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- PublicationSodium caprate-induced increases in intestinal permeability and epithelial damage are prevented by misoprostol(Elsevier, 2015-08)Epithelial damage caused by intestinal permeation enhancers is a source of debate over their safety. The medium chain fatty acid, sodium caprate (C10), causes reversible membrane perturbation at high dose levels required for efficacy in vivo, so the aim was to model it in vitro. Exposure of Caco-2 monolayers to 8.5mM C10 for 60min followed by incubation in fresh buffer led to (i) recovery in epithelial permeability (i.e. transepithelial electrical resistance (TEER) and apparent permeability coefficient (Papp) of [(14)C]-mannitol), (ii) recovery of cell viability parameters (monolayer morphology, plasma membrane potential, mitochondrial membrane potential, and intracellular calcium) and (iii) reduction in mRNA expression associated with inflammation (IL-8). Pre-incubation of monolayers with a mucosal prostaglandin cytoprotectant was attempted in order to further decipher the mechanism of C10. Misoprostol (100nM), inhibited C10-induced changes in monolayer parameters, an effect that was partially attenuated by the EP1 receptor antagonist, SC51322. In rat isolated intestinal tissue mucosae and in situ loop instillations, C10-induced respective increases in the [(14)C]-mannitol Papp and the AUC of FITC-dextran 4000 (FD-4) were similarly inhibited by misoprostol, with accompanying morphological damage spared. These data support a temporary membrane perturbation effect of C10, which is linked to its capacity to mainly increase paracellular flux, but which can be prevented by pre-exposure to misoprostol.
528Scopus© Citations 33
- PublicationPK/PD modelling of combed-shaped PEGylated salmon calcitonin conjugates of differing molecular weights(Elsevier, 2011-01-20)Salmon calcitonin (sCT) was conjugated via cysteine-1 to novel comb-shaped end-functionalised (poly(PEG) methyl ether methacrylate) (sCT-P) polymers, to yield conjugates of total molecular weights (MW) inclusive of sCT: 6.5, 9.5, 23 and 40 kDa. The conjugates were characterised by HPLC and their in vitro and in vivo bioactivity was measured by cAMP assay on human T47D cells and following intravenous (i.v.) injection to rats, respectively. Stability against endopeptidases, rat serum and liver homogenates was assessed. There were linear and exponential relationships between conjugate MW with potency and efficacy respectively, however the largest MW conjugate still retained 70% of Emax and an EC50 of 3.7 nM. In vivo, while free sCT and the conjugates reduced serum [calcium] to a maximum of 15–30% over 240 min, the half-life (T1/2) was increased and the area under the curve (AUC) was extended in proportion to conjugate MW. Likewise, the polymer conferred protection on sCT against attack by trypsin, chymotrypsin, elastase, rat serum and liver homogenates, with the best protection afforded by sCT-P (40 kDa). Mathematical modelling accurately predicted the MW relationships to in vitro efficacy, potency, in vivo PK and enzymatic stability. With a significant increase in T1/2 for sCT, the 40 kDa MW comb-shaped PEG conjugate of sCT may have potential as a long-acting injectable formulation.
400Scopus© Citations 24
- PublicationHigh content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs(Elsevier, 2011-08)Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates and/or food additives. They are present in many organisms including bacteria, insects, fish and mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs is required in order to predict potential failure at a later stage of development.Wedescribe a high-content analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software in order to understand multiple changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic-helical peptide, melittin, and selected structural analogs. The data shows that structural modification of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity.
1450Scopus© Citations 24
- PublicationHigh content analysis of cytotoxic effects of pDMAEMA on human intestinal epithelial and monocyte cultures(Elsevier, 2010-08)Poly(2-(dimethylamino ethyl)methacrylate) (pDMAEMA) is a cationic polymer with potential as an antimicrobial agent and as a non-viral gene delivery vector. The aim was to further elucidate the cytotoxicity of a selected pDMAEMA low molecular weight (MW) polymer against human U937 monocytes and Caco-2 intestinal epithelial cells using a novel multi-parameter high content analysis (HCA) assay and to investigate histological effects on isolated rat intestinal mucosae. Seven parameters of cytotoxicity were measured: nuclear intensity (NI), nuclear area (NA), intracellular calcium ([Ca2+]i), mitochondrial membrane potential (MMP), plasma membrane permeability (PMP), cell number (CN) and phospholipidosis. Histological effects of pDMAEMA on excised rat ileal and colonic mucosae were investigated in Ussing chambers. Following 24-72 hours exposure, 25-50 µg/ml pDMAEMA induced necrosis in U937 cells, while 100-250 µg/ml induced apoptosis in Caco-2. pDMAEMA increased NA and NI and decreased [Ca2+]i, PMP, MMP and CN in U937 cells. In Caco-2, it increased NI and [Ca2+]i, but decreased NA, PMP, MMP and CN. Phospholipidosis was not observed in either cell line. pDMAEMA (10 mg/ml) did not induce any histological damage on rat colonic tissue and only mild damage to ileal tissue following exposure for 60 min. In conclusion, HCA reveals that pDMAEMA induces cytotoxicity in different ways on different cell types at different concentrations. HCA potential for high throughput toxicity screening in drug formulation programmes.
925Scopus© Citations 51
- PublicationSafety and efficacy of sodium caprate in promoting oral drug absorption : from in vitro to the clinic(Elsevier, 2009-12-17)A major challenge in oral drug delivery is the development of novel dosage forms to promote absorption of poorly permeable Class III drugs across the intestinal epithelium. To date, no absorption promoter has been approved in a formulation specifically designed for oral delivery of Class III molecules. Promoters that are designated safe for human consumption have been licensed for use in a recently approved buccal insulin spray delivery system and also for many years as part of an ampicillin rectal suppository. Unlike buccal and rectal delivery, oral formulations containing absorption promoters have the additional technical hurdle whereby the promoter and payload must be co-released in high concentrations at the small intestinal epithelium in order to generate significant but rapidly reversible increases in permeability. The most advanced promoter in the clinic is the medium chain fatty acid (MCFA), sodium caprate (C10) , a compound already approved as a direct food additive. We discuss how it has evolved to a matrix tablet format suitable for administration to humans under the headings of mechanism of action at the cellular and tissue level and in vitro and in vivo efficacy and safety studies. In specific clinical examples, we review how C10-based formulations are being tested for oral delivery of bisphosphonates using Gastro Intestinal Permeation Enhancement Technology, GIPET (Merrion Pharmaceuticals, Ireland) and in a related solid dose format for anti-sense oligonucleotides (ISIS Pharmaceuticals, USA).
4225Scopus© Citations 175