Reduction in ocular complement factor B protein in mice and monkeys by systemic administration of factor B antisense oligonucleotide.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of permanent vision loss among the elderly in many industrialized countries, and the complement system plays an important role in the pathogenesis of AMD. Inhibition of complement factor B, a key regulator of the alternative pathway, is implicated as a potential therapeutic intervention for AMD. Here we investigated the effect of liver factor B reduction on systemic and ocular factor B levels. METHODS: Second-generation antisense oligonucleotides (ASOs) targeting mouse and monkey factor B mRNA were administered by subcutaneous injection to healthy mice or monkeys, and the level of factor B mRNA was assessed in the liver and the eye. In addition, the factor B protein level was determined in plasma and whole eyes from the treated animals. RESULTS: Mice and monkeys treated with factor B ASOs demonstrated a robust reduction in liver factor B mRNA levels with no change in ocular factor B mRNA levels. Plasma factor B protein levels were significantly reduced in mice and monkeys treated with factor B ASOs, leading to a dramatic reduction in ocular factor B protein, below the assay detection levels. CONCLUSIONS: The results add to the increasing evidence that the liver is the main source of plasma and ocular factor B protein, and demonstrate that reduction of liver factor B mRNA by an ASO results in a significant reduction in plasma and ocular factor B protein levels. The results suggest that inhibition of liver factor B mRNA by factor B ASOs would reduce systemic alternative complement pathway activation and has potential to be used as a novel therapy for AMD.

Optimization of Laboratory Ordering Practices for Complete Blood Count With Differential.

Objectives: To investigate the utilization of CBC and CBC with differential (CBC w/diff) tests at University of Alabama at Birmingham Hospital, and to determine if a reduction in CBC w/diff tests could be achieved without negatively impacting patient care. Methods: The quantity of testing and distribution of repeated tests before, during, and after an educational intervention were compared. Results: CBC w/diff tests were ordered 10-fold more frequently than CBC tests. The trauma burn intensive care unit ordered the most CBC w/diff tests, with repeat tests done every 4 or 12 hours. The educational intervention reduced the number of CBC w/diff tests ordered and tests repeated every 12 hours. Conclusions: The educational intervention changed the ordering practices of CBC w/diff and CBC tests. This was sustained after the intervention and no negative effects on patient care were noted. Similar interventions may lead to optimization of ordering practices of other laboratory tests.

Inhibition of the alternative complement pathway by antisense oligonucleotides targeting complement factor B improves lupus nephritis in mice.

Systemic lupus erythematosus is an autoimmune disease that manifests in widespread complement activation and deposition of complement fragments in the kidney. The complement pathway is believed to play a significant role in the pathogenesis and in the development of lupus nephritis. Complement factor B is an important activator of the alternative complement pathway and increasing evidence supports reducing factor B as a potential novel therapy to lupus nephritis. Here we investigated whether pharmacological reduction of factor B expression using antisense oligonucleotides could be an effective approach for the treatment of lupus nephritis. We identified potent and well tolerated factor B antisense oligonucleotides that resulted in significant reductions in hepatic and plasma factor B levels when administered to normal mice. To test the effects of factor B antisense oligonucleotides on lupus nephritis, we used two different mouse models, NZB/W F1 and MRL/lpr mice, that exhibit lupus nephritis like renal pathology. Antisense oligonucleotides mediated reductions in circulating factor B levels were associated with significant improvements in renal pathology, reduced glomerular C3 deposition and proteinuria, and improved survival. These data support the strategy of using factor B antisense oligonucleotides for treatment of lupus nephritis in humans.

Outcome of extremely preterm infants randomized at birth to different PaCO2 targets during the first seven days of life.

BACKGROUND: Ventilation with higher PaCO(2) goals may reduce lung injury and bronchopulmonary dysplasia (BPD). The effect may be enhanced by using a higher PaCO(2) goal than in previous trials. OBJECTIVE: To determine the clinical benefits and safety of higher PaCO(2) goals for ventilated preterm infants. STUDY DESIGN: Preterm infants with a gestational age between 23 and 28 completed weeks receiving mechanical ventilation within 6 h of birth were randomized to be managed with either a PaCO(2) target between 55 and 65 mm Hg (7.3- 8.7 kPa, minimal ventilation) or 35 and 45 mm Hg (4.7- 6.0 kPa, routine ventilation) for the first 7 days of life. The primary outcome measure was BPD, defined as need for mechanical ventilation or supplemental oxygen at 36 weeks postmenstrual age, or death. The neurodevelopmental status was assessed at 18-22 months corrected age. RESULTS: The trial was stopped early after enrolling 31% of the projected sample size. Enrolled infants had a median birth weight of 640 g. BPD or death occurred in 21/33 (64%) infants after minimal ventilation and 19/32 (59%) infants after routine ventilation. Minimal ventilation was associated with trends towards higher mortality and higher incidence of neurodevelopmental impairment, and a significantly increased combined outcome of mental impairment or death (p < 0.05). CONCLUSION: Minimal ventilation as performed in this study did not improve clinical outcome, and may have been associated with a worse neurodevelopmental outcome.

Solid-phase synthesis and screening of macrocyclic nucleotide-hybrid compounds targeted to hepatitis C NS5B.

