Paediatric drug development:reformulation, in vitro, genomic and in vivo evaluation

Angiotensin converting enzyme (ACE) inhibitors lisinopril and ramipril were selected from EMA/480197/2010 and the potassium-sparing diuretic spironolactone was selected from the NHS specials list for November 2011 drug tariff with the view to produce oral liquid formulations providing dosage forms targeting paediatrics. Lisinopril, ramipril and spironolactone were chosen for their interaction with transporter proteins in the small intestine. Formulation limitations such as poor solubility or pH sensitivity needed consideration. Lisinopril was formulated without extensive development as drug and excipients were water soluble. Ramipril and spironolactone are both insoluble in water and strategies combating this were employed. Ramipril was successfully solubilised using low concentrations of acetic acid in a co-solvent system and also via complexation with hydroxypropyl-β-cyclodextrin. A ramipril suspension was produced to take formulation development in a third direction. Spironolactone dosages were too high for solubilisation techniques to be effective so suspensions were developed. A buffer controlled pH for the sensitive drug whilst a precisely balanced surfactant and suspending agent mix provided excellent physical stability. Characterisation, stability profiling and permeability assessment were performed following formulation development. The formulation process highlighted current shortcomings in techniques for taste assessment of pharmaceutical preparations resulting in early stage research into a novel in vitro cell based assay. The formulations developed in the initial phase of the research were used as model formulations investigating microarray application in an in vitro-in vivo correlation for carrier mediated drug absorption. Caco-2 cells were assessed following transport studies for changes in genetic expression of the ATP-binding cassette and solute carrier transporter superfamilies. Findings of which were compared to in vitro and in vivo permeability findings. It was not possible to ascertain a correlation between in vivo drug absorption and the expression of individual genes or even gene families, however there was a correlation (R2 = 0.9934) between the total number of genes with significantly changed expression levels and the predicted human absorption.

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-β-cyclodextrin (HP-β-CD) and suspension development using xanthan gum. Systematic optimisation of formulation parameters for the different strategies resulted in the development of products stable for twelve months at long term stability conditions. In vivo evaluation showed CMAX of 10.48 µg/mL for co-solvent, 13.04µg/ml for the suspension and 29.58µg/mL for the cyclodextrin based ramipril solution. Interestingly, both ramipril solution (co-solvent) and the suspension showed a TMAX of 2.5h, however, cyclodextrin based ramipril produced TMAX at 0.75h following administration. The results presented in this study provide translatable products for oral liquid ramipril which offer preferential paediatric use over existing alternatives.

In-vitro and in-vivo antivenin activity of 2-[2-(5,5,8a-trimethyl-2- methylene-decahydro-naphthalen-1-yl)-ethylidene]-succinaldehyde against Ophiophagus hannah venom

Objectives Curcuma zedoaroides A. Chaveerach & T. Tanee, locally known as Wan-Paya-Ngoo-Tua-Mia, is commonly used in the North-Eastern part of Thailand as a 'snakebite antidote'. The aim of this study was to isolate the active compound from the rhizome of C. zedoaroides, to determine its structure and to assess its antagonistic activity in vitro and in vivo against King cobra venom. Methods The active compound was obtained from C. zedoaroides by extraction with acetone followed by purification using column chromatography; its X-ray structure was determined. Its inhibition of venom lethality was studied in vitro in rat phrenic nerve-hemidiaphragms and in vivo in mice. Key findings The acetone extract of the Curcuma rhizomes contained a C20 dialdehyde, [2-(5,5,8a-trimethyl-2-methylene-decahydro-naphthalen-1-yl)-ethylidene] -succinaldehyde, as the major component. The isolated curcuma dialdehyde was found active in vitro and in vivo for antivenin activity against the King cobra venom. Using isolated rat phrenic nerve-hemidiaphragm preparations, a significant antagonistic effect on the inhibition of neuromuscular transmission was observed in vitro. Inhibition on muscle contraction, produced by the 4 μg/ml venom, was reversed by 2-16 μg/ml of Curcuma dialdehyde in organ bath preparations over a period of 2 h. Mice intraperitoneally injected with 0.75 mg/kg venom and dialdehyde at 100 mg/kg had a significantly increased survival time. Injection of Curcuma dialdehyde (100 mg/kg) 30 min before the subcutaneous injection of the venom resulted in a 100% survival time after 2 h compared with 0% for the control group. Conclusions The in vitro and in vivo evaluation confirmed the medicinal use of traditional snake plants against snakebites. The bioactivity is linked to an isolated molecule and not a result of synergistic effects of a mixture. The active compound was isolated and the structure fully elucidated, including its stereochemistry. This dialdehyde is a versatile chemical building block and can be easily obtained from this plant source. © 2010 Royal Pharmaceutical Society of Great Britain.

