Adverse drug reactions (ADR) are a major concern for the pharmaceutical industry and health practitioners as they can cause morbidity and in severe cases mortality. ADRs are one of the major reasons why drugs fail during clinical trials so research directed at predicting ADRs to minimise failure is essential. The CDDO (2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oate) and the synthetic endoperoxide series are two promising classes that have potential for the treatment of cancers and malaria and may revolutionise treatment, within their fields, if approved for clinical use. The two main aims that are presented in this thesis are to; (i) design and synthesise novel analogues and chemical probes to identify potential molecular targets for both the CDDO and endoperoxide series (ii) develop appropriate in vitro test systems to help define the molecular mechanism of each class of drug. CDDO-Me (methyl 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oate) is one of the most potent inducers of Nrf2, a transcription factor that regulates the expression of numerous cell defence genes in mammalian cells. Nrf2 is sequestered in the cytosol by Keap1, which ‘senses’ chemical and oxidative stress via its 27 cysteine residues. Although CDDO-Me is one of the most potent inducers of Nrf2, the molecular target and chemical mechanism is still not defined. Current literature suggests that a reversible 1,4 conjugate addition to specific cysteine residue(s) located on the Keap1 protein results in an increase in Nrf2 levels. In order for SAR work to be performed a synthetic route to CDDO and analogues was developed which involved nine steps using oleanolic acid as starting material. Highlights of the chemistry included addition of the ketone using mCPBA and incorporation of the cyano group in steps 3 and 7 of the synthesis. In addition to preparing the target molecule CDDO a number of additional molecules were prepared to define the importance of functional groups in the A and C rings of CDDO. Genetically modified H4IIE rat hepatoma cells transiently transfected with the an Nrf2-sensitive luciferase reporter gene were used to screen the CDDO-Me analogues, including DDO-Me which lacks a cyano group on the A ring, for their ability to induce Nrf2. NMR studies with model thiols were performed to determine the ability of these compounds to form reversible or non-reversible adducts. Mass spectrometry (MS) was used to confirm the NMR data and interpretations. In total, four probes were identified that reacted in a non-reversible fashion: DDO-Me, DDO-Al and DDO-Az (click probe versions of the parent DDO-Me that can be used to facilitate proteomic studies) and CDDO-Epox (a probe with similar overall structure to CDDO-Me but can react at the β-carbon in a non-reversible fashion; this feature should aid proteomic approaches to reactive cysteine residue identification). To further investigate if these compounds were reactive to cysteine residues within a model protein, recombinant human GSTP1 was used as a model protein for chemically reactive molecules. Cys-47 located on GSTP1 has been shown to react with other electrophones and during our studies LCMS has confirmed that all four of the synthesised active probes were capable of attaching covalently to Cys-47 of GSTP1. The emergence of malaria parasite resistance to most available drugs, including the semi-synthetic artemisinin derivatives artemether and artesunate, has led to efforts to create new synthetic peroxides as potential antimalarial agents. Leading examples of synthetic endoperoxides include OZ277 (arterolane), a molecule in phase III clinical trials in combination with piperaquine, and OZ439, a second generation derivative with improved pharmacokinetics and enhanced in vivo antimalarial activity. 1,2,4,5-Tetraoxanes are another class of endoperoxide with proven excellent antimalarial profiles against both chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum and oral activity in murine models of the disease. It is currently widely accepted that endoperoxides have a similar antimalarial mechanism to artemisinin, whereby Fe2+ medicated generation of cytotoxic carbon-centred radicals, results in death of the parasite. It is presumed that C-radicals can react with important key proteins; however, the specific molecular target(s) that leads to eventual parasite death are still unknown. A chemical synthesis of tetraoxane probes that contain a UV chromophore was performed and analogues were subsequently screened for antimalarial activity. The most active tetraoxane identified was exposed to a range of Fe2+ salts and conditions developed to mimic the biological environment. Primary, secondary and novel carbon-centred radical derived products (surrogate markers of bioactivation) were purified using UV-HPLC, characterised and submitted as chemical probes and standards for biological studies. In order for proteomic studies to be initiated, an allyl or azide group was incorporated into a semi-synthetic artemisinin skeleton. The azide (and alkyne) functional group within these probes provides a handle for protein pull down via click chemistry. Azide and acetylenes were chosen over direct linkage to the biotin group to reduce steric hindrance in the semi-synthetic probe. The synthesised click probes were tested for antimalarial activity and were submitted for protein pull down and identification of potential molecular targets. Similarly DDO allyl and azide were synthesised and were tested for Nrf2 induction and further confirmed as viable probes via NMR experiments with simple thiols and GSTP1. In summary, novel CDDO non reversible probes were synthesised and have shown potential as chemical tools to identify the molecular targets/mechanisms by which these compounds activate Nrf2. Tetraoxanes also have been prepared along with artemisinin click probes and the latter have been submitted for click chemistry pull down experiments, within Plasmodium falciparum parasites, to identify potential molecular targets.

