) in MC3/5FU cells after MRP1 was downregulated through RNA interference (RNAi). Immunohistochemistry (IHC) staining of 127 cases of MEC tissues was scored with the expression index (EI). The EI of MDR1 and MRP1 (or nuclear MRP1) was analyzed with Spearman''s rank correlation analysis. Using multiple tumor tissue assays, the location of MRP1 in other tissues was checked by HIC. Luciferase reporter assays of MDR1 promoter was carried out to check the connection between MRP1 and MDR1 promoter.

Results

MRP1 downregulation led to a decreased MDR1 expression in MC3/5FU cells which was caused by decreased activity of MDR1 promoter. IHC study of 127 cases of MEC tissues demonstrated a strong positive correlation between nuclear MRP1 expression and MDR1 expression. Furthermore, IHC study of multiple tumor tissue array sections showed that although nuclear MRP1 widely existed in MEC tissues, it was not found in normal tissues or other tumor tissues.

Conclusions

Our findings indicate that nuclear MRP1 contributes to MDR mainly through regulating MDR1 expression in MEC. And the unique location of MRP1 made it an available target in identifying MEC from other tumors.     

Effects of Sertraline and Fluoxetine on P-Glycoprotein at Barrier Sites: In Vivo and In Vitro Approaches

Background and Purpose

Retention of substances from systemic circulation in the brain and testes are limited due to high levels of P-glycoprotein (P-gp) in the luminal membranes of brain and testes capillary endothelial cells. From a clinical perspective, P-gp rapidly extrudes lipophilic therapeutic agents, which then fail to reach efficacious levels. Recent studies have demonstrated that acute administration of selective serotonin reuptake inhibitors (SSRI) can affect P-gp function, in vitro and in vivo. However, little is known concerning the time-course of these effects or the effects of different SSRI in vivo.

Experimental Approach

The P-gp substrate, tritiated digoxin ([³H] digoxin), was co-administered with fluoxetine or sertraline to determine if either compound increased drug accumulation within the brains and testes of mice due to inhibition of P-gp activity. We undertook parallel studies in endothelial cells derived from brain microvessels to determine the dose-response and time-course of effects.

Key Results

In vitro, sertraline resulted in rapid and potent inhibition of P-gp function in brain endothelial cells, as determined by cellular calcein accumulation. In vivo, a biphasic effect was demonstrated. Brain accumulation of [³H] digoxin was increased 5 minutes after treatment with sertraline, but by 60 minutes after sertraline treatment, brain accumulation of digoxin was reduced compared to control. By 240 minutes after sertraline treatment brain digoxin accumulation was elevated compared to control. A similar pattern of results was obtained in the testes. There was no significant effect of fluoxetine on P-gp function, in vitro or in vivo.

Conclusions and Implications

Acute sertraline administration can modulate P-gp activity in the blood-brain barrier and blood-testes barrier. This clearly has implications for the ability of therapeutic agents that are P-gp substrates, to enter the brain when co-administered with SSRI.     

CYP1A1 induction in the colon by serum: involvement of the PPARα pathway and evidence for a new specific human PPREα site

Background

We previously showed that blood serum induced cytochrome P450 1A1 (CYP1A1) monooxygenase expression in vitro.

Objective

Our purpose was (i) to identify the molecular mechanism involved and (ii) to characterize the inducer compound(s) in serum involved at least in part.

Methods

Serum was fractionated on hydrophobic columns. PPARα involvement was demonstrated by gene reporter assays, DNA mutagenesis and EMSA. Gene expression was evaluated by qRT-PCR. Serum samples were analyzed using HS-SPME-GC-MS.

Results

The inductive effect of serum did not depend on the AhR pathway and was enhanced by cotransfection of PPARα cDNA. Mutations in the PPAR response elements of the CYP1A1 gene promoter suppressed this effect. One of the PPRE sites appeared highly specific for human PPARα, an unreported PPRE property. A link was found between CYP1A1 inducibility and serum hydrophobic compounds. Characterization of sera showed that hexanal, a metabolite produced by peroxidation of linoleic acid, was involved in CYP1A1 induction by serum, possibly along with other serum entities.

Conclusion

We demonstrate that serum induces CYP1A1 via the PPARα pathway and that hexanal is one of the serum inducers. The two PPRE sites within the CYP1A1 promoter are functional and one of them is specific for PPARα.     

