Publications by authors named "Kevin Brady"

33 Publications

Uniformed Services and the Field Hospital Experience During Coronovirus Disease 2019 (SARS-CoV-2) Pandemic: Open to Closure in 30 Days With 1,100 Patients: The Javits New York Medical Station.

Mil Med 2021 Feb 13. Epub 2021 Feb 13.

Department of Emergency Medicine, Brown University School of Medicine.

Introduction: The surge of SARS-CoV-2-virus infected (COVID-19) patients presenting to New York City (NYC) hospitals quickly overwhelmed and outnumbered the available acute care and intensive care resources in NYC in early March 2020. Upon the arrival of military medical assets to the Javits Convention Center in NYC, the planned mission to care for non-SARS-CoV-2 patients was immediately changed to manage patients with (SARS-CoV-2)COVID-19 and their comorbid conditions.Healthcare professionals from every branch of the uniformed services, augmented by state and local resources, staffed the Javits New York Medical Station (JNYMS) from April 2020.

Methods: The data review reported aggregated summary statistics and participant observations collected by N.Y. State and U.S. military officials.

Results: During the 28 days of patient intake at the JNYMS, 1,095 SARS-CoV-2-positive patients were transferred from NYC hospitals to the JNYMS. At its peak, the JNYMS accepted 119 patients in a single day, had a maximum census of 453, and had a peak intensive care unit census of 35. The median length of stay was 4.6 days (interquartile range: 3.1-6.9 days). A total of 103 patients were transferred back to local hospitals, and there were 6 deaths, with an overall mortality rate of 0.6% (95% CI, 0.3-1.2).

Discussion And Conclusions: This is the first report of the care provided at the JNYMS. Within 2 weeks, this multi-agency effort was able to mobilize to care for over 1,000 SARS-CoV-2 patients with varying degrees of illness in a 1-month period. This was the largest field hospital mobilization in the U.S. medical history in response to a non-wartime pandemic. Its success with huge patient throughput including disposition and low mortality relieved critical overcrowding and supply deficiencies throughout NYC hospitals. The downstream impact likely saved additional hundreds of lives and reduced stress on the system during this healthcare crisis.
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http://dx.doi.org/10.1093/milmed/usab003DOI Listing
February 2021

Meeting report: 1st workshop of the peptide ADME discussion group.

Xenobiotica 2021 Jan 19;51(1):122-125. Epub 2020 Feb 19.

Development ADME, Novo Nordisk A/S, Måløv, Denmark.

1. Challenges and opportunities within peptide ADME (absorption, distribution, metabolism and elimination) were presented and discussed at the 1st peptide workshop of the Peptide ADME Discussion Group in Gothenburg, Sweden (15th of October 2018). This article summarises the presentations and discussions from this 1st workshop. The following topics were covered: Background science presentation on peptidases Presentation of various peptide ADME packages Peptide drug-drug interactions (DDI).
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http://dx.doi.org/10.1080/00498254.2020.1729447DOI Listing
January 2021

Stirred Not Shaken: Facile Production of High-Quality, High-Concentration Graphene Aqueous Suspensions Assisted by a Protein.

ACS Appl Mater Interfaces 2020 Jan 9;12(3):3815-3826. Epub 2020 Jan 9.

Department of Chemistry , University of Connecticut , 55 North Eagleville Road , Storrs , Connecticut 06268 , United States.

A simple method to produce record concentrations (up to 10 mg mL) of high-quality aqueous graphene suspensions by using an ordinary benchtop magnetic stirrer is reported. The shear rates employed here are almost 10 times less than those in previous reports, and graphene is efficiently separated from unexfoliated graphite during the synthesis. Systematic optimization of synthesis parameters, such as pH, protein concentration, temperature, stirrer speed, and volume of solution, afforded efficient conversion (100%) of graphite to graphene-aqueous suspensions. The synthesis is readily scaled-up with a continuous flow reactor where the graphene is produced and separated 24/7, with little or no human intervention. Raman spectroscopy confirmed little to no sp or oxidative defects, and that the graphene nanosheets consisted of three to five layers. The graphene suspensions were coated on aluminum and tested for thermal conductivity applications. The thermal conductivity of our graphene sample was calculated to be 684 W m K, a value greater than that of a commercial sample. The activation energy measured for shear exfoliation by stirring was found to be over 45 billion times smaller than the corresponding thermal activation energy, affording physical insight into the process. We hypothesize that stirring selectively populates translational states that are necessary for exfoliation and thus requires far less energy than conventional exfoliation methods, where the energy is uniformly distributed among all available modes. Therefore, an efficient, convenient, and inexpensive method for graphene production in limited-resource settings is reported here.
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http://dx.doi.org/10.1021/acsami.9b15121DOI Listing
January 2020