Carboxylesterase 1 (CES1) is the main human liver esterase and is involved in the metabolism and disposition of numerous endogenous and pharmacological compounds. Some of the substrates of this enzyme are widely prescribed agents such as clopidogrel (Plavix®), methylphenidate (Ritalin®) and oseltamivir (Tamiflu®). However, there is much uncertainty regarding the genetic variability within CES1, and its regulation and involvement in drug-drug interactions (DDI). Polypharmacy is frequent in elderly, HIV and tuberculosis infected populations, and the risk of harmful DDIs is high, especially when these populations overlap. The role played by CES1 on the treatment of all these three pathologies and vice versa needs to be better characterized. In this thesis the role of CES1 genetic variability and its potential role in DDIs are explored both in isolation and in conjunction with other genetic, demographic, physio-pathological and iatrogenic factors. The impact of CES1 genetic variability was assessed on the anti-platelet effect of clopidogrel as well as on isoniazid pharmacokinetics in acute coronary syndrome (ACS) and HIV/Tuberculosis co-infected populations respectively. DDIs mediated by CES1 were explored in a HIV positive cohort treated with clopidogrel and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Also, in vitro experiments with primary hepatocytes were used to investigate CES1 intracellular expression in the presence of prototypical PXR inducers used in tuberculosis treatment. The results of this thesis show that the CES1 rs2244613 SNP does affect clopidogrel anti-aggregant activity and may contribute to treatment non-response. Another CES1 variant, rs3815583, was found to be associated with changes in isoniazid pharmacokinetics. The studies did not indicate that NNRTI coadministration with clopidogrel would impair the anti-platelet activity since no relevant changes in exposure of the antiplatelet agent were identified. In the same way, the results do not anticipate DDIs between CES1 substrates and rifamycins, since no induction of expression was identified after incubating primary human hepatocytes in vitro with rifampicin, rifabutin and rifapentine. In conclusion, the results shown in this thesis support the idea that CES1 genetic variability may play a bigger role than previously suspected in treatment response but may not be a mediator of clinically relevant DDIs.

Acute pancreatitis (AP) is a leading cause for hospitalisation and has significant quality of life implications for the patient and cost implications for the National Health Service. Although most episodes of AP are mild and self-limiting, the severe form of the disease is associated with a high mortality. In the absence of definitive treatment, management is mainly supportive. There is an urgent need to develop more effective biomarkers and drugs to manage AP. Genome-wide studies have demonstrated that proteins that bind to heparin (HBPs) form highly interconnected networks which are functionally important in health and disease. It was hypothesized that this is true in the pancreas and in AP. Testing this hypothesis, using mRNA as a proxy for protein, it was shown that HBPs constitute an important extracellular sub-proteome within the normal pancreas and in major pancreatic diseases that is likely to provide a rich repository of potential biomarkers and drug targets. Building upon this work, a proteomic analysis of HBPs in normal pancreas (NP) and in caerulein-induced mouse AP was undertaken. This has more than doubled the number of HBPs to 883, with 460 new HBPs identified. These may represent the most interconnected set of extracellular proteins and therefore with the greatest regulatory potential. Non canonical HBPs such as NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFA9, NDUFA10, NDUFA9 and NDUFA10 were identified and found to be underexpressed in AP as compared to NP. These may have potential moonlighting roles, not previously known. By virtue of being extracellular and binding to heparin, HBPs are accessible and are potential biomarkers and drug targets in AP. In addition to identifying existing biomarkers in AP such as pancreatic amylase, a number of HBPs with biomarkers potential such as HRG, CD14 and FN1 were identified and need further investigation. HBPs such as SERPINC1, VEGFA and PIP5K1C need further evaluation in drug development. These along with modified heparins, heparin mimetics and matrix therapy in AP provide exciting areas for future research.