Comparison and characterisation of ribozyme delivery in cultured glial and neuronal cells in vivo and in vitro

Ribozymes are short strands of RNA that possess a huge potential as biological tools for studying gene expression and as therapeutic agents to down-regulate undesirable gene expression. Successful application of ribozymes requires delivery to the target site in sufficient amounts for an adequate duration. However, due to their large size and polyanionic character ribozymes are not amenable to transport across biological membranes. In this study a chemically modified ribozyme with enhanced biological stability, targeted against the EGFR mRNA has been evaluated for cellular delivery to cultured glial and neuronal cells with a view to developing treatments for brain tumours. Cellular delivery of free ribozyme was characterised in cultured glial and neuronal cells from the human and rat. Delivery was very limited and time dependent with no consistent difference observed between glial and neuronal cells in both species. Cellular association was largely temperature and energy-dependent with a small component of non-energy dependent association. Further studies showed that ribozyme cellular association was inhibited with self and cross competition with nucleic and non-nucleic acid polyanions indicating the presence of cell surface ribozyme-binding molecules. Trypsin washing experiments further implied that the ribozyme binding surface molecules were protein by nature. Dependence of cellular association on pH indicated that interaction of ribozyme with cell surface molecules was based on ionic interactions. Fluoresence studies indicated that, post cell association, ribozymes were sequestered in sub-cellular vesicles. South-Western blots identified several cell surface proteins which bind to ribozymes and could facilitate cellular association. The limited cellular association observed with free ribozyme required the development and evaluation of polylactide-co-glycolide microspheres incorporating ribozyme for enhanced cellular delivery. Characterisation of microsphere mediated delivery of ribozyme in cultured glial and neuronal cells showed that association increased by 18 to 27-fold in all cell types with no differences observed between cell lines and species. Microsphere mediated delivery was temperature and energy dependent and independent of pH. In order to assess the potential of PLGA micro spheres for the CNS delivery of ribozyme the distribution of ribozyme entrapping microspheres was investigated in rat CNS after intracerebroventricular injection. Distribution studies demonstrated that after 24 hours there was no free ribozyme present in the brain parenchyma, however microsphere entrapped ribozyme was found in the CNS. Microspheres remained in the ventricular system after deposition and passed from the lateral ventricles to the third and fourth ventricle and in the subarachnoid space. Investigation of the influence of microsphere size on the distribution in CNS demonstrated that particles up to 2.5 and O.5f.lm remained in the ventricles around the choroid plexus and ependymal lining.

IN VIVO analysis of ocular morphological changes during phakic accommodation

The principal theme of this thesis is the in vivo examination of ocular morphological changes during phakic accommodation, with particular attention paid to the ciliary muscle and crystalline lens. The investigations detailed involved the application of high-resolution imaging techniques to facilitate the acquisition of new data to assist in the clarification of aspects of the accommodative system that were poorly understood. A clinical evaluation of the newly available Grand Seiko Auto Ref/ Keratometer WAM-5500 optometer was undertaken to assess its value in the field of accommodation research. The device was found to be accurate and repeatable compared to subjective refraction, and has the added advantage of allowing dynamic data collection at a frequency of around 5 Hz. All of the subsequent investigations applied the WAM-5500 for determination of refractive error and objective accommodative responses. Anterior segment optical coherence tomography (AS-OCT) based studies examined the morphology and contractile response of youthful and ageing ciliary muscle. Nasal versus temporal asymmetry was identified, with the temporal aspect being both thicker and demonstrating a greater contractile response. The ciliary muscle was longer in terms of both its anterior (r = 0.49, P <0.001) and overall length (r = 0.45, P = 0.02) characteristics, in myopes. The myopic ciliary muscle does not appear to be merely stretched during axial elongation, as no significant relationship between thickness and refractive error was identified. The main contractile responses observed were a thickening of the anterior region and a shortening of the muscle, particularly anteriorly. Similar patterns of response were observed in subjects aged up to 70 years, supporting a lensocentric theory of presbyopia development. Following the discovery of nasal/ temporal asymmetry in ciliary muscle morphology and response, an investigation was conducted to explore whether the regional variations in muscle contractility impacted on lens stability during accommodation. A bespoke programme was developed to analyse AS-OCT images and determine whether lens tilt and decentration varied between the relaxed and accommodated states. No significant accommodative difference in these parameters was identified, implying that any changes in lens stability with accommodation are very slight, as a possible consequence of vitreous support. Novel three-dimensional magnetic resonance imaging (MRI) and analysis techniques were used to investigate changes in lens morphology and ocular conformation during accommodation. An accommodative reduction in lens equatorial diameter provides further evidence to support the Helmholtzian mechanism of accommodation, whilst the observed increase in lens volume challenges the widespread assertion that this structure is incompressible due to its high water content. Wholeeye MRI indicated that the volume of the vitreous chamber remains constant during accommodation. No significant changes in ocular conformation were detected using MRI. The investigations detailed provide further insight into the mechanisms of accommodation and presbyopia, and represent a platform for future work in this field.