Do selective cyclo-oxygenase-2 inhibitors and traditional non-steroidal anti-inflammatory drugs increase the risk of atherothrombosis? Meta-analysis of randomised trials

Objective: To assess the effects of selective cyclo-oxygenase-2 (COX 2) inhibitors and traditional non-steroidal anti-inflammatory drugs (NSAIDs) on the risk of vascular events. Design: Meta-analysis of published and unpublished tabular data from randomised trials, with indirect estimation of the effects of traditional NSAIDs. Data sources: Medline and Embase (January 1966 to April 2005); Food and Drug Administration records; and data on file from Novartis, Pfizer, and Merck. Review methods: Eligible studies were randomised trials that included a comparison of a selective COX 2 inhibitor versus placebo or a selective COX 2 inhibitor versus a traditional NSAID, of at least four weeks' duration, with information on serious vascular events (defined as myocardial infarction, stroke, or vascular death). Individual investigators and manufacturers provided information on the number of patients randomised, numbers of vascular events, and the person time of follow-up for each randomised group. Results: In placebo comparisons, allocation to a selective COX 2 inhibitor was associated with a 42% relative increase in the incidence of serious vascular events (1.2%/year v 0.9%/year; rate ratio 1.42, 95% confidence interval 1.13 to 1.78; P = 0.003), with no significant heterogeneity among the different selective COX 2 inhibitors. This was chiefly attributable to an increased risk of myocardial infarction (0.6%/year v 0.3%/year; 1.86, 1.33 to 2.59; P = 0.0003), with little apparent difference in other vascular outcomes. Among trials of at least one year's duration (mean 2.7 years), the rate ratio for vascular events was 1.45 (1.12 to 1.89; P = 0.005). Overall, the incidence of serious vascular events was similar between a selective COX 2 inhibitor and any traditional NSAID (1.0%/year v 0.9/%year; 1.16, 0.97 to 1.38; P = 0.1). However, statistical heterogeneity (P = 0.001) was found between trials of a selective COX 2 inhibitor versus naproxen (1.57, 1.21 to 2.03) and of a selective COX 2 inhibitor versus non-naproxen NSAIDs (0.88, 0.69 to 1.12). The summary rate ratio for vascular events, compared with placebo, was 0.92 (0.67 to 1.26) for naproxen, 1.51 (0.96 to 2.37) for ibuprofen, and 1.63 (1.12 to 2.37) for diclofenac. Conclusions: Selective COX 2 inhibitors are associated with a moderate increase in the risk of vascular events, as are high dose regimens of ibuprofen and diclofenac, but high dose naproxen is not associated with such an excess.

The effects of copayment policies on adherence to prescription medicines in publicly insured populations