The ethical and practical issues of obtaining a blood sample pose a significant challenge to performing pharmacokinetic studies in children, infants and neonates. Dried blood spot analysis, based on the collection of a micro blood sample has potential to overcome these difficulties. There are at present a limited number of reports on the utility of dried blood spot analysis in clinical pharmacokinetic studies. The studies described in this thesis were undertaken to investigate the accuracy and precision of dried blood spot sampling coupled with mass spectrometry detection for drug quantification, and clinically validate the robustness and feasibility of this technique for pharmacokinetic studies in preterm neonates. Dried blood spot methods were developed for application to pharmacokinetic studies of test drugs dexamethasone and caffeine. Investigations were focused on the blood collection system, analyte recovery and optimisation of the detection system. In-vitro validation results indicated developed methods were precise, accurate and selective in accordance with the Food and Drug Administration regulatory guidelines on the assessment of bioanalytical methods. Results were not significantly affected by small variations in the blood volume spotted or the presence of petroleum jelly, which is often used on the sampling site during capillary blood collection in neonates. Variability in haematocrit was determined to be the single most important factor affecting assay accuracy. Stability assessments by comparison with freshly prepared samples verified the suitability of sample drying, storage and post sample extraction conditions. An investigation of method transferability between different analytical instruments was undertaken with caffeine to provide an assessment of the robustness of dried blood spot analysis. Results generated from a single and triple quadrupole mass spectrometer were comparable with an expected lower limit of quantification with the latter technique most likely due to a greater ionisation and detection efficiency. Intravenous dexamethasone pharmacokinetics was determined in 5 preterm neonates receiving treatment for chronic lung disease. Individual pharmacokinetic analyses were performed using a one compartment model to estimate primary pharmacokinetic parameters, clearance (mean, 0.18 l/h/kg) and volume of distribution (mean, 1.33 l/kg). The whole blood derived mean estimates were similar to previous plasma clearance and volume estimates of 0.14 l/h/kg and 1.91 l/kg, respectively reported in neonates (n=7). This highlights the potential for dried blood spot analysis as an alternative to conventional plasma based methods for dexamethasone dose optimisation studies in neonates. The population pharmacokinetics of oral / intravenous caffeine was determined in 67 preterm neonates. A one compartment model was used to describe the blood concentration-time data. Model evaluation using a bootstrapping technique confirmed the robustness and stability of the developed model. Pharmacokinetic parameters derived from dried blood spot drug measurements were estimated with precision (relative standard error < 10%) and were comparable to estimates of plasma clearance (mean, 7.3 vs. 7.0 ml/h/kg) and volume of distribution (mean, 593 vs. 851 ml/kg) from a previous population study in neonates (n=110). Weight and postnatal age were the most influential covariates in the clearance model which is in agreement with previous population studies. These results demonstrate that dried blood spot analysis is a practical technique, with significant potential as a robust method for use in clinical pharmacokinetic studies in vulnerable populations such as preterms. Haematocrit related effects on paper will need to be accounted for if this potential is to be realised. Further investigations to determine the reproducibility of capillary blood sampling in neonates and the impact of using blood drug measurements on pharmacokinetic parameter estimation will be necessary before widespread use of the technique is possible.

Transition metals have been used to mediate bioorthogonal reactions within a biological environment. In particular, applications of biocompatible palladium catalysis currently range from biomolecules modification to the in cellulo synthesis or activation of drugs. Here, the scope of palladium-mediated chemistry in living systems has been further extended with the development of a new homogenous palladium catalyst. This water-soluble, biocompatible, and traceable catalysts is based on a palladium-carbene complex coupled to a fluorescent labelled homing peptide for targeted delivery inside cells. This “SMART” catalyst is designed to activate both caged fluorophores and drugs through the cleavage of protecting groups or cross-coupling reactions. A second strategy for targeted delivery of a biocompatible palladium catalysis involves metal nanoparticles loaded onto a heterogeneous solid support. This “modular” catalyst can be implanted in vivo at the desired site of action, e.g. a tumour, and locally activate biomolecules. These two catalytic systems will allow us to selectively activate pro-drugs in vivo, with spatial control, thus minimising the side effects of the treatment on the whole body.

