In emergently ventilated trauma patients, low end-tidal CO2 and low cardiac output are associated and correlate with hemodynamic instability, hemorrhage, abnormal pupils, and death.

BACKGROUND: In a smaller experience, the authors previously demonstrated that end-tidal carbon dioxide (PetCO2) and cardiac output (CO) had a positive association in emergently intubated trauma patients during Emergency Department resuscitation. The aim of this larger study was to reassess the relationship of PetCO2 with CO and identify patient risk-conditions influencing PetCO2 and CO values. METHODS: The investigation consists of acutely injured trauma patients requiring emergency tracheal intubation. The study focuses on the prospective collection of PetCO2 and noninvasive CO monitor (NICOM®) values in the Emergency Department. RESULTS: From the end of March through August 2011, 73 patients had 318 pairs of PetCO2 (mm Hg) and CO (L/min.) values. Mean data included Injury Severity Score (ISS) ≥15 in 65.2%, Glasgow Coma Score of 6.4 ± 4.6, hypotension in 19.0%, and death in 34.3%. With PetCO2 ≤ 25 (15.9 ± 8.0), systolic blood pressure was 77.0 ± 69, CO was 3.2 ± 3.0, cardiac arrest was 60.4%, and mortality was 84.9%. During hypotension, CO was lower with major blood loss (1.9), than without major loss (5.0; P = 0.0008). Low PetCO2 was associated with low CO (P < 0.0001). Low PetCO2 was associated (P ≤ 0.0012) with ISS > 20, hypotension, bradycardia, major blood loss, abnormal pupils, cardiac arrest, and death. Low CO was associated (P ≤ 0.0059) with ISS > 20, hypotension, bradycardia, major blood loss, abnormal pupils, cardiac arrest, and death. CONCLUSIONS: During emergency department resuscitation, a decline in PetCO2 correlates with decreases in noninvasive CO in emergently intubated trauma patients. Decreasing PetCO2 and declining NICOM CO are associated with hemodynamic instability, hemorrhage, abnormal pupils, and death. The study indicates that NICOM CO values are clinically discriminate and have physiologic validity.

Emergency department noninvasive (NICOM) cardiac outputs are associated with trauma activation, patient injury severity and host conditions and mortality.

BACKGROUND: Anoninvasive cardiac output (CO) monitor (NICOM), using Bioreactance technology, has been validated in several nontrauma patient studies. We hypothesized that NICOM CO would have more significant associations with clinical conditions than would systolic blood pressure (sBP). METHODS: This is a prospective observational study of consecutive trauma activation patients during the first 10 to 60 minutes after emergency department arrival. RESULTS: Analysis includes 270 consecutive trauma activation patients with 1,568 observations. CO was decreased (p ≤ 0.002) with major blood loss, hypotension, red blood cell transfusion, Injury Severity Score (ISS) higher than 20, low PetCO2, abnormal pupils, elderly, preexisting conditions, low body surface area level, females, hypothermia, and death. CO was increased (p < 0.0001) with base deficit, ethanol positivity, and illicit drug positivity. The sBP was decreased (p ≤ 0.0005) with major blood loss, red blood cell transfusion, low PetCO2, low body surface area level, and illicit drug positivity. The sBP was increased (p e 0.01) with ISS higher than 20, elderly, and preexisting conditions. Total significant condition associations were CO 83% (15 of 18 patients) and sBP 47% (8 of 17 patients; p = 0.03). In hypotensive patients, CO was lower with major blood loss (3.3 ± 2.1 L/ min) than without (6.0 ± 2.2 L/min; p < 0.0001). Of survivors with ISS 15 or higher, NICOM patients experienced a shorter hospital length of stay (10.5 days) when compared with 2009 and 2010 patients (14.0 days; p = 0.03). CONCLUSION: The multiple associations of CO with patient conditions imply that NICOM provides an objective and clinically valid, relevant, and discriminate measure of cardiac function in acutely injured trauma activation patients. NICOM use may be associated with a shorter length of stay in surviving patients with complex injuries.

Discovery of 4'-azido-2'-deoxy-2'-C-methyl cytidine and prodrugs thereof: a potent inhibitor of Hepatitis C virus replication.

4'-Azido-2'-deoxy-2'-methylcytidine (14) is a potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase, displaying an EC(50) value of 1.2 µM and showing moderate in vivo bioavailability in rat (F=14%). Here we describe the synthesis and biological evaluation of 4'-azido-2'-deoxy-2'-methylcytidine and prodrug derivatives thereof.

The application of phosphoramidate ProTide technology to the potent anti-HCV compound 4'-azidocytidine (R1479).

We report the design, synthesis and evaluation of a family of ca 50 phosphoramidate ProTides of the potent anti-HCV compound 4'-azidocytidine (R1479), with variation on the ester, amino acid and aryl moiety of the ProTide. Sub-muM inhibitors of HCV emerge. The compounds are all non-cytotoxic in the replicon assay. We herein report detailed SARs for each of the regions of the ProTide.

