Histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated a wealth of biological effects, including anti-proliferative, anti-inflammatory, anti-parasitic, and cognition-enhancing activities. The recent FDA approvals of the inhibitors SAHA and FK-228 have validated HDACi clinical use in cutaneous T cell lymphoma, while numerous clinical trials are currently ongoing using HDACi against a variety of disease states. While the future of the HDAC field looks increasingly promising, there are lingering issues hindering broader use. Recent data point to dysregulation of specific HDAC isoforms in many disease states. However, most current HDACi are pan-inhibitors, lacking the specificity to target individual isoforms. Adding to this, there are currently 18 identified HDAC isoforms, and most lack a defined crystal structure, further complicating the task of designing isoform-specific inhibitors. Most importantly, HDACi have demonstrated a lack of efficacy against solid tumors in the clinic, a major obstacle to broader use in cancer therapy. Several of these issues could more fully be addressed through specific targeting of HDACi, and could bring HDACi into wider and more efficacious pharmaceutical use. Targeting the specific tissue or organelle where HDAC dysregulation occurs could confer greater efficacy in vivo. To this end, we have created four classes of compounds: (1) aryltriazolyl HDACi that potently inhibit HDAC activity and prostate cancer cell growth, (2) dual-targeted inhibitors of Topoisomerase II and HDAC and (3) dual-targeted inhibitors of Topoisomerase I and HDAC, both of which have potent inhibition against both target enzymes as well as cancer cell lines, and finally (4) macrocyclic HDACi that potently inhibit the growth of lung cancer cell lines and preferentially target lung tissue in vivo.

The research presented in this dissertation describes the creation and characterization of a novel antibody-vesicle conjugate modified with polyethylene glycol (PEG) that possesses enhanced binding to and uptake by inflammation-activated endothelial cells with improved storage stability and longer shelf-life and potential reduction in immunogenic potential compared to previous designs. Targeted drug delivery provides an effective means of delivering therapeutic concentrations of a drug to the site or organ of action. The drug is delivered using a niosome, a vesicle with an aqueous core and a bilayer membrane composed of non-ionic surfactants and cholesterol. Antibodies that recognize specific cell antigens are attached to the niosome to complete the targeting molecule, an immuno-niosome (IN). When functionalized PEG, a water soluble, biologically inert polymer, is attached to proteins, it can protect the protein, increasing its half-life in vivo. The immuno-niosome synthesis process is modified to include PEG incorporation into the niosome membrane, a process known as PEGylation. Since the vasculature connects the entire body, immuno-niosomes targeted to endothelial cells were used. When endothelial cells are activated during disease, stress and injury, certain receptors are expressed and upregulated. One such receptor, CD44, is upregulated in response to vascular inflammation associated with atherosclerosis. The research hypothesis is that the addition of polyethylene glycol to the drug delivery vesicle (immuno-niosome) using cyanuric chloride linking chemistry will improve colloidal stability and binding performance of the PEGylated immuno-niosomes to endothelial cell surface receptors expressed during an inflammatory response. The research presented in this dissertation provides the following evidence to support this hypothesis: Construction and characterization of a modified drug delivery vesicle using non-ionic surfactants conjugated with PEG and functionalized with antibodies against endothelial cell surface receptors (PEGylated immuno-niosomes) improves the colloidal stability over previously designed vesicles. Binding of PEGylated CD44-IN to activated endothelial cells is improved over previously designed vesicles. Binding of PEGylated CD44-IN to activated endothelial cells under physiological conditions (flow) is demonstrated. Uptake of PEGylated immuno-niosomes by activated or injured endothelial cells is demonstrated using confocal microscopy.

