The influence of hyperlipidemia on the pharmacodynamic and pharmacokinetic aspects of lipophilic drugs was explored. The antiarrhythmic, amiodarone, and the antifungal, ()-ketoconazole, were used as model drugs. Experimental hyperlipidemia was induced in rat using poloxamer 407 and two sensitive novel HPLC assays were developed. In a multiple dosing study, hyperlipidemia increased amiodarone plasma concentrations, heart concentrations and electrocardiographic changes. The amiodarone heart uptake could not be totally attributed to its unbound fraction, where the cardiac very low density lipoprotein receptors seemed to play a role in the uptake of bound drug. Amiodarone liver metabolism in presence and absence of hyperlipidemia was studied using isolated primary rat liver hepatocytes. The metabolism of amiodarone was lower in hepatocytes isolated from hyperlipidemic than those from normolipidemic rats. Hyperlipidemic serum resulted in a decrease in amiodarone metabolism and when coincubated, the expected decrease in unbound fraction seemed to resulted in greater inhibition of metabolism. ()-Ketoconazole showed stereoselectivity in its pharmacokinetics in rat with (+)-ketoconazole showing higher plasma concentrations than its antipode. This was attributed to its higher protein binding. There was no difference in the total bioavailability of the two enantiomers. Ketoconazole enantiomers exhibited nonlinear pharmacokinetics. In normolipidemic rat plasma ketoconazole enantiomers were more than 95% bound to lipoprotein deficient fraction. Hyperlipidemia resulted in shifting both enantiomers 20% to very low density and low density lipoprotein fractions. In a pharmacokinetic assessment, hyperlipidemia was found to increase ketoconazole enantiomer volume of distribution. Moreover, the stereoselectivity ratios of most pharmacokinetic parameters were changed. After oral dosing, the uptake of (-)-ketoconazole was significantly decreased. Since ketoconazole is used as a potent CYP3A inhibitor, alteration in liver concentrations of (-)-ketoconazole, the more potent inhibitory enantiomer, could decrease its CYP inhibitory potential. Hyperlipidemia potentiated the CYP-mediated interaction between ketoconazole and midazolam with significantly higher midazolam AUC and lower clearance. This was attributed to the inhibitory action of ketoconazole and the effect of hyperlipidemia on the binding of midazolam. Hyperlipidemia was found to unexpectedly decrease midazolam unbound fraction in plasma. In conclusion, the findings could explain some unexpected dose versus effect outcomes in hyperlipidemic patients receiving amiodarone or ketoconazole. / Pharmaceutical Sciences

A set of algorithms were designed, implemented and evaluated in order to, first, identifyclusters of conserved waters in binding pockets, i.e. hydration sites. Then, their contributionto the free energy of binding in a ligand-protein association was quantified by calculatingtheir enthalpy and entropy. The information obtained by using these algorithms couldcontribute to the development of new drugs by generating new ligands that target specifichigh-energy, unfavorable waters. Evaluation tests show that our algorithms can indeedprovide relevant data about how hydration sites influence ligand-protein binding.

The minimization of blood loss represents a significant clinical need in the arena of surgery, trauma, and emergency response medicine. Fibrinogen is our body's native polymer system activated in response to tissue and vasculature injury, and forms the foundation of the most widely employed surgical sealant and hemostatic agent. Non-covalent knob:hole interactions are central to the assembly of fibrin that leads to network and clot formation. This project exploits these affinity interactions as a strategy to direct fibrin polymerization dynamics and network structure so as to develop a temperature-triggered polymerizing fibrin mixture for surgical applications. Short peptides modeled after fibrin knob sequences have been shown to alter fibrin matrix structure by competing with native fibrin knobs for binding to the available holes on fibrinogen and fibrin. The fusion of such knob peptides to a non-native component should facilitate binding of the fused component to fibrinogen/fibrin, and may permit the concomitant modification of the fibrin matrix. We examined this hypothesis in a three-step approach involving (a) analyzing the ability of tetrapeptide knob sequences to confer fibrin(ogen) affinity on a non-fibrin protein, (b) investigating the effect of knob display architecture on fibrin(ogen) structure, and (c) designing a temperature-responsive knob-displaying construct to modulate fibrin(ogen) affinity at different temperature regimes, thus altering fibrin(ogen) structure.

