The Rutaceae family consists of 150 genera and 1,500 species, which are herbs, shrubs, and trees. The members of genus Glycosmis and Clausena are aromatic (contain volatile oils) and traditionally used for fever, swollen spleen, digestion, topical infections, skin itch, scabies, boils and ulcers. Accordingly from this family, Tetractomia roxburghiana, Glycosmis calcicola, and Clausena excavata were selected for systematic biological screening to exploit and identify compounds which may serve as subsequent leads for the treatment of skin diseases. While the initial aim of the programme had been to characterise these barely studied plants, the programme was subsequently extended to study their biological activity in order to justify their traditional use as medicines. During the course of this study, supporting analytical methodologies were used extensively and ultimately the evaluation of these methodologies contributed a significant proportion of the overall research programme. Initially crude extract was subjected to column chromatography and the compounds were isolated by overloading an analytical HPLC column. By the end of the programme, crude extracts were being analysed directly by gradient reversed-phase HPLC with subsequent direct scale up to preparative isolation on a (250 ´ 22) mm id column. Analytical reversed phase high performance liquid chromatography (HPLC) of the crude methanolic leaf extracts of T. roxbhurghina, G. calcicola, and C. excavata was carried out in order to qualitatively assess the number of constituents present in each fraction. Separation was achieved by using ACE-5-C18 (250 ´ 4.6 mm) with a flow rate of 1.5 ml/min, with the UV detection at 254 nm. Semi-preparative and preparative HPLC were also carried out in order to isolate components of these mixtures. Using spectral analysis, as swertisin, gallic acid, α-asarone and angelicin (furanocoumarin) were identified. In the same way, angelicin was identified from the methanolic leaf extract of Glycosmis calcicola and preparative HPLC of the methanolic leaf extract of Clausena excavata afforded three compounds, namely 2-(3,4-dimethoxy-phenyl)-5,7-dimethoxy-chromen-4-one, 2-(3,4-dimthoxy-phenyl) 3,7-dihydroxy-5-methoxy-chromen-4-one and 5,7-dihydroxy-2-phenyl-chromen-4-one (chrysin), which were confirmed by spectroscopic methods. Micellar electrokinetic chromatography (MEKC) was evaluated as a possible high-resolution technique for checking the purity of fractions isolated from preparative RP-HPLC. However it proved more effective to exploit orthogonal ( to RP-HPLC) modes of LC by using –NH2 and –SCX ion-exchange HPLC columns and/or, if resolution on analytical RP-HPLC was possible, structural elucidation was carried out using LC-NMR-MS. With respect to biological activity, a range of procedures that had been established at the University for checking activity against skin diseases was used. Free radical induced lipid peroxidation model has been selected for evaluation of antioxidant activity of the extract. The anti-oxidant activity of these extracts and compounds were assessed by free radical induced lipid peroxidation model. The results indicated that the methanolic leaf extract of Tetractomia roxburghiana showed marked anti-oxidant activity whereas methanolic leaf extract of Glycosmis calcicola and Clausena excavata showed moderate anti-oxidant activity. The IC50 value of the methanolic leaf extract of Tetractomia roxburghiana was found to be 201.3 µg/ml; whereas those for Glycosmis calcicola and Clausena excavata were found to be 450.6 µg/ml and 1106 µg/ml respectively. For all the extracts, no anti-bacterial activity was found against Staphylococcus aureus, Streptococcus pyogenes, Propionibacterium acne. Also no anti-fungal activity against Candida albicans was found. A total of three crude methanolic plant extracts, four isolated compounds and eleven semi-purified fractions were tested for in-vitro efficacy, using an agar incorporation method to determine the minimum inhibition concentration (MIC), against the dermatophyte species; Trichophyton rubrum, Trichophyton mentagrophytes and Epidermatophyton floccusum. The MIC value for crude methanolic extracts of Tetractomia roxbhurghiana and Glycosmis calcicola was found to be 62.5 µg/ml and 31.2 µg/ml against T.rubrum and T.mentagrophytes, whereas the methanol extract of Clausena excavata did not show any activity against dermatophytes. In conclusion, the anti-oxidant activity of Tetractomia roxburghiana was found to be comparable with that of propylgallate, which was used as a standard drug thus confirming, as anticipated, that Tetractomia roxburghiana might be a good source of anti-oxidant drugs. The extended degree of anti-oxidant activity displayed by methanolic extract of Tetractomia roxburghiana could be contributed to the presence of swertisin, gallic acid and angelicin, which are proven anti-oxidants. The anti-fungal activity of Glycosmis calcicola could be partly due to the presence of angelicin.

