Tissue-specific regulation of CYP3A by hydrolysable tannins in male pigs

1.?Little is known about the activities and regulation of cytochrome P4503A (CYP3A) enzymes in porcine colon in response to specific feeding components.

2.?We added hydrolyzable tannins to the diet of fattening boars and studied its effect on the expression of hepatic and intestinal CYP3A.

3.?In total, 51 Landrace?×?Large White boars were assigned to the following treatment groups: control (without the addition of hydrolysable tannins), T1 (diet-containing 1% hydrolysable tannin extract), T2 (diet-containing 2% hydrolysable tannin extract) and T3 (diet-containing 3% hydrolysable tannin extract). CYP3A expression and activity were measured in microsomes prepared from liver and colon tissue.

4.?CYP3A protein expression and activity were increased in the colon of pigs fed 2% and 3% tannins, while no changes were observed with lower tannin concentrations, or in the liver of any treatment groups. Also, it was demonstrated that colon mucosa possess CYP3A activity similar to that measured in the liver.

5.?The present results provide the first evidence that tannin supplementation can modulate CYP3A in porcine colon mucosa in vivo. The physiological significance of this finding for the health status of the individual animal needs further investigation.     

Prooxidant action of chebulinic acid and tellimagrandin I: Causing copper-dependent DNA strand breaks

The prooxidant activity of two hydrolysable tannins, chebulinic acid and tellimagrandin I, on plasmid DNA and genomic DNA of cultured MRC-5 human embryo lung fibroblasts was assessed. The results revealed that both hydrolysable tannins in combination with Cu(II) induced DNA strand breaks in pBR322 plasmid DNA in a concentration-dependent manner. Chebulinic acid and tellimagrandin I also induced genomic DNA strand breaks of MRC-5 human embryo lung fibroblasts in the presence of Cu(II). After treatment with chebulinic acid or tellimagrandin I alone, the pBR322 plasmid DNA and genomic DNA in MRC-5 cells kept intact. In addition, addition of Cu(I) reagent bathocuproinedisulfonic acid or catalase markedly inhibited the copper-dependent DNA strand breaks by both tannins. However, three typical hydroxyl radical scavengers, DMSO, ethanol and mannitol, did not inhibit the DNA strand breaks. Both tannins were able to reduce Cu(II) to Cu(I). These results indicated that chebulinic acid and tellimagrandin I induced the copper-dependent strand breaks of pBR322 plasmid DNA and MRC-5 genomic DNA with prooxidant action, in which Cu(II)/Cu(I) redox cycle and H₂O₂ were involved and hydroxyl radical formation is important in the hypothetical mechanism by which DNA strand breaks are formed.     

Inhibitory effects of Pinus massoniana bark extract on hepatitis C virus in vitro

Abstract

Context: Given that Pinus massoniana Lamb (Pinaceae) bark extract (PMBE) is a safe and non-toxic flavonoid found abundantly in nature, it was considered a promising novel candidate agent in the treatment of virus infection.

Object: Experiments were conducted to assay the antiviral character of PMBE against Hepatitis C virus (HCV).

Materials and methods: Assay of PMBE cytotoxicity, HCV replication, infectious HCV production, and its potential influence on the pathways for IFN-ISRE and NS3 protease were conducted.

Results: HCV replication was suppressed when the concentration of PMBE raised greater than 5?μg/mL and its EC₅₀ was 9.58?μg/mL. In the 10?μg/mL group, HCV replication was suppressed for 48?h. When the concentration increased to 40?μg/mL, HCV replication was significantly suppressed (luciferase activity was only 10% at 96?h). PMBE could inhibit HCV virus production efficiently (PMBE group was 5?FFU). Cell viability was affected by 40?μg/mL of PMBE. The F/R ratio ranged from 98% to 101%. The rate of OD₄₅₀ ranged from 96% to 102%. NS3 catalytic activity ranged from 5% (40?μg/mL PMBE) to 45% (5?μg/mL PMBE). Even when used in a low amount (5?μg/mL), NS3 catalytic activity was significantly inhibited (p?<?0.01).

Conclusions: The results suggest that PMBE is effective for use in the stabilization of HCV replication and active liver inflammation.     

