This study focused on two active pharmaceutical ingredients (APIs) that are used to treat two of the most notorious diseases in Africa, i.e. human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) and malaria. It is well known that many African countries lack effective regulatory control over medicines and patients are subsequently at risk of receiving sub-standard treatments. This study set out to investigate how the modification of the crystal packing (i.e. polymorphism) of these APIs may impact on their quality, safety and efficacy. Efavirenz (an antiretroviral) and Pyrimethamine (an antimalarial) were selected as the two model APIs for investigation during this study. It was found that a novel amorphous form (Form A) of Efavirenz had been prepared during this study through quench cooling. Form A was extensively characterised and compared to the preferred crystalline Form I, with the aim of providing a means of distinguishing between these two Efavirenz forms. In contrast to popular belief (that amorphous form should have improved dissolution and solubility properties over the crystalline counterpart), the powder dissolution of Form A was significantly lower than that of Form I. Further investigation indicated that this was due to the occurrence of agglomeration and phase-mediated transformation. This observation had led to the belief that Form A had poor thermodynamic stability. The glass transition temperature and the crystallisation activation energy, required for the recrystallisation of Form A, were subsequently determined in an attempt to elucidate its thermodynamic stability. The glass transition temperature of Form A was found to be unfeasibly low, hence confirming its tendency towards agglomeration. The crystallisation activation energy of Form A was determined by non-isothermal determinations, using differential scanning calorimetry (DSC), hot stage microscopy (HSM) and capillary melting point (CMP) analysis. These studies not only elucidated the required activation energy for the conversion of Form A into Form I, but it also found that the results from CMP were similar to those of the universally accepted DSC technique, allowing for the proposal of CMP as a cost-effective alternative to DSC for the quantitative measurement of the crystallisation of Efavirenz. Isothermal studies revealed that Form A had a short half-life, which, together with its poor dissolution performance, exemplified why this form was unsuitable for pharmaceutical use. The Pyrimethamine study focused on recrystallisation as a means of modifying its crystal packing and on an evaluation of the effect that such crystal modification may have on its safety and manufacturability. Anhydrous Pyrimethamine was recrystallised, using methanol, acetone, n-propanol, ethanol, N,N-dimethylformamide and N,N-dimethylacetamide. Ethanol, acetone and n-propanol altered the crystal habit of Pyrimethamine, without any modification of its crystal lattice. The different habits exhibited clear differences in flowability and compressibility, which could in turn affect manufacturing and therefore the quality of the finished pharmaceutical product (FPP). These habits were subsequently extensively characterised by means of in-silico molecular modelling predictions. It was found that recrystallisation from methanol, N,N-dimethylformamide and N,N-dimethylacetamide had resulted in solvatomorphism. These solvatomorphs contained their respective solvents in concentrations exceeding the allowed residual solvent limits, as set by the International Conference on Harmonisation (ICH) guidelines. These undesirable solvatomorphs were also comprehensively characterised as a service to the pharmaceutical industry, in order to identify the distinct characteristics that distinguish these forms from the preferred non-toxic form, and to provide techniques for transforming the toxic forms into the non-toxic form. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

This study focused on two active pharmaceutical ingredients (APIs) that are used to treat two of the most notorious diseases in Africa, i.e. human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) and malaria. It is well known that many African countries lack effective regulatory control over medicines and patients are subsequently at risk of receiving sub-standard treatments. This study set out to investigate how the modification of the crystal packing (i.e. polymorphism) of these APIs may impact on their quality, safety and efficacy. Efavirenz (an antiretroviral) and Pyrimethamine (an antimalarial) were selected as the two model APIs for investigation during this study. It was found that a novel amorphous form (Form A) of Efavirenz had been prepared during this study through quench cooling. Form A was extensively characterised and compared to the preferred crystalline Form I, with the aim of providing a means of distinguishing between these two Efavirenz forms. In contrast to popular belief (that amorphous form should have improved dissolution and solubility properties over the crystalline counterpart), the powder dissolution of Form A was significantly lower than that of Form I. Further investigation indicated that this was due to the occurrence of agglomeration and phase-mediated transformation. This observation had led to the belief that Form A had poor thermodynamic stability. The glass transition temperature and the crystallisation activation energy, required for the recrystallisation of Form A, were subsequently determined in an attempt to elucidate its thermodynamic stability. The glass transition temperature of Form A was found to be unfeasibly low, hence confirming its tendency towards