The coherent body of research described in the existing published work is concerned with new assay method development and validation using novel systematic approaches for pharmaceutical and diagnostic compounds. The first stage of the research was to study how analytical method development and validation are typically carried out at present and to formulate this into a simple step-by-step approach. Such a template and protocol was not only used as the foundation of this research programme but could also serve as a simple systematic guide for other practitioners and those new to the field. Furthermore, it was recognised that this protocol should satisfy the requirements of the most strategically important regulatory agencies. The second stage of this research involved evaluation and application of the above validation approach to new methods that were developed for a diverse range of analytes and samples. A new purity assay for 1,10-phenanthroline-5,6- dione and 4,7-phenanthroline-5,6-dione using high-performance liquid chromatography (HPLC) was developed and validated. Impurities in these compounds were identified by liquid chromatography-mass spectrometry (LCMS). Best practice in method development and validation is equally important in the analysis of both active components and excipients in formulated products. In the first case, a liquid chromatography assay method for determining the content of 2-(diethylamino)-N-(2,6-dimethylphenyl) acetamide in a gel formulation was developed and validated. In the second case, the individual contents of three phydroxy benzoic acid ester preservatives in a complex multi-component sample were determined following the development and validation of a liquid chromatography method. Finally, the validation approach was evaluated as applied to another analytical technique. Here, gas chromatography (GC) successfully used to develop a novel assay for p-cymene in tea tree oil formulations presented different analytical problems because of the very complex nature of this natural product. Stability study information to increase the shelf life of the product and validation data for the analytical method for p-cymene content was critically evaluated. iv In essence, the critical review of the requirements for method validation for various agencies and the subsequent preparation of guidelines on how to go about method validation have had a significant impact on how analytical practitioners worldwide go about method development and, more importantly, method validation. Further it was possible to apply these guidelines to conduct a series of effective, successful method validation for assays involving a range of typical pharmaceutical samples.

The use of API interfaces with quadrupole mass spectrometers has been shown to give rise to good sensitivity, selectivity, and robustness for the interfacing of LC to MS. Since their introduction in the 1990s the technique has rapidly become widespread, but at the outset of this research programme, there were still a number of problems associated with it, particularly when dealing with complex sample matrices. The aim of this research programme was to study illustrative examples of the kinds of problems associated with the analysis of biological samples using LC-API-MS in an attempt to arrive at strategies which could be employed to eliminate, or at least compensate for, the problems. Commonly reported problems include the occurrence of matrix effects - a change in response of the target analyte(s) as a result of the presence in the samples of co /late eluting interferences. An investigation which compared ESI with APCI ionisation illustrated a significant drawback in the accepted methodology for the elimination of matrix effects. Optimal LC conditions for a number of assays may use non-MS-friendly mobile phases. A simple and convenient solution to this problem was found to be the post column addition of organic modifier, which reproducibly and reliably enhanced sensitivity. This approach was initially used for a range of dihydropyridine calcium channel blockers and was subsequently applied to a range of chiral compounds from different therapeutic groups to illustrate that this was applicable as a generic technique for increasing sensitivity (typically by around an order of magnitude) in low organic mobile phases. Strategies to develop and validate methods for the determination of endogenous analytes in a biological fluid were investigated. This involved the use of a surrogate matrix, to develop a method for the determination of endogenous testosterone in human serum and the use of non-matrix calibration standards for the successful development and validation of a method for the analysis of indolyl 3 acryloylglycine (IAG) in human urine. As a result of observations suggesting promotion of ionisation of deltamethrin in liver tissue sample extracts, it was postulated that this was due to the presence of high concentrations of surfactants. After confirming the effect, a series of systematic investigations were performed to attempt to understand the mechanism to be able to utilise this as a general method for the enhancement of signal with low sensitivity analytes. It was found that the type of surfactant and concentration used was directly associated with an increased (or decreased) response. Although there remain a number of problems associated with the use of LC-API-MS, the work undertaken for this thesis has successfully demonstrated a number of techniques that can be applied to overcome these problems. Knowledge of the nature of the sample undergoing analysis, the required analytical conditions, and where required careful application of one of the techniques described will ensure that a robust method can be readily developed.