Hepatoprotective and antioxidant effect of ellagitannins and galloyl esters isolated from Melaleuca styphelioides on carbon tetrachloride-induced hepatotoxicity in HepG2 cells

Context In a previous study, the total extract of Melaleuca styphelioides Sm. (Myrtaceae) showed a significant hepatoprotective effect in a CCl₄-induced toxicity model in mice. However, the active components responsible for the activity of the extract were not identified.

Objective To determine the in vitro hepatoprotective activity of the isolated pure compounds from M. styphelioides leaves using the CCl₄-challenged HepG2 cell model.

Materials and methods The hepatoprotective activity of the compounds (at concentrations of 100, 50 and 25?μm), the total extract and silymarin (Sil) (100, 50 and 25?μg/ml) was determined by measuring the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) after pretreatment with the tested samples for one hour. Glutathione (GSH) and superoxide dismutase activity (SOD) were estimated to determine the mechanisms of the hepatoprotective activity.

Results Some compounds showed marked hepatoprotection, including tellimagrandin I, which produced 42, 36 and 31% decrease in ALT and 47, 43 and 37% decrease in AST, at the tested concentrations, respectively, pedunculagin (32, 32 and 30% decrease for ALT and 48, 48 and 45% for AST), tellimagrandin II (38, 32 and 26% decrease for ALT and 45, 40 and 34% for AST) and pentagalloyl glucose (30, 28 and 26% decrease for ALT and 45, 38 and 36% for AST). Tellimagrandin I and II showed the highest increase in GSH (113, 105 and 81% and 110, 103 and 79%, respectively), which was comparable to Sil. Pedunculagin produced the highest increase in SOD (497, 350 and 258%).

Conclusion This study highlights promising natural hepatoprotective candidates derived from M. styphelioides.     

Impact of pill burden on adherence to hepatitis C medication

Objective: To describe pill burden before and after hepatitis C virus (HCV) treatment initiation among patients newly treated for HCV infection, and to evaluate the association between HCV pill burden and gaps in HCV therapy.

Methods: This was a retrospective administrative claims study of patients treated with direct-acting antivirals (DAAs) for HCV from 1 November 2013 to 31 July 2016. HCV pill burden was defined as the pill count per day for the index HCV regimen. Mean overall pill burden (HCV medications plus non-HCV medications) was calculated in the 90?days before and after DAA initiation. Gaps in the index HCV regimen were assessed in the 6?months after DAA initiation. Multivariable logistic regression was used to compare the odds of a gap in HCV therapy across HCV pill burden categories (1 pill/day, 2 pills/day, and ≥3 pills/day).

Results: Among 9815 patients who met the study criteria, mean overall pill burdens before and after DAA treatment initiation were 5.4 and 7.7, respectively (p?<?.001). The adjusted odds ratio (OR) of a ≥15-day gap in HCV therapy was 1.75 (95% confidence interval [CI]?=?1.38–2.22) for patients with 2 HCV pills/day and 2.11 (95% CI?=?1.78–2.51) for patients with ≥3 pills/day, compared with patients with 1 HCV pill/day.

Conclusions: Patients with HCV have a substantial pill burden even before initiating HCV treatment. As higher HCV pill burden was associated with lower medication adherence, pill burden should be an important consideration in HCV treatment selection.     

Hepatitis C knowledge among new injection drug users

Aims: New injection drug users (IDUs) are vulnerable to hepatitis C infection from risky injection practices. This article considers the range of hepatits C virus (HCV) knowledge among participants in a 2-year study examining the behaviours of new IDUs.

Methods: Respondents (n = 36) were members of a cohort of new IDUs recruited in the New York City metropolitan area whose first injection occurred within the previous 18 months. Respondents were offered human immuno virus (HIV), hepatits B virus (HBV) and HCV testing. Two tested HIV positive and 12 tested HCV positive. During pre-test counselling they completed a qualitative interview focusing on aspects of HCV including: transmission, symptoms, long-term effects, prevention, treatment, concern regarding contracting HCV and how this concern impacted drug use and injection practices.

Findings: Substantial gaps were seen in HCV knowledge. While respondents were aware that HCV is a blood-borne virus, the majority had only approximate knowledge regarding transmission, symptoms and effects. Respondents reported little discussion of HCV with peers and minimal concern about contracting HCV, especially compared with HIV.

