Είναι γνωστό ότι τα λιποσώματα παρέχουν πολλά πλεονεκτήματα για τη χορήγηση και /ή τη στόχευση φαρμάκων (Lasic 1993, Gregoriadis 1988). Κατά τη χορήγηση λιποσωμικών φαρμακομορφών για τοπική (topical) εφαρμογή ή εφαρμογή σε επιθήλια (mucosal) (Rollan 1993, Schreier and Bouwstra 1994) είναι απαραίτητο οι ρεολογικές και βλεννοσυγκολητικές ιδιότητες των λιποσωμικών διασπορών να ρυθμίζονται ανάλογα με την επιδιωκόμενη οδό χορήγησης. Αυτό μπορεί εύκολα να επιτευχθεί με τη προσθήκη παραγόντων αύξησης ιξώδους (gelling agents) στις λιποσωμικές διασπορές. Συνεπώς προκύπτουν σύνθετες φαρμακοτεχνικές μορφές που από δομικής σύστασης είναι διασπορές λιποσωμικών μορφών φάρμακων σε συστήματα γελών (drug-in-liposome-in-gel). Ανάλογα με το βαθμό συγκράτησης του φαρμάκου στα λιποσώματα μετά τη διασπορά των λιποσωμάτων στην επιθυμητή φαρμακοτεχνική μορφή, μπορεί να τροποποιηθεί και ο ρυθμός απελευθέρωσης του φαρμάκου από τέτοια συστήματα. Αυτό συνδέεται σε μεγάλο βαθμό κυρίως με δύο ομάδες παραγόντων: • Πρώτον με τη σταθερότητα των λιποσωμάτων (ακεραιότητα μεμβράνης και μηχανική σταθερότητα) κατά τη διασπορά σε ημι-στερεές φαρμακοτεχνικές μορφές, κάτι το οποίο συνδέεται τόσο με την σκληρότητα (rigidity) της μεμβράνης του λιποσωμικού φορέα όσο και με τις φυσικές ιδιότητες του ημι-στερεού συστήματος (ιξώδες και ρεολογικές ιδιότητες). • Δεύτερον, με τις φυσικοχημικές ιδιότητες του προς μορφοποίηση φάρμακου. Όσο πιο λιπόφιλο είναι ένα φάρμακο και όσο μικρότερη η διαλυτότητα του στο νερό, τόσο μεγαλύτερος θα είναι λογικά ο χρόνος συγκράτησης του στις λιπιδικές διπλοστοιβάδες των λιποσωμάτων συγκριτικά με αμφίφιλα φάρμακα που έχουν σημαντικά υψηλότερη διαλυτότητα στο νερό. Επειδή σε ανάλογα σύνθετα (φάρμακο-σε-λιπόσωμα-σε-γέλες) συστήματα χορήγησης είναι πιθανόν να πρέπει να συνυπάρχουν εκτός από τον παράγοντα αύξησης του ιξώδους και άλλα έκδοχα, κυρίως παράγοντες που αυξάνουν τη διαλυτότητα διάφορων ουσιών ή επιφανειοδραστικά, ο σκοπός της παρούσας μελέτης ήταν η εξέταση της επίδρασης τέτοιων εκδοχών κατά την προσθήκη τους στα παραπάνω συστήματα. Πιο συγκεκριμένα μελετήθηκε η επίδραση των εξής εκδόχων: Transcutol, Propylene-glycol, Cremophor και Labrafac στη σταθερότητα λιποσωμικών μορφών φαρμάκων και στο ρυθμό απελευθέρωσης υδρόφιλων και λιπόφιλων μορίων από συστήματα διασποράς λιποσωμικών μορφών τέτοιων ουσιών σε γέλες (drug-in-liposome-in-gel). Τα έκδοχα αυτά μόνα ή σε συνδυασμό μεταξύ τους καθως και σε συνδυασμό με άλλους φορείς προσφέρονται για την παρασκευή κατάλληλων φαρμακοτεχνικών μορφών που αποβλέπουν στην αύξηση διαλυτότητας, απορρόφησης και τελικά της βιοδιαθεσιμότητας δυσδιάλυτων φαρμάκων Για την εκτίμηση του βαθμού επίδρασης των πιο πάνω αναφερθέντων εκδοχών στη σταθερότητα λιποσωμικών μορφών φαρμάκων και στο ρυθμό απελευθέρωσης τόσο των υδρόφιλων όσο και λιπόφιλων φάρμακων από συστήματα διασποράς λιποσωμικών μορφών τους σε γέλες (drug-in-liposome-in-gel). χρησιμοποιήθηκε ως μοντέλο υδρόφιλων μορίων η καλσεΐνη ενώ ως μοντέλο υδρόφοβων ουσιών η γκριζεοφουλβίνη. Εδώ ως παράγοντες αύξησης του ιξώδους για την παρασκευή των σύνθετων φαρμακευτικών μορφών, φάρμακο-σε-λιπόσωμα-σε-γέλες χρησιμοποιήθηκαν: (Ι) το πολυμερές του ακρυλικού οξέος, carbopol 974PNF, (ΙΙ) το πολυμερές υδροξυ-αιθυλ-κυτταρίνης (HEC-Hydroxyethylcellulose) καθως και (ΙΙΙ) μίγμα των δυο πολυμερών. Γενικά από τη μελέτη αυτή συμπεραίνουμε ότι: Η επίδραση των προς εξέταση παραγόντων στη σταθερότητα των λιποσωμάτων εξαρτάται από: τη λιπιδική σύσταση, την προσθήκη χοληστερόλης κατά την παρασκευή των λιποσωμάτων, τη λιποφιλικότητα του εκδόχου και τη συγκέντρωσή του. Σχετικά με τη σταθερότητα των λιποσωμάτων παρουσία των εκδόχων που μελετήθηκαν: 1. Tα PC λιποσώματα σπάνε πολύ εύκολα με και χωρίς την παρουσία των εκδοχών στο φορέα υδρογέλης. 2. Τα πιο ανθεκτικά λιποσώματα DSPC/Chol 1:1 φαίνεται να έχουν μεγαλύτερη σταθερότητα. Όμως και στις δυο περιπτώσεις (PC και DSPC/Chol) η σταθερότητα των λιποσωμάτων στους παράγοντες αυτούς μειώνεται ακολουθώντας την εξής σειρά: Transcutol ≈ Propylene-glycol < Cremophor < Labrafac. 