Understanding how memory, learning and reward work in unison to form adaptive and sometime maladaptive behaviour is at the forefront of modern neuroscience. The largest unmet need in treating maladaptive reward learning behaviours such as addiction is maintaining long-term abstinence and preventing relapse after re-exposure to drug-associated cues. Nicotinic acetylcholine receptors (nAChR) have been implicated in responses to drugs of abuse other than nicotine (Rahman et al., 2015) and the aim of this work was to characterise the role of α7 nAChRs in morphine reward learning using conditioned place preference (CPP). The α7 nAChR antagonist methyllycaconitine (MLA) was used to determine if these receptors contribute to specific stages of drug-paired learning, namely acquisition, expression, reconsolidation or reinstatement of morphine-CPP. In 7-8week old C57BL/6J mice MLA (4mg/kg, s.c), given 20 minutes prior to a conditioning dose of morphine (10mg/kg, i.p) or post-test trial, had no effect on the acquisition, reconsolidation or expression of morphine-CPP. However, when given 20 minutes prior to a priming dose of morphine (5mg/kg, i.p), MLA (4mg/kg, s.c) significantly inhibited drug-induced reinstatement. The mechanisms of this effect were investigated using glutamate receptor autoradiography. Changes in 2-Amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) and N-methyl-D-aspartate (NMDA) binding were examined in mice treated with either saline or MLA at morphine reinstatement. There were no significant changes in NMDA receptor binding (using [3H]MK-801) but morphine reinstatement significantly increased [3H]AMPA binding in the CA1/2 of the ventral but not dorsal hippocampus, or in any other brain regions examined (including mPFC, nucleus accumbens, amygdala and VTA). The selective increase in the hippocampus was partially antagonised by MLA, linking α7 nAChR activation to glutamatergic synaptic plasticity in the hippocampus. Intracranial infusions of MLA into the ventral but not the dorsal hippocampus or medial prefrontal cortex blocked reinstatement to morphine-CPP in male Wistar rats.

Navigation through the environment requires the ability to select relevant information from a multitude of irrelevant stimuli. Under conditions of processing conflict, attention and cognitive control processes bias sensory input based on internal goals. These processes are supported by the interplay of a fronto-parietal attention network that exerts a top-down influence on information processing and a superior temporal network that operates in parallel and that responds in a stimulus-driven manner to behaviorally salient stimuli. It is often reported that nicotine can enhance top-down attention control and reduce distraction. In experiments 1 and 2, the effects of increasing control demands on behavior were assessed using electrophysiological (EEG) and behavioral measures in an auditory number parity decision task with different levels of distraction. Participants made forced choice ‘odd’ or ‘even’ number decisions, while ignoring preceding or simultaneous novel distractors. A group of non-smokers was compared to overnight abstinent smokers (9 hours) and after nicotine intake via 2 mg nicotine tablet or via smoke-inhaled nicotine. The results revealed that preceding distractors impaired task performance due to orienting to and reorienting from the distractor. Simultaneous distractors did not cause orientation of attention (indicated by absence of a P3a Event-Related Potential) and produced smaller increments in response latencies. However, this type of complex novel stimulus initiated processes of memory updating that significantly impaired response sensitivity and accuracy. Nicotine withdrawal enhanced these distraction effects, whereas nicotine intake, particularly via smoking, normalized performance. In experiment 3, dichotic listening performance in a group of non-smokers was compared to abstinent smokers (12 hours) using behavioral, EEG and functional Magnetic Resonance Imaging (fMRI) measures. The perceptual salience of the stimuli was manipulated by systematically varying the Inter-aural Intensity Difference (IID) between them. The analysis pointed to distinct brain networks that differentially activate depending on the level of competition between sensory inputs and these effects were additionally modulated by nicotine withdrawal. Nicotine withdrawal impaired behavioral performance supported by evidence of enhanced use of memory and attention resources, and some evidence of task-independent default mode network activation. Overall, the findings suggest that withdrawal from nicotine, particularly in heavy smokers, is associated with impairments in cognitive control and that subsequent intake of nicotine serves mainly to normalize performance.