Conclusion: Accurate HCV information is failing to reach new IDUs, and new IDUs may not value the little information they do receive. More effective and innovative efforts are required to disseminate effective HCV prevention information to new IDUs.     

Burden of illness of hepatitis C from a managed care organization perspective

SUMMARY

Objectives: The objectives of this study were to: (1) determine the total hepatitis C virus (HCV)-related and total healthcare costs (HCV plus other co-morbidities) of patients with HCV in a managed care organization; (2) determine total healthcare costs of HCV patients with and without a human immunodeficiency virus (HIV) infection as a co-morbidity.

Methods: The study design was a retrospective analysis of a medical and pharmacy claims database of patients diagnosed with HCV in a 325000 member managed care organization. Patients diagnosed with HCV and 12 months of continuous eligibility in the managed care organization from January 1997 through December 1999 were included in the study. The main outcome measures of the study were the total healthcare costs and HCV-related healthcare costs and the impact of HIV as a co-morbidity on these costs.

Results: The study identified 614 patients meeting the inclusion criteria. The study population was 58% male and had a mean age of 46 (± 10.6)years. In patients receiving interferon-α, their median total healthcare costs exceeded $4600 and the median HCV-related costs exceeded $2470. The total healthcare costs of HCV patients with HIV as a co-morbidity were significantly larger than patients without this co-morbidity.

Conclusion: HCV represents a very important disease to managed care organizations. Patients with this disease require costly drug therapies and consume significant health care resources. Additional research is needed to more fully characterize future clinical and economic outcomes as new agents become available.     

Treatment of HBV/HCV coinfection

Importance of the field: Hepatitis B (HBV) and hepatitis C (HCV) virus infections are among the most common causes of advanced chronic liver disease worldwide. HBV/HCV coinfection is not uncommon with an estimated 7 – 20 million individuals affected worldwide. Patients with HBV/HCV coinfection have an increased risk for cirrhosis, hepatocellular carcinoma (HCC) and even death.

Areas covered in this review: The pathophysiology of HBV/HCV coinfection is complex, as different patterns of virological dominance may occur, which can even fluctuate over time. Recently, combination of pegylated interferon (PEG-IFN) plus ribavirin has been explored in HBV/HCV coinfected patients who are positive for HCV-RNA. HBV polymerase inhibitors may be indicated if HBV-DNA concentrations are above 2000 IU/ml. In this review, we summarize the epidemiology, viral interaction, its clinical features and the available treatment options.

What the reader will gain: Insights into viral interaction of HBV/HCV coinfection and treatment individualization strategies are provided in the review.

Take home message: Detailed serological and virological evaluations are required for HBV/HCV coinfected patients before initiation of antiviral therapy. At present, PEG-IFN-α plus ribavirin should be the treatment of choice in patients with dominant HCV replication. However, HBV rebound may occur after elimination of HCV, and thus close monitoring for both viruses is recommended even for patients with initially suppressed HBV-DNA.     

Preclinical development of TLR ligands as drugs for the treatment of chronic viral infections

Introduction: Toll-like receptors (TLRs) have been identified as key regulators of innate and adaptive immune responses in viral infection. Recent progress in this field revealed that there are significant interactions between the TLR system and pathogens in chronic viral infections. Therefore, TLR ligands have great potential for the treatment of chronic viral infections.

Areas covered: This review provides an overview of the methodology for preclinical testing of TLR ligands for three major viral infections: hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). TLR ligands have shown potent antiviral activity in different cell culture systems as well as animal models for these infections and induce the production of antiviral cytokines, modulated cellular immunological functions and antiviral effects in vivo.

Expert opinion: The recent progress in this field demonstrated that activation of a large number of TLR ligands is effective against viral infections in cell culture systems and animal models. Exploring these models, further in-depth elucidation of the molecular and immunological mechanisms of the antiviral activity of TLR ligands will be necessary to develop them into clinical useful drugs.     

Patent Evaluation: Antibacterial Condensed Carbapenems

Summary

Novelty: Novel condensed carbapenems are described. These compounds, containing an amidine group, are claimed to have antibacterial activity and are potentially useful in the treatment or prophylaxis of systemic or topical infections. A process for the synthesis of these tricyclic carbapenems is also claimed.