L’aérosolthérapie est une pratique ancienne de plusieurs siècles. Progressivement l’administration inhalée de bronchodilatateurs et de corticoïdes s’est imposé comme le traitement au long cours de référence des maladies pulmonaires obstructives. Plus récemment de nouvelles molécules, comme les antibiotiques ou les mucomodulateurs, ont été administrées avec succès par voie inhalée, particulièrement chez les patients atteints de mucoviscidose. Alors que ces succès de l’aérosolthérapie ont été obtenus chez les patients en ventilation spontanée et traités essentiellement dans le contexte ambulatoire, les données sont beaucoup plus parcellaires concernant l’aérosolthérapie chez les patients admis en réanimation et plus particulièrement ceux soumis à la ventilation artificielle. Néanmoins, l’effet physiologique des bronchodilatateurs et des corticoïdes a été documenté chez les patients soumis à la ventilation artificielle et plusieurs études expérimentales et de recherche clinique [ont] documenté la faisabilité de l’antibiothérapie inhalée chez ces patients. Cinq travaux ont été réalisés dans le cadre de la thèse, faisant appel à des méthodes de recherche expérimentale sur banc, d’expérimentation animale, de recherche clinique et épidémiologique concernant l’aérosolthérapie en réanimation. Les deux premiers travaux ont consisté en une enquête internationale par voie électronique auprès des huit cents médecins exerçant en réanimation et d’une étude prospective observationnelle durant deux semaines dans quatre-vingts services de réanimation. Les résultats principaux de ces travaux sont que l’aérosolthérapie était très fréquente en réanimation et concernait près d’un quart des patients. Les molécules administrées étaient essentiellement des bronchodilatateurs et des corticoïdes, mais l’antibiothérapie inhalée était également pratiquée. […] L’aérosolthérapie est apparue comme bien tolérée à court terme […]. Le troisième travail a consisté en une évaluation sur banc des systèmes de nébulisation pneumatique synchronisée intégrés dans quatre ventilateurs de réanimation. Les résultats principaux de ce travail sont que ces systèmes permettaient un bon contrôle du volume courant délivré au patient durant la nébulisation ; en revanche la synchronisation inspiratoire n’était pas optimale […]. Le quatrième travail a consisté en une étude clinique évaluant la pharmacocinétique sérique de l’amikacine après son administration inhalée à forte dose chez vingt-deux patients soumis à la ventilation artificielle et atteints de pneumonie nosocomiale. L’administration était réalisée à l’aide d’un système de nébulisation pneumatique innovant, adapté à la ventilation artificielle. Le résultat principal de ce travail a été que la nébulisation de 60 mg/Kg d’amikacine s’est avéré faisable chez le patient sous ventilation artificelle et que les concentrations sériques observées étaient inférieures à celles observées après perfusion intraveineuse. […] Enfin, le dernier travail a consisté en une étude animale comparant la pharmacocinétique sérique de l’amikacine après administration intraveineuse, nébulisation et aérosolisation in situ à l’extrémité de la sonde d’intubation de porcelets soumis à la ventilation artificielle avec des poumons non infectés. Le principal résultat est que l’aérosolisation in situ a permis d’administrer de grandes quantitiés d’amikacine en très peu de temps. Néanmoins, les concentrations intra-parenchymateuses étaient très hétérogènes après aérosolisation in situ et fréquemment faibles […]. / Aerosoltherapy is a centuries-old practice. Inhaled bronchodilatators and corticosteroids have become the long-term treatment of choice for obstructive lung diseases. More recently, new molecules, such as antibiotics or mucus modulators have been successfully administrated by inhalation, particularly in patients with cystic fibrosis. While the success of aerosoltherapy were obtained in patients breathing spontaneously and essentially treated in the outpatient setting, data are much scarcer concerning aerosoltherapy in patients admitted to intensive care and especially those undergoing mechanical ventilation. Nevertheless, the physiological effects of bronchodilators and corticosteroids have been documented in patients undergoing mechanical ventilation and several experimental and clinical researches documented the feasibility of inhaled antibiotic therapy in these patients. The thesis comprises five works carried out using methods of experimental bench research, animal experimentation so as clinical and epidemiological research concerning aerosoltherapy in the intensive care setting. The first two works consisted of an international electronic survey among eight hundred physicians working in intensive care and a two-week prospective cross-sectional study in eighty intensive care units. The main results of those works are that aerosoltherapy appeared very common in the intensive care unit and concerned about a quarter of all admitted patients. The molecules administrated were essentially bronchodilatators and corticosteroids, but inhaled antibiotic therapy was also practiced. Although implementation of aerosoltherapy, especially during mechanical ventilation, appeared frequently at odds with optimal practice, or even dangerous, it was well tolerated in the short term since only a hundred side effects were observed during the nine thousand aerosol administrations collected in the prospective study. The third work evaluated, in a bench model of mechanical ventilation, synchronized pneumatic nebulization systems integrated in four intensive care ventilators. The main results of this work are that these systems provided good control of the tidal volume during nebulization ; however inspiratory synchronization was not optimal since a significant proportion of nebulization occurred during expiration, due ti gas compression/decompression upstream of the nebulizer. The fourth work consisted of a clinical study evaluating serum pharmacokinetics of amikacin after high dose inhalation in twenty-two patients undergoing mechanical ventilation and suffering nosocomial pneumonia. Administration was carried out using an innovative pneumatic nebulization system adapted to mechanical ventilation. The main result of this work was that nebulization of 60 mg / kg of amikacin proved feasible in patients on mechanical ventilation and serum concentrations observed were lower than those observed after intravenous infusion. An increase in the dose and / or the yield of the nebulization system may be considered to promote greater lung deposition in order to favour improved efficacy. The presence or absence of a heated humidifier did not influence the results, thus allowing considering active humidification during prolonged nebulization within a high-dose strategy. Finally, the last work was an animal study comparing serum pharmacokinetics of amikacin after intravenous administration, nebulization and in situ aerosolisation at the end of the endotracheal tube in piglets with healthy lungs undergoing mechanical centilation. The main results are that in situ aerosolisation allowed administrating large amounts of amikacin in a very short time. Nevertheless, intra-parenchymal concentrations were very heterogeneous after in situ aerosolisation and often low, while nebulization allowed observing globally more homogeneous concentrations. Immuno-histological amikacin staining allowed observing lung deposition of amikacin at the tissue level.