Rapid Response to Pembrolizumab in a Critically Ill Mechanically Ventilated Patient with New Diagnosis of NSCLC.

J Thorac Oncol 2019 09;14(9):e193-e195

Division of Hematology/Oncology, University of Virginia School of Medicine, Charlottesville, Virginia. Electronic address:

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http://dx.doi.org/10.1016/j.jtho.2019.04.010DOI Listing
September 2019

Pericardial closure with extracellular matrix scaffold following cardiac surgery associated with a reduction of postoperative complications and 30-day hospital readmissions.

J Cardiothorac Surg 2019 Mar 15;14(1):61. Epub 2019 Mar 15.

Baptist Health, Jacksonville, FL, USA.

Background: A prospective, multi-center study (RECON) was conducted to evaluate the clinical outcomes of pericardial closure using a decellularized extracellular matrix (ECM) graft derived from porcine small intestinal submucosa.

Methods: Patients indicated for open cardiac surgery with pericardial closure using ECM were eligible for the RECON study cohort. Postoperative complications and readmission of the RECON patients were compared to the patient cohort in the Nationwide Readmissions Database (NRD). Inverse probability of treatment weighting was used to control the differences in patient demographics, comorbidities, and risk factors.

Results: A total of 1420 patients at 42 centers were enrolled, including 923 coronary artery bypass grafting (CABG) surgeries and 436 valve surgeries. Significantly fewer valve surgery patients in the RECON cohort experienced pleural effusion (3.1% vs. 13.0%; p < 0.05) and pericardial effusion (1.5% vs. 2.6%; p < 0.05) than in the NRD cohort. CABG patients in the RECON cohort were less likely to suffer bleeding (1.2% vs. 2.9%; p < 0.05) and pericardial effusion (0.2% vs. 2.2%, p < 0.05) than those in the NRD cohort. The 30-day all-cause hospital readmission rate was significantly lower among RECON patients than NRD patients following both valve surgery (HR: 0.34; p < 0.05) and CABG surgery (HR: 0.42; p < 0.05). In the RECON study, 14.4% of CABG patients and 27.0% of valve patients had postoperative atrial fibrillation as compared to previously reported risks, which generally ranges from 20 to 30% after CABG and from 35 to 50% after valve surgery.

Conclusions: Pericardial closure with ECM following cardiac surgery is associated with a reduction in the proportion of patients with pleural effusion, pericardial effusion, and 30-day readmission compared to a nationwide database.

Trial Registration: NCT02073331 , Registered on February 27, 2014.
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http://dx.doi.org/10.1186/s13019-019-0871-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6419853PMC
March 2019

A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease.

J Autoimmun 2018 12 13;95:1-14. Epub 2018 Nov 13.

JDRF/Wellcome Diabetes and Inflammation Laboratory, Wellcome Centre for Human Genetics, Nuffield Department of Medicine, NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford, UK; JDRF/Wellcome Diabetes and Inflammation Laboratory, Department of Medical Genetics, Cambridge Institute for Medical Research, NIHR Cambridge Biomedical Research Centre, University of Cambridge, Cambridge, CB2 OXY, UK. Electronic address:

Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and T function, making T-specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rβγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D). The reduced affinity of IgG-(IL-2N88D) for the IL-2Rβγ receptor resulted in a T-selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D) induced sustained preferential activation of T accompanied by a corresponding 10-14-fold increase in CD4 and CD8 CD25FOXP3 T; conditions that had no effect on CD4 or CD8 memory effector T cells. The expanded cynomolgus T had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D) increased T while wild-type IgG-IL-2 increased NK cells in addition to T. The expanded human T had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and T-selective IL-2 that activates and expands functional Tin vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures.
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http://dx.doi.org/10.1016/j.jaut.2018.10.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284106PMC
December 2018

Intraoperative Venovenous Extracorporeal Membrane Oxygenation as Rescue for a Patient With an Inhalational Burn and Iatrogenic Upper Airway Injury: A Case Report.