agglomeration. The crystallisation activation energy of Form A was determined by non-isothermal determinations, using differential scanning calorimetry (DSC), hot stage microscopy (HSM) and capillary melting point (CMP) analysis. These studies not only elucidated the required activation energy for the conversion of Form A into Form I, but it also found that the results from CMP were similar to those of the universally accepted DSC technique, allowing for the proposal of CMP as a cost-effective alternative to DSC for the quantitative measurement of the crystallisation of Efavirenz. Isothermal studies revealed that Form A had a short half-life, which, together with its poor dissolution performance, exemplified why this form was unsuitable for pharmaceutical use. The Pyrimethamine study focused on recrystallisation as a means of modifying its crystal packing and on an evaluation of the effect that such crystal modification may have on its safety and manufacturability. Anhydrous Pyrimethamine was recrystallised, using methanol, acetone, n-propanol, ethanol, N,N-dimethylformamide and N,N-dimethylacetamide. Ethanol, acetone and n-propanol altered the crystal habit of Pyrimethamine, without any modification of its crystal lattice. The different habits exhibited clear differences in flowability and compressibility, which could in turn affect manufacturing and therefore the quality of the finished pharmaceutical product (FPP). These habits were subsequently extensively characterised by means of in-silico molecular modelling predictions. It was found that recrystallisation from methanol, N,N-dimethylformamide and N,N-dimethylacetamide had resulted in solvatomorphism. These solvatomorphs contained their respective solvents in concentrations exceeding the allowed residual solvent limits, as set by the International Conference on Harmonisation (ICH) guidelines. These undesirable solvatomorphs were also comprehensively characterised as a service to the pharmaceutical industry, in order to identify the distinct characteristics that distinguish these forms from the preferred non-toxic form, and to provide techniques for transforming the toxic forms into the non-toxic form. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Crystal engineering has advanced the strategies of design and synthesis of organic solids with the main focus being on improving the properties of the developed materials. Research in this area has a significant impact on large-scale manufacturing as industrial processes may give rise, at various stages, to stress-induced transformations and product modification. This thesis investigates the solid-state properties at play in the case of the surface and structural reorganization which results from the stress within a crystal during the drying of labile multicomponent organic solids. Chapter 1 introduces various concepts in solid-state chemistry and explores their application in the manufacture of solid pharmaceuticals. The significance of stress-induced transformations during the drying process is illustrated by reactions associated with crystal decomposition processes such as dehydration, desolvation and sublimation. The chapter also introduces carbamazepine (CBZ) multicomponent materials as models for the studies of stress-induced transformations. Chapter 2 presents the experimental section of the work and describes the materials, methods and equipment used for the study. Chapter 3 presents the analysis of the various crystal structures of CBZ. The crystal forms are classified with an emphasis on a comparison of intermolecular interactions, coformer arrangement, crystal packing and the geometric parameters of slip/cleavage planes within the crystals. Chapter 4 details the experimental methods for preparation of the samples. Cooling solution crystallization was the standard method which has been selected, and crystal habit and surface variations have been studied as a function of the solution concentration and the crystallization environment. Attention is given, in particular, to the preparation of carbamazepine dihydrate and the specific cocrystals carbamazepine cocrystals formed with benzoquinone and oxalic acid. Chapter 5 is devoted to the dehydration of carbamazepine dihydrate for samples prepared and examined in approximate 1-gram laboratory scale quantities. It explores the effect of vacuum, temperature, humidity and seeding on the surface and bulk properties of the products. Chapter 6 presents the solid-state characterization results obtained for samples crystallized at a much larger scale (ca. kilogram quantities) with a particular emphasis placed on their mechanical properties. It explores the comparison of large scaled batches with laboratory scale samples in order to obtain a greater understanding of how small-scale laboratory studies may be extrapolated to more commercial processes. Chapter 7 present results on the stress-induced transformations of carbamazepine solvates and cocrystals. It details the effect of thermal decomposition on the surface and bulk properties of the products, possible seeding effects, and the interconversion between carbamazepine dihydrate and carbamazepine benzoquinone cocrystal. Chapter 8 combines the research findings concerning the structural analyses of the materials in the context of current literature. Limitations related to the use of carbamazepine as a model and to the experimental set-up are also explored. In the final chapter conclusions are presented which correlate observations made on the crystallization and decomposition of multicomponent materials operating at small-scale to effects appropriate to manufacturing of pharmaceuticals at large scale.