Polyfunctional pyrroles are interesting heterocyclic intermediates as they have a range of reactive centres and the chemoselectivity of their reactions under a range of conditions, is therefore, of much interest. Polyfunctionalised heterocycles are relatively difficult to prepare, but the reactions of these substituted pyrroles allow access to a wide variety of new substituted heterocyclic compounds via these intermediates. The aim of this project was to synthesise polyfunctional pyrroles in order to investigate their use in the preparation of libraries and compounds with known biological activity. The synthesis and initial investigation of the regioselectivity of polyfunctional pyrroles, such as 3,5-dichloro-1H-pyrrole-2,4-dicarboxaldehyde, has previously been described; this work investigated only nucleophilic substitutions. We have investigated the chemoselectivity of the reaction of these pyrroles with a range of reagents and a number of pyrrole derivatives were synthesised via selective functional group transformations. All new compounds were fully characterised by spectroscopic and elemental analysis. Another aim of this project was to discover novel agents that inhibit VEGF receptors using structure based drug design. We have identified hit compounds and synthesised them using regioselective reactions of functional groups present on the pyrrole ring. The compounds were tested for anti-proliferative activity against the HaCaT, human keratinocyte cell line, and also against HT29 and CaCo-2, human colon cell lines using the MTT assay.

Alzheimer’s Disease (AD), Parkinson’s Disease (PD) and Lewy Body Disease (LBD) are some of the many neurodegenerative disorders associated with dementia, for which there is no ultimate cure. It is widely accepted that central nervous system (CNS) nicotinic acetylcholine receptors (nAChRs) may be strongly implicated in the pathology of these devastating disorders, and that stimulation of nAChRs can enhance cognitive behaviour in animals and humans. Nicotine and other nicotinic receptor binding compounds have, over many years, been explored as potential therapies for disorders such as AD and PD. This thesis describes the preparation and pharmacological investigation of a series of 3- substituted and 3,5-disubstitued pyridine derivatives as potential novel and selective nictotinic receptor agonists. Chapter Two details the synthesis of targeted compounds using the generation of [(pyridin-3-yl)methyl]lithium and [(5-methylpyridin-3- yl)methyl]lithium, respectively and subsequent reaction with various electrophiles. Unsuccessful attempts at the synthesis of enantiomerically pure 4-substituted arylpyridin-3-yl-ethanol derivatives by reduction of prochiral 4-substituted arylpyridin-3-yl-ethanone derivatives were made using both catalytic and enzymatic approaches; however, a pair of enantiomerically pure alcohols were isolated via the resolution of diastereomeric esters (prepared by reaction with (S)-O-acetyl mandelic acid) and subsequent hydrolysis. iv Chapter Three explores the synthesis of targeted compounds using halogen-lithium exchange reactions of 3-bromopyridine using n-BuLi and ring-opening by the resultant pyridin-3-yllithium of 4-substituted aryl epoxides. As an extension, Sonogashira cross-coupling of 3- bromopyridine and 4-substituted arylacetylenes and subsequent hydration as an approach to 4-substituted pyridin-3-yl-ethanone derivatives is described. A series of indole derivatives were synthesised using identical approaches. Using methodology developed in previous Chapters, Chapter Four describes approaches to symmetrical and asymmetrical 3,5- bis(arylethynyl)pyridine derivatives, the corresponding bis(ketones), alcohols and 3,5-disubstituted keto-alcohol products. Chapter Five details preliminary pharmacological data (binding and functional assays) performed by our collaborators at Institut de Recherches Servier.

