PHIRST Trial - pharmacist consults: prioritization of HIV-patients with a referral screening tool.

The role of pharmacists in HIV outpatient clinics has greatly increased in the past decades. Given the limited resources of the health system, the prioritization of pharmacist consults is now a main concern. This study aimed to create a scoring system allowing for standardized prioritization of pharmacist consults for patients living with HIV. Data was retrospectively collected from 200 HIV patients attending the Chronic Viral Illness Service at the McGill University Health Center. An expert panel consisting of four pharmacists working in the field of HIV prioritized each patient individually, after which a consensus was established and was considered as the gold standard. In order to create a scoring system, two different methods (Delphi, statistical) were used to assign a weight to each characteristic considered to be important in patient prioritization. A third method (equal weight to each characteristic) was also evaluated. The total score per patient for each method was then compared to the expert consensus in order to establish the score cut-offs to indicate the appropriate categories of delay in which to see the patient. All three systems failed to accurately prioritize patients into urgency categories ("less than 48 h", "less than 1 month", "less than 3 months", "no consult required") according to expert pharmacist consensus. The presence of high level interactions between patient characteristics, the limited number of patients and the low prevalence of some characteristics were hypothesized as the main causes for the results. Creating a prioritization tool for pharmacy consults in HIV outpatient clinics is a complex task and developing a decision tree algorithm may be a more appropriate approach in the future to take into account the importance of combinations of patient characteristic.

Assay for evaluating ribonuclease H-mediated degradation of RNA-antisense oligonucleotide duplexes.

Ribonucleases H are complex enzymes whose functions are not clearly understood, further compounded by the fact that multiple forms of the enzyme are present in various organisms. They are known to recognize and degrade the ribonucleic acid (RNA) strand of numerous deoxyribonucleic acid (DNA)-RNA duplex substrates, and so may provide a unique mode of therapeutic intervention at the genetic level of virtually any disease. We have therefore set out detailed procedures for conducting routine assays with almost any one of this family of enzymes by a straightforward assay aimed at identifying novel enzyme-activating antisense oligonucleotides (AONs). The procedures described herein should enable easy identification of potent AON molecules, provided that the RNA is appropriately labeled for subsequent visualization following the guidelines set forth in this protocol.

Synthesis and properties of oligonucleotide chimeras containing 5'-amino-2'-deoxy-2'-fluoroarabinonucleosides.

Oligonucleotide analogues comprised of 2'-deoxy-2'-fluoro-beta-D-arabinose units joined via P3'-N5' phosphoramidate linkages (2'F-ANA(5'N)) were prepared for the first time. Among the compounds prepared were a series of 2'OMe-RNA-[GAP]-2'OMe-RNA 'chimeras', whereby the "GAP" consisted of DNA, DNA(5'N), 2'F-ANA or 2'F-ANA(5'N) segments. The chimeras with the 2'F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5'-amino derivatives, i.e., 2'F-ANA > DNA > 2'F-ANA(5'N) > DNA(5'N). Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E. coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2'F-ANA > 2'F-ANA(5'N) > DNA(5'N). The corresponding relative rates observed with the human enzyme were: gap DNA > 2'F-ANA(5'N) > 2'F-ANA > DNA(5'N).

Oligonucleotides comprised of alternating 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides and D-2'-deoxyribonucleosides (2'F-ANA/DNA 'altimers') induce efficient RNA cleavage mediated by RNase H.

Chimeric oligonucleotides comprised of alternating residues of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (2'F-ANA) and DNA were synthesized and evaluated for an important antisense property-the ability to elicit ribonuclease H (RNase H) degradation of complementary RNA. Experiments used both human RNase HII and Escherichia coli RNase HI. Mixed backbone oligomers comprising alternating three-nucleotide segments of 2'F-ANA and three-nucleotide segments of DNA were the most efficient at eliciting RNase H degradation of target RNA, and were significantly better than oligonucleotides entirely composed of DNA, suggesting that these mixed backbone oligonucleotides may be potent antisense agents.

Antisense inhibition of Flk-1 by oligonucleotides composed of 2'-deoxy-2'-fluoro-beta-D-arabino- and 2'-deoxy-nucleosides.

The design of new antisense oligomers with improved binding affinity for targeted RNA, while still activating RNase H, is a major research area in medicinal chemistry. RNase H recognizes the RNA-DNA duplex and cleaves the complementary mRNA strand, providing the main mechanism by which antisense oligomers elicit their activities. It has been shown that configuration inversion at the C2' position of the DNA sugar moiety (arabinonucleic acid, ANA), combined with the substitution of the 2'OH group by a fluorine atom (2'F-ANA) increases the oligomer's binding affinity for targeted RNA. In the present study, we evaluated the antisense activity of mixed-backbone phosphorothioate oligomers composed of 2'-deoxy-2'-fluoro-beta-D-arabinose and 2'-deoxyribose sugars (S-2'F-ANA-DNA chimeras). We determined their abilities to inhibit the protein expression and phosphorylation of Flk-1, a vascular endothelial growth factor receptor (VEGF), and VEGF biological effects on endothelial cell proliferation, migration, and platelet-activating factor synthesis. Treatment of endothelial cells with chimeric oligonucleotides reduced Flk-1 protein expression and phosphorylation more efficiently than with phosphorothioate antisenses (S-DNA). Nonetheless, these two classes of antisenses inhibited VEGF activities equally. Herein, we also demonstrated the capacity of the chimeric oligomers to elicit RNase H activity and their improved binding affinity for complementary RNA as compared with S-DNA.

Efficient RNase H-directed cleavage of RNA promoted by antisense DNA or 2'F-ANA constructs containing acyclic nucleotide inserts.

The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described. Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively. Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates. Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression. Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.