Targeted drug delivery is desired in cancer therapy since most of the side effects common to chemotherapy are related to the toxicity of the drug. Integrin over-expression has been shown in various cancer cells and can be exploited for targeted drug delivery. The goal of this study is to design amphiphilic conjugates with targeting motifs as a targeted drug delivery carrier. Toward this effort, novel amphiphilic conjugates of the Arg-Gly-Asp (RGD) peptide or GRGDS was linked to aliphatic acids of varying chain length. The hypothesis is that these novel amphiphilic conjugates, at concentrations above the critical micelle concentration (CMC), can form micelles in aqueous environment, encapsulate poorly-water soluble drugs, and target the α v β 3 integrin. The amphiphilic conjugate is also hypothesized to serve as targeting moiety in mixed micelle drug delivery system using Pluronic block copolymer. Synthesis of RGD amphiphilic conjugates was achieved by converting carboxylic acids into more reactive N-hydroxysuccinimidyl derivative and converting the carboxylic functional group of peptide into methyl ester. Then the activated NHS aliphatic ester was conjugated with methyl-protected peptide in the presence of organic base and methyl ester was removed in NaOH and subsequently neutralized. Intermediates and final products were characterized by MS, FTIR, and NMR. Micelle formation occurred in concentration of 0.015 to 0.12 mM for C 14 -RGD, C 16 -RGD, C 18 -RGD, and C 18 -GRGDS. Amphiphilic conjugate mixed with Pluronic L121 and Pluronic P104 (5% C 18 -RGD/L121 and 10% C 18 -GRGDS/P104) formed micelles at lower CMC of 0.0006 and 0.01 mM, respectively. Solubility of Taxol in water was improved by 87% when encapsulated in C 18 -RGD micelle above CMC. The solubility was increased 7 fold and 18 fold in mixed micelles of 5% C 18 -RGD/P104 and 5% C 18 -RGD/L121 above CMC. Three different drugs (DOX, Taxol, and etoposide) were used to evaluate the efficacy of the targeting C 18 -GRGDS micelle carrier alone or C 18 -RGD mixed with Pluronic block copolymers micelle. All 3 drugs significantly enhanced cytotoxicity toward cancer cells when loaded in micelle carrier above CMC. With same DOX concentration, C 18 -GRGDS micelle carrier significantly decreased percent of viable cells (12.9 ± 1.2%) above CMC when compared to concentrations below CMC (24.1 ± 1.0%). Mixed micelle of targeting amphiphile and Pluronic loaded with Taxol above CMC significantly decreased the percent of viable cells (38.3 ± 7.9%) when compared to non-targeting Pluronic block copolymer micelle (56.0 ± 2.8%). (Abstract shortened by UMI.)

This thesis is divided into two parts.Part I describes the organic extraction, separation, and liquid chromatographic-mass spectrometric analysis of chromophores from human and bovine retinal pigment epithelium. Flurorophores in the retinal pigment epithelium have been implicated in age related macular degeneration. In addition, the synthesis and characterization of a number of bis-retinoid type compounds that may potentially be found in such extracts, or that may be used for insight into pyridinium bis-retinoid reactivity, was accomplished.Part II describes a study of pyridinium bis-retinoid-folic acid coupling with respect to linker type, linker length, and nature of the linkage. Folic acid has been used as a targeting compound for a variety of cancer types. Development of HPLC and UV-Vis conditions suitable for the analysis of this new type of macromolecule was performed.