This integrative doctoral report describes how I have used my underpinning experience as a practising pharmacist to make a substantial contribution to pharmacy education. Using separate models for undergraduate and postgraduate students I have planned and delivered high quality innovative programmes which prepare undergraduate pharmacy students for practice and postgraduate professional students for advanced practice. Using an action research methodology I have planned and led a team in the development of a suite of M.Sc. programmes (modular master’s degrees) which matches stakeholder requirements (i.e. students and employers). These programmes offer flexible learning opportunities requiring limited contact. With support systems in place, this model allows the busy healthcare professional to work in a full time capacity whilst studying for a postgraduate qualification. Using this model I have developed short courses and led a team to successfully operate them both in the UK and in Hong Kong. Taking an early lead in the development of supplementary and then independent prescribing courses, has allowed our graduates to develop to meet their potential and allow these practitioners to specialise in their chosen clinical fields. My work with the Centre for Excellence in Healthcare Professional Education (CETL4HealthNE) is perhaps one of the most important and major suggested changes to pharmacy undergraduate education for many years. This is the introduction of Inter-Professional Education (IPE) and practice-based learning. I am a firm advocate for IPE and practice-placements being at the heart of, and becoming a substantial component of, undergraduate pharmacy education. This allows clinical patient-focused teaching to be maximised. I believe that this is of critical importance to ensure that new graduates have both underpinning theoretical knowledge and practical application ability. This is all with the same goal: for the safe and effective care of patients. From my own experiences gained from my collaborative research work and CETL4HealthNE, I propose a model where pharmacists work together with other healthcare professionals, both in practice and in IPE, for the benefit of patient care.

Recently, hydroxypropyl methylcellulose (HPMC) has been made available as an alternative to gelatin for the manufacture of two-piece hard capsules. Hard Capsules manufactured from HPMC with carrageenan as a gelling agent have demonstrated rapid and comparable in-vivo disintegration times to gelatin and overcome some of the disadvantages gelatin capsules present. Gelatin becomes brittle when stored at low humidity and shell dehydration may occur with hygroscopic fillings. In addition, the presence of aldehyde groups in the filling material can reduce the solubility of the gelatin capsule shell by crosslinking. HPMC capsule shells demonstrate lack of brittleness even at moisture levels below 2%, no cross-linking and improved chemical stability, however there is a lack of information relating to the physico- chemical properties of HPMC capsule shells and their dissolution behaviour. The aims of this work were to develop and use different techniques to investigate the physicochemical properties of HPMC and gelatin hard capsule shells and to study the possible interactions between the capsule shell (after different storage conditions) and dissolution media (composition, pH and ionic strength) with and without filling materials. Thermal analysis was undertaken using MDSC to determine and compare the glass transition temperatures of gelatin and three batches of HPMC, which gave an insight into their fundamental physico-chemical properties. Rheological studies were undertaken using DMA, which is a novel method that has not been used previously on capsule shells, to investigate and compare the different viscoelastic properties of the capsules. These included: static scans to study the elastic modulus, linear creep to determine the behaviour of the capsule shells under stress, and dynamic scans to determine the storage modulus and viscosity. The influence of storage RH and time (35% and 53% RH for 24 hours and 3 days), dissolution media composition, ionic strength and pH on the shell dissolution time, and drug release properties of the capsules (using theophylline as the model drug) was also investigated. The findings show that the gelatin capsules became