Advances in combinatorial chemistry have facilitated the production of large chemical libraries that can be used as tools to discover biological probes and therapeutics. High-throughput screening (HTS) strategies have emerged as the standard method to assess the biological activity of small molecules. These screens involve the individual analysis of each small molecule in multi-well plates, often requiring expensive automated methods and development of robust assays that may not translate to physiologically relevant contexts. This problem of evaluating large numbers of reagents in physiologically relevant cell and animal models has been addressed for genetic reagents such as RNAi, CRISPR, and cDNA by creating barcoded retroviral libraries that can be used to infect target cells in culture or in animal models. Using these tools, effective reagents can be selected and decoded using a rapid and inexpensive procedure compared to testing of individual reagents one at a time in an arrayed fashion. In order to more efficiently analyze small molecules, a pooled approach would similarly be useful. This dissertation describes the studies towards developing a pooled screening strategy for small molecules in cellular contexts. Through an initial screen, we set to phenotypically profile small molecule biological activity in a pooled fashion, while simultaneously gain insight about an individual, active molecule’s mechanism of action. I first describe the design of the pooled screen and define the goals necessary for successful application. Next, I outline the steps taken and challenges encountered during the invention of each component of the technology. Finally, I discuss a computational, target-based approach to design small molecules appropriate for future applications of the new screening technology.

Polymorphism and crystallinity are now recognised as important issues in drug development. This is shown by the increased amount of research in this area over recent years. In pharmaceutical development milling is an important unit operation for size reduction to improve powder handling, processing and dissolution rate. The aim of this thesis was to investigate the effect of ball milling (and cryo-milling) on the solid state properties, including amorphous drug formation, of pharmaceutical solids. Milling was carried out using an oscillatory ball mill (Mixer Mill MM301, Retsch GmbH & Co., Germany). In cryo-milling the milling jars were immersed in liquid nitrogen for three min before milling. XRPD was used as the main technique to evaluate the milled samples. Ranitidine hydrochloride (RAN) and indomethacin (INDO) were the model drugs used in this study. It was found that upon milling, RAN form 1 converts to RAN form 2 via an amorphous phase. A faster amorphization rate was observed when the crystalline samples were cryo-milled. Amorphous ranitidine hydrochloride was characterized to have a glass transition (T[g]) range of 13 to 30 �C and a crystallization exotherm (T[c]) between 30 and 65 �C. Conversion was found to occur faster when the temperature of the solid powder was greater than the T[c]. Under various storage conditions, similarly, crystallization of the amorphous phase mainly led to RAN form 2. However, some form 1 and amorphous phase was also detected on the XRPD diffractograms. Using partial least squares regression, the amount of solid form components in the ternary RAN mixtures were successfully quantified. RAN form 2 did not convert to form 1 under any milling (including cryo-milling) or storage conditions used in this study. Overall, RAN form 2 was found to be the thermodynamically stable form and the two (RAN) polymorphs are likely to be a monotropic pair. In a co-milling study of INDO and RAN, the two crystalline drugs were successfully converted into a single amorphous phase after 60 min of co-milling in a cold room (4 �C). The T[g] range (26-44 �C) was also characterized for these mixtures. DRIFTS spectra of the co-milled amorphous samples indicated an interaction had occurred between the carboxylic acid carbonyl (HO-C=O) and benzonyl amide (NC=O) of the INDO molecule with the aci-nitro (C=NO₂) of RAN. Depending on the ratio of INDO to RAN, in general, the amorphous mixtures were stable at 4 �C after 30 days of storage. Crystallization was faster when the binary mixtures were stored at higher temperature or contained higher amounts of RAN in the mixture. Although XRPD and DRIFTS suggested an interaction between the two drugs, co-crystal formation was not observed between INDO and RAN. Ball milling can be used to produce amorphous drug. The rate and extent of amorphization is dependent on the milling conditions. A faster rate of amorphization was observed when the crystalline drugs were cryo-milled. Amorphous drug formation can be made either alone or in combination with another crystalline drug. Amorphization could offer a significant improvement on the dissolution profile and the bioavailability of the poorly water soluble drug - indomethacin. Furthermore, ball milling can also be used to produce a homogenous mix between solids. The �goodmix� effect can be used for seed-induced crystallization or, when the XRPD or Raman data were combined with partial least squares regression, to create a reliable calibration model for quantitative analysis.

