Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Pharmaceutical Science." Includes bibliographical references.

Thesis (Ph. D.)--School of Pharmacy. University of Missouri--Kansas City, 2005. / "A dissertation in pharmaceutical sciences and pharmacology." Advisor: Ashim K. Mitra. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed June 26, 2006. Includes bibliographical references (leaves 174-192). Online version of the print edition.

The National Cancer Institution reported over 80,000 diagnoses of bladder cancer (BCa) in the United States in 2018. Despite these numbers, minimal research toward developing new diagnostic techniques and treatment options are underway. Evidence suggests a significant increase in non-specific a-naphthyl acetateesterase levels in BCa patient’s urine. There has been little research focused on identification of the esterase present. It is also suggested that elevated oxidative stress resulting in production of reactive oxygen species (ROS) is common in tumorigenic bladder cells as a result of increased metabolic activity. Oxidized protein hydrolase (OPH) is an 80kD serine protease, previously found to be elevated in many other types of cancer. OPH degrades proteins damaged by ROS and also exhibits a highly specific esterase activity toward (AcApNA) N-acetyl-alanyl-p-nitroanilide and ANAA (α-naphthyl N-acetylalaninate) containing substrate. Investigation of OPH expression in BCa could result in development of new diagnostic techniques and possible application toward prodrugs targeting cells with elevated ROS and/or OPH. Due to lack of commercial OPH, a positive control for this protein is needed for testing. To do this E. coli(BL-21 DE-3) were cultured and inserted with pET-21a (+) plasmids containing a human OPH gene insert prior to a His7 tag. After being selectively grown on ampicillin media, the bacteria were induced by IPTG and digested using lysozyme. The soluble rOPH suspended in the supernatant was separated from the pellet by centrifugation and further purified using Ni-NTA resin chromatography columns specific for the His7 tag sequence. The UM-UC-3 bladder cancer cell line, commonly used in published research to screen efficiency of chemotherapeutics, were cultured in accordance to ATCC. These cells were then compared against none tumorigenic bladder cancer cells and rOPH in a series of tests. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) were transferred for western blot analysis using antibodies specific for human OPH to investigate the expression levels present in cells. Native-PAGE electrophoresis showed OPH esterase activity across these cells using S-ANAA substrate as a specific esterase colorimetric stain. With these results, possible treatment options can be investigated with use of novel prodrug chemotherapy specifically targeting OPH in BCa cells, ultimately leading to apoptosis in effected cells. These events may also lead to possible biomarkers used for easier and earlier diagnosis of BCa across various spectrums.

Yes / The archetypal prodrug of the imidazotetrazine class is the anticancer agent temozolomide (TMZ). The prodrug activation kinetics of TMZ show an unusual pH dependence: it is stable in acid and rapidly hydrolyses in alkali (Denny, B.J., et al. Biochemistry 1994, 33, 9045–9051). The incipient drug MTIC has the opposite properties—relatively stable in alkali but unstable in acid. In this study, the mechanism of prodrug activation was probed in greater detail to determine whether the reactions are specific or general acid or base catalysed. Three prodrugs and drugs were investigated, TMZ, MTIC and the novel dimeric imidazotetrazine EA27. Hydrolysis in a range of citrate-phosphate buffers (pH 8.0, 7.4, 4.0) was measured by UV spectrophotometry. Reaction of TMZ and MTIC obeyed single-phase, pseudo-first order kinetics (Figure 1). EA27 was more complex, showing biphasic but approximately pseudo-first order kinetics, Figure. General acid or base catalysis indicates that protonation or deprotonation is the rate-limiting step (rls). In biological milieu, the nature and concentration of other acidic or basic solutes may affect the prodrug activation reaction. In contrast, specific acid or base catalysis indicates that protonation or deprotonation occurs before the rls, so catalysis depends only on the local concentration of hydroxide or hydronium ion (i.e., pH) so the reaction kinetics are not expected to change appreciably in a biological medium.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 93-115.

New imidazotetrazinones have been synthesised that possess features in their structures to release aziridinium ions upon ring opening. Unstable 2-aminoethylisocyanates were required in this preparation, which were synthesized with BOC-protection of the amino group to counteract the reactivity of the amine towards the isocyanate group in the case of aliphatic amines; in contrast, anilinoethylisocyanates were synthesized unprotected. Substituents with a range of electron-withdrawing and electron-releasing properties were introduced at the p-position of the aniline ring. A 13C-labelled study confirmed the release of the aziridinium ion by these imidazotetrazinones in neutral pH buffer solution. Furthermore the kinetics of the hydrolysis in neutral aqueous solution of some these new tetrazines were similar to temozolomide, in addition to useful acid stability. Other imidazotetrazinones were synthesised for the purpose of releasing alcohols and phenols. Their synthesis was performed with a one-carbon linker between the imidazotetrazinone 3-position and the alcohols or phenols to be released. The release of alcohol and phenol through the hydrolysis of the intermediate diazonium ions to the unstable hemiacetals that decomposed to the alcohol and phenol was confirmed by 1H NMR. The kinetics of the hydrolysis of these tetrazines in neutral aqueous solution showed a faster reaction rate compared with temozolomide (t1/2 = 0.53 and 0.36 h compared with temozolomide 1.4 h).