A A Pract 2018 Sep;11(5):115-117

From the Department of Anesthesia and Operative Services.

Venovenous extracorporeal membrane oxygenation (VV-ECMO) is a well-established alternative oxygenation method for critically ill patients. A 58-year-old male was transferred to our level 1 trauma and burn center after sustaining an inhalational injury from a carburetor explosion, with subsequent iatrogenic tracheal injury and emergent cricothyrotomy before arrival. During attempted surgical airway stabilization, our ability to ventilate and oxygenate was compromised. Intraoperative VV-ECMO enabled rescue from severe hypoxemia and subsequent recovery without lasting neurologic sequelae. This case highlights the utility of VV-ECMO for acute intraoperative rescue.
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http://dx.doi.org/10.1213/XAA.0000000000000747DOI Listing
September 2018

Brain Shuttle Antibody for Alzheimer's Disease with Attenuated Peripheral Effector Function due to an Inverted Binding Mode.

Cell Rep 2018 01;22(1):149-162

Pharma Research and Early Development (pRED), Neurodegeneration and Regeneration, Roche Innovation Center, Basel, Switzerland. Electronic address:

Receptors show promise for the transport of monoclonal antibodies (mAbs) across the blood-brain barrier. However, safety liabilities associated with peripheral receptor binding and Fc effector function have been reported. We present the Brain Shuttle-mAb (BS-mAb) technology, and we investigate the role of Fc effector function in vitro and in an Fcγ receptor (FcγR)-humanized mouse model. Strong first infusion reactions (FIRs) were observed for a conventional mAb against transferrin receptor (TfR) with a wild-type immunoglobulin G1 (IgG1) Fc. Fc effector-dead constructs completely eliminated all FIRs. Remarkably, no FIR was observed for the BS-mAb construct with a native IgG1 Fc function. Using various BS-mAb constructs, we show that TfR binding through the C-terminal BS module attenuates Fc-FcγR interactions, primarily because of steric hindrance. Nevertheless, BS-mAbs maintain effector function activity when binding their brain target. Thus, mAbs with full effector function can be transported in a stealth mode in the periphery while fully active when engaged with their brain target.
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http://dx.doi.org/10.1016/j.celrep.2017.12.019DOI Listing
January 2018

From the Cover: The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides.

Toxicol Sci 2017 05;157(1):112-128

Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Copenhagen, Hørsholm, Denmark.

Non-human primates (NHPs) are currently considered to be the non-rodent species of choice for the preclinical safety assessment of single-stranded oligonucleotide (SSO) drugs. We evaluated minipigs as a potential alternative to NHPs to test the safety of this class of compounds. Four different phosphorothioated locked nucleic acid-based SSOs (3 antisense and 1 anti-miR), all with known safety profiles, were administered to minipigs using similar study designs and read-outs as in earlier NHP studies with the same compounds. The studies included toxicokinetic investigations, in-life monitoring, clinical and anatomic pathology. In the minipig, we demonstrated target engagement by the SSOs where relevant, and a similar toxicokinetic behavior in plasma, kidney, and liver when compared with NHPs. Clinical tolerability was similar between minipig and NHPs. For the first time, we showed similar and dose-dependent effects on the coagulation and complement cascade after intravenous dosing similar to those observed in NHPs. Similar to NHPs, morphological changes were seen in proximal tubular epithelial cells of the kidney, Kupffer cells, hepatocytes, and lymph nodes. Minipigs appeared more sensitive to the high-dose kidney toxicity of most of the selected SSOs than NHPs. No new target organ or off-target toxicities were identified in the minipig. The minipig did not predict the clinical features of human injection site reactions better than the NHPs, but histopathological similarities were observed between minipigs and NHPs. We conclude that there is no impediment, as default, to the use of minipigs as the non-rodent species in SSO candidate non-clinical safety packages.
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http://dx.doi.org/10.1093/toxsci/kfx025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414856PMC
May 2017

Postinfarct VSD management using 3D computer printing assisted percutaneous closure.

