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Pharmaceuticals, Volume 6, Issue 3 (March 2013) – 7 articles , Pages 287-439

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548 KiB  
Review
Protein Kinase C Inhibitors as Modulators of Vascular Function and Their Application in Vascular Disease
by Raouf A. Khalil
Pharmaceuticals 2013, 6(3), 407-439; https://doi.org/10.3390/ph6030407 - 21 Mar 2013
Cited by 21 | Viewed by 10546
Abstract
Blood pressure (BP) is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM) and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases [...] Read more.
Blood pressure (BP) is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM) and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases in vascular resistance and hypertension (HTN). In VSM, activation of surface receptors by vasoconstrictor stimuli causes an increase in intracellular free Ca2+ concentration ([Ca2+]i), which forms a complex with calmodulin, activates myosin light chain (MLC) kinase and leads to MLC phosphorylation, actin-myosin interaction and VSM contraction. Vasoconstrictor agonists could also increase the production of diacylglycerol which activates protein kinase C (PKC). PKC is a family of Ca2+-dependent and Ca2+-independent isozymes that have different distributions in various blood vessels, and undergo translocation from the cytosol to the plasma membrane, cytoskeleton or the nucleus during cell activation. In VSM, PKC translocation to the cell surface may trigger a cascade of biochemical events leading to activation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK), a pathway that ultimately increases the myofilament force sensitivity to [Ca2+]i, and enhances actin-myosin interaction and VSM contraction. PKC translocation to the nucleus may induce transactivation of various genes and promote VSM growth and proliferation. PKC could also affect endothelium-derived relaxing and contracting factors as well as matrix metalloproteinases (MMPs) in the extracellular matrix further affecting vascular reactivity and remodeling. In addition to vasoactive factors, reactive oxygen species, inflammatory cytokines and other metabolic factors could affect PKC activity. Increased PKC expression and activity have been observed in vascular disease and in certain forms of experimental and human HTN. Targeting of vascular PKC using PKC inhibitors may function in concert with antioxidants, MMP inhibitors and cytokine antagonists to reduce VSM hyperactivity in certain forms of HTN that do not respond to Ca2+ channel blockers. Full article
(This article belongs to the Special Issue Protein Kinase Inhibitors)
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Article
Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection
by Melissa Hamm, Yang Wang and Richard R. Rustandi
Pharmaceuticals 2013, 6(3), 393-406; https://doi.org/10.3390/ph6030393 - 21 Mar 2013
Cited by 27 | Viewed by 13667
Abstract
The N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through their interaction [...] Read more.
The N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase) and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF) to analyze a recombinant mAb produced in murine myeloma (NS0) cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC) high performance liquid chromatography (HPLC) to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS) analysis. Full article
(This article belongs to the Special Issue Biologics)
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Review
Inactivation of Caliciviruses
by Raymond Nims and Mark Plavsic
Pharmaceuticals 2013, 6(3), 358-392; https://doi.org/10.3390/ph6030358 - 21 Mar 2013
Cited by 21 | Viewed by 8408
Abstract
The Caliciviridae family of viruses contains clinically important human and animal pathogens, as well as vesivirus 2117, a known contaminant of biopharmaceutical manufacturing processes employing Chinese hamster cells. An extensive literature exists for inactivation of various animal caliciviruses, especially feline calicivirus and murine [...] Read more.
The Caliciviridae family of viruses contains clinically important human and animal pathogens, as well as vesivirus 2117, a known contaminant of biopharmaceutical manufacturing processes employing Chinese hamster cells. An extensive literature exists for inactivation of various animal caliciviruses, especially feline calicivirus and murine norovirus. The caliciviruses are susceptible to wet heat inactivation at temperatures in excess of 60 °C with contact times of 30 min or greater, to UV-C inactivation at fluence ≥30 mJ/cm2, to high pressure processing >200 MPa for >5 min at 4 °C, and to certain photodynamic inactivation approaches. The enteric caliciviruses (e.g.; noroviruses) display resistance to inactivation by low pH, while the non-enteric species (e.g.; feline calicivirus) are much more susceptible. The caliciviruses are inactivated by a variety of chemicals, including alcohols, oxidizing agents, aldehydes, and β-propiolactone. As with inactivation of viruses in general, inactivation of caliciviruses by the various approaches may be matrix-, temperature-, and/or contact time-dependent. The susceptibilities of the caliciviruses to the various physical and chemical inactivation approaches are generally similar to those displayed by other small, non-enveloped viruses, with the exception that the parvoviruses and circoviruses may require higher temperatures for inactivation, while these families appear to be more susceptible to UV-C inactivation than are the caliciviruses. Full article
(This article belongs to the Special Issue Biologics)
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Review
A Review of Therapeutic Aptamer Conjugates with Emphasis on New Approaches
by John G. Bruno
Pharmaceuticals 2013, 6(3), 340-357; https://doi.org/10.3390/ph6030340 - 19 Mar 2013
Cited by 94 | Viewed by 13193
Abstract
The potential to emulate or enhance antibodies with nucleic acid aptamers while lowering costs has prompted development of new aptamer-protein, siRNA, drug, and nanoparticle conjugates. Specific focal points of this review discuss DNA aptamers covalently bound at their 3' ends to various proteins [...] Read more.