No description available.

In this thesis will be described research towards the development of bioorthogonal bond-cleavage reactions, and their applications in targeted drug delivery (Figure 1). The first project relates to the development of a palladium mediated bond-cleavage or "decaging" reaction which can cause a propargyl carbamate to decompose and release an amine. This was further developed by the incorporation of a protein modification handle which allowed an amine-bearing drug to be covalently ligated to a protein by a palladium-cleavable linker. This chemistry was demonstrated by the conjugation of the anticancer drug doxorubicin to a tumour targeted anti-HER2 nanobody. The drug could then be delivered to cancer cells upon addition of a palladium complex. The second project relates to the development of a platinum mediated bond-cleavage reaction. This was developed with the aim of using platinum-containing anticancer drugs - such as cisplatin - as a catalyst to cause drug release reactions in tumours. In this reaction an alkyne-containing amide can decompose to release an amine upon addition of platinum complexes, and was applied to the release of prodrugs of the cytotoxins monomethylauristatin E and 5-fluorouracil in cancer cells. A cisplatin-cleavable antibody-drug conjugate was designed and synthesised, and progress towards its biological evaluation will be discussed.

Nanoparticles were synthesized and modified for target drug delivery. The research involved the aqueous synthesis of near infrared (NIR) sensitive Au-Au₂S nanoparticles. An anti-cancer drug (<i>cis-platin</i>) was subsequently adsorbed onto the Au-Au₂S nanoparticle surface via the 11-mercaptoundecanoic acid layers. The results showed that the degree of adsorption of cis-platin onto Au-Au₂S nanoparticles was controlled by the pH value of solution, and the rate of drug release was sensitive to NIR irradiation. The results of the synthesis, drug-release properties and nanoparticle-cell interactions will be discussed. / Singapore-MIT Alliance (SMA)

Protein-protein interactions (PPIs) are attractive targets because of their therapeutic potential. One approach to design small molecules that can disrupt the PPIs is to use structural information of proteins. With this approach, triazole-based peptidomimetics that mimic beta-turn hot-spot regions in neurotrophins were synthesized. The monovalent mimics were assembled into bivalent mimics via a combinatorial method. Three different bivalent mimics were prepared for different studies. Bivalent mimics with long-linkers bound to TrkA or TrkC receptor and showed partial antagonism for the receptors. Other mimics were conjugated with cytotoxic compounds and they were used for TrkC targeted drug delivery. The last group of bivalent mimics previously showed targeted delivery effects for pancreatic cancer cells. In this study, we synthesized Eu-chelated bivalent mimics to perform a competitive binding assay for pancreatic cancer cells. Previous research in our group focused on design of secondary structures' mimics on rigid scaffolds as "minimalist mimics." We sought to establish structural design criteria for the minimalist mimics, and we wanted to propose that sets of such compounds could mimic local pairs of amino acids in any secondary structures as "universal peptidomimetics." Thus, we designed five compounds, such as oxazoline-, pyrrole-, dyine- "kinked" and "linear" bistrizole-based peptidomimetics, and performed molecular modelings, DFT calculations, and QMD for them to validate our hypothesis. On the concepts of "minimalist mimics" and "universal peptidomimetics," we developed the C alpha ? C beta vector matching program to evaluate preferred orientations of C alpha - C beta coordinates for secondary structures. We applied the program to omegatides and pyrrolinone-pyrrolidine oligomers. The compounds matched better with strands than for helices. We expanded the C alpha ? C beta vector matching idea to a method that ranks preferred conformations of small molecules on any combination of three interface side-chains in all structurally characterized PPIs. We developed a PDB mining program (explores key orientation, EKO) to do this, and EKO applied to pyrrolinone-pyrrolidine oligomers to find targets. EKO found several interesting targets, such as AICAR Tfase, GAPDH, and HIV-1 protease. HIV-1 dimerization inhibition and Zhang-Poorman kinetic assays were performed to validate our hypothesis, and the results showed that pyrrolinone-pyrrolidine derivatives inhibited HIV-1 dimerization.

No description available.