A convergent strategy for the synthesis of cyclic nucleotide-hybrid molecules on controlled pore glass is reported. A major advantage of the approach is the lack of restrictions on the sequence and structural variation, allowing the incorporation of modified ribonucleosides (such as 2'-OMe-ribonucleotides), as well as threoninol derivatives. This methodology allows a fully automated assembly by means of standard phosphoramidite chemistry and is based on a recently published procedure for the preparation of cyclic oligodinucleotides in the DNA series (M. Smietana, E. T. Kool, Angew. Chem. 2002, 114, 3856-3859; Angew. Chem. Int. Ed. Engl. 2002, 41, 3704-3707). A library of potential cyclic hybrid inhibitor compounds targeting hepatitis C virus NS5B enzyme (the replicating polymerase of HCV) was generated by means of the parallel-pool strategy. Screening of the library revealed that cyclic hybrid c(C(OME)EthenodA) was a significant inhibitor of NS5B, with an IC(50) of 40 microM. Preliminary structure-activity studies of this lead compound are described.

Novel P4 truncated tripeptidyl alpha-ketoamides as HCV protease inhibitors.

We describe herein the synthesis and evaluation of two series of P-4 truncated tripeptidyl alpha-ketoamides as HCV serine protease inhibitors. The most promising compound disclosed in this communication 7b demonstrated enzyme binding affinity (K(i)) at 0.27 uM.

Identification of a C-terminal regulatory motif in hepatitis C virus RNA-dependent RNA polymerase: structural and biochemical analysis.

The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-A resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to approximately 50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.

Comparative characterization of two DEAD-box RNA helicases in superfamily II: human translation-initiation factor 4A and hepatitis C virus non-structural protein 3 (NS3) helicase.

Eukaryotic initiation factor 4A (eIF4A) is an ATP-dependent RNA helicase and is homologous to the non-structural protein 3 (NS3) helicase domain encoded by hepatitis C virus (HCV). Reported here is the comparative characterization of human eIF4A and HCV NS3 helicase in an effort to better understand viral and cellular helicases of superfamily II and to assist in designing specific inhibitors against HCV infections. Both eIF4A and HCV NS3 helicase domain were expressed in bacterial cells as histidine-tagged proteins and purified to homogeneity. Purified eIF4A exhibited RNA-unwinding activity and acted on RNA or RNA/DNA but not DNA duplexes. In the absence of cellular cofactors, eIF4A operated unwinding in both the 3' to 5' and 5' to 3' directions, and was able to unwind blunt-ended RNA duplex, suggesting that bidirectionality is an intrinsic property of eIF4A. In contrast, HCV NS3 helicase showed unidirectional 3' to 5' unwinding of RNA and RNA/DNA, as well as of DNA duplexes. With respect to NTPase activity, eIF4A hydrolysed only ATP or dATP in the presence of RNAs, whereas HCV NS3 helicase could hydrolyse all ribo- and deoxyribo-NTPs in an RNA-independent manner. In parallel, only ATP or dATP could drive the unwinding activity of eIF4A whereas HCV NS3 could function with all eight standard NTPs and dNTPs. The observed differences in their substrate specificity may prove to be useful in designing specific inhibitors targeting HCV NS3 helicase but not human eIF4A.

P1 and P3 optimization of novel bicycloproline P2 bearing tetrapeptidyl alpha-ketoamide based HCV protease inhibitors.

With the aim of discovering potent and selective HCV protease inhibitors, we synthesized and evaluated a series of 1a based tetrapeptidyl ketoamides with additional modification(s) at P1', P1, and P3 positions. As a result of this effort, we found that replacement of the P3 valine with tert-leucine resulted in the discovery of a series of inhibitors (e.g., 3a, 3c, and 4c) endowed with improved enzyme and/or cellular activity relative to 1a. When dosed to F-344 rats orally at 50mg/kg, 3a achieved 2.5x higher liver and plasma exposure in comparison to that detected with 1a.

P4 cap modified tetrapeptidyl alpha-ketoamides as potent HCV NS3 protease inhibitors.

We describe herein the design, syntheses, and biological evaluation of new series of P4 tetrazole and adipic acid, ester, amide capped tetrapeptidyl alpha-ketoamide based HCV protease inhibitors.

Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase.

The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K(d), of approximately 22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 A. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.

Expression and purification of untagged full-length HCV NS5B RNA-dependent RNA polymerase.

The NS5B encoded by the hepatitis C virus genome is a RNA-dependent RNA polymerase essential to viral replication. The entire NS5B protein contains a catalytic domain followed by a regulatory motif and a membrane-anchor domain at its C-terminus. Reported here is the molecular cloning and expression of the full-length NS5B polymerase (NS5B-FL) in bacterial cells as a non-fusion protein. The non-tagged NS5B-FL was purified to homogeneity using sequential chromatographic columns and its identity was confirmed using anti-NS5B peptide antibodies and amino acid sequencing. Purified NS5B-FL demonstrated RNA-dependent RNA polymerase activity and was able to replicate a HCV RNA genome fragment through both copy-back and de novo mechanisms. Its biochemical properties were further characterized in comparison with a truncated form of NS5B polymerase with a deletion of 51 residues from its C-terminus.