Introduction: The effect of renal impairment (RI) on systemic exposure is assessed in phase I with RI studies and/or in phase III with population pharmacokinetic analysis. Regulatory review has indicated that the estimated effect of RI from the two methods may differ. Aim: To map the estimated effect of RI on systemic exposure based on phase I and III data, to investigate if the estimated effect based on the two data sources differ and to investigate causes to this potential discrepancy. Methods: Marketing authorisation applications (MAA) were scrutinised with focus on impact of RI on systemic exposure estimated based on phase I and III data. In addition, a simulation-estimation study was performed to explore causes to discrepancies. Phase I and III data were simulated and analysed with non-compartmental analysis (NCA) and population analysis. The phase III data were simulated under several alternative conditions thought to be potential sources for discrepancies,  such as uncertainty in creatinine clearance (CLCR) measurements and varying number of subjects. Results: Six examples were found in MAAs in which a discrepancy was observed, where phase III tended to estimate a lower effect of RI compared with phase I. In the simulation-estimation study, the NCA of phase I data over-predicted the effect of RI on systemic exposure, while the population analysis of phase III data estimated the effect of RI without bias. Uncertainty in CLCR measurement in the phase III data resulted in under-prediction of the effect of RI on systemic exposure. Conclusions: A discrepancy in the estimated effect of RI on systemic exposure between phase I and III was observed in existing MAAs. The NCA of phase I RI study and uncertain CLCR measurements were identified as possible reasons to the discrepancy.

Sildenafil citrate (SC) is a phosphodiesterase-5 inhibitor that is used to treat pulmonary hypertension (PH) in paediatric patients. The purpose of these studies was to develop a formulation and manufacture an orodispersible tablet (ODT) that can be easily administered to neonates and children with PH. The advantages of ODT dosage forms include ease of administration, rapid dissolution of the API, SC. Furthermore the dosage form can be taken without water which is beneficial to patients without immediate access to potable fluids. A simple, rapid, accurate, precise and selective reversed-phase HPLC method was developed and validated in accordance with International Conference on Harmonization (ICH) guidelines and was successfully used for the analysis of SC as raw material and in SC containing pharmaceutical dosage forms. Preformulation studies were performed on SC, alone and in combination with potential excipients that could be used to make tablets. Investigations into potential interactions between SC and the excipients were performed using Differential Scanning Calorimetry (DSC) and Infrared Spectroscopy (IR). DSC results revealed that SC was compatible with all potential excipients except mannitol and magnesium stearate. However these interactions were not observed with IR and therefore it was concluded that the interactions were induced by the high temperatures that DSC operates at. Particle size and shape was also established by use of Scanning Electron Microscopy (SEM) and flow properties were monitored by calculating Carr’s Index (CI) and the Hausner Ratio (HR). Direct compression was used as the method of manufacture for SC tablets as this approach is simple and the most economic production approach. The powder blends were assessed for bulk and tapped density and the CI and HR were used to determine the flowability of the blends. The quality attributes of the resultant tablets that were monitored included uniformity of weight, friability, crushing strength, tensile strength, disintegration, wetting and in vitro dispersion times. Design of Experiments is an efficient statistical approach that has become a popular tool used in the pharmaceutical industry to optimize formulation compositions, as it allows for the investigation of several input factors at the same time whilst not using the tedious and traditional “ modification of one variable at a time” approach. A Central composite experimental design was chosen as the most appropriate means to optimize the formulation as it produces more accurate results as opposed to other experimental designs approaches as input factors are investigated at five different levels. Through the use of mathematical modelling, optimum concentrations of disintegrant(s) and an appropriate blending time were established. Analysis of the data from the experimental design and mathematical modelling studies reveal that no changes in disintegrant concentration or blending time altered the disintegration time of the formulation to any significant extent. This result is most likely due to the fact that the critical disintegrant concentration has been reached and increasing the disintegrant concentration further has no effect on disintegration time. It was also established that a change in the concentration of CMS and CRP altered the wetting time of the tablet significantly. Finally it was noted that there was a linear relationship between blending time and the uniformity of content of the tablets produced in these studies. The optimized product was a white tablet with a diameter of 7.31 mm with a thickness of 2.80mm.The dosage form had no visible cracks or evidence of picking or sticking. The tablet exhibits suitable friability and tensile strength while exhibiting a disintegration time of only 8s. Therefore an orodispersible tablet containing SC intended for paediatric use has been successfully developed, manufactured and optimized through the use of preformulation studies, appropriate quality control monitoring and mathematical modelling. These formulations require further optimization in respect of addition of flavours and or additional sweetening agents.