The design, synthesis, and antiviral activity of monofluoro and difluoro analogues of 4'-azidocytidine against hepatitis C virus replication: the discovery of 4'-azido-2'-deoxy-2'-fluorocytidine and 4'-azido-2'-dideoxy-2',2'-difluorocytidine.

The discovery of 4'-azidocytidine (3) (R1479) (J. Biol. Chem. 2006, 281, 3793; Bioorg. Med. Chem. Lett. 2007, 17, 2570) as a potent inhibitor of RNA synthesis by NS5B (EC(50) = 1.28 microM), the RNA polymerase encoded by hepatitis C virus (HCV), has led to the synthesis and biological evaluation of several monofluoro and difluoro derivatives of 4'-azidocytidine. The most potent compounds in this series were 4'-azido-2'-deoxy-2',2'-difluorocytidine and 4'-azido-2'-deoxy-2'-fluoroarabinocytidine with antiviral EC(50) of 66 nM and 24 nM in the HCV replicon system, respectively. The structure-activity relationships within this series were discussed, which led to the discovery of these novel nucleoside analogues with the most potent compound, showing more than a 50-fold increase in antiviral potency as compared to 4'-azidocytidine (3).

The application of the phosphoramidate ProTide approach confers micromolar potency against hepatitis C virus on inactive agent 4'-azidoinosine: kinase bypass on a dual base/sugar modified nucleoside.

Novel phosphoramidate ProTides derived from 4'-azidoinosine have been prepared and evaluated in the replicon assay against hepatitis C Virus (HCV). The parent nucleoside analogue is inactive in this assay, while the ProTides are active at low microM levels in some cases. This is a rare example of an inosine nucleoside analogue with potent antiviral activity and further supports the notion of ProTides as a drug discovery motif.

The design, synthesis, and antiviral activity of 4'-azidocytidine analogues against hepatitis C virus replication: the discovery of 4'-azidoarabinocytidine.

4'-Azidocytidine 3 (R1479) has been previously discovered as a potent and selective inhibitor of HCV replication targeting the RNA-dependent RNA polymerase of hepatitis C virus, NS5B. Here we describe the synthesis and biological evaluation of several derivatives of 4'-azidocytidine by varying the substituents at the ribose 2' and 3'-positions. The most potent compound in this series is 4'-azidoarabinocytidine with an IC(50) of 0.17 microM in the genotype 1b subgenomic replicon system. The structure-activity relationships within this series of nucleoside analogues are discussed.

2'-deoxy-4'-azido nucleoside analogs are highly potent inhibitors of hepatitis C virus replication despite the lack of 2'-alpha-hydroxyl groups.

RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2'-alpha-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of alpha-hydroxy moieties. 2'-deoxy-2'-beta-fluoro-4'-azidocytidine (RO-0622) and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC(50) = 171 +/- 12 nM and 24 +/- 3 nM for RO-9187 and RO-0622, respectively; CC(50) >1 mM for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4'-azidocytidine) or 2'-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC(50) values 8-150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2'-alpha-deoxy-4'-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection.

Ultrasound-guided fine-needle aspiration versus fine-needle capillary sampling biopsy of thyroid nodules: does technique matter?

OBJECTIVE: Both fine-needle aspiration (FNA) and fine-needle capillary (FNC) sampling of palpable thyroid nodules have been advocated. The appropriate technique for biopsy of nonpalpable nodules now sampled under ultrasound guidance has not been assessed. The objective of this study was to determine cytologic adequacy rates of ultrasound-guided FNA and FNC sampling. METHODS: Ultrasound-guided fine-needle biopsy was performed on 180 thyroid nodules. Nodules were measured and characterized (mixed cystic-solid or solid) on diagnostic sonography. The technique used for biopsy (FNA versus FNC sampling) alternated sequentially per patient. Four 25-gauge fine-needle samples were obtained from each nodule. The sampling adequacy and cytopathologic diagnoses were recorded. The significance of differences in cytologic adequacy rates between techniques was then assessed with a repeated measures logistic regression model. RESULTS: Fine-needle aspiration and FNC sampling biopsies were performed in 88 and 92 nodules, respectively. Fine-needle aspiration was diagnostic (ie, satisfactory) in 78 (89%) of 88 nodules and nondiagnostic (less than optimal or unsatisfactory) in 10 (11%) of 88 nodules; FNC sampling was diagnostic in 80 (87%) of 92 nodules and nondiagnostic in 12 (13%) of 92 nodules. Fine-needle aspiration sampling was diagnostic in 38 (86%) of 44 solid nodules and 40 (91%) of 44 mixed cystic-solid nodules; FNC sampling was diagnostic in 48 (81%) of 59 solid nodules and 32 (97%) of 33 mixed cystic-solid nodules. These percentages were not significantly different. CONCLUSIONS: Ultrasound-guided FNA and FNC sampling result in comparable diagnostic cytologic adequacy rates. Equivalent diagnostic yields and the technical ease of capillary sampling may prompt adoption of FNC sampling at high-volume endocrine/radiology practices.