3. To Labrafac διαταράσσει στο μεγαλύτερο βαθμό τις λιπιδικές διπλοστοιβάδες προκαλώντας τη μεγαλύτερη διαρροή των εγκλωβισμένων μορίων προκαλώντας σημαντικές δομικές μεταβολές στα λιποσώματα. 4. H χοληστερόλη αυξάνει την σταθερότητα των λιποσωμάτων κυρίως παρουσία των εκδοχών με μια σχετική ασθενή δράση αλλά όχι σημαντικά παρουσία του εκδόχου Labrafac. Σχετικά με τη κινητική απελευθέρωσης ουσιών από λιποσωμικές τους μορφές που διασπείρονται σε γέλες παρουσία των εκδόχων που μελετήθηκαν: 1. Ένα σημαντικό εύρημα είναι ότι υπάρχει μεγάλη διαφορά στη συμπεριφορά απελευθέρωσης λιπόφιλων και υδρόφιλων μορίων από λιποσωμικές μορφές τους όταν τα τελευταία διασπείρονται σε συστήματα υδρογέλων παρουσία και απουσία των εκδόχων καθως και στη μεταξύ τους απελευθέρωση. 2. Σε γενικές γραμμές η απελευθέρωση των εγκλωβισμένων στα λιποσώματα ουσιών, όταν αυτά διασπείρονται στις γέλες που περιέχουν έκδοχα, φαίνεται να συνδέεται-επηρεάζεται από τη σταθερότητα των λιποσωμάτων παρουσία αυτών των εκδόχων, όπως ευρέθηκε στο πρώτο μέρος της μελέτης. Η κινητική απελευθέρωσης λιπόφιλων φαρμάκων από σύνθετα συστήματα όπως αυτά που μελετήθηκαν μπορεί να επιβραδύνεται σημαντικά όταν μέσα στις μορφές προστίθενται έκδοχα με υψηλή λιπόφιλα (Labrafac- γκριζεοφουλβίνη) (πιθανή κατανομή της γκριζεοφουλβίνης σε λιπόφιλες περιοχές). Κλείνοντας επισημαίνουμε ότι η ανακάλυψη νέων συστημάτων χορήγησης φαρμάκων αποτελεί σήμερα τη μεγαλύτερη πρόκληση για τους επιστήμονες. Οι πιο πάνω φορείς συγκαταλέγονται ανάμεσα στα πιο σπουδαία συστήματα χορήγησης φαρμάκων όχι μόνο λόγω συμβατότητας τους ως προς το ανθρώπινο οργανισμό αλλά και εξαιτίας της μεγάλης δυνατότητας εφαρμογών που προσφέρουν. Απώτερος σκοπός αυτής της μελέτης είναι η καταχώρηση στη βιβλιογραφία νέων στοιχείων σχετικά με την επίδραση αυτών των εκδοχών και συνεπώς η διευκόλυνση κατά την επιλογή των καταλληλότερων συνδυασμών φορέων-εκδοχών στο σχεδιασμό επιθυμητών φαρμακοτεχνικών μορφών. / It is well known that liposomes offer many advantages for the delivery and/or targeting of drugs (Lasic 1993, Gregoriadis 1988). When mucosal or topical delivery of liposomal formulations is considered (Rollan 1993, Schreier and Bouwstra, 1994), it is essential that rheological and/or mucoadhesive properties of the liposome dispersions are adjusted accordingly, depending on the intended route of administration. This can be easily dome by adding gelling agents in the liposomal dispersions. Thereby, eventually a drug-in-liposome-in gel complex formulation is developed. Depending on the retention of the drug in the liposomes after the liposomes are dispersed in the preferred formulation, the release rate of the drug may be modified. This is highly related with two major groups of factors; • First the stability of the liposomes (membrane integrity and mechanical stability) during dispersion in the semi-solid formulation that may be related to the vesicle-membrane rigidity as well as the semisolid system physical properties (viscosity and rheological properties). • Second, the physicochemical properties of the drug formulated. A more lipophilic drug with low aqueous solubility should be logically retained for longer time periods in liposome lipid bilayers when compared with amphiphillic drugs with considerable high aqueous solubility (that will be the driving force to move drug molecules to partition in the aqueous environments until saturation occurs). In some cases, in addition to the gelling agents it is essential to have also other excipients in the complex drug-in-liposome-in-gel systems, as solubilizers, surfactants, co-surfactants et.c. Herein, we evaluate the effect of such excipients on the release of drugs from complex systems. We chose to use trancutol, propylene glycol, cremophor and labrafac-hydro, that are commonly used excipients in pharmaceutical formulations. In order to evaluate the extent to which these