Inhibitors of the Hepatitis C Virus Polymerase; Mode of Action and Resistance

The hepatitis C virus (HCV) is a pandemic human pathogen posing a substantial health and economic burden in both developing and developed countries. Controlling the spread of HCV through behavioural prevention strategies has met with limited success and vaccine development remains slow. The development of antiviral therapeutic agents has also been challenging, primarily due to the lack of efficient cell culture and animal models for all HCV genotypes, as well as the large genetic diversity between HCV strains. On the other hand, the use of interferon-α-based treatments in combination with the guanosine analogue, ribavirin, achieved limited success, and widespread use of these therapies has been hampered by prevalent side effects. For more than a decade, the HCV RNA-dependent RNA polymerase (RdRp) has been targeted for antiviral development. Direct acting antivirals (DAA) have been identified which bind to one of at least six RdRp inhibitor-binding sites, and are now becoming a mainstay of highly effective and well tolerated antiviral treatment for HCV infection. Here we review the different classes of RdRp inhibitors and their mode of action against HCV. Furthermore, the mechanism of antiviral resistance to each class is described, including naturally occurring resistance-associated variants (RAVs) in different viral strains and genotypes. Finally, we review the impact of these RAVs on treatment outcomes with the newly developed regimens.     

Purification and characterization of RHP (factor H) and study of its interactions with the first component of complement.

RHP has been purified from the plasma of both normal individuals and patients with rheumatoid arthritis (RA). RHP from both these sources was shown to be identical with Factor H by reaction with antisera and N-terminal amino acid sequence analysis. Factor H, from both normal and RA sera, inhibited the solubilization of immune precipitates but did not affect prevention of immune precipitation. Factor H was shown to inhibit the haemolytic activity of fluid-phase C1, but unlike C1-inhibitor, it had little effect on C1 bound to EA (EAC1). Factor H was shown to complex with intact C1, to isolated C1q and to the C1r:C1s tetramer. However, binding of factor H to C1 did not dissociate the C1 macromolecule. A C1-Factor H complex was detected in the serum and plasma from normal individuals and patients with systemic lupus erythematosus and RA. Serum levels of this complex were reduced, by EDTA-treatment of serum and by activation of complement by the classical pathway.     

Anti‐envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection

In rare cases, individuals with a history of long‐term injecting drug use remain seronegative and aviraemic, despite prolonged and likely repeated exposure to Hepatitis C virus (HCV) through high‐risk behaviour. We describe anti‐HCV Envelope (E) antibody responses in a prospective cohort of carefully defined highly exposed but uninfected subjects (HESN) and comparison subjects who were also high risk and uninfected, but rapidly became HCV infected (Incident). Longitudinally collected samples from HESN cases (n = 22) were compared to Incident controls (n = 22). IgG, IgM and IgA from sera were tested by ELISA to genotype 1a and 3a E glycoproteins, and recombinant genotype 1a E2 antigen. IgG subclass isotyping was performed for those positive for IgG. Virus‐neutralizing activity was assessed on HCV pseudoparticles, and HCV E–specific B cells analysed using flow cytometry. A significant minority of HESN cases (n = 10; 45%) had anti‐E, predominantly in the IgG2 subclass, which was not found in the pre‐infection time point of the Incident cases (n = 1; 5%). A subset of the HESN subjects also had neutralizing activity and HCV‐specific B cells detected significantly more than Incident cases pre‐infection. In conclusion, the HESN phenotype is associated with IgG2 anti‐E antibodies, neutralization activity and HCV E–specific memory B cells. These findings suggest that HESN subjects may be resistant to HCV infection through humoral immune‐mediated mechanisms.     

Preparation,physicochemical characterization and biological evaluation of cefodizime metal ion complexes

Objectives Cefodizime is a broad spectrum cephalosporin belonging to the third generation agents. In this study, attention has been paid to the preparation, physicochemical characterization and biological evaluation of new Cu²⁺, Zn²⁺, Fe³⁺, Co²⁺ and Al³⁺ complexes of cefodizime. Methods The stoichiometrics and the mode of bonding of the complexes were deduced from their elemental and metal analysis, electrical conductivity measurements, UV‐vis, infrared and Raman spectroscopic investigations. Study of the stoichiometry of these complexes referred to the formation of 1: 1 ratios of metal to ligand. Antimicrobial activity of the complexes was determined using two strains of Gram‐positive (Bacillus subtilis and Proteus vulgaris) and two strains of Gram‐negative (Escherichia coli W3110 and Pseudomonas putida) bacteria. The minimal inhibitory concentration was determined as the lowest concentration inhibiting bacterial growth on solid Luria Bertani medium. Key findings The spectra gave evidence as to the position of binding. In addition, the aqueous solubility of cefodizime was strongly reduced by complexation. Conclusions The antibacterial activity of cefodizime was not affected by complexation with Al³⁺ but it was reduced by complexation with the other tested metal ions against the bacteria under study.     

Evolution of the surgical management of primary aldosteronism.

During the past two decades 50 patients were operated on for primary aldosteronism. Diagnosis was based on high aldosterone excretion or secretion during a high sodium intake and, more recently, low stimulated plasma renin activity. Computed tomography and adrenal venography with selective adrenal vein catheterization for determination of aldosterone/cortisol ratios were helpful in the distinction between adenoma and bilateral hyperplasia. As a result of preoperative localization, unilateral posterior or flank approach to the adrenal has replaced transabdominal as the approach of choice. Overall in-hospital mortality in this series was 10% and occurred exclusively with a transabdominal approach in the early part of the series. Adrenalectomy has been curative in 66% of patients with adenoma and in 38% of patients with hyperplasia which includes patients with adenomatous (dominant macroscopic adenoma, 1 cm or greater) hyperplasia when the cure rate was 75%. Currently, only patients who have unilateral adrenal hyperfunction, who respond to spironolactone with a fall in blood pressure, and who are a good operative risk are considered for operation by posterior or flank approach. These guidelines for the management of primary aldosteronism, used since 1974, have been associated with an excellent response (92%), zero mortality and reduced morbidity.     