Early predictions of a potential drug candidate’s time-course of effect and side-effects, based on models describing drug concentrations, drug effects and disease progression, would be valuable to make drug development more efficient. Pharmacodynamic modeling can incorporate and propagate prior knowledge and be used for simulations of different scenarios. In this thesis three population pharmacodynamic models were developed to describe the antipsychotic effects and the side-effects prolactin elevation and Extra Pyramidal Symptoms (EPS) following administration of D2-receptor antagonists, commonly used in the treatment of schizophrenia. Model parameter estimates of prolactin elevating potencies of six compounds correlated with in vitro values of receptor affinities, and parameters related to diurnal prolactin variation and tolerance were similar for the different compounds. The developed prolactin model can thereby be used to predict the time-course of prolactin elevation in patients for a drug candidate using information on in vitro affinity to the D2-receptor. Furthermore, the clinical antipsychotic effect and the prolactin elevation was found to correlate on the individual level for the three antipsychotic compounds investigated and a quantitative relation between D2-receptor occupancy in the brain and prolactin elevation was established. These results support the use of prolactin concentrations as a biomarker in drug development or for individual dose adjustments in clinical care. The developed model for spontaneously reported EPS adverse events, following treatment with one of five antipsychotics drugs, characterized both the duration and severity of EPS. The model successfully described both the proportions and number of transitions between severity grades and was shown to adequately simulate longitudinal categorical EPS data. Complex pharmacodynamic models are often associated with long estimation times and non-normal distributions of individual parameters. A method for shortening computation times by substituting differential equations for difference equations was evaluated and shown to be valuable for some models. In addition, transformation of distributions allowed for non-normal distributions of between-subject variability to be better characterized and thereby simulation properties were improved. In conclusion, population pharmacodynamic models for a range of D2-receptor antagonists were developed and together with the investigated methods the models can facilitate prediction of effects and side-effects in drug development.

Targeting angiogenic vasculature has been validated as a viable approach for cancer imaging and therapy. The tumour vasculature-specific ligand asparagine-glycine-arginine (NGR) peptide targets the isoform of aminopeptidase N (CD13) expressed on endothelial cells lining angiogenic vessels. CD13 has become widely recognized as a rational target for therapeutic development and several NGR-conjugated agents are now in pre-clinical and clinical development. In the current study, a CT image-based approach is used to evaluate the in vivo performance of several NGR-conjugated liposome formulations that vary in terms of NGR density and PEG spacer arm length. Indeed, for the first time it is demonstrated that CT imaging can be used for quantitative and longitudinal assessment of the pharmacokinetics and biodistribution of an actively targeted liposome formulation. In comparison to conventional methods, CT imaging enables visualization of the intratumoural distribution of liposomes and quantification of the fraction of tumour occupied by the vesicles over time.

For bone resorption, vacuolar-type H+-ATPases (V-ATPases) on the plasma membranes of osteoclasts acidifies the extracellular millieu adjacent to the bone surface. The V-ATPase a3 and d2 subunits are enriched in osteoclasts. B2 subunit is also expressed on the osteoclast plasma membrane. Disruption of genes encoding subunits a3 and d2 impairs bone resorption. In this study, we have shown an interaction between the a3-B2 and a3-d2 subunits. Luteolin and KM91104 were found to be effective inhibitors of the a3-d2 and a3-B2 interactions respectively. Secondary assays revealed luteolin and KM91104 were not toxic to cells, did not affect osteoclastogenesis yet inhibited bone resorption. Furthermore luteolin did not affect V-ATPase subunit formation or assembly. Inhibitors of osteoclast resorption that do not affect osteoclast viability, preserve osteoclast–osteoblast signalling are desirable than existing anti-resorptives. Therefore, V-ATPase a3–d2 and a3-B2 interactions are viable targets for anti-resorptive therapeutics for osteoporosis.

Targeting angiogenic vasculature has been validated as a viable approach for cancer imaging and therapy. The tumour vasculature-specific ligand asparagine-glycine-arginine (NGR) peptide targets the isoform of aminopeptidase N (CD13) expressed on endothelial cells lining angiogenic vessels. CD13 has become widely recognized as a rational target for therapeutic development and several NGR-conjugated agents are now in pre-clinical and clinical development. In the current study, a CT image-based approach is used to evaluate the in vivo performance of several NGR-conjugated liposome formulations that vary in terms of NGR density and PEG spacer arm length. Indeed, for the first time it is demonstrated that CT imaging can be used for quantitative and longitudinal assessment of the pharmacokinetics and biodistribution of an actively targeted liposome formulation. In comparison to conventional methods, CT imaging enables visualization of the intratumoural distribution of liposomes and quantification of the fraction of tumour occupied by the vesicles over time.

