Paul J White, Pascal Hickey, Linda Sze Tu, Stephen J Headey, Martin J Scanlon, Ben J Boyd, Colin W Pouton[show abstract] [hide abstract]
ABSTRACT: We recently reported that dense gas processing of the protein ovalbumin (OVA) resulted in the formation of particles that were insoluble in water and which retained their immunogenicity in vivo. In the present study, the colloidal properties of these pure protein particles were investigated to in part inform rational formulation approaches. The colloidal properties of the particles, in terms of size, zeta potential and pH-dependent surface and solution properties, were examined. In phosphate-buffered saline (pH7.4), flocculation of the particles was observed, which was prevented when particles were suspended in acetate buffer at pH lower than 4. The resulting particle size was 300nm with low polydispersity and zeta potential of 22.9±3.1mV (mean±SEM, n=3) at pH3. Dense gas OVA particles were also prevented from flocculation using steric stabilisation with Pluronic F127. In this form the particles were stable in Krebs-Henseleit solution for 48h at room temperature. These findings indicate that insoluble pure protein particles produced by dense gas processing have desirable characteristics as particulate vaccines, including consistency of particle size under controlled conditions and high colloid stability.The Journal of pharmacy and pharmacology. 10/2012; 64(10):1386-93.
Celine Valant, Luigi Aurelio, Vijay B Urmaliya, Paul White, Peter J Scammells, Patrick M Sexton, Arthur Christopoulos[show abstract] [hide abstract]
ABSTRACT: Despite the identification of 2-amino-3-benzoylthiophenes (2A3BTs) as the first example of small-molecule allosteric potentiators of agonist function at a G protein-coupled receptor (GPCR)-the adenosine A(1) receptor-their mechanism of action is still not fully understood. We now report the mechanistic basis for the complex behaviors noted for 2A3BTs at A(1) receptors. Using a combination of membrane-based and intact-cell radioligand binding, multiple signaling assays, and a native tissue bioassay, we found that the allosteric interaction between 2A3BTs and the agonists 2-chloro-N(6)-[(3)H]cyclopentyladenosine or (-)-N(6)-(2-phenylisopropyl)adenosine (R-PIA) or the antagonist [(3)H]8-cyclopentyl-1,3-dipropylxanthine is consistent with a ternary complex model involving recognition of a single extracellular allosteric site. However, when allowed access to the intracellular milieu, 2A3BTs have a secondary action as direct G protein inhibitors; this latter property is receptor-independent as it is observed in nontransfected cells and also after stimulation of another GPCR. In addition, we found that 2A3BTs can signal as allosteric agonists in their own right but show bias toward certain pathways relative to the orthosteric agonist, R-PIA. These results indicate that 2A3BTs have a dual mode of action when interacting with the A(1) receptor and that they can engender novel functional selectivity in A(1) signaling. These mechanisms need to be factored into allosteric ligand structure-activity studies.Molecular pharmacology 09/2010; 78(3):444-55. · 4.53 Impact Factor
Article: Enhanced extravasation, stability and in vivo cardiac gene silencing via in situ siRNA-albumin conjugation.[show abstract] [hide abstract]
ABSTRACT: A potential barrier to progression of siRNA therapeutics to the clinic is the ability of these agents to cross the vascular endothelium to reach target cells. This study aimed to bypass the endothelial barrier by harnessing the extravasation capability of the serum protein albumin to allow siRNA to reach cardiomyocytes. A strategy for conjugating siRNA to albumin in vivo was developed that involved activating 3'-amine, 2'-O-methyl, phosphorothioate modified siRNA with succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to yield maleimide-functionalized siRNA ("activated siRNA"); this thiol-reactive species can then irreversibly link to the single surface-exposed cysteine residue of endogenous albumin following intravenous administration. An IGF-I-receptor (IGF-IR) siRNA sequence which was effective in vitro was used to test the ability of the siRNA-albumin conjugate to bypass the endothelial barrier in Balb/C mice and produce silencing. In situ conjugation of maleimide-functionalized siRNA to albumin in mouse serum occurred within minutes of addition, and the resulting conjugate was found to be nuclease stable by SDS-PAGE analysis. In