Biology: The specific compound is shown to have antibacterial activity against Gram-positive bacteria (MIC 0.1-1 μg/ml), Gram-negative bacteria (E. coli 1852E 0.5 μg/ml, Ps. aeruginosa 1911E 8 μg/ml) and also anaerobes (B. fragilis 2017E 0.2 μg/ml). No adverse effects were observed in toxicological studies in mouse and rat at 500 mg/kg iv.

Chemistry: The syntheses of seven intermediates and two examples by modification of known methods are fully detailed. Only one compound is specifically claimed, (4S,8S,9R,10S,12R)-4-(N-methylformamidino)-10-(1-hydroxyethyl)-11-oxo-1-azatricyclo[7.2.0.0³⁸]undec-2-ene-2-carboxylic acid.     

Investigational drugs for hepatitis C

Importance of the field: Hepatitis C virus (HCV) infection affects 180 million people worldwide. Standard anti-HCV therapy combines effect of pegylated IFN-alpha on immune response and antiviral activity of ribavirin. Sustained virological response rate achieved with this standard of care medication is around 50% for HCV genotype 1, the most prevalent worldwide genotype. So there is an obvious need to enhance efficacy of treatment.

Areas covered in this review: We describe novel treatment options studied within the recent few years, which are focused on inhibitors of HCV-specific enzymes such as NS3/4 protease and NS5B polymerase or influence host-virus interactions.

What the reader will gain: According to the most recent data, triple therapies consisted of pegylated interferons, ribavirin and protease inhibitors demonstrate significant improvement of treatment efficacy in comparison to current standard of care medication. Moreover, new forms of interferons and ribavirin, more effective, less toxic, and more convenient, become probably a fundamental component of anti-HCV therapy in the near future.

Take home message: In coming years, we can expect that triple therapy becomes a standard medication, and treatment without interferon and/or ribavirin becomes a new studied therapeutic scenario.     

Advances in the treatment of hepatitis B virus/hepatitis C virus coinfection

Introduction: Patients with chronic hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection are at a high risk of developing liver cirrhosis and hepatocellular carcinoma, and consequently, warrant effective treatment.

Areas covered: Effective treatment should eradicate HCV infection and inhibit HBV replication but without serious adverse reactions. Careful evaluation of disease progression, predominance of one virus over another, comorbidities and concomitant hepatitis delta virus and/or HIV infection are essential for better therapy choices. In the case of HCV predominance, Peg-interferon plus ribavirin with or without a first-generation directly acting antiviral (DAA) should be the first choice, but future treatments will be DAA-based and interferon-free. In the case of HBV predominance, tenofovir or entecavir should be part of treatment. Patients should be closely monitored for early identification and treatment of HCV or HBV reactivation.

Expert opinion: High potency and high genetic barrier nucleos(t)ide analogues to inhibit HBV replication have been used for years, with no urgency for new drugs. Several DAAs for interferon-free therapy for HCV eradication will be available in the near future. We hope that the high cost of these drugs will not be a limitation to their use in developing countries. Further investigation of HBV/HCV interaction is needed before and during the administration of new therapies.     

Dasabuvir: a new direct antiviral agent for the treatment of hepatitis C

Introduction: Treatment of hepatitis C virus (HCV) infection with direct-acting antivirals (DAAs) has revolutionized the care of infected patients. Among these novel compounds are non-nucleoside analogs, which bind viral RNA-dependent RNA polymerase resulting in a conformational change inhibiting RNA synthesis.

Areas covered: Efficacy and tolerability of treatment regimens containing the non-nucleoside analog polymerase inhibitor dasabuvir (ABT-333).

Expert opinion: Dasabuvir-containing regimens achieve high rates of sustained virologic response in HCV genotype 1a and 1b–infected patients when combined with other DAAs, namely paritaprevir (ABT-450), ritonavir and ombitasvir (ABT-267). In the populations studied, dasabuvir seems to be well tolerated and safe. The major limitations of this novel drug are its genotype-restricted activity, the necessity to include ribavirin for HCV genotype 1a and the emergence of resistance if not combined with other DDAs.     

cis-trans-Isomerization of [E]-5-(2-bromovinyl)-2,2′-anhydrouridine in vivo in rats

1. [E]-5-(2-bromovinyl)-2,2′-anhydrouridine ([E]BVANUR) has considerable antiviral activity against herpes simplex virus type 1 (HSV-1).