compounds can affect the release rate of drugs from drug-in-liposome-in-gel systems we followed the release of two model compounds, one hydrophilic dye (calcein) and one lipophilic drug (griseofulvin). The effect of the rigidity of liposomal membranes was evaluated by testing two different liposome lipid compositions for each case of encapsulated compounds, one that is known to be what is called “leaky”, which is the plain PC (egg lecithin) composition, and one which is very rigid, which is the DSPC/Chol (1:1 mol/mol) composition. Additionally, we also evaluated the effect of the gel composition and characteristics, by studying the release of both drugs from liposomes that have been dispersed in systems with different properties. For this we used gels composed of the acrylic acid based polymer Carbopol 974, which is a substance of many commercially available semisolid formulations, at two different concentrations (same rheological properties and different viscosity) as well as a cellulose based gel, composed of hydroxyethyl-cellulose (different rheological properties). Additionally we evaluated also a mixture of the two polymers, which has been proposed as a formulation base with several advantages, for vaginal delivery of drugs. In addition, we studied the stability of liposomes during incubation in the presence of the plain excipients (in 2 different concentrations, 10% and 25%) in order to see if this can be correlated on the effect of the same agents on drug release from the complex systems. The general conclusion extracted from the experimental results of this study is that the effect of the excipients on liposome stability depend on (i) the lipid composition of the liposomal membranes, (ii) the inclusion of cholesterol in the liposomes or not, (iii) the lipophilicity of the excipient and (iv) the concentration of the excipient. More analytically: In respect to liposome stability: 1. PC liposomes are easily disrupted with or without the presence of the excipients in the gels. 2. The very rigid DSPC/Chol (1:1 mol/mol) liposomes demonstrate higher stability. 3. In both cases the effect of the excipients follows the sequence: Transcutol ≈ Propylene-glycol < Cremophor < Labrafac. 4. Labrafac has the highest effect on liposome stability it practically dissolves liposomal membranes. 5. Cholesterol inclusion in liposomal membranes results in increased stability, however even the most stable DSPC/Chol (1:1) liposomes are very unstable in presence of Labrafac. In respect to the release of liposomal drugs from the complex systems in presence of the excipients studied: 3. A significant finding is that there is a difference in the release pattern and rate between lipophilic and hydrophilic liposome-encapsulated molecules, in the presence of excipient or not (control studies).. 4. In general, there seems to be a correlation between the stability of liposomes in presence of excipients and the effect that the specific excipient has on the release kinetics of liposome encapsulated molecules from complex drug-in-liposome-in-gel systems. 5. The release of lipophilic molecules (that are encapsulated in the lipospmes) from complex systems can be substantially sustained when lipophilic excipients are added in the gel (as is the case of griseofulvin and labrafac). Concluding, we stress the fact that the invention of new drug delivery systems with better performance is a great challenge for scientists involved in pharmaceutics. The systems studied here are offering many advantages due to their biocompatible nature and the extended number of applications they may have. The final scope of this study is the entry of new data about the effect of excipients on the properties of complex drug delivery systems. As the availability similar data in the related literature increases, it will become more easy to make the best selection of excipients during the development of better formulations for existing of new drugs.