Effects of GSTM1/GSTT1 Gene Polymorphism and Fruit & Vegetable Consumption on Antioxidant Biomarkers and Cognitive Function in the Elderly: A Community Based Cross-Sectional Study

Background

It was reported that Glutathione S-transferase (GST) gene polymorphism and fruit and vegetable (FV) intake were associated with body antioxidant capacity. The oxidative/anti-oxidative imbalance played an important role in the pathogenesis of AD. However, the association of GST genotype, dietary FV consumption with body antioxidant biomarkers and cognitive function in the elderly is not clear.

Objective

The aim of the present study was to determine the association of GST genotype, and dietary FV intake, with antioxidant biomarkers and cognitive function in the elderly.

Methods

Food frequency questionnaire was used to collect data of dietary FV intakes in 504 community dwelling elderly aged from 55 to 75 years old. GSTM1 and GSTT1 genotypes were determined by using multiple-PCR method. Plasma and erythrocyte antioxidant biomarkers were measured. Cognitive function was measured by using Montreal Cognitive Assessment. Statistical analysis was applied for exploring the association of GST genotype and FV intake with antioxidant biomarkers level and cognitive function in the elderly.

Results

Individual GSTM1 or GSTT1 gene deletion affects body antioxidant biomarkers levels, including erythrocyte GST activity, plasma total antioxidant capacity, and glutathione levels. GSTM1and/or GSTT1 gene deletion have no effects on cognitive function in the surveyed participants. The effect of GST genotype on antioxidant biomarkers are FV intake dependent. There is interaction of FV intake and GST genotype on cognitive function in the elderly.

Conclusion

GST genotype or daily FV consumption impact body antioxidant biomarkers, but not cognitive function in the elderly. There were combined effects of GST genotype and FV consumption on cognitive function in the elderly population. Large scale perspective population study is required to explore the association of GST genetic polymorphism, FV consumption and antioxidant biomarkers and cognitive function in the elderly.     

Multidrug resistance-associated protein-1 (MRP1) genetic variants,MRP1 protein levels and severity of COPD

Background

Multidrug resistance-associated protein-1 (MRP1) protects against oxidative stress and toxic compounds generated by cigarette smoking, which is the main risk factor for chronic obstructive pulmonary disease (COPD). We have previously shown that single nucleotide polymorphisms (SNPs) in MRP1 significantly associate with level of FEV₁ in two independent population based cohorts. The aim of our study was to assess the associations of MRP1 SNPs with FEV₁ level, MRP1 protein levels and inflammatory markers in bronchial biopsies and sputum of COPD patients.

Methods

Five SNPs (rs212093, rs4148382, rs504348, rs4781699, rs35621) in MRP1 were genotyped in 110 COPD patients. The effects of MRP1 SNPs were analyzed using linear regression models.

Results

One SNP, rs212093 was significantly associated with a higher FEV₁ level and less airway wall inflammation. Another SNP, rs4148382 was significantly associated with a lower FEV₁ level, higher number of inflammatory cells in induced sputum and with a higher MRP1 protein level in bronchial biopsies.

Conclusions

This is the first study linking MRP1 SNPs with lung function and inflammatory markers in COPD patients, suggesting a role of MRP1 SNPs in the severity of COPD in addition to their association with MRP1 protein level in bronchial biopsies.     

Inhibition of P-Glycoprotein by HIV Protease Inhibitors Increases Intracellular Accumulation of Berberine in Murine and Human Macrophages

Background

HIV protease inhibitor (PI)-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR), a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER) stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp) in HIV PI-mediated accumulation of BBR in macrophages.

Methodology and Principal Findings

Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT) and human P-gp transfected (MDCK/P-gp) cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123) efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp.

Conclusion and Significance

HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.     

Pharmacogenetic Study of Drug-Metabolising Enzyme Polymorphisms on the Risk of Anti-Tuberculosis Drug-Induced Liver Injury: A Meta-Analysis

Background

Three first-line antituberculosis drugs, isoniazid, rifampicin and pyrazinamide, may induce liver injury, especially isoniazid. This antituberculosis drug-induced liver injury (ATLI) ranges from a mild to severe form, and the associated mortality cases are not rare. In the past decade, many investigations have focused the association between drug-metabolising enzyme (DME) gene polymorphisms and risk for ATLI; however, these studies have yielded contradictory results.