Emulsification/internal gelation as a method for preparation of diclofenac sodium–sodium alginate microparticles

Emulsification/internal gelation has been suggested as an alternative to extrusion/external gelation in the encapsulation of several compounds including non-steroidal anti-inflammatory drugs such as diclofenac sodium. The objective of the present study was a trial to formulate diclofenac sodium as controlled release microparticles that might be administered once or twice daily. This could be achieved via emulsification/internal gelation technique applying Box-Behnken design to choose these formulae. Box-Behnken design determined fifteen formulae containing specified amounts of the independent variables, which included stirring speed in rpm (X1), drug:polymer ratio (X2) and the surfactant span 80% (X3). The dependent variables studied were cumulative percent release after two hours (Y1), four hours (Y2) and eight hours (Y3). The prepared microparticles were characterized for their production yield, sizes, shapes and morphology, entrapment efficiency and Diclofenac sodium in vitro release as well. The results showed that the production yield of the prepared diclofenac sodium microparticles was found to be between 79.55% and 97.41%. The formulated microparticles exhibited acceptable drug content values that lie in the range 66.20–96.36%. Also, the data obtained revealed that increasing the mixing speed (X1) generally resulted in decreased microparticle size. In addition, scanning electron microscope images of the microparticles illustrated that the formula contains lower span concentration (1%) in combination with lower stirring speed (200 rpm) which showed wrinkled, but smooth surfaces. However, by increasing surfactant concentration, microspheres’ surfaces become smoother and slightly porous. Kinetic treatment of the in vitro release from drug-loaded microparticles indicated that the zero order is the drug release mechanism for the most formulae.     

Characterization and activity of cephalosporin metal complexes

Semi-synthetic cephalosporin antibiotics have structures similar to that of penicillins, and both groups of compounds are characterized by similar properties and determined by the same methods. Most antibiotics, including cephalosporins and their decomposition products, contain electron donor groups that can bind naturally occurring metal ions in vivo. Cephalosporin antibiotics exhibit a change in their toxicological properties and biological performance when they were tested as metal complexes. The proposed reason for such a behavior is the capability of chelate binding of the cephalosporins to the metals. In an attempt to understand the coordination mode of metals with cephalosporins, different spectroscopic techniques such as IR, UV-visible, NMR spectroscopy and voltammetric measurements were carried out to elucidate the structure of the metal-cephalosporin complexes. Synthesis, characterization and biological screening of the cephalosporins and of the cephalosporin-metal complexes are discussed in this review. However, little information is available on the influence of the metal ions on the pharmacokinetics of the cephalosporin derivatives.     

Measurement of complement activation products in patients with chronic rheumatic diseases

Summary Measurement of the complement activation products C1s:C1-inh, C3bP and C5b-9 by ELISA in plasma samples from normals, rheumatoid arthritis (RA) and systemic lupus erythematosis (SLE) patients showed significantly elevated levels in the two patient groups (P<0.0001 for C1s:C1-inh, C3bP and C5b-9) compared to normals. In seropositive RA patients there were significant correlations between the levels of the three complement activation complexes and IgM-RF, IgG-RF and IgA-RF. However, IgM-RF did not interfere with any of the ELISA systems. Mean levels of C1s:C1-inh, C3bP and C5b-9 were the same in paired plasma and synovial fluids; however, C3bP levels in the paired samples did not correlate with one another by rank. Our conclusions are that: (a) elevated plasma levels of these complement activation products are detectable in rheumatic diseases; (b) plasma levels of these complement activation products are related to Rheumatoid factor (RF) levels in seropositive RA patients; and (c) IgM-RF does not influence these solid-phase ELISA procedures.     

Prevalence of renal artery stenosis in high-risk veterans referred to cardiac catheterization

OBJECTIVE: The prevalence of atherosclerotic renal artery stenosis (RAS) varies depending on patient selection with no specific guidelines on indications for selective renal angiography in patients referred for coronary angiography. The goal of this study is to determine the prevalence and predictors of renal artery stenosis in hypertensive veterans referred for coronary angiography. DESIGN: Prospective study. SETTING: Tertiary care veterans' administration facility in the USA. PATIENTS, PARTICIPANTS: A total of 90 veterans referred for coronary angiography with an initial ascending aortic pressure > 135 mmHg. INTERVENTIONS: Selective renal angiography was performed following coronary angiography. RESULTS: We found that 28% of the patients had single RAS (> or = 50% stenosis), while 16% had single RAS > or = 70% stenosis, 10% had bilateral RAS >or = 50% and 6% had bilateral RAS > or = 70%. Significant positive univariate predictors of RAS (> or = 50%) were age, peripheral vascular disease (PVD), creatinine level (Cr) and myocardial infarction. Significant multivariate predictors of RAS (> or = 50%) were age > 65 years [relative risk (RR), 3.6; 95% confidence interval (CI), (1.2-10.6)], PVD [RR 3.2, 95% CI (1.1-9.1)] and Cr > 1 mg/dl [RR 4.9, 95% CI (1.53-15.9)]. No complications related to renal angiography were noted. CONCLUSIONS: Selective renal angiography during routine coronary angiography in hypertensive veterans with coronary artery disease is safe and uncovers RAS in many older patients with PVD and renal insufficiency.