Indian Heart J 2015 Nov-Dec;67(6):581-5. Epub 2015 Oct 28.

Banner University Medical Center - Phoenix Campus, United States. Electronic address:

Postinfarct VSD (PIVSD) carries a grim prognosis. The mainstay of management has been surgical repair. The advent of septal occluder devices has offered an attractive alternative to surgical repair. Most PIVSD have serpiginous tracts with necrotic tissue, which makes assessing the defect challenging. 3D computer printing has become useful in preprocedure planning of complex surgical procedures in multiple subspecialties.
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http://dx.doi.org/10.1016/j.ihj.2015.09.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699954PMC
January 2017

HIPAA's place in court-ordered discovery.

J AHIMA 2014 Sep;85(9):40-2

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September 2014

Implementation of the National Breast and Cervical Cancer Early Detection Program: the beginning.

Cancer 2014 Aug;120 Suppl 16:2540-8

Office on Women's Health, Office of the Assistant Secretary for Health, US Department of Health and Human Services, District of Columbia.

In 1990, Congress passed the Breast and Cervical Cancer Mortality Prevention Act because of increases in the number of low-income and uninsured women being diagnosed with breast cancer. This act authorized the Centers for Disease Control and Prevention (CDC) to establish the National Breast and Cervical Cancer Early Detection Program (NBCCEDP) to provide high-quality and timely breast and cervical cancer screening and diagnostic services to low-income, uninsured women. The program started in 1991, and, in 1993, Congress amended the act to allow the CDC to fund American Indian and Alaska Native tribes and tribal organizations. By 1996, the program was providing cancer screening across the United States. To ensure appropriate delivery and monitoring of services, the program adopted detailed policies on program management, evidence-based guidelines for clinical services, a systematized clinical data system to track service quality, and key partnerships that expand the program's reach. The NBCCEDP currently funds 67 programs, including all 50 states, the District of Columbia, 5 US territories, and 11 tribes or tribal organizations.
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http://dx.doi.org/10.1002/cncr.28820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4481738PMC
August 2014

Whether a bill becomes a law.

Authors:
Kevin Brady

Chest 2014 Feb;145(2):206-208

United States House of Representatives, (Texas, 8th District, R) Washington, DC. Electronic address:

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http://dx.doi.org/10.1378/chest.13-2789DOI Listing
February 2014

Virtual medical scribes: making electronic medical records work for you.

J Med Pract Manage 2013 Sep-Oct;29(2):133-6

Physicians Angels, Inc., Toledo, OH, USA.

There is increasing buzz around the term "medical scribe" in healthcare today. Medical scribes help meet the growing electronic medical record (EMR) data entry challenge healthcare providers face. Medical scribes reduce providers' paperwork burden, increase a medical practice's net margins, and reduce stress levels for doctors and their staff. They do this by charting patient encounters in real-time during patient examinations, thus reducing significantly the data entry workload that EMRs place on providers. Medical scribes can work onsite or offsite from a HIPAA-secure location, the latter being known as "virtual medical scribes." This article explores the uses and benefits of scribes to give you the background to employ them effectively in your clinic or hospital.
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January 2014

Disposition of biologics.

Adv Pharmacol 2012 ;63:257-77

Bicycle Therapeutics Ltd., Cambridge, United Kingdom.