Methods

PubMed, EMBASE, ISI web of science and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. A meta-analysis was performed to examine the association between polymorphisms from 4 DME genes (NAT2, CYP2E1, GSTM1 and GSTT1) and susceptibility to ATLI. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Heterogeneity among articles and their publication bias were also tested.

Results

38 studies involving 2,225 patients and 4,906 controls were included. Overall, significantly increased ATLI risk was associated with slow NAT2 genotype and GSTM1 null genotype when all studies were pooled into the meta-analysis. Significantly increased risk was also found for CYP2E1*1A in East Asians when stratified by ethnicity. However, no significant results were observed for GSTT1.

Conclusions

Our results demonstrated that slow NAT2 genotype, CYP2E1*1A and GSTM1 null have a modest effect on genetic susceptibility to ATLI.     

Molecular Analysis and Phenotypic Study in 14 Chinese Families with Bietti Crystalline Dystrophy

Purpose

To investigate the clinical features and cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2) gene mutations in 14 Chinese families with Bietti crystalline dystrophy (BCD).

Methods

Seventeen patients from 14 unrelated Chinese families with BCD were recruited for complete clinical ophthalmic examination and genetic study. The 11 exons of CYP4V2 were amplified from genomic DNA of all patients and their family members by polymerase chain reaction (PCR) and then sequenced. Exons of TIMP3 were also sequenced in BCD patient associated with choroidal neovascularization (CNV). One hundred and seventy unrelated healthy Chinese subjects were screened for mutations in CYP4V2.

Results

All 17 patients with BCD had mutations in CYP4V2; one of these mutations was novel (c.219T>A, p.F73L) and four other mutations had been reported. The p.F73L mutation was a commonly detected mutation in our study (seven out of 34 alleles), either in the homozygous state or in the heterozygous state. Among the patients, considerable phenotypic variability was detected, both within and between families. Screening of TIMP3 did not find any mutation in the BCD patient associated with CNV.

Conclusion

The novel CYP4V2 c.219T>A (p.F73L) mutation may be another recurrent mutation in Chinese patients with BCD. Our study expands the mutation spectrum of CYP4V2 and characterizes novel genotype–phenotype associations in Chinese patients with BCD.     

Safety and immunogenicity of rSh28GST antigen in humans: phase 1 randomized clinical study of a vaccine candidate against urinary schistosomiasis

Background

Treatment of urinary schistosomiasis by chemotherapy remains challenging due to rapid re-infection and possibly to limited susceptibility to praziquantel treatment. Therefore, therapeutic vaccines represent an attractive alternative control strategy. The objectives of this study were to assess the safety and tolerability profile of the recombinant 28 kDa glutathione S-transferase of Schistosoma haematobium (rSh28GST) in healthy volunteers, and to determine its immunogenicity.

Methodology

Volunteers randomly received 100 µg rSh28GST together with aluminium hydroxide (Alum) as adjuvant (n = 8), or Alum alone as a comparator (n = 8), twice with a 28-day interval between doses. A third dose of rSh28GST or Alum alone was administered to this group at day 150. In view of the results obtained, another group of healthy volunteers (n = 8) received two doses of 300 µg of rSh28GST, again with a 28-day interval. A six-month follow-up was performed with both clinical and biological evaluations. Immunogenicity of the vaccine candidate was evaluated in terms of specific antibody production, the capacity of sera to inhibit enzymatic activity of the antigen, and in vitro cytokine production.

Principal Findings

Among the 24 healthy male participants no serious adverse events were reported in the days or weeks after administration. Four subjects under rSh28GST reported mild reactions at the injection site while a clinically insignificant increase in bilirubin was observed in 8/24 subjects. No hematological nor biochemical evidence of toxicity was detected. Immunological analysis showed that rSh28GST was immunogenic. The induced Th2-type response was characterized by antibodies capable of inhibiting the enzymatic activity of rSh28GST.

Conclusions

rSh28GST in Alum did not induce any significant toxicity in healthy adults and generated a Th2-type immune response. Together with previous preclinical results, the data of safety, tolerability and quality of the specific immune response provide evidence that clinical trials with rSh28GST could be continued in humans as a potential vaccine candidate against urinary schistosomiasis.     