Introduction: Copayments for prescriptions are associated with decreased adherence to medicines resulting in increased health service utilisation, morbidity and mortality. In October 2010 a 50c copayment per prescription item was introduced on the General Medical Services (GMS) scheme in Ireland, the national public health insurance programme for low-income and older people. The copayment was increased to €1.50 per prescription item in January 2013. To date, the impact of these copayments on adherence to prescription medicines on the GMS scheme has not been assessed. Given that the GMS population comprises more than 40% of the Irish population, this presents an important public health problem. The aim of this thesis was to assess the impact of two prescription copayments, 50c and €1.50, on adherence to medicines.Methods: In Chapter 2 the published literature was systematically reviewed with meta-analysis to a) develop evidence on cost-sharing for prescriptions and adherence to medicines and b) develop evidence for an alternative policy option; removal of copayments. The core research question of this thesis was addressed by a large before and after longitudinal study, with comparator group, using the national pharmacy claims database. New users of essential and less-essential medicines were included in the study with sample sizes ranging from 7,007 to 136,111 individuals in different medication groups. Segmented regression was used with generalised estimating equations to allow for correlations between repeated monthly measurements of adherence. A qualitative study involving 24 individuals was conducted to assess patient attitudes towards the 50c copayment policy. The qualitative and quantitative findings were integrated in the discussion chapter of the thesis. The vast majority of the literature on this topic area is generated in North America, therefore a test of generalisability was carried out in Chapter 5 by comparing the impact of two similar copayment interventions on adherence, one in the U.S. and one in Ireland. The method used to measure adherence in Chapters 3 and 5 was validated in Chapter 6. Results: The systematic review with meta-analysis demonstrated an 11% (95% CI 1.09 to 1.14) increased odds of non-adherence when publicly insured populations were exposed to copayments. The second systematic review found moderate but variable improvements in adherence after removal/reduction of copayments in a general population. The core paper of this thesis found that both the 50c and €1.50 copayments on the GMS scheme were associated with larger reductions in adherence to less-essential medicines than essential medicines directly after the implementation of policies. An important exception to this pattern was observed; adherence to anti-depressant medications declined by a larger extent than adherence to other essential medicines after both copayments. The cross country comparison indicated that North American evidence on cost-sharing for prescriptions is not automatically generalisable to the Irish setting. Irish patients had greater immediate decreases of -5.3% (95% CI -6.9 to -3.7) and -2.8% (95% CI -4.9 to -0.7) in adherence to anti-hypertensives and anti-hyperlipidaemic medicines, respectively, directly after the policy changes, relative to their U.S. counterparts. In the long term, however, the U.S. and Irish populations had similar behaviours. The concordance study highlighted the possibility of a measurement bias occurring for the measurement of adherence to non-steroidal anti-inflammatory drugs in Chapter 3. Conclusions: This thesis has presented two reviews of international cost-sharing policies, an assessment of the generalisability of international evidence and both qualitative and quantitative examinations of cost-sharing policies for prescription medicines on the GMS scheme in Ireland. It was found that the introduction of a 50c copayment and its subsequent increase to €1.50 on the GMS scheme had a larger impact on adherence to less-essential medicines relative to essential medicines, with the exception of anti-depressant medications. This is in line with policy objectives to reduce moral hazard and is therefore demonstrative of the value of such policies. There are however some caveats. The copayment now stands at €2.50 per prescription item. The impact of this increase in copayment has yet to be assessed which is an obvious point for future research. Careful monitoring for adverse effects in socio-economically disadvantaged groups within the GMS population is also warranted. International evidence can be applied to the Irish setting to aid in future decision making in this area, but not without placing it in the local context first. Patients accepted the introduction of the 50c charge, however did voice concerns over a rising price. The challenge for policymakers is to find the ‘optimal copayment’ – whereby moral hazard is decreased, but access to essential chronic disease medicines that provide advantages at the population level is not deterred. This evidence presented in this thesis will be utilisable for future policy-making in Ireland.

Streptococcus thermophilus APC151 strain is suitable for the manufacture of naturally GABA-enriched bioactive yogurt

Consumer interest in health-promoting food products is a major driving force for the increasing global demand of functional (probiotic) dairy foods. Yogurt is considered the ideal medium for delivery of beneficial functional ingredients. Gamma-amino-butyric acid has potential as a bioactive ingredient in functional foods due to its health-promoting properties as an anti-stress, anti-hypertensive, and anti-diabetic agent. Here, we report the use of a novel Streptococcus thermophilus strain, isolated from the digestive tract of fish, for production of yogurt naturally enriched with 2 mg/ml of gamma-amino-butyric acid (200 mg in a standard yogurt volume of 100 ml), a dose in the same range as that provided by some commercially available gamma-amino-butyric acid supplements. The biotechnological suitability of this strain for industrial production of yogurt was demonstrated by comparison with the reference yogurt inoculated with the commercial CH1 starter (Chr. Hansen) widely used in the dairy industry. Both yogurts showed comparable pH curves [ΔpH/Δt = 0.31-0.33 h-1], viscosity [0.49 Pa-s], water holding capacity [72–73%], and chemical composition [moisture (87–88%), protein (5.05–5.65%), fat (0.12–0.15%), sugar (4.8–5.8%), and ash (0.74–1.2%)]. Gamma-amino-butyric acid was not detected in the control yogurt. In conclusion, the S. thermophilus APC151 strain reported here provides a natural means for fortification of yogurt with gamma-amino-butyric acid.