First example of phosphoramidate approach applied to a 4'-substituted purine nucleoside (4'-azidoadenosine): conversion of an inactive nucleoside to a submicromolar compound versus hepatitis C virus.

We report on the synthesis of the anti hepatitis C virus (HCV) agent 4'-azidoadenosine (1) and the application of the phosphoramidate ProTide technology to this nucleoside. The synthesis of 1 was achieved through an epoxide intermediate followed by regio- and stereoselective ring opening by azidotrimethylsilane in the presence of a Lewis acid. Compound 1 did not inhibit HCV replication in cell culture at concentrations up to 0.1 mM. However, a submicromolar active agent could be derived from 1 by the application of the ProTide technology. All the phosphoramidates prepared were L-alanine derivatives with variations in the aryl moiety and in the ester part of the amino acid. The benzyl ester and the l-naphthyl phosphate (18) had the best activity in replicon assay. Phosphoramidates (18-21) achieved a significant improvement in antiviral potency over the parent nucleoside (1) with no increase in cytotoxicity.

Application of the phosphoramidate ProTide approach to 4'-azidouridine confers sub-micromolar potency versus hepatitis C virus on an inactive nucleoside.

We report the application of our phosphoramidate ProTide technology to the ribonucleoside analogue 4'-azidouridine to generate novel antiviral agents for the inhibition of hepatitis C virus (HCV). 4'-Azidouridine did not inhibit HCV, although 4'-azidocytidine was a potent inhibitor of HCV replication under similar assay conditions. However 4'-azidouridine triphosphate was a potent inhibitor of RNA synthesis by HCV polymerase, raising the question as to whether our phosphoramidate ProTide approach could effectively deliver 4'-azidouridine monophosphate to HCV replicon cells and unleash the antiviral potential of the triphosphate. Twenty-two phosphoramidates were prepared, including variations in the aryl, ester, and amino acid regions. A number of compounds showed sub-micromolar inhibition of HCV in cell culture without detectable cytotoxicity. These results confirm that phosphoramidate ProTides can deliver monophosphates of ribonucleoside analogues and suggest a potential path to the generation of novel antiviral agents against HCV infection. The generic message is that ProTide synthesis from inactive parent nucleosides may be a warranted drug discovery strategy.

Design, synthesis, and antiviral properties of 4'-substituted ribonucleosides as inhibitors of hepatitis C virus replication: the discovery of R1479.

A series of 4'-substituted ribonucleoside derivatives has been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell culture. The most potent and non-cytotoxic derivative was compound 28 (4'-azidocytidine, R1479) with an IC(50) of 1.28 microM in the HCV replicon system. The triphosphate of compound 28 was prepared and shown to be an inhibitor of RNA synthesis mediated by NS5B (IC(50)=320 nM), the RNA polymerase encoded by HCV. Data on related analogues have been used to generate some preliminary requirements for activity within this series of nucleosides.

The novel nucleoside analog R1479 (4'-azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture.

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC(50) = 1.28 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K(i) of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.

Activity of the isolated HIV RNase H domain and specific inhibition by N-hydroxyimides.

This report describes a procedure to generate enzymatically active, isolated HIV RNase H domain. In contrast to previously described preparations, the RNA cleavage activity of the untagged RNase H domain was surprisingly similar to that of the full-length HIV-RT protein. Signature cleavages at 18 and 9 nucleotides downstream of a recessed RNA 5'-end were retained with the isolated RNase H domain. Activity was strongly decreased by deletion of 3 amino acids from the C-terminus, consistent with an important structural or functional role of the C-terminal alpha-helix. A prototype N-hydroxyimide (2-hydroxy-4H-isoquinoline-1,3-dione) was found to inhibit the activity of the isolated HIV RNase H domain as well as the RNase H activity of full-length HIV reverse transcriptase. In contrast, the compound did not significantly inhibit the structurally closely related Escherichia coli RNase HI. Specific binding of N-hydroxyimide compounds to the isolated RNase H domain was observed by protein fluorescence quenching.

Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors.

Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.

Use of a pharmacophore model to discover a new class of influenza endonuclease inhibitors.

Data from both our own and literature studies of the biochemistry and inhibition of influenza virus endonuclease was combined with data on the mechanism of action and the likely active site mechanism to propose a pharmacophore. The pharmacophore was used to design a novel structural class of inhibitors, some of which were found to have activities similar to that of known influenza endonuclease inhibitors and were also antiviral in cell culture.