PEGylation has been used for decades to enhance the pharmacokinetic properties of protein therapeutics. This method has been effective at increasing the serum half-life of these drugs, but the mechanism of how it does this is unclear. Chapter 1 is an introduction to the methods of PEGylation. In chapter 2 we show that the effect of PEGylation on the conformational stability of the WW domain differs based on amino acid linker and conjugation site. We show that all positions in the WW domain that were tested can be stabilized by at least one amino acid linker. The rate of proteolysis is proportional to the degree of conformational stability. Chapter 3 shows that PEG-based desolvation can increase the strength of the interaction between two salt bridge residues, though the effect of structural context is unclear. A crystal structure shows that PEG occupies the space between the PEGylation site and the salt bridge, displacing water. In Chapter 4 we discuss the effect that PEGylation has on the interaction strength of a solvent exposed hydrophobic patch. When the c Log P of the hydrophobic patch increases, PEG increases the conformational stability of the WW domain more dramatically. Chapter 5 is about the effect of PEG based desolvation on the strength of an NH-π hydrogen bond in the WW domain between Trp11 and Asn26. When Trp11 is mutated to Phe, Tyr and naphthylalanine (Nal), the melting temperatures correlate with the calculated interaction energies between the sidechain arene of the hydrogen bond acceptor and formamide. When Asn26 is PEGylated in the presence of each of these amino acids, the effect that PEG has on the conformational stability of the WW domain correlates with the melting temperature of the nonPEGylated variants, the calculated interaction energies, the arene molecular polarizability, and the arene molar volume.

No description available.

<p>The intricate interplay of biomolecules acting together, rather than alone, provides insight into the most basic of cellular functions, such as cell signaling, metabolism, defense, and, ultimately, the creation of life. Inherent in each of these processes is an evolutionary tendency towards increased efficiency by means of biolgocial synergy-- the ability of individual elements of a system to produce a combined effect that is different and often greater than the sum of the effects of the parts. Modern biochemists are challenged to find model systems to characterize biological synergy.</p><p>We discuss the multicomponent, enzyme complex the cellulosome as a model system of biological synergy. Native cellulosomes comprise numerous carbohydrate-active binding proteins and enzymes designed for the efficient degradation of plant cell wall matrix polysaccharides, namely cellulose. Cellulosomes are modular enzyme complexes, comparable to enzyme "legos" that may be readily constructed into multiple geometries by synthetic design. Cellulosomal enzymes provide means to measure protein efficiency with altered complex geometry through assay of enzymatic activity as a function of geometry.</p><p>Cellulosomes are known to be highly efficient at cellulose depolymerization, and current debates on the molecular origins of this efficiency suggest two related effects provide this efficiency: i) substrate targeting, which argues that the localization of the enzyme complex at the interface of insoluble cell wall polysaccharides facilitates substrate depolymerization; and ii) proximity effects, which describe the implicit benefit for co-localizing multiple enzymes with divergent substrate preferences on the activity of the whole complex.</p><p>Substrate targeting can be traced to the activity of a single protein, the cellulosomal scaffoldin cellulose binding module CBM3a that is thought to uniquely bind highly crystalline, insoluble cellulose. We introduce methods to develop a molecular understanding of the substrate preferences for CBM3a on soluble and insoluble cellulosic substrates. Using pivaloylysis of cellulose triacetate, we obtain multiple soluble cello-oligosaccharides with increasing degree of glucose polymerization (DP) from glucose (DP1) to cellodecaose (DP10) in high yield. Using calorimetry and centrifugal titrations, cello-oligosacharides were shown to not bind Clostridial cellulolyticum CMB3a. We developed AFM cantilever functionalization protocols to immobilize CBM3a and then probe the interfacial binding between CBM3a and a cellulose nanocrystal thin film using force spectroscopy. Specific binding at the interface was demonstrated in reference to a control protein that does not bind cellulose. The results indicate that i) CBM3a specifically binds nanocrystalline cellulose and ii) specific interfacial binding may be probed by force spectroscopy with the proper introduction of controls and blocking agents.</p><p>The question of enzyme proximity effects in the cellulosome must be answered by assaying the activity of cellulosomal cellulases in response to cellulosome geometry. The kinetic characterization of cellulases requires robust and reproducible assays to quantify functional cellulase content of from recombinant enzyme preparations. To facilitate the real-time routine assay of cellulase activity, we developed a custom synthesis of a fluorogenic cellulase substrate based on the cellohexaoside of Driguez and co-workers (vide infra). Two routes to synthesize a key thiophenyl glycoside building block were presented, with the more concise route providing the disaccharide in four steps from a commercial starting material. The disaccharide building blocks were coupled by chemical activation to yield the fully protected cellohexaoside over additional six steps. Future work will include the elaboration of this compound into an underivatized FRET-paired hexasaccharide and its subsequent use in cellulase activity assays.