2. [E]BVANUR is not a substrate of pyrimidine nucleoside phosphorylases, but it is an inhibitor of uridine phosphorylase (K_i=450 nM).

3. [E]BVANUR (trans-isomer, parent compound) undergoes isomerization to [Z]BVANUR (cis-isomer), the only metabolite in rat, which was identified by?h.p.l.c., mass spectra and n.m.r. spectroscopy.

4. Absorption of the drug from the gastrointestinal tract after oral administration is minimal. Absorption of [E]BVANUR from the abdominal cavity after i.p. administration was slow.     

Alcyonacean Metabolites VII – Chemical Constituents of Lobophytum denticulatum and Lobophytum strictum of the Indian Ocean

Abstract

A rare cembranoid diterpene, (7E,11E,1R,2S,3R,4R,14S)-14-acetoxy-3,4-epoxycembra-7,11,15-triene-17,2-olide (1), was isolated from Lobophytum denticulatum and a new polyhydroxysterol, 7α-hydroxyandamansterol (9) has been identified as peracetyl derivative from Lobophytum strictum. Several known polyhydroxysterols have also been isolated from these organisms. 1 exhibited moderate antibacterial activity.     

Photoprotective effects of cranberry juice and its various fractions against blue light-induced impairment in human retinal pigment epithelial cells

Context: Cranberry has numerous biological activities, including antioxidation, anticancer, cardioprotection, as well as treatment of urinary tract infection (UTI), attributed to abundant phenolic contents.

Objective: The current study focused on the effect of cranberry juice (CJ) on blue light exposed human retinal pigment epithelial (ARPE-19) cells which mimic age-related macular degeneration (AMD).

Materials and methods: Preliminary phytochemical and HPLC analysis, as well as total antioxidant capacity and scavenging activity of cranberry ethyl acetate extract and different CJ fractions (condensed tannins containing fraction), were evaluated. In cell line model, ARPE-19 were irradiated with blue light at 450?nm wavelength for 10?h (mimic AMD) and treated with different fractions of CJ extract at different doses (5–50?μg/mL) by assessing the cell viability or proliferation rate using MTT assay (repairing efficacy).

Results: Phytochemical and HPLC analysis reveals the presence of several phenolic compounds (flavonoids, proanthocyanidin, quercetin) in ethyl acetate extract and different fractions of CJ. However, the condensed tannin containing fraction of ethyl acetate extract of CJ displayed the greater (p?p?p?Discussion and conclusion: In conclusion, this study distinctly proved that condensed tannin containing fraction of CJ probably exhibits better free radicals scavenging activity and thereby effectively protected the ARPE-19 cells and thus, hampers the progress of AMD.     

Co-expression of hepatitis C virus polytope–HBsAg and p19-silencing suppressor protein in tobacco leaves

Context: Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition.

Objective: We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine.

Materials and methods: A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc–HBsAg was also evaluated by co-infiltration of a p19 expression vector.

Results: Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc–HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants.

Discussion and conclusion: The results indicated the possibility of expression of HCVpc–HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.     

Grazoprevir + elbasvir for the treatment of hepatitis C virus infection

Introduction: Hepatitis C virus (HCV)-related liver disease is a cause of significant morbidity and mortality worldwide. Currently, direct-acting antiviral drugs (DAAs) are associated with an increased sustained virologic response (SVR) and are the gold standard for treating HCV infection.

Areas covered: The new combination of grazoprevir, an inhibitor of HCV NS3/4A, and elbasvir, an inhibitor of HCV NS5A, once daily will be available for the treatment of HCV infection. This combination therapy has a high efficacy in HCV genotype 1 and 4 infections, inducing a SVR up to 95%, even in difficult to treat patients such as cirrhotic, HIV co-infected, or dialysis-dependent patients, and patients with stage 4–5 chronic kidney disease or those who failed previous therapy. The safety of grazoprevir combined with elbasvir is very good and without significant adverse effects in phase 2 or 3 studies. For patients who failed prior DAA therapy, in vitro and in vivo studies showed that the grazoprevir and elbasvir combination is fully active against resistance to NS3/4A protease inhibitors. Resistance to NS5B inhibitors is least susceptible to grazoprevir or elbasvir.

Expert opinion: This new combination of gazoprevir with elbasvir offers an opportunity to cure HCV infection with short interferon-free therapy, even in difficult to treat patients.