Meta-Analysis of Cytochrome P-450 2C9 Polymorphism and Colorectal Cancer Risk

Background

CYP2C9 encodes a member of the cytochrome P450 superfamily of enzymes which play a central role in activating and detoxifying many carcinogens and endogenous compounds thought to be involved in the development of colorectal cancer (CRC). In the past decade, the relationship between CYP2C9 common polymorphisms (R144C and I359L) and CRC has been reported in various ethnic groups; however, these studies have yielded contradictory results. To investigate this inconsistency, we performed this meta-analysis.

Methods

Databases including Pubmed, EMBASE, Web of Science and China National Knowledge Infrastructure (CNKI) were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association.

Results

A total of 13 articles involving 9,463 cases and 11,416 controls were included. Overall, the summary odds ratio of CRC was 0.98 (95% CI: 0.89−1.06) and 0.99 (95% CI: 0.87−1.14) for CYP2C9 144C and 359L alleles, respectively. No significant results were observed using dominant or recessive genetic model for these polymorphisms. In the stratified analyses according to ethnicity and sex, no evidence of any gene-disease association was obtained.

Conclusions

This meta-analysis suggests that the CYP2C9 may not be associated with colorectal cancer development.     

Pharmacokinetic genes do not influence response or tolerance to citalopram in the STAR*D sample

Background

We sought to determine whether clinical response or tolerance to the Selective Serotonin Reuptake Inhibitor (SSRI) citalopram is associated with genetic polymorphisms in potentially relevant pharmacokinetic enzymes.

Methodology

We used a two-stage case-control study design in which we split the sample of 1,953 subjects from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) trial into a discovery (n = 831) and validation set (n = 1,046). Fifteen polymorphisms from five (CYP2D6, ABCB1, CYP2C19, CYP3A4, and CYP3A5) pharmacokinetic genes were genotyped. We examined the associations between these polymorphisms and citalopram response and tolerance. Significant associations were validated in the second stage for those polymorphism found to be statistically significant in the first stage.

Conclusions

No genetic polymorphism in the pharmacokinetic genes examined was significantly associated with our response or tolerance phenotypes in both stages. For managing pharmacological treatment with citalopram, routine screening of the common pharmacokinetic DNA variants that we examined appears to be of limited clinical utility.     

Sorafenib Metabolism Is Significantly Altered in the Liver Tumor Tissue of Hepatocellular Carcinoma Patient

Background

Sorafenib, the drug used as first line treatment for hepatocellular carcinoma (HCC), is metabolized by cytochrome P450 (CYP) 3A4-mediated oxidation and uridine diphosphate glucuronosyl transferase (UGT) 1A9-mediated glucuronidation. Liver diseases are associated with reduced CYP and UGT activities, which can considerably affect drug metabolism, leading to drug toxicity. Thus, understanding the metabolism of therapeutic compounds in patients with liver diseases is necessary. However, the metabolism characteristic of sorafenib has not been systematically determined in HCC patients.

Methods

Sorafenib metabolism was tested in the pooled and individual tumor hepatic microsomes (THLMs) and adjacent normal hepatic microsomes (NHLMs) of HCC patients (n = 18). Commercial hepatic microsomes (CHLMs) were used as a control. In addition, CYP3A4 and UGT1A9 protein expression in different tissues were measured by Western blotting.

Results

The mean rates of oxidation and glucuronidation of sorafenib were significantly decreased in the pooled THLMs compared with those in NHLMs and CHLMs. The maximal velocity (V_max) of sorafenib oxidation and glucuronidation were approximately 25-fold and 2-fold decreased in the pooled THLMs, respectively, with unchanged K_m values. The oxidation of sorafenib in individual THLMs sample was significantly decreased (ranging from 7 to 67-fold) than that in corresponding NHLMs sample. The reduction of glucuronidation in THLMs was observed in 15 out of 18 patients’ samples. Additionally, the level of CYP3A4 and UGT1A9 expression were both notably decreased in the pooled THLMs.

Conclusions

Sorafenib metabolism was remarkably decreased in THLMs. This result was associated with the down regulation of the protein expression of CYP3A4 and UGT1A9.