https://www.um.edu.mt/library/oar/handle/123456789/31983 2026-04-11T14:17:44Z https://www.um.edu.mt/library/oar/handle/123456789/33676 Title: Pharmacogenetic implications in clopidogrel therapy : a pharmacist-Ied management approach Abstract: Pharmacists are in a position to take a leading role in the clinical implementation of pharmacogenetics as regards genotyping and interpretation of results as well as proposing recommendations to personalise therapy. The cytochrome P (CYP) 450 2C19 enzyme is the principal enzyme involved in clopidogre1 metabolism and is encoded by a highly polymorphic gene. Presence of the CYP2C 19 loss-of-function (LoF) *2 variant allele is associated with reduced CYP2C19-mediated activity which impairs clopidogrel bioactivation and increases the risk of adverse cardiac events after percutaneous coronary intervention (PCI). The principal aim of this research was to apply pharmacist-led CYP2C19 genotyping to support consultant cardiologists in the individualisation of antip1ate1et therapy prescribed in patients undergoing PCI. The study protocol was approved by the University Research Ethics Committee. A patient data collection form was developed and psychometrically evaluated. Two hundred and fifty-two patients undergoing PCI with stent deployment for stable angina or acute coronary syndrome (ACS) were recruited by non-probability sampling from the Cardiac Catheterisation Suite at Mater Dei Hospital (MDH). Patients included were ≥ 18 years and prescribed dual antiplatelet therapy with aspirin and clopidogrel. Exclusion criteria were patients with coagulation disorders, platelet disorders, chronic liver disease and/or history of stroke/transient ischaemic attack. Infonned written consent was obtained from each patient at the time of recruitment. 5mL of peripheral blood was collected into a purple-top ethylenediaminetetraacetic acid (EDT A) tube and genomic DNA (gDNA) extraction was performed with the QIAamp@ DNA Mini QIAcube kit (Qiagen). CYP2Cl9 genotyping for the *2 (rs4244285) and *17 (rsI2248560) variant alleles was undertaken using the laboratory-based TaqMan@ SNP Drug Metabolism Assays (Life Technologies) on the 7500 real-time PCR system (Applied Biosystems). Genotypes identified were categorised into four metaboliser phenotypes according to the 'Clinical Pharmacogenetics Implementation Consortium guidelines for CYP2C19 genotype and clopidogre1 therapy' namely; 'extensive' metabo1isers (EMs; *1/*1), 'ultra-rapid' metabo1isers (UMs; *11*17, *17/*17), 'intermediate' metabolisers (IMs; * 1/*2, *2/* 17) or 'poor' metabolisers (PMs; *2/*2). The patients were followed-up 6 to 12 months post-PCI for stent thrombosis (ST) and in-stent restenosis (ISR). Thirty-four out of the 252 patients were genotyped for the LoF *2 allele with two alternative genotyping assays namely, the Spartan™ RX system (Spartan Bioscience), a rapid point-of-care (POC) assay using gDNA obtained from a buccal swab, and the laboratory-based GenID® reverse-dot blot (RDB) hybridisation assay (Autoimmun Diagnostika GmbH) using gDNA obtained from whole blood. Comparison between the three assays was carried out. Prevalence of the *2 and * 17 variant alleles in Maltese patients was compared to 12 populations bordering the Mediterranean Sea from Southern Spain to Tunisia. Eighty-two out of the total 252 patients had a history of previous PCI with stent deployment and were divided into patients who presented with angiography-confirmed ISR at time of recruitment and those who did not. Retrospective analysis was undertaken to investigate any association between various predictors including CYP2C19 *2 allele carrier status and ISR and ST. The financial impact of CYP2C19*2 genotyping to personalise antip1ate1et therapy to limit ISR and ST was estimated for the three assays using the retrospective data. Baseline characteristics of the 252 patients were: Gender (75% male), age (mean 65 years, range 26-89 years), ethnicity (99% Caucasian), weight (mean 81 kg, range 47- 134 kg), positive family history of IHD (65%), active smoking (32%), hypertension (70%), dyslipidaemia (68%) and diabetes mellitus (46%). The majority of patients (55%) were undergoing PCI for stable angina and 45% were undergoing PCI after being admitted with ACS. Polyphannacy was common and most patients (64%) were prescribed between 6 and 10 chronic medications. Distribution of CYP2C19 genotypes was divided into EMs (n=129, 51 %), UMs (n=58, 23%) and IMs (n=65, 26%). No patients in the study population were PMs. Total allele frequency of *2 and *17 was 13% and 15% respectively. Percentage agreement in genotype results between the TaqMan® and GenID® RDB assays was 100% while percentage agreement in genotype results between the Spartan ™ RX assay and both the TaqMan® and GenID® RDB assays was 97%. This difference resulted since 1 patient who was genotyped as a carrier of two *2 alleles with the Spartan ™ RX assay was genotyped as a carrier of one *2 allele with the other two assays. When the *2 allele frequency in Maltese patients was compared to the 12 populations bordering the Mediterranean Sea, prevalence in the Maltese population was in accordance (p>0.05) with all these populations. Retrospective analysis of the 82 patients with prior PCI identified 29 (35%) patients who presented with ISR at the time of recruitment and were undergoing repeat PCI. In 13 of these 29 patients the stent was deployed ≤ 1 year before recruitment into the study and the patients were still on maintenance c1opidogrel therapy at the time of repeat PCI. Univariate analysis showed a statistically significant association (p<0.05) between coronary ISR and CYP2C 19 *2 allele carrier status. Ten (12%) of the 82 patients presented with ST at time of recruitment and were undergoing repeat PCI. In 3 