Treatment trials for neonatal seizures: the effect of design on sample size

Neonatal seizures are common in the neonatal intensive care unit. Clinicians treat these seizures with several anti-epileptic drugs (AEDs) to reduce seizures in a neonate. Current AEDs exhibit sub-optimal efficacy and several randomized control trials (RCT) of novel AEDs are planned. The aim of this study was to measure the influence of trial design on the required sample size of a RCT. We used seizure time courses from 41 term neonates with hypoxic ischaemic encephalopathy to build seizure treatment trial simulations. We used five outcome measures, three AED protocols, eight treatment delays from seizure onset (Td) and four levels of trial AED efficacy to simulate different RCTs. We performed power calculations for each RCT design and analysed the resultant sample size. We also assessed the rate of false positives, or placebo effect, in typical uncontrolled studies. We found that the false positive rate ranged from 5 to 85% of patients depending on RCT design. For controlled trials, the choice of outcome measure had the largest effect on sample size with median differences of 30.7 fold (IQR: 13.7–40.0) across a range of AED protocols, Td and trial AED efficacy (p<0.001). RCTs that compared the trial AED with positive controls required sample sizes with a median fold increase of 3.2 (IQR: 1.9–11.9; p<0.001). Delays in AED administration from seizure onset also increased the required sample size 2.1 fold (IQR: 1.7–2.9; p<0.001). Subgroup analysis showed that RCTs in neonates treated with hypothermia required a median fold increase in sample size of 2.6 (IQR: 2.4–3.0) compared to trials in normothermic neonates (p<0.001). These results show that RCT design has a profound influence on the required sample size. Trials that use a control group, appropriate outcome measure, and control for differences in Td between groups in analysis will be valid and minimise sample size.

Use of fluorescence lifetime imaging microscopy (FLIM) as a timer of cell cycle S phase

Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2’-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.

Design, synthesis and evaluation of novel ellipticine derivatives and analogues as anti-cancer agents

This thesis describes work carried out on the synthesis of novel 5- and 11-substituted ellipticines and derivatives of the ellipticine analogues, isoellipticine and deazaellipticine, followed by investigation of their potential as anti-cancer agents. Preparation of the key 5- and 11-substituted ellipticine targets involved the development of regiospecific, sequential alkylation reactions with alkenyllithium and Grignard reagents. Investigation of these novel reactions resulted in a new route towards 5-substituted ellipticines via Grignard reaction with vinylmagnesium bromide. These novel 5-vinylellipticine derivatives were further functionalised in an ozonolysis reaction, followed by oxidation to give a range of novel 5-substituted ellipticines. Less success was encountered in the 11-substituted ellipticine series, however preparation of these derivatives using a previously published route was accomplished, and the resulting 11-formylellipticine was further derivatised to give a panel of novel 9- and 11-substituted ellipticines, incorporating amide, carboxylate, imine and amine functionality. The successful route towards 5-substituted ellipticines was applied to the preparation of a range of novel 11-substituted isoellipticines and 6-substituted deazaellipticines, the first time substantial synthesis has been undertaken with these analogues. In addition to this, the first preparation of isoellipticinium salts is described, and a panel of novel isoellipticinium, 7 formylisoellipticinium and 7-hydroxyisoellipticinium salts were synthesised in good yields. Biological evaluation of a panel of 43 novel ellipticine, isoellipticine and deazaellipticine derivatives was accomplished with a topoisomerase II decatenation assay and submission to the NCI 60-cell line screen. Four novel isoellipticine topoisomerase II inhibitors were identified from the decatenation assay, with strong activity at 10 μM. In addition to this, NCI screening identified five highly cytotoxic ellipticine and isoellipticine compounds with remarkable selectivity profiles for different cancer types. These novel lead compounds represent new templates for further research and synthesis.