The bacterial RNA binding protein Hfq is a key component for bacterial sRNA mediated riboregulation of mRNA expression. A kinetic and thermodynamic analysis of Hfq binding to its sRNA targets DsrA, RprA, and OxyS, and to its mRNA target rpoS was carried out. The ability of Hfq to significantly enhance the stability of the DsrA-rpoS and RprA-rpoS complex was demonstrated, and the entire untranslated leader region of rpoS was shown to be important for Hfq binding and in Hfq facilitated sRNA-mRNA duplex formation. Hfq was not shown to enhance OxyS binding to rpoS. DsrA and OxyS were shown to bind mostly to the proximal surface region of Hfq, while RprA bound to both proximal and distal surface regions. The rpoS leader region was shown to possess at least two distinct Hfq binding sites, with one site binding the proximal region and the other to the distal region of Hfq. These sites were shown to be important for Hfq to stimulate DsrA-rpoS binding. The outer-circumference region and the C-terminal tail of Hfq does not play a major role in binding DsrA, RprA, OxyS and rpoS, and in stimulating DsrA-rpoS binding. Evidence was obtained implicating Hfq to bind DsrA, RprA, OxyS, and oligo rA18 in a 1:1 protein to RNA stoichiometry. Binding properties of Hfq to E. coli genomic DNA were examined. Double stranded DNA was shown to bind mostly on the distal surface region and the C-terminal tail of Hfq with an affinity 10 fold less than Hfq targeted RNA. Single stranded DNA binds Hfq more tightly than double stranded DNA and binding seems to be sequence specific. Evidence indicates Hfq binds certain sequences of the E. coli genome.

Polyelectrolyte complexation is a rapidly growing field with applications in functional multilayer (PEM) and nanoparticle (PEC) generation, where PEM films are deposited using Layer-by-Layer technique initiated by Decher and PECs are prepared using mixing-centrifugation technique initiated by our group. Its advantages (e.g. easy preparation) result in various applications in aqueous solution, especially in pharmaceutical and biomedical fields. Therefore, the objectives in this study are to explore interesting applications of polyelectrolyte complexation in the field of low molecular chiral compound and high molecular protein binding. Due to the rapidly growing demands for preparing optically pure compounds in the pharmaceutical field, herein, enantiospecific PEM and PEC were prepared using chiral polyelectrolytes (e.g. homo-polypeptide) and their ability of chiral recognition was investigated by ATR-FTIR, UV/Vis etc.. Chiral PEM and PEC showed pronounced enantiospecificity for both small (amino acids, vitamin) and large (protein) chiral compounds. This chiral recognition is performed by a diffusion process of chiral compounds into PEM based on the structures of chiral selector (PEM, PEC) and chiral probes (chiral compounds). However, the influences, e.g. pH value, ionic strength, surface orientation etc., were found to affect significantly the enantiospecificity. Beside planar substrates, porous membranes (e.g. PTFE) were modified using chiral PEM and successfully applied in enantiospecific permeation. Additionally, protein binding properties of PEC particle dispersions or PEC particle films were also studied. Due to the properties of polyelectrolytes used for PEC (e.g. molecular weight, charge density) and proteins (e.g. isoelectric point, size, hydrophobicity), PEC showed different uptake characteristics towards different proteins. Electrostatic and hydrophobic interaction as well as counterion release force were considered as possible driving forces for protein binding.

No description available.