Metal ions such as silver (Ag+), mercury (Hg2+), zinc (Zn2+) and copper (Cu+/Cu2+) have a long history of antimicrobial usage and some, such as Cu+/Cu2+, Ag+ and Zn2+ compounds are still used as antimicrobials. Prior to the introduction of antibiotics, Ag+ was arguably the most important antimicrobial and with the rapid emergence of antibiotic resistance, interest in Ag+ and its compounds as alternative antimicrobials have recently been revived. However, resistance to Ag+-based compounds has been emerging, with initial reports of carriage of silver resistance on a Salmonella enetrica serovar Typhimurium multi-resistance plasmid pMG101 isolated from burns patients in 1975. The proposed model for the mechanism of Ag+ resistance encoded by the sil genes from pMG101 involves export of Ag+ ions via an ATPase (SilP), an RND family effluxer (SilCFBA) and a periplasmic chaperone of Ag+ (SilE). SilE is a periplasmic protein predicted to be intrinsically disordered until it binds Ag+ ions. This hypothesis was tested using structural and biophysical studies which showed that SilE is an intrinsically disordered and unstructured protein in its free apo-form, but folds to a compact, defined structure upon optimal binding of six Ag+ ions in its holo-form. Sequence analyses and site-directed mutagenesis established the importance of histidine and methionine containing motifs for Ag+-binding, and identified a nucleation core that initiates Ag+-mediated folding of SilE. The data show that SilE is a molecular metal sponge absorbing up to a maximum of eight Ag+ ions.

Η φαρμοκογονιδιωματική είναι η μελέτη του πως οι γενετικές διαφορές επηρεάζουν τις ποικίλες αποκρίσεις των ασθενών σε φάρμακα. Τα τελευταία χρόνια, έχουν γίνει πολλές μελέτες για την επιλογή φαρμακογονιδιωματικών δεικτών σε διάφορους πληθυσμούς της Ευρώπης, αλλά και παγκοσμίως. Εξετάσαμε και συγκρίναμε τη δο-μή του γονιδιώματος του ελληνικού πληθυσμού όσον αφορά τους φαρμακογονιδι-ωματικούς δείκτες και τη συχνότητα εμφάνισης τους σε αυτόν σε σχέση με τους κεντροευρωπαίους (CEU),όπως έχουν μελετηθεί και καταταχθεί από το πρόγραμμα HapMap. Μια γενική μελέτη 1936 φαρμακογονιδιωματικών δεικτών σε 225 γονίδια με φαρμακογονιδιωματικό ενδιαφέρον διεξήχθη σε 50 υγιείς Έλληνες εθελοντές, χρησιμοποιώντας την DMET+ μικροσυστοιχίες (Affymetrix, Santa Clara,CA,USA). Η στατιστική ανάλυση αποκάλυψε 46 φαρμακογονιδιωματικούς δείκτες που παρου-σιάζουν σημαντική στατιστική διαφορά ανάμεσα στους δύο πληθυσμούς (p<0.05). Η μελέτη μας επεκτάθηκε σε συνολικά 10 πληθυσμούς, οι οποίοι χωρίστηκαν σε υποομάδες σύμφωνα με ιστορικά και γεωγραφικά κριτήρια, δηλαδή, Βαλκάνια (Ελ-λάδα, Κροατία, Σερβία, Σλοβενία), Κεντρική Ευρώπη (Ουγγαρία, Σλοβενία, Πολωνία, Τσεχία, Γερμανία), Νότια Ευρώπη (Ελλάδα, Μάλτα, Τουρκία). Σε αυτή την περίπτω-ση εστιάσαμε το ενδιαφέρον μας στη σύγκριση φαρμακογονιδιωματικών δεικτών που δεν εμφανίζονται ούτε πολύ σπάνια, αλλά ούτε και πολύ συχνά σε αυτές τις πληθυσμιακές ομάδες, δηλαδή σε ποσοστό εμφάνιση από 20% έως και 50%. Τελι-κά, αποδείχθηκε ότι 123 σημαντικοί φαρμακογονιδιωματικοί δείκτες στην υποομάδα της Νότιας Ευρώπης, 103 στην υποομάδα των Βαλκανίων και 106 στην υποομάδα της Κεντρικής Ευρώπης συγκλίνουν ως προς τις συχνότητες εμφάνισης τους. Συ-νεπώς, η κύρια και μελλοντική συνεισφορά αυτής της μελέτης είναι η συμβολή της στον εξορθολογισμό της φαρμακευτικής αγωγής σε αυτές τις χώρες με πιθανή μεί-ωση των ιατροφαρμακευτικών εξόδων. Ένα επιπρόσθετο (προαιρετικό) βήμα είναι η ανάπτυξη της DruGeVar (http://drugevar.genomicmedicinealliance.org), μιας βάσης δεδομένων τριγωνισμού φαρμάκων, γονιδίων και φαρμακογονιδιωματικών δεικτών, σε μια προσπάθεια κα-τασκευής μιας κατανοητής βάσης δεδομένων που θα μπορούσε να εξυπηρετεί την κλινική εφαρμογή της φαρμακογονιδιωματικής. Συνεπώς, ένα απλό και προγνωστικό προφίλ φαρμακευτικής απόκρισης, το οποίο θα παρέχει πληροφορίες για την πιθανότητα αποτελεσματικότητας