For bone resorption, vacuolar-type H+-ATPases (V-ATPases) on the plasma membranes of osteoclasts acidifies the extracellular millieu adjacent to the bone surface. The V-ATPase a3 and d2 subunits are enriched in osteoclasts. B2 subunit is also expressed on the osteoclast plasma membrane. Disruption of genes encoding subunits a3 and d2 impairs bone resorption. In this study, we have shown an interaction between the a3-B2 and a3-d2 subunits. Luteolin and KM91104 were found to be effective inhibitors of the a3-d2 and a3-B2 interactions respectively. Secondary assays revealed luteolin and KM91104 were not toxic to cells, did not affect osteoclastogenesis yet inhibited bone resorption. Furthermore luteolin did not affect V-ATPase subunit formation or assembly. Inhibitors of osteoclast resorption that do not affect osteoclast viability, preserve osteoclast–osteoblast signalling are desirable than existing anti-resorptives. Therefore, V-ATPase a3–d2 and a3-B2 interactions are viable targets for anti-resorptive therapeutics for osteoporosis.

Natural products and small molecules play a major role in drug development. However, using natural products as a source of medicine comes with many challenges, such as lack of natural abundance and difficulty in isolation. Consequently, synthetic organic chemistry is a solution in order to access these compounds in usable quantities. However, synthetic chemisty comes with its own challenges such as efficiency, chemoselectivity, stereoselectivity and enantioselectivity. Therefore, synthetic tools that addresses these challenges are required solve these limitations. This thesis discusses new methodologies using strained carbocycles (cyclopropanes and cyclopropenes) as the reactive subunit for the construction of different carbocyclic and heterocyclic compounds. The homo-Nazarov cyclization of alkenyl and heteroaryl cyclopropyl ketones was used in order to construct cyclohexenones, cyclohexenols, heteroaryl ring-fused cyclohexenones, dihydrofurans, furans and furanones in a mild and efficient manner. Benzofused heteroaromatic compounds were achieved via the Lewis acid-catalyzed cycloisomerization of cyclopropene-3,3-dicarbonyls and furan-3-carboxylates. These heteroaromatic compounds can be applied to medicinal chemistry and material science.

Recombinant antibodies and antibody fragments have become powerful tools for therapy, in vivo and in vitro diagnostics, and laboratory research. However, the production of antibody fragments in high yield for preclinical and clinical trials can be a serious bottleneck in drug discovery. This dissertation describes the development of novel screening systems for isolating antibody fragments and alternatively, E. coli genes, that facilitate expression in E. coli. In the first part of this work, we have developed a screening system for isolating Fab mutants exhibiting 4~5 fold higher expression level at 37oC compared to the parental Fab, by utilizing the APEx 2-hybrid system and multi-color FACS as a screening tool. In the APEx 2-hybrid system, the bacterial periplasm constitutes the milieu for the association of membrane-anchored bait protein and solubly expressed, epitope-tagged prey protein. Upon disruption of the outer membrane, only prey proteins that bind to the bait remain cell-associated and are detected by flow cytometry using fluorescently labeled anti-epitope antibodies. In the second part of this work we developed a new strategy to engineer scFv that can be expressed in soluble and active form in the absence of disulfide bonds. This was achieved using a strain incapable of forming disulfide bonds in proteins expressed in its periplasm in combination with the APEx 2-hybrid system. The selected clones exhibited higher solubility, activity, and stability than that of the wild type scFv in the reducing condition of the cytoplasm. Finally, we sought to isolate E. coli gene fragments that can enhance IgG production in the periplasm of E. coli by a newly developed screening system based on soluble expression of IgG and E. coli genomic fragments. The isolated gene fragments, which are located between moeA and iaaA in the E. coli chromosome, improved the total expression of polypeptides of IgG and also assembly of IgG. / text

The escalation with overdose control (EWOC) method is a popular modelbased dose finding design for phase I clinical trials. Dose finding for combined drugs has grown rapidly in oncology drug development. A two-dimensional EWOC design is proposed for dose finding with two agents in combination based on a four-parameter logistic regression model. During trial conduct, the posterior distribution of the maximum tolerated dose (MTD) combination is updated continuously in order to find the appropriate dose combination for each cohort of patients. The probability that the next dose combination exceeds the MTD combination can be controlled by a feasibility bound, which is based on a prespecified quantile for the MTD distribution such as to reduce the possibility of over-dosing. Dose escalation, de-escalation or staying at the same doses is determined by searching the MTD combination along rows and columns in a two-drug combination matrix. Simulation studies are conducted to examine the performance of the design under various practical scenarios, and illustrate it with a trial example. / published_or_final_version / Statistics and Actuarial Science / Master / Master of Philosophy