Acidic amino acids as counterions of ciprofloxacin: Effect on growth and pigment production in Staphylococcus aureus NCTC 8325 and Pseudomonas aeruginosa PAO1.

Antimicrobial resistance (AMR) is emerging as a global threat to public health. One of the strategies employed to combat AMR is the use of adjuvants which act to enhance or reinstate antimicrobial activity by inhibiting resistance mechanisms. However, these adjuvants are themselves not immune to selecting resistant phenotypes. Thus, there is a need to utilise mechanisms which are either less likely to or unable to trigger resistance. One commonly employed mechanism of resistance by microorganisms is to prevent antimicrobial uptake or efflux the antibiotic which manages to permeate its membrane. Here we propose amino acids as antimicrobial adjuvants that may be utilizing alternate mechanisms to fight AMR. We used a modified ethidium bromide (EtBr) efflux assay to determine its efflux in the presence of ciprofloxacin within Staphylococcus aureus (NCTC 8325) and Pseudomonas aeruginosa (PAO1). In this study, aspartic acid and glutamic acid were found to inhibit growth of both bacterial species. Moreover, a reduced production of toxic pigments, pyocyanin and pyoverdine by P. aeruginosa was also observed. As evident from similar findings with tetracycline, these adjuvants, may be a way forward towards tackling antimicrobial resistance.

Profiling gene expression dynamics underpinning conventional testing approaches to better inform pre-clinical evaluation of an age appropriate spironolactone formulation.

There is a need to accelerate paediatric formulation evaluation and enhance quality of early stage data in drug development to alleviate the information pinch point present between formulation development and clinical evaluation. This present work reports application of DNA microarrays as a high throughput screening tool identifying markers for prediction of bioavailability and formulation driven physiological responses. With a focus on enhancing paediatric medicine provision, an oral liquid spironolactone suspension was formulated addressing a paediatric target product profile. Caco-2 cells cultured on transwell inserts were implemented in transport assays in vitro and DNA microarrays were used to examine gene expression modulation. Wistar rats were used to derive in vivo bioavailability data. In vitro, genomic, and in vivo data sets were concurrently evaluated linking drug transport and the genomic fingerprint generated by spironolactone formulation exposure. Significant changes in gene expression are reported as a result of formulation exposure. These include genes coding for ATP-binding cassette (ABC) transporters, solute carrier (SLC) transporters, cytochrome P450 (CYP) enzymes, and carboxylesterase enzymes. Genomic findings better inform pre-clinical understanding of pharmacokinetic and pharmacodynamic responses to spironolactone and its active metabolites than current in vitro drug transport assays alone.

Evaluation of anti-biofilm activity of acidic amino acids and synergy with ciprofloxacin on Staphylococcus aureus biofilms.

Acidic amino acids, aspartic acid (Asp) and glutamic acid (Glu) can enhance the solubility of many poorly soluble drugs including ciprofloxacin (Cip). One of the mechanisms of resistance within a biofilm is retardation of drug diffusion due to poor penetration across the matrix. To overcome this challenge, this work set to investigate novel counter ion approach with acidic amino acids, which we hypothesised will disrupt the biofilm matrix as well as simultaneously improve drug effectiveness. The anti-biofilm activity of D-Asp and D-Glu was studied on Staphylococcus aureus biofilms. Synergistic effect of combining D-amino acids with Cip was also investigated as a strategy to overcome anti-microbial resistance in these biofilms. Interestingly at equimolar combinations, D-Asp and D-Glu were able to significantly disperse (at 20 mM and 40 mM) established biofilms and inhibit (at 10 mM, 20 mM and 40 mM) new biofilm formation in the absence of an antibiotic. Moreover, our study confirmed L-amino acids also exhibit anti-biofilm activity. The synergistic effect of acidic amino acids with Cip was observed at lower concentration ranges (<40 mM amino acids and <90.54 µM, respectively), which resulted in 96.89% (inhibition) and 97.60% (dispersal) reduction in CFU with exposure to 40 mM amino acids. Confocal imaging indicated that the amino acids disrupt the honeycomb-like extracellular DNA (eDNA) meshwork whilst also preventing its formation.

Paediatric drug development of ramipril: reformulation, in vitro and in vivo evaluation.