και ασφάλειας ενός φαρμάκου για συγκεκριμένο ασθενή, θα αλλάξει την πρακτική και την οικονομία της ιατρικής. / Pharmacogenomics is the study of how genetic differences influence the variability in patients’ responses to drugs. In recent years there have been many studies on the selection of pharmacogenomic biomarkers, in different populations in Europe and worldwide. We examined and compared the genome structure of the Greek population in regards to pharmacogenomic biomarkers and their incidence in that population to HapMap CEU population. A general study of 1936 pharmacogenomic biomarkers in 235 genes with pharmacogenomic interest was conducted in 50 healthy Greek individuals by using DMET+ microarray (Affymetrix, Santa Clara,CA,USA). Statistical analysis revealed 46 pharmacogenomic biomarkers showing statistical significance among those two populations (p<0.05). We expanded our study to ten European populations dividing them into subgroups according to the history and geography standards, i.e. the Balkans (Greece, Croatia, Serbia, and Slovenia), Central (Hungary, Slovenia, Poland, Czech Republic and Germany) and Southern Europe (Greece, Malta, Turkey). In that case our interest was channeled in the comparison of pharmacogenomic biomarkers among 20%-50% frequency of appearance. Finally, i) 123 important pharmacogenomic biomarkers in the subgroup of South Europe, ii) 103 in the subgroup of Balkans and iii) 106 in the sub-group of Central Europe were posing vast convergence among these countries.So with this study we expect to contribute to the rationalization of medication in these countries with possible reductions in medical costs. An additional step was the development of DruGeVar (http://drugevar.genomicmedicinealliance.org), an online database triangulating drugs with genes and pharmacogenomics biomarkers in an effort to build a comprehensive database that could serve clinical pharmacogenomics. The medical significance and economic value of a simple, predictive medicine response profile, which will provide information on the likelihood of efficacy and safety of a drug for an individual patient, will change the practice and economics of medicine.

Ο σκοπός της παρούσης εργασίας είναι να εξετάσουμε την κινητική απελευθέρωσης υδρόφιλων και λιπόφιλων μορίων που εγκλωβίζονται σε λιποσώματα, όταν τα λιποσώματα διασπείρονται σε συστήματα φορέων- υδρογελών. Στην παρούσα εργασία μελετάται η απελευθέρωση της υδατοδιαλυτής ουσίας καλσεΐνης και της λιποδιαλυτής γκριζεοφλουβίνη τόσο από σταθερά λιποσώματα (DSPC:Chol) όσο και από ασταθή (PC) διεσπαρμένα σε φορείς υδρογελών. / Release of calcein and griseofulvin (GRF) from control (gels in which solutes are dissolved in) and liposomal gels was studied using agarose-assisted immobilization as a technique to separate gels from drug-receptor compartments.Results show that calcein release from liposomal gels is slower compared to control gels, and can be further retarded by using rigid-membrane liposomes (faster release from PC-liposome compared to DSPC/Chol-liposome gels).Additionally, calcein release is not affected by the lipid amount loaded (in the range from 2 to 8 mg/ml), therefore solute loading can be controlled according to needs. Oppositely, GRF release from liposomal gels is determined by drug loading. At high drug loading levels (compared to GRF aqueous solubility), GRF is released with constant rate from liposomal gels irrespective of liposome type (PC or DSPC/Chol). Thereby, for amphiphilic/lipophilic drugs, drug properties (solubility, log P) determine the system behavior. Calcein and GRF release from control carbopol gels is faster compared to HEC and mixture gels. The same is true for calcein in liposomal gels. Carbopol gel rheological properties were found to be significantly different (compared to the other gels), implying that these characteristics are important for drug diffusion from gels.