La squalénisation est une méthode de vectorisation sous forme nanoparticulaire qui consiste àcoupler de manière covalente un dérivé du squalène à des principes actifs hydrophiles tels que lesanalogues nucléosidiques. Les conjugués amphiphiles obtenus sont capables de s’auto-organiserspontanément dans l’eau en nanoparticules d’une centaine de nanomètres de diamètre etpossèdent des activités anticancéreuses ou antivirales remarquables. Notre objectif était d’étendrecette stratégie à différentes classes d’antitumoraux, comme les antimétabolites, les antimitotiqueset les agents alkylants. Différents dérivés du squalène ont ainsi été synthétisés puis couplés à cesprincipes actifs pour former les bioconjugués squalénisés correspondants. Il a été montré que cesprodrogues étaient capables de s’auto-assembler en nanoparticules spontanément en milieuaqueux, que le principe actif soit hydrophile ou hydrophobe. Les suspensions nanoparticulaires deces prodrogues se sont montrées actives in vitro sur différentes lignées cellulaires cancéreuseshumaines et murines et in vivo chez la Souris sur des modèles de cancer. La squalénisation a donc étéétendue à diverses familles de composés anticancéreux confirmant qu’il s’agit d’une méthodegénérale de vectorisation pourvue d’un fort potentiel thérapeutique. / Squalenoylation is a strategy of vectorization consisting in coupling squalene derivatives tohydrophylic drugs as nucleoside analogues. The amphiphilic conjugates obtained are able to selfassembleinto nanoparticles with a diameter of 100 nm in water. In addition those nanoparticleshave shown impressive anticancer and antiviral activities. Our objective was to extend this strategyto different anticancer drugs as antimetabolites, antimitotics and alkylating agents. Differentsqualenoyl derivatives have been synthesised and then coupled to drugs to furnish thecorresponding squalenoylated biconjugates. It has been shown that those prodrugs were able toself-assemble into nanoparticles in water. Nanoparticles of the bioconjugates are active in vitro ondifferent human cancer cell lines and in vivo in Mice on different cancer models. Squalenoylation hasfinally been extended to numerous anticancer drugs, proving that this strategy is a general methodof vectorization with a high therapeutic potential.

Purpose: Combretastatin A4 phosphate (CA4P) and its structural analog, combretastatin A1 phosphate (CA1P), are soluble prodrugs capable of interacting with tubulin and causing rapid vascular shutdown within tumors. CA4P has completed Phase I clinical trials, but recent preclinical studies have shown that CA1P displays a greater antitumor effect than the combretastatin A4 (CA4) analog at equal doses. The aim of this study, therefore, is to compare pharmacokinetics and metabolism of the two compounds to determine whether pharmacokinetics plays a role in their differential activity. Experimental Design: NMRI mice bearing MAC29 tumors received injection with either CA4P or CA1P at a therapeutic dose of 150 mg·kg-1, and profiles of both compounds and their metabolites analyzed by a sensitive and specific liquid chromatography/mass spectroscopy method. Results: The metabolic profile of both compounds is complex, with up to 14 metabolites being detected for combretastatin A1 (CA1) in the plasma. Many of these metabolites have been identified by liquid chromatography/mass spectroscopy. Initial studies, however, focused on the active components CA4 and CA1, where plasma and tumor areas under the curve were 18.4 and 60.1 µg·h·ml-1 for CA4, and 10.4 and 13.1 µg·h·ml-1 for CA1, respectively. In vitro metabolic comparisons of the two compounds strongly suggest that CA1 is metabolized to a more reactive species than the CA4. Conclusions: Although in vitro studies suggest that variable rates of tumor-specific prodrug dephosphorylation may explain these differences in pharmacokinetics profiles, the improved antitumor activity and altered pharmacokinetic profile of CA1 may be due to the formation of a more reactive metabolite.

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2007. / "A dissertation in pharmaceutical sciences and chemistry." Advisor: Ashim K. Mitra. Typescript. Vita. Description based on contents viewed July 16, 2008; title from "catalog record" of the print edition. Includes bibliographical references (leaves 173-182). Online version of the print edition.

Chemotherapy is widely used to treat various forms of cancer. However, some chemotherapeutic drugs, due to their antineoplastic properties, also act upon healthy cells which normally replicate rapidly causing a plethora of undesirable side effects. One rising and promising therapeutic strategy is the development of prodrugs. Prodrugs are derivatives of the pharmaceutically active drugs but require an enzymatic or biochemical transformation within a certain biological space in order for it to become activated and capable of exerting the desired pharmacological effect. As a novel prodrug approach, this thesis describes the pioneering use of a bioorthogonal organometallic (BOOM) activation strategy to develop spatially-controlled anticancer treatments. Bioorthogonal reactions are selective chemical processes between two abiotic reagents in a biological system that do not interfere with the system’s biotic components. In BOOM reactions, one of the reagents is a metal catalyst, which if immobilized, could in principle allow for the local transformation of a continuous flow of a bioorthogonal chemo-substrate indefinitely. To exploit the benefits of this paradigm in anticancer therapy, this thesis reports the design, synthesis and screening of a set of prodrugs masked with bioorthogonal protecting groups sensitive to activation by a catalysts-based “activating device”. Specifically, it describes the synthesis of palladium (Pd0) functionalized resins (the activating device) capable of activating cytotoxic pyrimidine analogue prodrugs masked with Pd0-labile protecting groups. Both the Pd0 functionalized resins and the BOOM-activated prodrugs are independently non-cytotoxic. However, once in combination together, the Pd0 is capable of mediating the removal of the masking groups in situ and rendering the drugs in their cytotoxic state with comparable antiproliferative properties to the unmodified parental drugs in vitro. The Pd0 resins also display biocompatibility and local catalytic activity inside zebrafish embryos. This approach is intended to generate a more targeted therapeutic treatment regime while minimizing harm to normal healthy tissues through the local generation of prodrugs which are not dependent on intrinsic biological activators but by an external activating device, thus reducing the systemic presence of the drug.