The purpose of this thesis is to explore the interaction between antibiotics at sub-lethal doses, and E.coli. Initially we focussed on pairwise antibiotic interaction, and the potential to exploit these interactions to minimise antibiotic resistance. In testing the hypothesis that antagonism will slow adaptation by reducing selection for resistance we determined that there are conditions in which this fails to be the case. We furthermore caution against treating drug interactions as anything other than a dynamic property of the bacteria-drug interaction, by showing that the relationship between two drugs may be both synergistic and antagonistic depending on a variety of factors. Whilst exploring the adaptive response to drug combinations we discovered a highly unusual effect of Doxycycline to act as a growth stimulant to E.coli AG100. Chapter 3 and 4 are devoted to determining the nature and mechanism of this stimulation, and analysing any potential genomic changes using whole genome re-sequencing. Having shown that dose response is not always a monotone function of increasing drug dose, in chapter 5 we also look at the dose response in a diffusive context, using a custom built imaging system to show the common non-monotonicity of disk diffusion type assays, that manifest themselves as bullseye patterns of growth. We use a mathematical model to explore the ecological and adaptive reasons for such patterns. Finally in chapter 6 we look at the coevolutionary history of phage and E.coli REL606 strains, by determining trade-offs caused by lambda phage and the sole carbon source maltotriose both utilising the same porin (lamB) for cell entry.

No description available.

In this thesis, a multi-scale approach is provided to a pharmacokinetic and a pharmacodynamic problem. The first part of this research provides a realistic mathematical physiological model of the liver to predict drug drug interactions (DDIs). The model describes the geometry of a lobule (liver unit) and integrates the exchange processes, diffusion and active transport, between the hepatocytes and the blood and possible drug-drug interactions such as; reversible inhibition, mechanistic based inhibition (MBI) and enzyme induction. The liver model is subsequently integrated into a PBPK model with 7 compartments (artery blood, venous blood, gut, liver, kidney, lung, rest of the body). To assess the efficiency of the model to predict DDIs, 77 clinical DDI studies were compared to the model. These 77 clinical studies represent 5 victim drugs (midazolam, simvastatin, triazolam, cerivastatin and nifedipine) and 30 perpetrator drugs. The reversible inhibition, MBI and induction parameters for the majority of the perpetrators were estimated with in vitro experiments and adjusted for the human liver size. The PK parameters, such as clearance and absorption rate, and the physiological parameters were obtained from the literature. The DDIs were measured as the ratio of the AUC (Area Under the Curve of the blood concentration) or the ratio of the maximum concentration Cmax of the victim drug administered with and without the perpetrator drug. The predicted ratios were compared with the clinical observation by calculating the geometric fold error GMFE. The GMFE for AUCratio and Cmax,ratio were calculated to be 1.54 and 1.34, respectively. Moreover, the PBPK model excluding the gut compartment under-predicts both inhibition (lower AUCratio) and induction (higher AUCratio) which strongly suggests that the gut DDI component can not be neglected for accurate clinical prediction. However, the static combined model by Fahmi et al. [1, 2] without the gut component fortuitously predicts the clinical AUCratio better than inclusion with the gut component. To conclude, the model predicts DDIs relatively well as it is in the lower range of errors reported in the literature (1.47-2.00 [1, 2]). Moreover, the model is able to predict the pharmacokinetics of drugs and provides a dynamic description of the DDIs, such as the enzyme level and spatial distribution within a lobule. Furthermore, the perpetrator dose regimen can be changed or the error in the in vitro parameters can be integrated to observe their influences on the AUC ratio. The second part of this research explored the Warburg effect in a avascular tumour growth model incorporating a cell shedding term to account for tumour shrinkage. The tumour model was based on an extension of the Ward and King model [3], where two sub-populations; living cells and dead cells are considered. Three diffusion equations for glucose, lactate and the drug are considered and included into the model for growth rate, natural death rate and a death rate due to the drug. The simulation of the model without a drug shows similar behaviour to the original model by Ward and King despite the presence of the shedding term and predicts an extracellular pH of 6.8. However, when a drug treatment is added, the model is able to simulate the shrinkage of the tumour unlike the original model. Moreover, two scenarios with a basic, neutral and acidic drug were explored, assuming similar efficiency at physiological pH to assess the effect of changes in the extracellular pH. Acidic or weak base drugs seem to be more efficient in low pH environment as the fraction of neutral form is greater and therefore more drug is available to cross the cell membrane to reach its target.