The potential to emulate or enhance antibodies with nucleic acid aptamers while lowering costs has prompted development of new aptamer-protein, siRNA, drug, and nanoparticle conjugates. Specific focal points of this review discuss DNA aptamers covalently bound at their 3' ends to various proteins for enhanced stability and greater pharmacokinetic lifetimes in vivo. The proteins can include Fc tails of IgG for opsonization, and the first component of complement (C1q) to trigger complement-mediated lysis of antibiotic-resistant Gram negative bacteria, cancer cells and possibly some parasites during vulnerable stages. In addition, the 3' protein adduct may be a biotoxin, enzyme, or may simply be human serum albumin (HSA) or a drug known to bind HSA, thereby retarding kidney and other organ clearance and inhibiting serum exonucleases. In this review, the author summarizes existing therapeutic aptamer conjugate categories and describes his patented concept for PCR-based amplification of double-stranded aptamers followed by covalent attachment of proteins or other agents to the chemically vulnerable overhanging 3' adenine added by Taq polymerase. PCR amplification of aptamers could dramatically lower the current $2,000/gram cost of parallel chemical oligonucleotide synthesis, thereby enabling mass production of aptamer-3'-protein or drug conjugates to better compete against expensive humanized monoclonal antibodies. Full article
(This article belongs to the Special Issue Aptamer-Based Therapeutics)
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Article
Cell Penetration Properties of a Highly Efficient Mini Maurocalcine Peptide
by Céline Tisseyre, Eloi Bahembera, Lucie Dardevet, Jean-Marc Sabatier, Michel Ronjat and Michel De Waard
Pharmaceuticals 2013, 6(3), 320-339; https://doi.org/10.3390/ph6030320 - 18 Mar 2013
Cited by 121 | Viewed by 7285
Abstract
Maurocalcine is a highly potent cell-penetrating peptide isolated from the Tunisian scorpion Maurus palmatus. Many cell-penetrating peptide analogues have been derived from the full-length maurocalcine by internal cysteine substitutions and sequence truncation. Herein we have further characterized the cell-penetrating properties of one [...] Read more.
Maurocalcine is a highly potent cell-penetrating peptide isolated from the Tunisian scorpion Maurus palmatus. Many cell-penetrating peptide analogues have been derived from the full-length maurocalcine by internal cysteine substitutions and sequence truncation. Herein we have further characterized the cell-penetrating properties of one such peptide, MCaUF1-9, whose sequence matches that of the hydrophobic face of maurocalcine. This peptide shows very favorable cell-penetration efficacy compared to Tat, penetratin or polyarginine. The peptide appears so specialized in cell penetration that it seems hard to improve by site directed mutagenesis. A comparative analysis of the efficacies of similar peptides isolated from other toxin members of the same family leads to the identification of hadrucalcin’s hydrophobic face as an even better CPP. Protonation of the histidine residue at position 6 renders the cell penetration of MCaUF1-9 pH-sensitive. Greater cell penetration at acidic pH suggests that MCaUF1-9 can be used to specifically target cancer cells in vivo where tumor masses grow in more acidic environments. Full article
(This article belongs to the Special Issue Cell-penetrating Peptides 2012)
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Article
Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers
by Luiza I. Hernandez, Katie S. Flenker, Frank J. Hernandez, Aloysius J. Klingelhutz, James O. McNamara and Paloma H. Giangrande
Pharmaceuticals 2013, 6(3), 295-319; https://doi.org/10.3390/ph6030295 - 14 Mar 2013
Cited by 65 | Viewed by 9015
Abstract
Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA [...] Read more.
Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method (“QUSIM”) and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo. Full article
(This article belongs to the Special Issue RNAi-Based Therapeutics)
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Review
RNAi Therapeutics in Autoimmune Disease
by Kaleb M. Pauley and Seunghee Cha
Pharmaceuticals 2013, 6(3), 287-294; https://doi.org/10.3390/ph6030287 - 05 Mar 2013
Cited by 21 | Viewed by 6910
Abstract
Since the discovery of RNA interference (RNAi), excitement has grown over its potential therapeutic uses. Targeting RNAi pathways provides a powerful tool to change biological processes post-transcriptionally in various health conditions such as cancer or autoimmune diseases. Optimum design of shRNA, siRNA, and [...] Read more.
Since the discovery of RNA interference (RNAi), excitement has grown over its potential therapeutic uses. Targeting RNAi pathways provides a powerful tool to change biological processes post-transcriptionally in various health conditions such as cancer or autoimmune diseases. Optimum design of shRNA, siRNA, and miRNA enhances stability and specificity of RNAi-based approaches whereas it has to reduce or prevent undesirable immune responses or off-target effects. Recent advances in understanding pathogenesis of autoimmune diseases have allowed application of these tools in vitro as well as in vivo with some degree of success. Further research on the design and delivery of effectors of RNAi pathway and underlying molecular basis of RNAi would warrant practical use of RNAi-based therapeutics in human applications. This review will focus on the approaches used for current therapeutics and their applications in autoimmune diseases, including rheumatoid arthritis and Sjögren’s syndrome. Full article
(This article belongs to the Special Issue RNAi-Based Therapeutics)
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