Ramipril is used mainly for the treatment of hypertension and to reduce incidence of fatality following heart attacks in patients who develop indications of congestive heart failure. In the paediatric population, it is used most commonly for the treatment of heart failure, hypertension in type 1 diabetes and diabetic nephropathy. Due to the lack of a suitable liquid formulation, the current study evaluates the development of a range of oral liquid formulations of ramipril along with their in vitro and in vivo absorption studies. Three different formulation development approaches were studied: solubilisation using acetic acid as a co-solvent, complexation with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and suspension development using xanthan gum. Systematic optimisation of formulation parameters for the different strategies resulted in the development of products stable for 12 months at long-term stability conditions. In vivo evaluation showed C(max) of 10.48 µg/ml for co-solvent, 13.04 µg/ml for the suspension and 29.58 µg/ml for the cyclodextrin-based ramipril solution. Interestingly, both ramipril solution (co-solvent) and the suspension showed a T(max) of 2.5 h, however, cyclodextrin-based ramipril produced T(max) at 0.75 h following administration. The results presented in this study provide translatable products for oral liquid ramipril which offer preferential paediatric use over existing alternatives.

A novel anionic dendrimer for improved cellular delivery of antisense oligonucleotides.

The optimal design of hybridisation-competent antisense oligonucleotides (ODNs) coupled with an efficient delivery system appear to be important prerequisites for the successful use of antisense reagents for gene silencing. We selected an antisense ODN complementary to an accessible region of the epidermal growth factor receptor (EGFR) mRNA with the aid of an antisense oligonucleotide scanning array. The scanning array comprised 2684 antisense ODN sequences targeting the first 120 nts in the coding region of EGFR mRNA. The array-designed antisense ODN was covalently conjugated to a novel anionic dendrimer using a pentaerythritol-based phosphoroamidite synthon via automated DNA synthesis and the ability of this conjugate to effectively deliver and down-regulate EGFR expression in cancer cells was evaluated. Each dendrimeric structure had nine ODN molecules covalently linked to a common centre at their 3' termini. This dendrimer conjugate was markedly more stable to serum nucleases compared to the free ODNs and the cellular uptake of ODN-dendrimer conjugates was up to 100-fold greater as compared to mannitol, a marker for fluid phase endocytosis, and up to 4-fold greater than naked ODN in cancer cells. ODN-dendrimer uptake was energy-dependent and mediated, at least in part, via binding to cell surface proteins; a process that was inhibited by self-competition and by competition with free ODN, salmon sperm DNA, heparin and dextran sulphate. Fluorescent microscopy studies showed a combination of punctate and more diffuse cytosolic distribution pattern for fluorescently labelled ODN-dendrimer conjugate in A431 cells implying internalization by endocytosis followed by release and sequestration of the conjugate into the cytosol. Little or no conjugate appeared to be present in the nuclei of A431 cells. In vitro RNase H-mediated cleavage assays confirmed that covalently conjugated antisense ODNs in the dendrimer conjugate were able to hybridize and cleave the array-defined hybridisation target site within the EGFR mRNA without the need for ODN dissociation from the conjugate. In cell culture, ODN-dendrimer conjugates were effective in inhibiting cancer cell growth that correlated with a marked knockdown in EGFR protein expression. These data highlight a novel anionic dendrimer delivery system for gene silencing oligonucleotides that improved their biological stability, cellular delivery and antisense activity in cultured cancer cells.

Co-delivery of an antisense oligonucleotide and 5-fluorouracil using sustained release poly (lactide-co-glycolide) microsphere formulations for potential combination therapy in cancer.

Antisense oligonucleotides (AODNs) can selectively inhibit oncogene expression by Watson-Crick hybridisation to target mRNA and are being increasingly considered for use in combination with conventional drugs for potential anticancer therapy. Combination therapy of AODNs and cytotoxic agents using biodegradable polymeric delivery systems potentially offers several advantages including site-specific or organ-directed targeting, protection from digesting enzymes, and improved pharmacokinetics/pharmacodynamics resulting from sustained delivery of the entrapped drugs. Using a model AODN targeting the epidermal growth factor receptor (that is over-expressed in several cancers including breast and brain cancer) and the commonly used cytotoxic agent, 5-fluorouracil (5-FU), we have examined the use of poly (lactide-co-glycolide) (P(LA-GA)) microsphere formulations for co-delivery of these agents. Both agents were either co-entrapped in a single microsphere formulation or individually entrapped in two separate microsphere formulations and release profiles determined in vitro. Using a double emulsion method for preparing the P(LA-GA) microspheres suitable entrapment and sustained release over 35 days was observed in both types of formulation. Release of AODN and 5-FU from all formulations appeared to be biphasic. However, the release rates of the two agents were significantly slower when co-entrapped as a single microsphere formulation compared to those obtained with the separate formulations. Electrophoretic mobility shift assays suggested that this might be, in part, due to an interaction of 5-FU with the oligodeoxynucleotide (ODN). Further, our data suggest that by mixing individual formulations of 5-FU and ODNs at different mass ratios allowed greater flexibility in achieving the desired release profile as well as avoiding potential drug-drug interactions. Thus, co-administration of individual P(LA-GA) microsphere formulations of AODNs and 5-FU, at appropriate mass ratios, appears worthy of further investigation for the potential co-delivery of these anti-cancer agents in vivo.