Sprague-Dawley rats, activated siRNA showed a significantly enhanced elimination half-life (75.9 ± 18.2 min) compared to unactivated siRNA (5.1 ± 0.2 min). Intravenous injection of this activated siRNA (1 mg/kg daily for four days) significantly reduced left ventricle IGF-IR mRNA to 64.1 ± 4.1% of that in vehicle-treated animals (mean ± SEM), while the control siRNA (unconjugated) had no effect (n = 4, P > 0.05). Imaging of microvessels from mice treated with fluorescein-labeled activated siRNA showed clear evidence of extravasation and cellular uptake in capillary endothelial cells, cardiomyocytes and vascular smooth muscle cells for mice treated with the activated siRNA but not mice treated with the unactivated siRNA. siRNA-albumin constructs are therefore capable of extravasation to the myocardium resulting in silencing in this otherwise silencing-resistant organ.Molecular Pharmaceutics 12/2011; 9(1):71-80. · 4.78 Impact Factor
Article: Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds.[show abstract] [hide abstract]
ABSTRACT: Conjugation of siRNA to macromolecules such as serum albumin has multiple potential benefits, including enhanced extravasation via albumin-mediated transcytosis across endothelial cells and reduced renal clearance. In attempting to conjugate siRNA to albumin, we used commercially sourced amine-modified siRNA and reacted it with the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group suitable for conjugation to the thiol group of the surface-exposed cysteine residue (Cys 34) within albumin. We found the conjugation of the SMCC-treated siRNA to bovine serum albumin (BSA) to be very inefficient and investigated the cause of the low yield of conjugate. Ultrafiltration with phosphate-buffered saline prior to activation with SMCC dramatically increased the yield of siRNA-albumin conjugate (~15-fold). Communication with the commercial supplier revealed that ammonium acetate buffer was used in a desalting step as part of the siRNA purification process prior to supply, likely resulting in ammonium counterions to the siRNA polyanion, which would interfere with conjugation by consuming the SMCC. After ultrafiltration, a greatly reduced amount of SMCC could be used to affect conjugation, without significant reduction in yield. These data indicate that amine-modified siRNA sourced commercially may require ultrafiltration or dialysis prior to use in conjugation reactions.Journal of nucleic acids 01/2011; 2011:154609.
Article: Tissue dependent differences in G-protein coupled receptor kinases associated with 5-HT4 receptor desensitization in the rat gastro-intestinal tract.[show abstract] [hide abstract]
ABSTRACT: Desensitization of 5-HT(4) receptors is regulated by G-protein coupled receptor kinases (GRKs). However, the specific GRK(s) that regulates the desensitization of 5-HT(4) receptors in the in vivo setting is unknown. We investigated the in situ expression of 5-HT(4) receptors and the GRKs in the rat gastrointestinal tract using immunohistochemistry and their interaction using coimmunoprecipitation. 5-HT(4) receptors were expressed in the tunica muscularis mucosae of the oesophagus, longitudinal muscle, myenteric plexus, circular muscle, submucosal plexus and muscularis mucosae of both the proximal and distal colon. GRK2 was expressed in longitudinal muscle and occasionally in myenteric plexus whilst GRK5 showed limited expression in the nerve endings of the myenteric plexus and submucosal plexus of the colon. GRK3 was expressed in the tunica muscularis mucosae of the oesophagus, circular muscle, submucosal plexus and muscularis mucosae of the colon. GRK6 was expressed in the tunica muscularis mucosae of the oesophagus, longitudinal muscle, circular muscle, and muscularis mucosae of the colon. Stimulation of tunica muscularis mucosae of the oesophagus and distal colon using the 5-HT(4) receptor agonist, tegaserod, followed by analysis of the 5-HT(4) receptor antibody immunoprecipitate, revealed the coimmunoprecipitation of GRK6 with 5-HT(4) receptors in the tunica muscularis mucosae of oesophagus while GRK2 and GRK6 were coimmunoprecipitated with 5-HT(4) receptors in the distal colon. This study indicates that GRK6 may be involved in the regulation of the desensitization of 5-HT(4) receptors in the rat oesophagus whilst GRK2 and GRK6 may be involved in regulation of the desensitization of 5-HT(4) receptors in the distal colon.Biochemical pharmacology 01/2011; 81(1):123-33. · 4.25 Impact Factor