Healthcare associated infections are a major concern within the health services as they inflict a significant financial burdens and time constraints on the healthcare system. Effective skin antisepsis prior to incision of the skin, for example, during surgery, is essential in preventing subsequent infection. Current evidence-based guidelines recommend the use of 2 % (w/v) chlorhexidine digluconate (CHG), preferably in 70 % (v/v) isopropyl alcohol (IPA) prior to incision of the skin. However, many antimicrobial agents poorly permeate into the skin and microorganisms residing in the deeper layers and around hair follicles, may survive the procedure and cause infection. Lipid-based nanocarriers are promising drug delivery system with the potential to improve chemical stability, control drug release and alter drug pharmacokinetics. In present study, the ability of lipid-based nanocarriers to enhance the skin retention of antimicrobial agents was accessed. The solid lipid nanoparticles (SLNs) and nanoemulsions (NEs) of triclosan (TSN) and chlorhexidine digluconate (CHG) were prepared and compared based on their physicochemical parameter and better skin retention properties. SLNs of TSN was prepared using glyceryl behenate (GB) and glyceryl palmitostearate (GP) solid lipids, while NEs of TSN and CHG were prepared using eucalyptus oil (EO) and olive oil (OO) with combination of surfactants Tween® 80 and Span® 80. Characterisation and optimisation of SLN and NE formulations to find better skin retention ability is described with various other studies within this thesis. Skin permeation of TSN and CHG was subsequently investigated by in vitro Franz diffusion model using artificial membrane and full thickness porcine ear skin and the penetration profile were determined by differential stripping technique to quantify the amount of drug retained within skin. In both SLN and NE formulations, no detectable level of TSN and CHG was found in receiver medium through full thickness porcine ear skin in 24 h, which is advantageous for topical drug delivery system. SLNs prepared with GP, as solid lipid was able to produce smaller size formulation along with better skin penetration compared with GB-SLNs formulation. SLNs and NEs of TSN was analysed and compared for enhanced skin retention properties. The results demonstrated a significantly enhanced skin penetration of TSN for NE formulations compared to SLNs, which might be due to difference in composition and physical state of lipids and physicochemical parameter of formulations. In case of CHG-loaded NEs, the results demonstrated EO show better skin penetration compared to OO formulations due to its skin penetration enhancing property, which might be beneficial for skin antisepsis prior to invasive procedure to reduce the microorganisms on and within the skin. However, further studies are required to study antibacterial effects of nanoformulations against various skin microorganisms, to analyse skin permeation and retention ability of prepared nanoformulations in in vivo diffusion studies and further studies to analyse toxicity and skin tolerance of EO alone or in combination with antimicrobial agents.

Efavirenz displays many desirable pharmacokinetic properties such as a long half-life allowing once daily dosing and potency against HIV. Despite these favourable properties efavirenz-containing therapy is associated with the development of central nervous system (CNS) toxicities. Current investigations indicate that high plasma concentrations of efavirenz play a putative role in the development of CNS side effects, but there is a current paucity of data relating to the underlying mechanisms of toxicity. Various nanotechnologies have been explored in attempts to mitigate some of the limitations with efavirenz. While there has been progress in increasing the bioavailability of efavirenz there has been no attempt to assess the impact of increased exposure to efavirenz on CNS toxicity. The body of work presented in this thesis aimed firstly to investigate the underlying mechanism of efavirenz CNS toxicity and secondly to assess uptake and CNS toxicity of efavirenz and a novel solid drug nanoformulation (SDN) of efavirenz. The work presented in this thesis utilised a variety of in vitro, in vivo and in silico methodologies. Chapter 2 utilised allelic discrimination polymerase chain reaction in order to investigate the association of single nucleotide polymorphism (SNPs) in the gamma aminobutyric acid receptor with early treatment discontinuation of efavirenz. In order to assess the effects of SDN efavirenz on the occurrence of CNS toxicities, an in vivo model of anxiety (elevated plus maze) was employed (chapter 3). Chapter 4 detailed the development of a robust and sensitive liquid chromatography tandem mass spectrometer assay for the detection of efavirenz in multiple matrices. The uptake of efavirenz and SDN efavirenz in the CNS was investigated utilising cellular uptake and inhibition studies (chapter 5). Physiologically based pharmacokinetic (PBPK) simulations were used to investigate the distribution of efavirenz in plasma, cerebrospinal fluid (CSF) and brain tissue (chapter 6). Despite an initial trend with Rs211014 and Rs6556547 (univariate analysis) of the training cohort, these SNPs were not found to be significant in the multivariate analysis or in either analysis of the test cohort. Following multiple doses rats treated with efavirenz, but not SDN efavirenz, exhibited anxiety-like behaviour in the EPM. The profile of changes indicated