</p><p>This dissertation also covers an experimental system for the evaluation of methonium desolvation thermodynamics. Methonium (-N+Me3, Am) is an organic cation widely distributed in biological systems. The appearance of methonium in biological transmitters and receptors seems at odds with the large unfavorable desolvation free energy reported for tetramethylammonium (TMA+), a frequently utilized surrogate of methonium. We report an experimental system that facilitates incremental internalization of methonium within the molecular cavity of cucurbit[7]uril (CB[7]).</p><p>Using a combination of experimental and computational studies we show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by a remarkably small desolvation enthalpy of just 0.5±0.3 kcal*mol-1, a value significantly less endothermic than those values suggested from gas-phase model studies (+49.3 kcal*mol-1). More surprisingly, the incremental withdrawal of methonium surface from water produces a non- monotonic response in desolvation enthalpy. A partially desolvated state exists, in which a portion of the methonium group remains exposed to solvent. This structure incurs an increased enthalpic penalty of ~3 kcal*mol-1 compared to other solvation states. We attribute this observation to the pre- encapsulation de-wetting of the methonium surface. Together, our results offer a rationale for the wide biological distribution of methonium and suggest limitations to computational estimates of binding affinities based on simple parameterization of solvent-accessible surface area.</p> / Dissertation

<p>Molecular recognition is arguably the most elementary physical process essential for life that arises at the molecular scale. Molecular recognition drives events across virtually all length scales, from the folding of proteins and binding of ligands, to the organization of membranes and the function of muscles. Understanding such events at the molecular level is massively complicated by the unique medium in which life occurs: water. In contrast to recognition in non-aqueous solvents, which are driven largely by attractive interactions between binding partners, binding reactions in water are driven in large measure by the properties of the medium itself. Aqueous binding involves the loss of solute-solvent interactions (desolvation) and the concomitant formation of solute-solute interactions. Despite decades of research, aqueous binding remains poorly understood, a deficit that profoundly limits our ability to design effective pharmaceuticals and new enzymes. Particularly problematic is understanding the energetic consequences of aqueous desolvation, an area the Toone and Beratan groups have considered for many years.</p><p> In this dissertation, we embark on a quest to shed new light on aqueous desolvation from two perspectives. In one component of this research, we improve current computational tools to study aqueous desolvation, employing quantum mechanics (QM), molecular dynamics (MD) and Monte Carlo (MC) simulations to better understand the behavior of water near molecular surfaces. In the other, we use a synthetic host, cucurbit[7]uril (CB[7]), in conjunction with a de novo series of ligands to study the structure and thermodynamics of aqueous desolvation in the context of ligand binding with atomic precision, a feat hitherto impossible. A simple and rigid macrocycle, CB[7] alleviates the drawbacks of protein systems for the study of aqueous ligand binding, that arise from conformational heterogeneity and prohibitive computational costs to model.</p><p> </p><p> We first constructed a novel host-guest system that facilitates internalization of the trimethylammonium (methonium) group from bulk water to the hydrophobic cavity of CB[7] with precise (atomic-scale) control over the position of the ligand with respect to the cavity. The process of internalization was investigated energetically using isothermal titration microcalorimetry and structurally by nuclear magnetic resonance (NMR) spectroscopy. We show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by an unfavorable desolvation enthalpy of just 0.49±0.27 kcal*mol-1, a value significantly less endothermic than those values suggested from previous gas-phase model studies. Our results offer a rationale for the wide distribution of methonium in biology and demonstrate important limitations to computational estimates of binding affinities based on simple solvent-accessible surface area approaches.</p><p> To better understand our experimental results, we developed a two-dimensional lattice model of water based on random cluster structures that successfully reproduces the temperature-density anomaly of water with minimum computational cost. Using reported well-characterized ligands of CB[7], we probed water structure within the CB[7] cavity and identified an energetically perturbed cluster of water. We offer both experimental and computational evidence that this unstable water cluster provides a significant portion of the driving force for encapsulation of hydrophobic guests.</p><p> The studies reported herein shed important light on the thermodynamic and structural nature of aqueous desolvation, and bring our previous understanding of the hydrophobic effect based on ordered water and buried surface area into question. Our approach provides new tools to quantify the thermodynamics of functional group desolvation in the context of ligand binding, which will be of tremendous value for future research on ligand/drug design.</p> / Dissertation

No description available.