brittle at low moisture content and show some degree of aging upon storage, this was not seen for HPMC. Capsules made form HPMC were more elastic than gelatin and gelatin/Polyethylene glycol, with a greater degree of recovery to an applied stress (e.g. the Young’s Modulus for New HPMC and gelatin capsules was 0.728 MPa vs 1.092 MPa respectively after storage at 53%RH/3 days, and % JR for both capsules was 99.26% vs 98.47% respectively after storage at 35%RH/24 hr). It was also found that storage conditions showed no significant effect on the capsule shell dissolution time, and pH had minimal effect on shell dissolution time of the HPMC capsules. The Changes seen with change in pH were attributed to dissolution media composition, salt concentration and ionic strength of the different dissolution media. For HPMC shells, dissolution time in Sörensen phosphate buffer decreased by 13 – 22 % as pH increased from 5 to 8, however, in citro-phosphate buffer there was a 19 – 36 % increase in dissolution time from pH 5 to 7, In acetate buffer, HPMC shells did not dissolve in pH 6, whereas gelatin dissolution time increased from 2.0 to 3.5 mins as pH increased. Drug dissolution rate was highest from HPMC capsules in all 0.1 M 3 dissolution media compared with gelatin and was affected by the presence of high concentrations of K+ and Na+ ions, whereas gelatin capsules were influenced mostly by the presence of Na+ ions. For example in 0.1 M potassium phosphate buffer (KPB) 100% drug release from HPMC capsules occurred after 35 mins, however, this occurred after 180 mins for gelatin capsules, and in 0.1 M sodium phosphate buffer (NaPB) 100% drug release from HPMC and gelatin capsules occurred after 27 mins and 120 mins respectively. After 5 hours, < 4 % and 10 % drug release was obtained in 0.5 M KPB and NaPB respectively. The mechanism of drug release from gelatin capsules was found to be different in both basic and acidic media. This was constant for the HPMC capsules, showing that the change in pH did not affect the release mechanism. These investigations support work that has been previously reported concerning the properties of the HPMC capsules, and provide new information In terms of their viscoelasticity, interactions with various ions present in different dissolution media, drug dissolution behaviour before and after storage for prolonged periods, and the mechanisms of drug release.

Psoriasis is a chronic inflammatory skin disorder that affects around two percent of the population. There are many treatments available for the management of psoriasis including topical therapy, systemic agents and phototherapy. Despite the number of treatments available, however, there are still problems in the management of psoriasis. It is suggested here that the thioredoxin enzyme system may play a role in the pathology of psoriasis. Using specific molecular modelling techniques, a lead compound, RDP00060, was identified as a potential inhibitor of thioredoxin reductase, a key enzyme in the thioredoxin system. In vitro RDP00060 showed moderate inhibitory activity against the thioredoxin enzyme system with an IC50 value of 1.4 mM. RDP00060 also showed powerful activity in an MTT assay using a human papilloma virus immortalized keratinocyte (HPV-16) cell line. To increase the inhibitory activity towards thioredoxin reductase, molecular modelling techniques were used to identify analogues of RDP00060 with a high binding affinity for thioredoxin reductase. Several novel compounds were then synthesized, characterized and evaluated for inhibitory activity towards the thioredoxin system. One of the compounds, N-(3,4-bis-(toluene-4- sulfonylamino)phenyl)-2-furamide (33f) showed good inhibitory activity against the thioredoxin enzyme with an IC50 value of 37 μM. It is anticipated that N-(3,4- bis-(toluene-4-sulfonylamino)phenyl)-2-furamide (33f) binds to thioredoxin reductase irreversibly through a 1,4-conjugate addition mechanism. This compound also showed powerful activity in the MTT assay using an HPV-16 immortalized keratinocyte cell line. Further testing revealed that N-(3,4-bis-(toluene-4-sulfonylamino)phenyl)-2- furamide (33f) also showed apoptotic and antiproliferative properties in human Tcells. As a result of this work, N-(3,4-bis-(toluene-4-sulfonylamino)phenyl)-2- furamide (33f) has been selected for further investigation as a potential antipsoriatic agent.

