http://hdl.handle.net/10197/2747 2013-05-21T22:55:08Z 2013-05-21T22:55:08Z Ryan, Sinead M. Frías, Jesús M. Wang, Xuexuan Sayers, Claire T. Haddleton, David M. Brayden, David James http://hdl.handle.net/10197/3020 2011-07-19T13:41:40Z 2011-01-20T00:00:00Z

PK/PD modelling of combed-shaped PEGylated salmon calcitonin conjugates of differing molecular weights Ryan, Sinead M.; Frías, Jesús M.; Wang, Xuexuan; Sayers, Claire T.; Haddleton, David M.; Brayden, David James 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.

2011-01-20T00:00:00Z Walsh, Edwin G. Maher, Sam Devocelle, Marc O'Brien, Peter J. Baird, Alan W. Brayden, David James http://hdl.handle.net/10197/3018 2012-03-29T13:32:53Z 2011-08-01T00:00:00Z

High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs Walsh, Edwin G.; Maher, Sam; Devocelle, Marc; O'Brien, Peter J.; Baird, Alan W.; Brayden, David James 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.

2011-08-01T00:00:00Z Rawlinson, Lee-Anne Betty O'Brien, Peter J. Brayden, David James http://hdl.handle.net/10197/3009 2011-07-11T15:32:45Z 2010-08-01T00:00:00Z

High content analysis of cytotoxic effects of pDMAEMA on human intestinal epithelial and monocyte cultures Rawlinson, Lee-Anne Betty; O'Brien, Peter J.; Brayden, David James 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.

2010-08-01T00:00:00Z Maher, Sam Leonard, Thomas W Jacobson, Jette Brayden, David James http://hdl.handle.net/10197/2908 2011-04-28T13:42:20Z 2009-12-17T00:00:00Z

Safety and efficacy of sodium caprate in promoting oral drug absorption : from in vitro to the clinic Maher, Sam; Leonard, Thomas W; Jacobson, Jette; Brayden, David James 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).

2009-12-17T00:00:00Z Wang, Xuexuan Maher, Sam Brayden, David James http://hdl.handle.net/10197/2761 2011-07-12T11:33:28Z 2010-07-01T00:00:00Z

Restoration of rat colonic epithelium after in situ intestinal instillation of the absorption promoter, sodium caprate Wang, Xuexuan; Maher, Sam; Brayden, David James Sodium caprate (C10) is an oral absorption promoter that is currently in clinical trials as a component of solid dosage forms for poorly permeable small molecules and peptides. Clinical data with zoledronic acid tablets suggest that significant delivery along with acceptable safety can be achieved from a once-a-week dosing regime. C10 has surfactant-like properties at the high doses used in vivo and therefore we examined its effects on rat intestinal epithelium following intestinal instillation. Results: Addition of 100 mM concentrations of C10 with the paracellular flux marker, fluorescein isothiocyanate-dextran 4 kDa, permitted a bioavailability of 33% to be achieved. When C10 was added 10, 30 and 60 min in advance of fluorescein isothiocyanate-dextran 4 kDa, enhancement still occurred, but was progressively reduced. Histology revealed that the permeability increase was likely related in part to superficial epithelial damage caused in the first few minutes of exposure, which was rapidly repaired within 30–60 min. Conclusions: Design of optimized dosage forms containing C10 should co-release the payload and promoter close to the epithelium in high concentrations. While C10 induces some epithelial damage, its remarkable capacity for epithelial repair may render this effect insignificant in vivo.

2010-07-01T00:00:00Z Rawlinson, Lee-Anne Betty Ryan, Sinead M. Mantovani, Giuseppe Syrett, Jay A. Haddleton, David M. Brayden, David James http://hdl.handle.net/10197/2755 2011-02-16T09:56:58Z 2010-02-08T00:00:00Z

Antibacterial effects of poly(2-(dimethylamino ethyl)methacrylate) against selected Gram-positive and Gram-negative bacteria Rawlinson, Lee-Anne Betty; Ryan, Sinead M.; Mantovani, Giuseppe; Syrett, Jay A.; Haddleton, David M.; Brayden, David James Antimicrobial coatings can reduce the occurrence of medical device-related bacterial infections. Poly(2-(dimethylamino ethyl)methacrylate)) (pDMAEMA) is one such polymer that is being researched in this regard. The aims of this study were to (1) elucidate pDMAEMA’s antimicrobial activity against a range of Gram-positive and Gram-negative bacteria and (2) to investigate its antimicrobial mode of action. The methods used include determination of minimum inhibitory concentration (MIC) values against various bacteria and the effect of pH and temperature on antimicrobial activity. The ability of pDMAEMA to permeabilise bacterial membranes was determined using the dyes 1-N-phenyl-naphthylamine (NPN) and Calcein-AM. Flow cytometry was used to investigate pDMAEMA’s capacity to be internalised by bacteria and to determine effects on bacterial cell cycling. pDMAEMA was bacteriostatic against Gram-negative bacteria with MIC values between 0.1–10 mg/ml. MIC values against Gram-positive bacteria were variable. pDMAEMA was active against Gram-positive bacteria around its pKa and at lower pH values, while it was active against Gram-negative bacteria around its pKa and at higher pH values. pDMAEMA inhibited bacterial growth by binding to the outside of the bacteria, permeabilising the outer membrane and disrupting the cytoplasmic membrane. By incorporating pDMAEMA with erythromycin, it was found that the efficacy of the latter was increased against Gram-negative bacteria. Together, the results illustrate that pDMAEMA acts in a similar fashion to other cationic biocides.

2010-02-08T00:00:00Z