PEGylation can improve the pharmacokinetic properties of protein therapeutics via decreasing renal clearance and shielding the protein surface from proteases, antibody neutrailization, and aggregation. Conformational stability enhancement can provide criteria for the identification of optimal sites for PEGylation, but how PEG influence the noncovalent interactions from the surface of proteins has not been well illustrated. Macrocyclization can effectively enhance the conformational stability of small peptides and large proteins. Combination of PEG-based conformational stability enhancement and macrocyclization-based conformational constraint has not been explored. Macrocycliziation has been employed to stabilize protein tertiary structures, but there are no general guidelines for interhelical staple to stabilize coiled-coil motifs of proteins. Chapter 1 is an introduction to peptide stapling and macrocyclization of proteins. Chapter 2 describes our test of the hypothesis that PEG increases the conformational stability of proteins by desolvating nearby salt bridges. In chapter 3, we explore the combination of PEG-based conformational stability enhancement with macrocyclization on WW domain, and find that the most important criteria for PEG stapling is ensuring the side chains cross-linked by PEG are distant in primary sequence but close in tertiary structure. In chapter 4, we further apply this macrocyclization criteria to another -sheet-based protein, SH3 domain of the chicken Src protein, and to a disulfide-bonded parallel coiled-coil heterodimer derived from the yeast transcription factor GCN4. In chapter 5, we explore the determinants of PEG-staple-based stabilization by changing the distance of the staple to the terminal interhelical disulfide bond, varying the length of staple, exploring different solvent exposed positions for stapling and employing heterochiral residues for stapling. We further apply the interhelical PEG staple to a HER-2 affibody, and find that PEG-stapling increases the conformational stability and proteolytic resistance of the stapled affibody relative to its non-stapled counterpart and to the native unmodified affibody.

Information about protein structures is important in many areas of life sciences, including structure-based drug design. Gas phase methods, like electrospray ionization and mass spectrometry are powerful tools for the analysis of molecular interactions and conformational changes which complement existing solution phase methods. Novel techniques such as single particle imaging with X-ray free electron lasers are emerging as well. A requirement for using gas phase methods is that we understand what happens to proteins when injected into vacuum, and what is the relationship between the vacuum structure and the solution structure. Molecular dynamics simulations in combination with experiments show that protein structures in the gas phase can be similar to solution structures, and that hydrogen bonding networks and secondary structure elements can be retained. Structural changes near the surface of the protein happen quickly (ns-µs) during transition from solution into vacuum. The native solution structure results in a reasonably well defined gas phase structure, which has high structural similarity to the solution structure. Native charge locations are in some cases also preserved, and structural changes, due to point mutations in solution, can also be observed in vacuo. Proteins do not refold in vacuo: when a denatured protein is injected into vacuum, the resulting gas phase structure is different from the native structure. Native structures can be protected in the gas phase by adjusting electrospray conditions to avoid complete evaporation of water. A water layer with a thickness of less than two water molecules seems enough to preserve native conditions. The results presented in this thesis give confidence in the continued use of gas phase methods for analysis of charge locations, conformational changes and non-covalent interactions, and provide a means to relate gas phase structures and solution structures.