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Human cells possess multiple nucleoside transporters (NTs) that belong to either the human equilibrative or concentrative NT (hENT: hENT1/2/3/4; hCNT: CNT1/2/3) families. In the kidney, coupling of apical hCNT3 activities to basolateral hENT1/2 activities is hypothesized to mediate renal nucleoside proximal tubular absorption while apical ENT1 may have a role in secretion. The overall aim of this research was to increase understanding of the roles of hENTs and hCNTs in renal handling of physiological nucleosides and anti-cancer nucleoside analog drugs. This was achieved by investigating the distribution of hENTs and hCNTs in human kidney tissue and the function of hENTs and hCNTs in cellular uptake and transepithelial fluxes of nucleosides in cultured human renal proximal tubule cells (hRPTCs). Immunolocalization of hCNT3 and hENT1 in human kidney tissue revealed that hENT and hCNT3 were present in apical membranes of proximal tubules. Production and characterization of adherent hRPTC cultures demonstrated endogenous hCNT3, hENT1, and hENT2 activities. These results provided evidence for the involvement of hCNT3, hENT1, and hENT2 in renal handling of nucleosides. Comparison of adherent hRPTC cultures derived from kidneys from different individuals demonstrated that hCNT3 activities varied between cultures. Also, the extent of cellular uptake of fludarabine, an anti-cancer nucleoside drug, and degree of cytotoxicity was reflected in the different hCNT3 activities observed between cultures. These results suggested that hCNT3 plays an important role in fludarabine renal handling and is a determinant of potential renal toxicities. Production of polarized monolayer cultures of hRPTCs on transwell permeable inserts enabled the functional localization of hCNT3 and hENT1 to apical membranes and hENT2 to basolateral membranes. Transepithelial flux studies demonstrated that (i) apical-to-basolateral fluxes of adenosine were mediated by apical hCNT3 and basolateral hENT2, (ii) basolateral-to-apical fluxes of 2′-deoxyadenosine were mediated, in part, by apical hENT1 and basolateral hOATs, and (iii) apical-to-basolateral fluxes of fludarabine, cladribine, and clofarabine were mediated by apical hCNT3. These studies showed that coupling of apical hCNT3 to basolateral hENT2 mediates proximal tubular nucleoside reabsorption, that coupling of basolateral human organic anion transporters (hOATs) to apical hENT1 mediates proximal tubular nucleoside secretion, and that hCNT3 is a key determinant of fludarabine proximal tubular reabsorption and cytoxicity.

(a) The Robins reagent [2-acetamido-6-O-(diphenylcarbamoyl)purine] was utilized for glycosylation under Lewis acid conditions. Regioselectivity of glycosylation depends on the glycosyl donor and its 2-O- or 2-N-protecting group. Regioselective N9 glycosylation of 2-acetamido-6-O-(diphenylcarbamoyl)purine with problematic glucosamine has been accomplished by protecting the amino function as a phthalimido group with consequent stabilization of the oxocarbenium cation, and lowering the activation energy by introduction of trichloroacetimidate at the anomeric carbon. (b) 6-Heteroaryl functions [6-(1,2,4-triazol-4-yl) and 6-(imidazol-1-yl)] were introduced into purine derivatives for regioselective N9 alkylation. The regiospecificity of alkylation mainly results from steric effects due to the coplanar conformation of the two linked heterocyclic rings governed by conjugation. Several of the obtained acyclic derivatives showed antiviral and antitumor activities. (c) Glycosylation of purine derivatives with 2-deoxy-3,5-di-O-(p-toluoyl)-a-D-erythro-pentofuranosyl chloride using the sodium salt method usually gave a mixture of both anomers. Lipophilic groups were introduced into the imidazole ring of 6-(imidazol-1-yl)purine derivatives to increase the solubility of the sodium salts in moderately polar solvents. Differential solvation effects in binary solvent mixtures were utilized to improve the stereoselectivity of glycosylation. The stereoselectivity varied with the sizes of lipophilic groups and the polarity of solvents. With the propyl group, and in CH3CN/toluene (1:1) and/or CH3CN/CH2Cl2 (1:1), regiospecfic and highly stereoselective glycosylation of purines with 2-deoxy-3,5-di-O-(p-toluoyl)-a-D-erythro-pentofuranosyl chloride was achieved. (d) Using the above method, a low cost and efficient synthesis of 2-chloro-2'-deoxyadenosine (2-CdA, cladribine) was accomplished with an overall yield of 48% from inexpensive guanosine and 57% from 2,6-dichloropurine. 2-Chloro-6-(2-propylimidazol-1-yl)purine was prepared either from guanosine in a yield of 61% in 5 steps or from 2,6-dichloropurine in a yield of 72% in one step. Coupling of this 2-chloro-6-heteroarylpurine with 2-deoxy-3,5-di-O-(p-toluoyl)-α-D-erythro-pentofuranosyl chloride in binary solvent mixtures, followed by activation of imidazolyl as a better leaving group via benzylation at N3 and then ammonolysis gave cladribine in good yield (79%) for 3 steps. Analogs of purine derivatives with lipophilic groups (butyl, pentyl and 2-phenylpropyl) worked almost as well.