some clear behavioural effects that are likely to be linked to drug-related CNS effects. In particular, a tendency of efavirenz to increase time spent on the central platform may be indicative of anxiogenesis. Cellular accumulation of efavirenz was reduced significantly by montelukast and amantadine, with the reduction in accumulation by prazosin bordering on significance (indicating efavirenz may be a substrate for OCT1 and an SLCO transporter). Additionally, cellular accumulation of SDN efavirenz particles was reduced by dynasore, indicating dynamin-mediated uptake. PBPK simulations predicted efavirenz accumulation in brain tissue, with a tissue to plasma ratio 15.8. The natural occurrence of conditions such as depression involves a complex interplay of factors influencing neurotransmission. This makes identifying single predictors of efavirenz CNS toxicity more difficult. The data presented in this thesis may be built upon to understand the mechanisms governing efavirenz disposition in the CNS and factors influencing the occurrence of CNS toxicity.

Synthetic cannabinoid receptor agonists or more commonly known as synthetic cannabinoids (SCs) were originally created to obtain the medicinal value of THC but they are an emerging social problem. SCs are mostly produced coated on herbal materials or in powder form and marketed under a variety of brand names, e.g. “Spice”, “K2”. Despite many SCs becoming controlled under drug legislation, many of them remain legal in some countries around the world. In Scotland, SCs are controlled under the Misuse of Drugs Act 1971 and Psychoactive Substances Act 2016 that only cover a few early SCs. In Saudi Arabia, even fewer are controlled. The picture of the SCs-problem in Scotland is vague due to insufficient prevalence data, particularly that using biological samples. Whilst there is evidence of increasing use of SCs throughout the world, in Saudi Arabia, there is currently no data regarding the use of products containing SCs among Saudi people. Several studies indicate that SCs may cause serious toxicity and impairment to health therefore it is important to understand the scale of use within society. A simple and sensitive method was developed for the simultaneous analysis of 10 parent SCs (JWH-018, JWH-073, JWH-250, JWH-200, AM-1248, UR-144, A-796260, AB-FUBINACA, 5F-AKB-48 and 5F-PB-22) in whole blood and 8 corresponding metabolites (JWH-018 4-OH pentyl, JWH-073 3-OH butyl, JWH-250 4-OH pentyl, AM-2201 4-OH pentyl, JWH-122 5-OH pentyl, JWH-210 5-OH pentyl, 5F-AKB-48 (N-4 OH pentyl), 5F-PB-22 3-carboxyindole)in urine using LLE and LC-MS/MS. The method was validated according to the standard practices for method validation in forensic toxicology (SWGTOX, May 2013). All analytes gave acceptable precision, linearity and recovery for analysing blood and urine samples. The method was applied to 1,496 biological samples, a mixture of whole blood and urine. Blood and/or urine samples were analysed from 114 patients presenting at Accident and Emergency in Glasgow Royal Infirmary, in spring 2014 and JuneDecember 2015. 5F-AKB-48, 5F-PB-22 and MDMB-CHMICA were detected in 9, 7 and 9 cases respectively. 904 urine samples from individuals admitted to/liberated from Scottish prisons over November 2013 were tested for the presence of SCs. 5F-AKB-48 (N-4 OH pentyl) was detected in 10 cases and 5F-PB-22 3-carboxyindole in 3 cases. Blood and urine samples from two post-mortem cases in Scotland with suspected ingestion of SCs were analysed. Both cases were confirmed positive for 5F-AKB-48. A total of 463 urine samples were collected from personnel who presented to the Security Forces Hospital in Ryiadh for workplace drug testing as a requirement for their job during July 2014. The results of the analysis found 2 samples to be positive for 5F-PB-22 3carboxyindole. A further study in Saudi Arabia using a questionnaire was carried out among 3 subpopulations: medical professionals, members of the public in and around smoking cafes and known drug users. With regards to general awareness of Spice products, 16%, 11% and 22% of those participants of medical professionals, members of the public in and around smoking cafes and known drug users, respectively, were aware of the existence of SCs or Spice products. The respondents had an overall average of 4.5% who had a friend who used these Spice products. It is clear from the results obtained in both blood and urine testing and surveys that SCs are being used in both Scotland and Saudi Arabia. The extent of their use is not clear and the data presented here is an initial look into their prevalence. Blood and urine findings suggest changing trends in SC use, moving away from JWH and AM SCs to the newer 5F-AKB-48, 5-F-PB-22 and MDMBCHMICA compounds worldwide. In both countries 5F-PB-22 was detected. These findings clarify how the SCs phenomenon is a worldwide problem and how the information of every country regarding what SCs are seized can help and is not specific for that country. The analytes included in the method were selected due to their apparent availability in both countries, however it is possible that some newer analytes have been used and these would not have been detected. For this reason it is important that methods for testing SCs are updated regularly and evolve with the ever-changing availability of these drugs worldwide. In addition, there is little published literature regarding the concentrations of these drugs found in blood and urine samples and this work goes some way towards understanding these.