Tuberculosis (TB) is an infectious, deadly disease, caused by Mycobacterium tuberculosis (M.tb). In 2010, there were 8,8 million incident cases of TB globally. South Africa currently has the third highest TB incident cases worldwide. In an attempt to address the challenges facing TB chemotherapy, among which frequent dosing and long duration of therapy resulting in poor patient compliance, a novel poly(DL-lactic-co-glycolic) acid (PLGA) nanoparticulate drug delivery system (DDS) encapsulating anti-TB drugs was developed. It is hypothesised that this nanoparticulate DDS will address the challenges mentioned by enabling decreased dosing frequency, shortening duration of therapy and minimising adverse side effects. Therefore, favourable modification of pharmacodynamic (PD) and pharmacokinetic (PK) properties of the conventional anti-TB drugs was demonstrated. Furthermore, the nanoparticles will provide a platform for drug delivery to macrophages that serve as hosts for M.tb. The study design was based on determining specific physicochemical properties of the nanoparticulate DDS to elucidate the hypothesis. Spray-dried PLGA nanoparticles were prepared using the double emulsion solvent evaporation technique. In vivo analysis of macrophage uptake and possible immunological response in mice were evaluated. In vitro protein-binding assays of PLGA nanoparticles encapsulating anti-TB drugs isoniazid (INH) and rifampicin (RIF) were performed with subsequent in vivo tissue distribution assays to support protein-binding data generated. Finally, PK/PD analyses were conducted to evaluate the effect of nanoencapsulation on the anti-TB drugs. These involved in vitro assays to determine if sufficient drug was released from the nanoparticles to exhibit minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC). Furthermore, in vivo drug distribution and drug release kinetics assays of encapsulated RIF, INH, pyrazinamide (PZA) and ethambutol (ETB) in a mouse model were performed. The results confirmed that the PLGA nanoparticles (<250 nm, low positive zeta potential) were taken up by macrophages in vivo with no significant immunological effect. Furthermore the nanoparticles were present in the brain, heart, kidneys, lungs, liver and spleen for up to 7 days following once-off oral dosing at 13.23± 0.11%, 16.81± 0.11%, 54.89± 0.95%, 15.61± 1.15%, 48.48± 2.28% and 5.73± 0.21%, respectively. This was further confirmed by drug analysis demonstrating the presence of INH, RIF and ETB at different time points up to 7 days in the lungs, kidneys, liver and spleen. However, PZA was not detected. Nanoencapsulated RIF and INH exhibited MICs and MBCs in vitro over 14 days and these drugs were also observed in plasma for up to 7 days post once-off oral dosing. ETB and PZA were observed up to 3 days. From the results generated, it can be concluded that the nanoparticles were taken up by macrophages without eliciting an immune response. This provides a platform for drug delivery to specific sites. Furthermore, the nanoparticulate DDS exhibited sustained drug release in vitro and in vivo over a number of days above the MIC for the drugs analysed. Sustained drug distribution was also observed. It can therefore be concluded that the hypothesised reduction in dose frequency and duration of therapy for this DDS is a possibility / Thesis (PhD (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013

Tuberculosis (TB) is an infectious, deadly disease, caused by Mycobacterium tuberculosis (M.tb). In 2010, there were 8,8 million incident cases of TB globally. South Africa currently has the third highest TB incident cases worldwide. In an attempt to address the challenges facing TB chemotherapy, among which frequent dosing and long duration of therapy resulting in poor patient compliance, a novel poly(DL-lactic-co-glycolic) acid (PLGA) nanoparticulate drug delivery system (DDS) encapsulating anti-TB drugs was developed. It is hypothesised that this nanoparticulate DDS will address the challenges mentioned by enabling decreased dosing frequency, shortening duration of therapy and minimising adverse side effects. Therefore, favourable modification of pharmacodynamic (PD) and pharmacokinetic (PK) properties of the conventional anti-TB drugs was demonstrated. Furthermore, the nanoparticles will provide a platform for drug delivery to macrophages that serve as hosts for M.tb. The study design was based on determining specific physicochemical properties of the nanoparticulate DDS to elucidate the hypothesis. Spray-dried PLGA nanoparticles were prepared using the double emulsion solvent evaporation technique. In vivo analysis of macrophage uptake and possible immunological response in mice were evaluated. In vitro protein-binding assays of PLGA nanoparticles encapsulating anti-TB drugs isoniazid (INH) and rifampicin (RIF) were performed with subsequent in vivo tissue distribution assays to support protein-binding data generated. Finally, PK/PD analyses were conducted to evaluate the effect of nanoencapsulation on the anti-TB drugs. These involved in vitro assays to determine if sufficient drug was released from the nanoparticles to exhibit minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC). Furthermore, in vivo drug distribution and drug release kinetics assays of encapsulated RIF, INH, pyrazinamide (PZA) and ethambutol (ETB) in a mouse model were performed. The results confirmed that the PLGA nanoparticles (<250 nm, low positive zeta potential) were taken up by macrophages in vivo with no significant immunological effect. Furthermore the nanoparticles were present in the brain, heart, kidneys, lungs, liver and spleen for up to 7 days following once-off oral dosing at 13.23± 0.11%, 16.81± 0.11%, 54.89± 0.95%, 15.61± 1.15%, 48.48± 2.28% and 5.73± 0.21%, respectively. This was further confirmed by drug analysis demonstrating the presence of INH, RIF and ETB at different time points up to 7 days in the lungs, kidneys, liver and spleen. However, PZA was not detected. Nanoencapsulated RIF and INH exhibited MICs and MBCs in vitro over 14 days and these drugs were also observed in plasma for up to 7 days post once-off oral dosing. ETB and PZA were observed up to 3 days. From the results generated, it can be concluded that the nanoparticles were taken up by macrophages without eliciting an immune response. This provides a platform for drug delivery to specific sites. Furthermore, the nanoparticulate DDS exhibited sustained drug release in vitro and in vivo over a number of days above the MIC for the drugs analysed. Sustained drug distribution was also observed. It can therefore be concluded that the hypothesised reduction in dose frequency and duration of therapy for this DDS is a possibility / Thesis (PhD (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013