One of the primary contributing factors to the escalating costs of health care is the high cost of innovator pharmaceutical products. As a consequence, health authorities in various countries and in particular in the developing world have identified generic prescribing and generic substitution as possible strategies to contain the escalating costs of health care provision. There is therefore a need for formulation scientists in developing countries to invest more time in the research and development of generic formulations. Clobetasol 17-propionate (CP) generic cream formulations containing 0.05% w/w of the drug were manufactured and characterized using in vitro testing. Formulation development studies were preceded by the development and validation of an RP-HPLC with UV detection for the quantitation and characterization of CP in innovator and generic cream formulations during formulation development and assessment studies. Furthermore the in vitro release ates of CP release from innovator and generic cream formulations were monitored using a validated in vitro release test method developed in these studies. The formulation of CP cream products was accomplished using a variety of commercially available mixed primary emulsifiers, such as Estol® 1474, Ritapro® 200, Emulcire® 61 WL and Gelot® 64. Successful formulations were selected based on their ability to remain physically stable immediately after manufacture and for 24 hours after storage at room temperature (22°C). Estol® 1474 was found to produce an unstable cream and was therefore not investigated further. The other three emulgents produced stable creams, but only the in vitro release profile of CP from a cream manufactured to contain Gelot® 64 was found to be statistically similar to that of the innovator formulation. Therefore the cream containing Gelot® 64 was selected as the most appropriate prototype generic cream formulation and was characterized in vitro in terms of CP content, viscosity, pH and in vitro release rate. Data generated from these studies were compared to those of the innovator product, Dermovate® cream, using statistical methods. The CP content, pH and in vitro release rate data of the CP formulation were similar to those of the innovator product, however the intrinsic viscosity of Dermovate® cream was almost three (3) times greater than the intrinsic viscosity of the test formulation developed using Gelot® 64. The CP cream formulation developed in these studies was stored for 4 weeks at 40 ± 2°C and 25 ± 5% RH in an incubator and the formulation was found to be stable. A formulation has been developed and assessed and found to be suitable for use as a topical semi-solid dosage form for CP.

Plasmodium falciparum 1–deoxy–D–xylulose–5 phosphatereductoisomerase (PfDXR) plays a role in isoprenoid biosynthesis in the malaria parasite and is absent in the human host, making this parasite enzyme an attractive target for antimalarial drug design. To characterize PfDXR, it is necessary to produce large quantities of the enzyme in a soluble and functional form. However, the over–production of malarial proteins in prokaryotic host systems often results in the formation of truncated proteins or insoluble protein aggregates. A heterologous expression system was developed for the production of active PfDXR using codon harmonization and tight control of expression in the presence of lac repressor. Yields of up to 2 mg/l of enzyme were reported using the optimised expression system, which is 8 to 10– fold greater than previously reported yields. The kinetic parameters Km, Vmax and kcat were determined for PfDXR; values reported in this study were consistent with those reported in the literature for other DXR enzymes. A three–dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure–checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non–apicomplexan DXR proteins. Residues Val44 and Asn45, essential for NADPH binding; and catalytic hatch residues Lys224 and Lys226, which are unique to the species of Plasmodium, were mutated to resemble those of E. coli DXR. Interestingly,these mutations resulted in significant reductions in substrate affinity, when compared to the unmutated PfDXR. Mutant enzymes PfDXR(VN43,44AG) and PfDXR(KK224,226NS) also demonstrated a decreased ability to turnover substrate by 4–fold and 2–fold respectively. This study indicates a difference in the role of the catalytic hatch of PfDXR with regards to the way in which it captures substrates. The study also highlights subtle differences in cofactor binding to PfDXR, compared with the well characterized EcDXR enzyme. The validated PfDXR model was also used to develop a novel efficient in silico screening method for potential tool compounds for use in the rational design of novel DXR inhibitors. Following in silico screening of 46 potential DXR inhibitors, a two–tiered in vitro screening approach was undertaken. DXR inhibition was assessed for the 46 novel compounds using an NADPH– ependant DXP enzyme inhibition assay and antimalarial potential was assessed using P.falciparum–infected erythrocyte growth assays. Select compounds were tested in human cells in order to determine whether they were toxic to the host. From the parallel in silico and in vitro drug screening, it was evident that only a single compound demonstrated reasonable potential binding to DXR (determined using in silico docking), inhibited DXR in vitro and inhibited P. falciparum growth, without being toxic to human cells. Its potential as a lead compound in antimalarial drug development is therefore feasible. Two outcomes were evident from this work. Firstly, analogues of known antimalarial natural products can be screened against malaria, which may then lead towards the rational design of novel compounds that are effective against a specific antimalarial drug target enzyme, such as PfDXR. Secondly, the rational design of novel compounds against a specific antimalarial drug target enzyme can be untaken by adopting a coupled in silico and in vitro approach to drug discovery.