L’endothélium joue un rôle primordial dans la régulation de l’homéostasie vasculaire, en partie, par la libération de facteurs relaxants [le monoxyde d’azote (NO), le facteur hyperpolarisant dérivé de l’endothélium (EDHF) et la prostacycline (PGI2)] et contracturants (les prostanoïdes). Le vieillissement vasculaire est associé à une dysfonction endothéliale caractérisée par une diminution des composantes relaxantes mais également par une augmentation des réponses contractiles dépendantes de l’endothélium. Le but de ce travail était d’évaluer les effets préventifs et curatifs de produits naturels riches en polyphénols (extraits du vin rouge et d’aubépine) contre la dysfonction endothéliale liée au vieillissement chez le rat et l’induction d’une sénescence réplicative dans des cellules endothéliales en culture. Ces produits naturels ont des propriétés préventives et curatives vis-à-vis de la dysfonction endothéliale liée au vieillissement qui se manifestent par une amélioration des composantes NO et EDHF de relaxation et par une diminution des réponses contractiles dépendantes de l’endothélium. Ces effets protecteurs sont vraisemblablement dus à une diminution du stress oxydant et une normalisation du système angiotensine dans la paroi artérielle. Les polyphénols retardent la sénescence réplicative des cellules endothéliales probablement par augmentation de l’expression de la eNOS, de la formation de NO et la diminution de l’expression et de l’activité de p21. Les résultats de ce travail de recherche confirment les données cliniques indiquant que la consommation régulière d’aliments riches en polyphénols diminue le risque cardiovasculaire et augmente l’espérance de vie. / The endothelium plays a crucial role in the regulation of vascular homeostasis, in part, by the release of relaxing factors [nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) and prostacyclin (PGI2)] and contracting factors (prostanoids). Vascular aging is associated with an endothelial dysfunction characterized by decreased endothelium-dependent relaxation, but also by increased endothelium-dependent contractile responses. The purpose of this research was to evaluate the preventive and curative effects of polyphenols-rich natural products (red wine and Crataegus extracts) against both aging-related endothelial dysfunction in rat mesenteric artery and replicative senescence in porcine coronary artery endothelial cells. Polyphenols have preventive and curative properties against the aging-related endothelial dysfunction. They improved NO- and EDHF-mediated relaxations and decreased endothelium-dependent contractile responses. These protective effects are associated to a decrease in oxidative stress and normalization of angiotensin system in the arterial wall. Polyphenols delay replicative senescence in endothelial cell most likely by preventing the downregulation of eNOS expression and activity, and the upregulation of p53/p21 pathway. The results of this research confirm the clinical data indicating that regular consumption of foods rich in polyphenols reduces cardiovascular risk and increased life expectancy.