Inhibitor of growth (ING) proteins have been classified as type II tumour suppressor proteins due to their ability to facilitate cellular events such as chromatin remodelling, apoptosis, angiogenesis, DNA replication, DNA repair, cell cycle progression, cell senescence and hormone response regulation. These processes are all associated with combating oncogenesis; conversely, recent evidence suggesting that ING proteins also function as oncogenes in certain cancers has spurred the investigation of ING proteins as potential anticancer targets. In order to better understand the complex role ING proteins play in the cell, the mechanisms that direct ING proteins to the chromatin template require extensive study. This dissertation investigates the role the chromatin environment plays in recruiting ING proteins by characterizing the interaction between ING proteins and chromatin. ING proteins have been shown to interact with the histone H3 lysine 4 trimethylated (H3K4me3) epigenetic mark through binding studies between peptides comprising the ING plant homeodomain (PHD) finger and the H3 N-terminal tail. However, these studies do not take into account the effect of organizing H3 into a nucleosome or the effect of the remaining ING protein structural domains. In order to address these elements, this dissertation describes binding studies between the PHD finger of Yng1 (Yng1PHD) and H3K4me3 in the context of a nucleosome, and between full-length Xenopus laevis ING1 (xING1) and H3K4me3 in the context of a nucleosome. A 6XHis tagged xING1 protein was purified, Yng1PHD was obtained from Dr. Leanne Howe, and an analog of H3K4me3 (H3KC4me3) was installed into recombinant H3 protein and used to reconstitute nucleosomes. Affinity-tag based anti-Yng1PHD and anti-xING1 pull-down assays were then used to display an in vitro H3K4 methylation-dependent interaction between Yng1PHD / xING1 and H3KC4me3 containing nucleosomes. In addition, analytical ultracentrifuge (AUC) analysis of the xING1 protein displayed the presence of 3 species containing sedimentation coefficients consistent with those that would be expected from monomeric, dimeric and tetrameric forms of xING1. Several studies have focused on the interaction between ING proteins and DNA binding proteins such as transcription factors and hormone receptors which recruit ING proteins to specific genes. However, little knowledge is available regarding the role chromatin plays in recruiting ING proteins with the exception of the interaction between the ING PHD fingers and H3K4me3. This dissertation addresses this gap in knowledge by investigating the nature of chromatin bound by the human ING1b (hING1b) protein. For this purpose, HEK293 cells were transfected with a Flag-hING1b construct. Upon fractionation of the HEK293 chromatin, Flag-hING1b was found to localize exclusively to the “Pellet” fraction. ChIP analysis of the HEK293 chromatin showed that Flag-hING1b bound nucleosomes were deprived of H3K9me3, H3K27me3 and H3S10P, contained no enrichment for H3K4me3 and H3K36me3, and were significantly enriched for H2A.Z. Lastly, a hING1b-GFP construct was transiently transfected into SKN-SH human neuroblastoma cells and found to be evenly distributed throughout the nucleus with moderate enrichment on chromatin and within the nucleolus. / Graduate

The P22 c2 repressor protein (P22R) binds to DNA sequence-specifically and helps direct the temperate lambdoid bacteriophage P22 to the lysogenic developmental pathway. To gain insight into its DNA binding mechanism, we solved the 1.6 Å x-ray structure of the N-terminal domain (NTD) of P22R in a complex with a DNA fragment containing the synthetic operator sequence [d(ATTTAAGATATCTTAAAT)]2 This operator has an A-T at position 9L and T-A at position 9R and is termed DNA9T. Van der Waals interactions between protein and DNA appear to confer sequence-specificity. The structure of the P22R NTD – NA9T complex suggests that sequence-specificity arises substantially from interaction of a valine with a complementary binding cleft on the major groove surface of DNA9T. The cleft is formed by four methyl groups on sequential base pairs of 5' TTAA 3'. The valine cleft is intrinsic to the DNA sequence and does not arise from protein-induced DNA conformational change. Protein-DNA hydrogen bonding plays a secondary role in specificity.