Drug development is a complex process, requiring scientific and regulatory input at almost all stages from multiple groups of expertise. Small molecule development issues are covered in other parts of this volume. This chapter is devoted to discussing the large molecules, or biologics, and the particular nuances involved in developing these molecules as medicines. Our definition of biologic, for the purposes of this chapter, differs from that described by the regulatory bodies. Where regulators state that a biologic is a molecule produced by a living organism, be it a mammalian, insect, yeast or bacteria cell, or whole animal, we prefer to include molecules such as oligonucleotides and peptides here, which are usually chemically synthesized. So our definition is that of a molecule whose composition mostly entails naturally occurring amino acids, sugars or nucleotide bases. There are modifications made chemically to oligonucleotides and peptides to improve their drug-like properties, but for this volume, we class them as biologics. The aim of this chapter is to describe some of the differences, complexities and paradoxically, simplifications in the pharmacokinetics and ADME sciences during drug development of biologics when compared to the more familiar small molecule drug development process. The impact of the particular pharmacokinetics and ADME sciences of biologics on toxicological and pharmacological end points will be discussed.
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http://dx.doi.org/10.1016/B978-0-12-398339-8.00007-0DOI Listing
November 2012

Safe, long-term hepatic expression of anti-HCV shRNA in a nonhuman primate model.

Mol Ther 2012 Sep 26;20(9):1737-49. Epub 2012 Jun 26.

Tacere Therapeutics, San Jose, California, USA.

The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a "single-shot " therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.
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http://dx.doi.org/10.1038/mt.2012.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437581PMC
September 2012

Kappa agonist CovX-Bodies.

Bioorg Med Chem Lett 2012 Jun 25;22(12):4173-8. Epub 2012 Apr 25.

Worldwide Medicinal Chemistry, Pfizer Ramsgate Road, Sandwich, Kent CT13 9NJ, UK.

Small peptidic kappa agonists were covalently linked to the reactive lysine of the CovX antibody to create compounds having potent activity at the kappa receptor with greatly extended half-life when compared to the parent peptide as exemplified by compound 20.
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http://dx.doi.org/10.1016/j.bmcl.2012.04.040DOI Listing
June 2012

In vitro characterization of the activity of PF-05095808, a novel biological agent for hepatitis C virus therapy.

Antimicrob Agents Chemother 2012 Mar 27;56(3):1364-75. Epub 2011 Dec 27.

Pfizer Global Research and Development, Sandwich, Kent, United Kingdom.

PF-05095808 is a novel biological agent for chronic hepatitis C virus (HCV) therapy. It comprises a recombinant adeno-associated virus (AAV) DNA vector packaged into an AAV serotype 8 capsid. The vector directs expression of three short hairpin RNAs (shRNAs) targeted to conserved regions of the HCV genome. These shRNAs are processed by the host cell into the small interfering RNAs which mediate sequence-specific cleavage of target regions. For small-molecule inhibitors the key screens needed to assess in vitro activity are well defined; we developed new assays to assess this RNA interference agent and so to understand its therapeutic potential. Following administration of PF-05095808 or corresponding synthetic shRNAs, sequence-specific antiviral activity was observed in HCV replicon and infectious virus systems. To quantify the numbers of shRNA molecules required for antiviral activity in vitro and potentially also in vivo, a universal quantitative PCR (qPCR) assay was developed. The number of shRNA molecules needed to drive antiviral activity proved to be independent of the vector delivery system used for PF-05095808 administration. The emergence of resistant variants at the target site of one shRNA was characterized. A novel RNA cleavage assay was developed to confirm the spectrum of activity of PF-05095808 against common HCV clinical isolates. In summary, our data both support antiviral activity consistent with an RNA interference mechanism and demonstrate the potential of PF-05095808 as a therapeutic agent for chronic HCV infection.
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http://dx.doi.org/10.1128/AAC.05357-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294929PMC
March 2012

40th anniversary Drug Metabolism Discussion Group Open Meeting.

Authors:
Kevin Brady

Bioanalysis 2012 Jan;4(1):13-5

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http://dx.doi.org/10.4155/bio.11.287DOI Listing
January 2012

Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense.

Mol Ther 2011 Dec 4;19(12):2163-8. Epub 2011 Oct 4.

Biotherapeutics, Pfizer Global Research and Development, Sandwich, UK.

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.
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http://dx.doi.org/10.1038/mt.2011.206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3242665PMC
December 2011

Quantitation of locked nucleic acid antisense oligonucleotides in mouse tissue using a liquid-liquid extraction LC-MS/MS analytical approach.

Bioanalysis 2011 Sep;3(17):1911-21

Pharmacokinetics, Dynamics & Metabolism Department, Pfizer Global Research & Development, Sandwich, UK.