Theophylline is a dimethylated xanthine similar in structure to caffeine which is commonly found in tea, coffee and cola beverages (Hendeles and Weinberger, 1983; Rall, 1985). Clinically, its most important pharmacological action is the ability to relax bronchial smooth muscle throughout the bronchial tree (Persson, 1986). This effect has found extensive use in the treatment of asthma with the drug being recommended as the first line agent for chronic asthma (la/rate et ai, 1986). The observation that both beneficial effects as well as toxicity correlate with serum concentrations and that the drug displays a narrow therapeutic window (Finn et al, 1981; Hendeles and Matthay, 1986) has resulted in the recommendation that theophylline dosing be guided by serum concentration measurements (Hendeles and Weinberger, 1980; Whiting et al, 1984; Fitzpatrick and Moss-Barclay, 1985; Barlow et. al, 1988). However, this recommendation appears to have been largely ignored locally. In 1986, one of the first local Therapeutic Drug Monitoring Clinics for theophylline was established at R K Khan Provincial Hospital in Chatsworth, Durban. Preliminary results from this clinic confirmed the widespread use of standard theophylline dosing regimens and revealed that 68% (n = 44) of patients given these regimens had serum theophylline concentrations below the generally accepted therapeutic range (Pillai and Miller, 1988). Previous studies have assessed the influence of Therapeutic Drug Monitoring programmes in terms of the attainment of 'therapeutic' serum concentrations (Whiting et aI, 1984; Fitzpatrick and Moss-Barclay, 1985). This approach has been criticised and it has been recommended that clinical assessment should be the criterion. The purpose of this study was to investigate the influence of serum concentration monitoring on theophylline utilisation at the R K Khan Hospital in terms of clinical control of asthma symptoms. A secondary purpose of this study was to determine population pharmacokinetic parameters in Indian patients. In order to interpret the serum concentrations and make recommendations on dosage design for individual patients, the Bayesian technique of drug dose optimisation is used (Sheiner et aI, 1972). This technique has been shown to be accurate, precise and easy to use (Sheiner and Beal, 1982; Hurley and McNeil, 1988) particularly with currently available computer software. It has been emphasised, however, that for satisfactory performance of this technique, good initial estimates of the population parameter distributions are important (Whiting et al, 1986). Since this information is not available for the Indian population this study was undertaken. A knowledge of population pharmacokinetics can help one to choose initial dosage, to modify dosage appropriately in response to observed drug levels, to make rational decisions regarding drug regulatory requirements and toinvestigate and elucidate certain research questions in pharmacokinetics (Sheiner, 1984). The NONMEM approach (Sheiner et aI, 1972; 1977), currently the mostsatisfactory method of population pharmacokinetic data analysis is utilised in this study. / Thesis (M.Pharm.)--University of Durban-Westville, 1989.

The main objective of the present study was to determine the feasibility of obtaining aqueous polymer-coated pellet formulations using EudragitR NE 30 D dispersion and chlorpheniramine maleate as the model drug. Many factors influence the rate of drug release from coated beads including, the substrate, the coating formulation and the coating process. A drug release profile that was comparable to that of the reference standard, DykatussR Capsules was obtained with a formulation employing 8.3% EudragitR NE 30 D, 0.5% talc and 1% polyethylene glycol. In vitro dissolution tests on this formulation showed drug release to be predictable, reproducible and independent of the dissolution methods or media. Short term storage confirmed the stability at room temperature (20°C) and low temperature (5C). Scanning electron micrographs of pellets stored at elevated temperatures i.e. 37°C with 80% relative humidity and 40°C illustrated the phenomenon of 'further gradual coalescence' which corresponded to the decrease in release of drug from the pellets. / Thesis (M.Sc.)-University of Natal, Durban, Westville, 1991.