Physiologically-based pharmacokinetic (PBPK) modelling is traditionally employed to predict drug concentration-time profiles in plasma and tissues using information from physiology/biology, in vitro experiments and in silico predictions. Model-based analysis of population pharmacokinetic (PK) data is rarely performed in such a mechanistic framework, as empirical compartmental models are mainly utilised for this purpose. However, the combination of traditional PBPK methodologies with parameter estimation techniques and non-linear mixed effects modelling is an approach with progressively increasing impact due to the significant advantages it offers. Therefore, the general aim of this thesis is to illustrate, explore and thus further facilitate the application of physiologically-based pharmacokinetic models in the context of population data analysis. In order to pursue this aim, this work firstly particularly focuses on the population pharmacokinetics of simvastatin (SV) and its active metabolite, simvastatin acid (SVA). The complex simvastatin pharmacokinetics and their clinical significance, due to the association with simvastatin-induced myopathy, provide an excellent case to illustrate the advantages of a mechanistically sound population model. In the current work, both conventional and physiologically-based population models were developed using clinical PK data for SV and SVA. Specifically, the developed model-based approaches successfully quantified the impact of demographics, genetic polymorphisms and drug-drug interactions (DDIs) on the SV/SVA pharmacokinetics. Therefore, they can be of significant application either in the clinic or during drug development in order to assess myopathy and DDI risk. Secondly, in this work the following advantages offered by integrated population PBPK modelling were clearly illustrated through specific applications: 1) prediction of drug concentrations at the tissue level, 2) ability to extrapolate outside the studied population and/or conditions and 3) ability to guide the design (sample size) of prospective clinical studies. Finally, in the current work, further methodological aspects related to the application of this integrated population PBPK modelling approach were explored. Of specific focus was the parameter estimation process aided by prior distributions and the derivation of the latter from different in vitro/in silico sources. In addition, specific methodology is illustrated in this work that allows the incorporation of stochastic population variability in the structural parameters of such models without neglecting the underlying physiological constraints.

Nucleoside analogues have been extensively used in medication. The nucleoside analogue cordycepin is the principal bioactive compound found in the caterpillar fungi (Cordyceps and Ophiocordyceps). It has been shown to have biological activity, including anti-inflammatory, immunomodulatory and anti-proliferative activity in many kinds of malignant cells. Intracellular drug interactions at the nucleotide level can be explained by understanding the intracellular metabolism of nucleoside analogues as well as their plasma metabolism since their efficacy of therapy or toxicity does not associate with the plasma level of nucleoside. Therefore, investigation of the metabolism of nucleoside analogues is required for a full understanding of their pharmacological activity and toxicity. For that reason, here an ion pairing LC-MS/MS method has been developed for quantitative analysis of the nucleoside analogue cordycepin and the metabolites and its application to cell culture, using in vitro and in vivo studies. Several HPLC parameters and extraction techniques have been optimised, followed by optimisation of the mass spectrometry method by examining the fragmentation of nucleotides. The method was then validated and applied to study the metabolism of cordycepin in vitro and in vivo, to investigate the effect of the cordycepin treatment with or without pentostatin on the intracellular level of endogenous nucleotides, and to examine the intracellular metabolism of nucleoside analogue 4-thiouridine and the effect of its metabolite on the metabolic balance of adenine and uridine nucleotides. The study on the intracellular metabolism of cordycepin in MCF7 and HeLa cells shows that cordycepin was rapidly metabolized into the deaminated form by adenosine deaminase (ADA) in culture medium as well as in cancer cells; therefore combination with pentostatin, an ADA inhibitor, resulting in the highly accumulated phosphorylated metabolite intracellularly. In contrast, cordycepin in C. militaris extracts showed much lower degradation in non-heat-treated serum compared with pure cordycepin that indicates a strong evidence of the presence of a deaminase inhibitor in the extract of Cordyceps. Moreover, the determination of concentrations of cordycepin and the metabolites in the plasma and liver of rats dosed with cordycepin proves that the half-life of cordycepin and its metabolites are very short in the plasma; nevertheless they are accumulated in the liver with repeated administration. Treatment using cordycepin initially caused an increase in the intracellular concentrations of nucleoside triphosphate, but in the long term, the active metabolite of cordycepin likely induced a long term change in the cell resulting in a drop in nucleotide levels. Pentostatin on its own reduced nucleoside triphosphates levels in the long term and combination with cordycepin increased the effect of cordycepin on nucleotide concentrations. High levels of the accumulated cordycepin triphosphate led to a massive decline in nucleotide levels. A study on the intracellular metabolism of nucleoside analogue 4-thiouridine has shown that generally the uptake of 4-thiouridine into NIH 3T3 cells was fast and the phosphorylated metabolite rapidly was developed only after two min labelling. However, it was also shown that its phosphorylation was not very efficient, but the level of the phosphorylated metabolite increased in serum-stimulated cells likely because the enzyme was upregulated in the presence of growth factor. Moreover, the present study provides additional evidence that 4-thiouridine and its metabolite have no adverse effect on the metabolic balance of adenine and uridine nucleotides. This study confirms that pharmacological activity of nucleosides analogues and their cytotoxicity highly rely on the accumulation of their phosphorylated metabolites. Consequently, the activity and the level of the enzymes involved in their metabolism are highly influential on their pharmacological action as well as their toxicity.