of these 10 patients the stent was deployed ≤ 1 year before recruitment and the patients were still on maintenance clopidogre1 therapy at the time of repeat PCI. Univariate analysis showed a statistically significant association (p<O.05) between *2 allele carrier status and ST ≤ 1 year. The financial analysis indicated cost savings using all three CYP2C19*2 genotyping assays. The findings have important clinical implications for clopidogre1 use in Malta. According to evidence-based guidelines for CYP2C19 genotype and clopidogre1 therapy, an alternative P2Y 12-receptor inhibitor such as prasugre1 is recommended in carriers of the CYP2C 19 LoF *2 allele (26% in this study). The statistically significant association observed between CYP2C19 *2 allele carrier status and ST and ISR despite clopidogre1 therapy, indicates that carriers of the *2 alelle may benefit from treatment with prasugre1 to reduce risk of post-PCI complications. Compared to the 1aboratorybased assays, the POC Spartan™ RX assay provides very rapid results, is accurate and reliable, non-invasive and operator-friendly. Implementation of pharmacist-led antip1ate1et therapy individualisation guided by CYP2C19*2 genotyping in patients undergoing PCI has the potential to limit the incidence of ST and ISR, improve patients' quality of life and reduce healthcare costs . Keywords: clopidogre1, percutaneous coronary intervention, CYP2C 19 10ss-of-function *2 allele, stent thrombosis, coronary in-stent restenosis, laboratory-based CYP2C19 genotyping, rapid point-of-care CYP2C19 genotyping, pharmacist-1ed approach, individualised antiplatelet therapy, cost-effectiveness Description: PH.D 2015-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/32361 Title: Distribution of ciprofloxacin in the peripheries Abstract: The principal objective of this study was to develop and validate innovative, simple, rapid and effective High Performance Liquid Chromatography (HPLC) methods for the quantification of either ciprofloxacin or clindamycin, to be used in the blood and tissue of patients suffering from Peripheral Arterial Disease (PAD). The antibiotic used for this study was selected according to which suitable method of analysis could be developed and validated to be then used for high throughput screening in the local clinical setting. This method was used on blood and tissue samples of patients suffering from varying degrees of PAD. Other aims included evaluating factors influencing the distribution of the chosen antibiotic in the peripheries of patients suffering from PAD, establishing a 3-predictor regression model in relation to such a distribution and trying to predict the concentration of the chosen antibiotic present in the infected tissue. The regression model developed helped predict whether the chosen antibiotic reaches the infected area in concentrations which are high enough to treat the infection. Ciprofloxacin was chosen and innovative methods for its determination and quantification in blood and tissue were validated following method development. These methods were used to quantify ciprofloxacin in blood and tissue of patients suffering from PAD. Blood and tissue samples were collected from 50 patients (33 male; 17 female) who were admitted to hospital for debridement or amputation procedures. The majority of these patients (n= 49) were suffering from diabetes. Thirty- five patients were suffering from severe PAD. A 3-predictor regression model was developed from the data collected. It was found that the concentration of ciprofloxacin reaching the infected tissue was dependent on the severity of P AD, the number of medications that the patient was taking and on the concentration of ciprofloxacin in plasma. The regression model was then tested on samples of IO of these patients. The calculated value for ciprofloxacin concentrations obtained using the regression model was compared to the already determined ciprofloxacin concentration by HPLC. The regression model was also used on other blood samples of patients from whom debrided or amputated tissue samples could not be collected. This was done to help determine how much ciprofloxacin reached the ischemic peripheries. The innovative HPLC methods which were developed and validated for the determination of ciprofloxacin in plasma and tissue can be effectively used for high throughput analysis in a clinical setting where results can be attained in a short period of time for a large number of patient samples. The regression model developed can help predict and help calculate a more tailored individualised dose of ciprofloxacin for patients suffering from infections in the peripheries. This can lead to adequate dosing in these patients with less undesirable effects and better treatment outcomes avoiding unwanted complications and achieving higher levels of therapeutic success in the process. Keywords: ciprofloxacin, peripheral arterial disease, high performance liquid chromatography, blood, tissue Description: PharmD 2015-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/32265 Title: Chiral pharmacokinetics of fluoxetine Abstract: BACKGROUND Fluoxetine is a selective serotonin reuptake inhibitor and has been analysed in pharmacokinetic studies mainly by chromatographic techniques. DESIGN A novel, robust and reproducible chiral gas chromatographic method for the separation and measurcmcnt of the cnantiomcrs of fluoxetine and norfluoxetine, in urine and plasma, was developed. The method was applied in cumulative urine excretion studies and oral saliva population pharmacokinetic studies. METHODS The technique involved the use of GC/MS instrumentation for the acquisition of data in the electron impact, selective-ion monitoring mode. Three male Maltese patients and two female Maltese patients (n = 5), with a