This research has focused on the development of novel substrate mimics as potential DXR inhibitors of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), an essential enzyme in the mevalonate-independent pathway for the biosynthesis of isoprenoids in Plasmodium falciparum. DXR mediates the isomerisation and reduction of 1-deoxy-D-xylulose-5-phosphate (DOXP) into 2C-methyl-D-erithrytol 4-phosphate (MEP) and has been validated as an attractive target for the development of novel anti-malarial chemotherapeutic agents. Reaction of various amines with specially prepared 4-phosphonated crotonic acid in the presence of the peptide coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), has afforded a series of amido-phosphonate esters in moderate to good yields (48% - 73%) which, using a RuCl₃/CeCl₃/NaIO₄ catalyst system, have been dihydroxylated to furnish the dihydroxy-amido phosphonate ester pro-drugs; subsequent hydrolysis under microwave irradiation has afforded the corresponding phosphonic acids. A second series of potential inhibitors viz., 3-substituted aniline-derived phosphonate esters, their corresponding phosphonic acids and mono-sodium salts, have also been successfully synthesised. In these compounds, the essential functional groups are separated by one, two, three or four methylene groups, Deprotonation of the 3-substituted aniline substrates, followed by reaction with the appropriate ω-chloroalkanoyl chloride produced the ω-chloroamide intermediates, which were subjected to the Michaelis-Arbuzov reaction to afford the diethyl phosphonate esters in moderate to good yields (48% - 74%). Microwave-assisted TMSBrmediated cleavage of the phosphonate esters furnished the phosphonic acids, neutralisation of which afforded the mono-sodium salts. Furan-derived phosphate esters and phosphonic acids have been prepared as conformationally-restricted DOXP analogues. Functionalization at C-5 of the trityl-protected furan was achieved using the Vilsmeier-Haack formylation and Friedel-Crafts acylation reactions and, following de-tritylation, phosphorylation and oximation, using hydroxylamine hydrochloride, the novel oxime derivatives have been isolated as a third series of potential DXR inhibitors in very good yields (87% - 96%). Finally, in order to exploit an additional binding pocket in the PƒDXR active site, a series of N-benzylated phosphoramidic derivatives were obtained in seven steps from the starting material, diethyl phosphoramidate. The known inhibitors, fosmidomycin and its acetyl derivative FR900098, were also successfully synthesised as standards for STD-NMR binding and inhibition assays. In all, over 200 compounds (136 novel) have been prepared and appropriately characterised using 1-and 2-D NMR and IR spectroscopic analysis and, where necessary, HRMS or combustion analysis. Saturation Transfer Difference (STD) protein-NMR experiments, undertaken using selected compounds, have revealed binding of most of the ligands examined to EcDXR. Computersimulated docking studies have also been used to explore the preferred ligand-binding conformations and interactions between the ligands and essential DXR active-site residues, while DXR-enzyme inhibition assays of selected synthesised ligands have revealed certain patterns of inhibitory activity.