Background: A significant challenge of oligonucleotide bioanalysis is the selective extraction from complex tissue samples, where the molecules that distribute into the intracellular space are extensively protein bound and sit amongst a high concentration of endogenous nucleic acid material. Published analytical methodology currently purports extensive sample preparation requirements that include cell lysis steps, homogenization and dual cleanup with liquid-liquid extraction and solid-phase extraction, prior to injection.

Results: We have developed a simple liquid-liquid extraction approach to rapidly isolate antisense oligonucleotides from biological tissues with high recovery and combined these preparative steps with a robust monolithic column LC-MS/MS setup. The platform showed improved chromatographic resolution and detection sensitivity over standard reversed-phase columns and required a low sample volume.

Conclusion: The high-throughput method was sufficient to accurately quantify multiple antisense oligonucleotides in mouse tissue and plasma down to low ng/g and ng/ml levels, respectively, for pharmacokinetic determination, and exhibited a high degree of specificity.
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http://dx.doi.org/10.4155/bio.11.100DOI Listing
September 2011

The Drug Metabolism Discussion Group in 2011.

Xenobiotica 2011 Aug;41(8):603-4

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http://dx.doi.org/10.3109/00498254.2011.581090DOI Listing
August 2011

Selection, optimization, and pharmacokinetic properties of a novel, potent antiviral locked nucleic acid-based antisense oligomer targeting hepatitis C virus internal ribosome entry site.

Antimicrob Agents Chemother 2011 Jul 18;55(7):3105-14. Epub 2011 Apr 18.

Internal Medicine, Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, United Kingdom.

We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC(50)]) and cytotoxic (50% cytotoxic concentration [CC(50)]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC(50) = 4 nM) and low cytotoxicity (CC(50) >880 nM) targeting the 25- to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25- to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25- to 40-nt region have no antiviral effect, revealing important regulatory features of the 25- to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.
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http://dx.doi.org/10.1128/AAC.00222-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122463PMC
July 2011

Complementary PCAF-coenzyme A mutagenesis: chemoenzymatic synthesis of a novel enlarged coenzyme A analogue and evaluation of its biological activity.

Chembiochem 2010 Oct;11(15):2100-3

University of Nottingham, School of Biomedical Sciences, Nottingham, NG7 2UH, UK.

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http://dx.doi.org/10.1002/cbic.201000286DOI Listing
October 2010

The synthetic cannabinoid R(+)WIN 55,212-2 inhibits the interleukin-1 signaling pathway in human astrocytes in a cannabinoid receptor-independent manner.

J Biol Chem 2005 Oct 16;280(43):35797-806. Epub 2005 Aug 16.

UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.

R(+)WIN 55,212-2 is a synthetic cannabinoid that controls disease progression in models of multiple sclerosis. This is associated with its ability to reduce migration of leukocytes into the central nervous system. Because leukocyte migration is dependent on induction of adhesion molecules and chemokines by pro-inflammatory cytokines, we examined the effects of R(+)WIN 55,212-2 on their expression. Using 1321N1 astrocytoma and A-172 glioblastoma as cell models we show that R(+)WIN 55,212-2, but not its inactive chiral form S(-)WIN 55,212-2, strongly inhibits the interleukin-1 (IL-1) induction of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the chemokine IL-8. This inhibition is not mediated via the CB1 or CB2 cannabinoid receptors, because their selective antagonists and pertussis toxin failed to affect the inhibitory effects of R(+)WIN 55,212-2. Furthermore reverse transcription-PCR analysis did not detect the expression of either receptor in 1321N1 cells. R(+)WIN 55,212-2 was shown to inhibit adhesion molecule and chemokine expression at the level of transcription, because it strongly inhibited the IL-1 induction of ICAM-1, VCAM-1, and IL-8 mRNAs and blocked the IL-1 activation of their promoters. The NFkappaB pathway was then assessed as a lead target for R(+)WIN 55,212-2. NFkappaB was measured by expression of a transfected NFkappaB-regulated reporter gene. Using this assay, R(+)WIN 55,212-2 strongly inhibited IL-1 activation of NFkappaB. Furthermore R(+)WIN 55,212-2 inhibited the ability of overexpressed Myd88, Tak-1, and IKK-2 to induce the reporter gene suggesting that R(+)WIN 55,212-2 acts at or downstream of IKK-2 in the IL-1 pathway. However R(+)WIN 55,212-2 failed to inhibit IL-1-induced degradation of IkappaBalpha, excluding IKK-2 as a direct target. In addition electrophoretic mobility shift and chromatin immunoprecipitation assays showed that R(+)WIN 55,212-2 does not regulate the IL-1-induced nuclear translocation of NFkappaB or the ability of the latter to bind to promoters regulating expression of ICAM-1 and IL-8. These data suggest that R(+)WIN 55,212-2 blocks IL-1 signaling by inhibiting the transactivation potential of NFkappaB.
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http://dx.doi.org/10.1074/jbc.M507959200DOI Listing
October 2005

DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings.

Nucleic Acids Res 2004 1;32(10):2977-86. Epub 2004 Jun 1.

Centre for Biomolecular Sciences (CBS), School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

Loading of the replicative ring helicase onto the origin of replication (oriC) is the final outcome of a well coordinated series of events that collectively constitute a primosomal cascade. Once the ring helicase is loaded, it recruits the primase and signals the switch to the polymerization mode. The transient nature of the helicase-primase (DnaB-DnaG) interaction in the Escherichia coli system has hindered our efforts to elucidate its structure and function. Taking advantage of the stable DnaB-DnaG complex in Bacillus stearothermophilus, we have reviewed conflicting mutagenic data from other bacterial systems and shown that DnaG interacts with the flexible linker that connects the N- and C-terminal domains of DnaB. Furthermore, atomic force microscopy (AFM) imaging experiments show that binding of the primase to the helicase induces predominantly a 3-fold symmetric morphology to the hexameric ring. Overall, three DnaG molecules appear to interact with the hexameric ring helicase but a small number of complexes with two and even one DnaG molecule bound to DnaB were also detected. The structural/functional significance of these data is discussed and a speculative structural model for this complex is suggested.
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http://dx.doi.org/10.1093/nar/gkh628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC434434PMC
June 2004

Synapsis and DNA cleavage in phiC31 integrase-mediated site-specific recombination.

Nucleic Acids Res 2004 11;32(8):2607-17. Epub 2004 May 11.

Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK.

The Streptomyces phage phiC31 encodes an integrase belonging to the serine recombinase family of site-specific recombinases. The well studied serine recombinases, the resolvase/invertases, bring two recombination sites together in a synapse, and then catalyse a concerted four-strand staggered break in the DNA substrates whilst forming transient covalent attachments with the recessed 5' ends. Rotation of one pair of half sites by 180 degrees relative to the other pair occurs, to form the recombinant configuration followed by ligation of the DNA backbone. Here we address the nature of the recombination intermediates formed by phiC31 integrase when acting on its substrates attP and attB. We have identified intermediates containing integrase covalently attached to cleaved DNA substrates, attB or attP, by analysis of complexes in gels and after treatment of these complexes with proteinases. Using a catalytically inactive integrase mutant, S12A, the synaptic complexes containing integrase, attP and attB were identified. Furthermore, we have shown that attB mutants containing insertions or deletions are blocked in recombination at the stage of strand cleavage. Thus, there is a strict spacing requirement within attB, possibly for correct positioning of the catalytic serine relative to the scissile phosphate in the active site. Finally, using integrase S12A we confirmed the inability of attL and attR or other combinations of sites to form a stable synapse, indicating that the directionality of integrative recombination is determined at synapsis.
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http://dx.doi.org/10.1093/nar/gkh538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC419440PMC
June 2004

Characterization of antibody-antigen interactions by fluorescence spectroscopy.

Methods Mol Biol 2004 ;248:431-41

Department of Chemistry, The Open University, Milton Keynes, UK.

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http://dx.doi.org/10.1385/1-59259-666-5:431DOI Listing
March 2004

Antibody purification by column chromatography.

Methods Mol Biol 2004 ;248:379-88

School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, UK.

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http://dx.doi.org/10.1385/1-59259-666-5:379DOI Listing
March 2004