<p></p> <p><strong><em>Background </em></strong></p> <p>Oral pharmacotherapy has the potential to offer multiple sclerosis patients improved clinical outcomes compared to traditional therapies.</p> <p><strong><em>Objectives </em></strong></p> <p>This review assesses the effects of oral therapies compared to placebo and interferon beta-1a in adults with relapsing-remitting multiple sclerosis (RRMS).</p> <p><strong><em> </em></strong></p> <p><strong><em>Search methods </em></strong></p> <p>We searched the MEDLINE, EMBASE, Cochrane Library, Web of Science (January 1980 to April 2011) and clinincaltrials.gov (April 2011) databases and reference lists of articles. The FDA website was also searched.</p> <p><strong><em>Selection criteria </em></strong></p> <p>Double-blind, placebo-controlled, randomized trials of RRMS patients who were treated with fingolimod, cladribine, laquinimod or interferon beta-1a.</p> <p><strong><em>Data collection and analysis </em></strong></p> <p>Two reviewers independently assessed articles for inclusion. Data extraction and quality assessment was completed by one reviewer and verified for accuracy. Meta-analysis and indirect treatment comparison methods were used to estimate relative measures of efficacy.</p> <p><strong><em>Results </em></strong></p> <p>Although 11 trials involving 7,127 participants were included in this review, only 2,109 (30%) and 1,738 (24%) participants contributed to the direct and indirect estimates respectively, for the primary outcome, annualized relapse rate. Oral therapy and interferon beta-1a had a significantly different rate of relapse compared to placebo (Mean difference [MD] -0.21, 95% confidence interval [CI] -0.27 to -0.16 , p < 0.00001 and MD -0.33 95% CI -0.65 to -0.01). There was a significant risk reduction of 37% and 19% in the number of patients with at least one relapse for oral therapy and interferon beta-1a compared to placebo respectively. Safety analysis favoured placebo for both sets of trials (p=0.002 and p=0.04). Indirect estimates were not significant for all three outcomes however; comparability between direct evidence was noted.</p> <p><strong><em>Conclusions </em></strong></p> <p>Oral pharmacotherapy and interferon beta-1a are effective compared to placebo in controlling relapse rate in patients with RRMS. The indirect measures of effect provide initial estimates of comparative efficacy and incorporation of future evidence will be necessary.</p> / Master of Science (MSc)

Treatment of patients with multiple sclerosis (MS) and development of new therapies have been challenging due to the disease complexity and slow progression, and the limited sensitivity of available clinical outcomes. Modeling and simulation has become an increasingly important component in drug development and in post-marketing optimization of use of medication. This thesis focuses on development of pharmacometric models for characterization and quantification of the relationships between drug exposure, biomarkers and clinical endpoints in relapse-remitting MS (RRMS) following cladribine treatment. A population pharmacokinetic model of cladribine and its main metabolite, 2-chloroadenine, was developed using plasma and urine data. The renal clearance of cladribine was close to half of total elimination, and was found to be a linear function of creatinine clearance (CRCL). Exposure-response models could quantify a clear effect of cladribine tablets on absolute lymphocyte count (ALC), burden of disease (BoD), expanded disability status scale (EDSS) and relapse rate (RR) endpoints. Moreover, they gave insight into disease progression of RRMS. This thesis further demonstrates how integrated modeling framework allows an understanding of the interplay between ALC and clinical efficacy endpoints. ALC was found to be a promising predictor of RR. Moreover, ALC and BoD were identified as predictors of EDSS time-course. This enables the understanding of the behavior of the key outcomes necessary for the successful development of long-awaited MS therapies, as well as how these outcomes correlate with each other. The item response theory (IRT) methodology, an alternative approach for analysing composite scores, enabled to quantify the information content of the individual EDSS components, which could help improve this scale. In addition, IRT also proved capable of increasing the detection power of potential drug effects in clinical trials, which may enhance drug development efficiency. The developed nonlinear mixed-effects models offer a platform for the quantitative understanding of the biomarker(s)/clinical endpoint relationship, disease progression and therapeutic response in RRMS by integrating a significant amount of knowledge and data.