Using a mechanism-based approach to drug discovery, efforts have been directed towards developing novel ATP mimics that can act as GS inhibitors. The purine-based systems, adenosine, adenine and allopurinol, were identified as possible scaffolds for potential ATP mimics, while various meta-disubstituted benzenoid compounds, 3-aminobenzonitrile, 3-aminophenol, resorcinol, 3-aminobenzyl alcohol, 3-hydroxybenzoic acid and 3-aminobenzoic acid have been explored as adenine analogues. These compounds were treated with different alkylating and acylating agents. Allylation of all the substrates was achieved using allyl bromide and N-9 alkylation of protected allopurinol was effected using a number of specially prepared Baylis-Hillman adducts. Acylation of the benzenoid precursors with chloroacetyl chloride, acetoxyacetyl chloride, acryloyl chloride and specially prepared 2,3,4,5,6-pentaacetylgluconoyl chloride afforded the corresponding mono- and /or diacylated products in varying yields (4-96%). Elaboration of the alkylated and acylated products has involved the reaction of hydroxy systems with diethyl chloro phosphate and chloro derivatives with triethyl phosphite in Arbuzov-type reactions to afford phosphorylated products. In all cases, products were fully characterized using 1- and 2-D NMR analysis and, where appropriate, high-resolution mass spectrometry. The application of Modgraph and ChemWindow NMR prediction programmes has been explored and the resulting data have been compared with experimental chemical shift assignments to confirm chemical structures and, in some cases, to establish the position of allylation or acylation. Experimental assignments were found to be generally comparable with the Modgraph data, but not always with the ChemWindow values. The docking of selected products in the 'active-site' of GS and their structural homology with ATP, both in their free and bound conformations have been studied using the ACCELERYS Cerius² platform. All the selected ATP mimics exhibit some form of interaction with the 'active-site' residues, and a number of them appear to be promising GS ligands.

Magister Pharmaceuticae - MPharm / Artemisia Afra, a popular South African traditional herbal medicine is commonly administered as a tea infusion of the leaves. However, clinical trials proving it safety and efficacy are lacking mainly due to the absence of good quality dosage forms and credible placebos for the plant. The objectives of this study were to prepare a standardized preparation of the plant leaves and freeze-dried aqueous extract powder of the leaves, in a tea bag dosage form and to design and prepare credible placebos for these plant materials. / South Africa

A amiodarona é um dos fármacos mais usados para o tratamento de arritmias cardíacas, tanto ventriculares como supraventriculares. Apesar de sua eficácia, o uso da amiodarona está associado a vários efeitos adversos, incluindo a toxicidade pulmonar. O mecanismo pelo qual a amiodarona causa lesão nas células pulmonares humanas não é inteiramente conhecido, mas estudos em cultura de células hepáticas humanas e pulmonares de ratos têm sugerido que a disfunção mitocondrial e o estresse oxidativo têm um papel importante na citotoxicidade da amiodarona. Os compostos fenólicos, incluindo catequina e epicatequina são amplamente distribuídos na natureza e conhecidos por sua capacidade de reduzir o estresse oxidativo. Além disso, alguns compostos fenólicos são capazes de modular a atividade mitocondrial. Em vista disso, o objetivo deste trabalho foi avaliar a capacidade dos compostos fenólicos catequina e epicatequina em a disfunção mitocondrial e os danos oxidativos causados pela amiodarona em células de fibroblasto de pulmão humano (MRC-5). Para atingir os objetivos as células MRC-5 foram tratadas com diferentes concentrações de catequina e epicatequina e após foram expostas a amiodarona 100 μM. A disfunção mitocondrial foi determinada através da atividade do complexo I da cadeia de transporte de elétrons e a biossíntese de ATP usando kits específicos. A viabilidade celular foi avaliada através do ensaio de 3-[4,5- dimetiltiazol 2-il]-2,5 difenil brometo de tetrazolina. A atividade das enzimas superóxido dismutase e catalase foram determinadas espectrofotometricamente. Os danos oxidativos a lipídeos e proteínas foram verificados através dos ensaios de substâncias reativas ao acido tiobarbitúrico e a proteínas carboniladas, respectivamente, e os níveis de óxido nítrico foram avaliados usando o método de Griess. Os resultados mostraram que a amiodarona inibiu