mean age of 45.9±14.6 years donated a single sample of urine and oral fluid, one hour after a morning dose of racemic fluoxetine and the samples were analysed by the developed method. Algorithms were developed either mathematically or by software in order to carry out pharmacokinetic studies. RESULTS Calibration curves for enantiomers were linear with high correlation values ranging between 0.88 and 0.97. In cumulative studies, mean half-lives equalled 97.47±0.14h for (S)-fluoxetine, 97.52± 0.34h for (R)-fluoxetine, 113.61±0.19h for (S)-norfluoxetine and 113.55±0.39h for (R)norfluoxetine. In population studies, mean half-lives equalled 42.64±11.65h for (S)-fluoxetine, 47.63±2.61h for (R)-fluoxetine, 197.81±0.32h for (S)-norfluoxetine and 199.72±1.00h for (R)norfluoxetine. DISCUSSION The results showed that the methodology was robust enough for application in pharmacokinetic studies. The pharmacokinetic studies generated results that conformed to literature. CONCLUSION The studies could be applied in therapeutic drug monitoring of the enantiomers. Description: PharmD 2015-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/32026 Title: An evaluation of a pharmaceutical care model in patients with rheumatological disorders Abstract: Pharmaceutical care offered to patients with chronic musculoskeletal rheumatic diseases within a multidisciplinary team approach definitely improves patient outcomes and financial constraints on the patients and their families as well as the national health system. There is however an increasing need for the health professionals to offer and continuously upgrade the quality of service offered and pharmaceutical care is no exception. Out of all the rheumatic diseases, rheumatoid arthritis, besides being the commonest form, is also the condition for which the pharmaceutical industry has contributed enormously towards effective but costly drug armamentarium. Achieving remission or at least treatment to target using timely medications individualised according to patients' lifestyle, co-morbidites and disease condition is the core of effective patient management and is why this research focuses on rheumatoid arthritis. The aim of this research was to develop and implement valid instruments which systematically assess pharmacotherapy adherence to evidence based medicine, incorporating these tools within the philosophy of pharmaceutical care models. The main outcome of this research was the development and clinical implementation of medication assessment tool specific to rheumatoid arthritis, RhMAT, providing a quality system loop. Latest evidence based guidelines, recommendations and standards on rheumatoid arthritis and its management as set out by the American College of Rheumatology (ACR), the European League against Rheumatism (EULAR), the British Society for Rheumatology (BSR) and the National Institute for Health and Care Excellence (NICE) were used to develop the RhMAT. The summary of product characteristics for each drug included in the RhMAT were used as reference for criteria related to pharmacological properties of the respective drugs. The RhMAT was designed in the form of a table which includes the criterion assessed and its cross reference. The RhMA T allows the user to determine whether each criterion tested is being adhered to (Yes) or not (No). If the criterion is not met, the user must determine whether the ''No'' response is justified or not justified. The user can also choose between not applicable or insufficient data to determine an appropriate choice. A general instruction sheet was compiled as a reference guide aiming for standardisation of the correct use of the RhMA T. The RhMA T was validated by an expert panel who assessed applicability of the tool to the practical scenario, presentation, robustness and validity of the criteria provided within the RhMAT. Inter-rater variability tests were conducted and the RhMA T was implemented in a secondary care ambulatory setting. The developed RhMAT consists of 11 separate sections targeting general aspects of rheumatoid arthritis, use of analgesics, methotrexate, su1phasa1azine, hydroxych10roquine, 1eflunomide, parenteral sodium aurothioma1ate, general pre-bio10gic screening, biologic therapy, use of glucocorticoids and remission cases. Following validation by the expert panel the RhMAT was piloted and tested for inter-rater variability and thereafter implemented. The RhMAT was run twice, Phase 1 (at baseline) and Phase 2 (at 12 months +/-3 months) on a total of 78 patients. An adherence rate to the RhMAT was calculated at both phases of the study. The validated Health Assessment Questionnaire (HAQ) and the Medication Compliance questionnaire were used to support the RhMAT in a clinical scenario. Study findings indicate that locally the total RhMAT adherence rate achieved was 82%, defined as high adherence. At Phase 2, the total adherence rate statistically significantly (p value <0.05) increased to 85% following the pharmacist's intervention. The clinical contribution of the innovative RhMAT was two fold. First the RhMAT is able to detect the adherence rate to evidence based medicine. It is therefore able to show quantitatively the quality of pharmacotherapeutic management offered within a multidisciplinary service. Secondly the RhMAT is able to identify gaps which lead to lack of adherence to evidence based medicine. Together with the clinician and the patient the pharmacist can act on the identified gaps through the RhMA T further improving the quality of service offered. The characteristics demonstrated by the RhMAT were high inter-rater level of agreement (Cohen Kappa value of 0.916) and an application time of 15-20 minutes making it practical in a busy ambulatory care setting. Description: PharmD 2015-01-01T00:00:00Z