a atividade do complexo I da cadeia de transporte de elétrons em 53% e a biossíntese de ATP em 9,5% e tanto a catequina como a epicatequina foram capazes de evitar estes efeitos em todas as concentrações (5, 10, 20 μM) testadas. Verificou-se que a amiodarona reduziu a atividade das enzimas superóxido dismutase e catalase (indicando produção de superóxido e peróxido de hidrogênio) e aumentou os danos oxidativos a lipídeos e proteínas. Os compostos fenólicos catequina e epicatequina foram capazes de minimizar as alterações no metabolismo redox induzidos pela amiodarona e aumentar a viabilidade nas células MRC-5. Catequina e epicatequina reduziram a depleção de óxido nítrico causada pela amiodarona. Este trabalho mostrou, pela primeira vez, que o mecanismo de toxicidade da amiodarona em células MRC-5 está associado à disfunção mitocondrial, principal causa de geração de dano oxidativo celular e que estes efeitos tóxicos são em parte reduzidos pela catequina e epicatequina. Embora outros estudos sejam necessários, estes dados abrem novas perspectivas para estudos visando o desenvolvimento de medicamentos que minimizem os efeitos tóxicos da amiodarona. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES. / Amiodarone is among the most widely used drugs for the treatment of ventricular and supraventricular cardiac arrhythmias. However, the use of amiodarone is associated with several side effects including pulmonary toxicity. The mechanism of amiodarone toxicity is not well known, but studies in human liver cells and rats lung cells have been suggested that mitochondrial dysfunction and oxidative stress play important role in the amiodarone cytotoxicity. Phenolic compounds, including catechin and epicatechin are widespread in nature and known for their ability to reduce oxidative stress. In addition, some phenolic compounds are able to modulate mitochondrial activity. Therefore, the objective of this study was to evaluate the ability of phenolic compounds catechin and epicatechin to minimize the mitochondrial dysfunction and oxidative damage induced by amiodarone in human lung fibroblast cells (MRC-5). To achieve the objectives, MRC-5 cells were treated with different concentrations of catechin and epicatechin and then amiodarone 100 μM. Mitochondrial dysfunction was determined by the activity of complex I of the electron transport chain and ATP biosynthesis using specific kits. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The activity of the enzymes superoxide dismutase and catalase were determined spectrophotometrically. The oxidative damage to lipids and proteins have been verified through the test substances reactive to the thiobarbituric acid and carbonyl protein, respectively, and nitric oxide levels were evaluated using the Griess method. The results showed that amiodarone inhibit 53% of the activity of complex I of the electron transport chain and 9.5% of ATP biosynthesis and both catechin and epicatechin were able to avoid these effects in all concentrations (5 10, 20 mM) tested. It was found that amiodarone reduced the superoxide dismutase and catalase activities (indicating the production of radicals superoxide and hydrogen peroxide) and increased oxidative damage to lipids and proteins. Phenolic compounds catechin and epicatechin were able to minimize alterations in the redox metabolism and increase in viability of MRC-5 cells. Furthermore, catechin and epicatechin reduced nitric oxide depletion caused by amiodarone. This study showed, for the first time, that toxicity of amiodarone in human lung cultured cells is associated, at least, in part, with mitochondrial dysfunction which was avoided by catechin and epicatechin. Although further studies are needed, these data open new perspectives for studies aiming the development of drugs that minimize the toxic effects of amiodarone.

The diploma thesis is focused on the analysis and evaluation of risks in the introducing of new drug in the case of unexpected mass infections from the point of view of black swans. This thesis acquaints itself with the current state of the problematics, explains the terminology and meaning of black swans and gives examples of biological black swans. On the basis of the analysis and the available informations is proposed a risk assessment approach together with proposals how to reduce or eliminate possible risks.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12801号 / 論医博第2073号 / 新制||医||1001(附属図書館) / 80845 / 京都大学大学院薬学研究科創薬科学専攻 / (主査)教授 川上 浩司, 教授 松原 和夫, 教授 今中 雄一 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM