Specific interactions of ligands with receptors is one of the approaches for active targeting of anticancer drugs to cancer cells. Over expression of integrin receptors is a physiological manifestation in several cancers and is associated with cancer progression and metastasis, which makes it an attractive target for cancer chemotherapy. The peptide sequence for this integrin recognition is the Arg-Gly-Asp (RGD). Self-assembly offers a unique way of presenting ligands to target receptors for recognition and binding. This study focuses on development of integrin specific peptide amphiphile self-assemblies as carriers for targeted delivery of paclitaxel to α v β 3 integrin overexpressing cancers. Amphiphiles composed of conjugates of different analogs of RGD (linear, cyclic or glycosylated) and aliphatic fatty acid with or without 8-amino-3,6-dioxaoctanoic acid (ADA) as linker were synthesized and characterized. The amphiphiles exhibited Critical Micellar Concentration in the range of 7-30 μM. Transmission electron microscopy images revealed the formation of spherical micelles in the size range of 10-40 nm. Forster Resonance Energy Transfer studies revealed entrapment of hydrophobic dyes within a tight micellar core and provided information regarding the cargo exchange within micelles. The RGD micelles exhibited competitive binding with 55% displacement of a bound fluorescent probe by the cyclic RGD micelles. The internalization of fluorescein isothiocynate (FITC) loaded RGD micelles was significantly higher in A2058 melanoma cells compared to free FITC within 20 minutes of incubation at 37°C. The same micelles showed significantly lower internalization at 4°C and on pretreatment with 0.45M sucrose confirming endocytotic uptake of the RGD micellar carriers. The IC50 of paclitaxel in A2058 melanoma cells was lower when treated within RGD micelles as compared to treatment of free drug. On the other hand, IC50 values increased by 2 to 9 fold for micellar treatment in comparison to free drug in Detroit 551 cells. In A2058 melanoma xenograft mice model, the Paclitaxel-RGD micelles exhibited a significant inhibition of tumor growth in comparison to control treatment for both alternate day and twice weekly treatments. The studies showed the feasibility of using the non covalent peptide based self-assemblies as vehicles for targeted delivery in cancer.

Acquired tamoxifen resistance develops in the majority of hormone responsive breast cancers and frequently involves overexpression of the PI3K/AKT axis. Here, breast cancer cells, with elevated endogenous AKT or overexpression of activated AKT exhibited tamoxifen-stimulated cell proliferation and enhanced cell motility. To gain mechanistic insight on AKT-induced endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of AKT overexpression. Consistent with the biological outcome, many of these transcripts function in cell proliferation and cell motility networks and were quantitatively validated in a larger panel of breast cancer cells. Moreover, ribonucleotide reductase M2 (RRM2) was revealed as a key contributor to AKT-induced tamoxifen resistance. Inhibition of RRM2 by RNAi-mediated approaches significantly reversed the tamoxifen-resistant cell growth, inhibited cell motility, and activated pro-apoptotic pathways. In addition, treatment of tamoxifen-resistant breast cancer cells with the small molecule RRM2 inhibitor Didox significantly reduced cell growth in vitro and in vivo. To further establish a functional association between RRM2 expression and tamoxifen resistance in breast cancer cells, gain of function studies were performed by overexpressing RRM2 in MCF-7 cells. Overexpression of RRM2 profoundly reduced tamoxifen sensitivity and down-regulated ER-&agr; in otherwise tamoxifen sensitive breast cancer cells. Furthermore, breast cancer cells with high RRM2 had elevated Her-2 and EGFR expression, modulated ER-&agr; signaling and NFκB expression. These findings also indicate that it may be possible to use RRM2 as a prognostic factor in breast cancer patients under tamoxifen therapy, and can be considered a potential therapeutic target in tumors that have acquired resistance to tamoxifen. Finally, inhibition of RRM2 by drug Didox effectively eradicates the tamoxifen resistant population, revealing a potential beneficial effect of combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance. In conclusion, the findings of this work delineate the important role of RRM2 in Akt induced and acquired tamoxifen resistance in breast cancer. It also provides a preclinical rationale for evaluating tamoxifen in combination with Didox for breast cancer treatment.

Oil in water (o/w) nanoemulsion is a two-phase dispersed system in which the oil can incorporate poorly water soluble drugs to form a liquid dosage form. The enhancement of bioavailability with a use of nanoemulsion has often been reported empirically and speculated to be a result of the enhanced dissolution due to a larger surface area, however, the mechanism of nanoemulsion permeation was yet to be explored. The goal of this dissertation was to understand the mechanism of nanoemulsion permeation and to control permeation and bioavailability. The first objective was to delineate the effect of thermodynamic activities of the drug in nanoemulsion on the permeation through a barrier. The flux from nanoemulsion depended on the thermodynamic activities of both ionized and unionized species in the aqueous phase of nanoemulsion. A simple nanoemulsion was not favorable to enhance the permeation over the saturated solution due to the reduced thermodynamic activity. Thus, the second objective was to elucidate the role of transient supersaturation on permeation enhancement using nanoemulsion. In vitro permeation using self-nanoemulsifying drug delivery system (SNEDDS) was enhanced over the saturated solution due to the transient supersaturation; however the enhancement of bioavailability in rats was not due to the enhanced passive permeation. Therefore there was a need to increase or prolong the supersaturation. The third objective was to control the supersaturation to enhance in vitro permeation by formulation approaches. The optimum drug loading was determined based on the precipitation kinetics; however the ability to modulate the thermodynamic activity to enhance permeation by changing the drug loading was limited. The precipitation inhibitor, hydroxypropylmethyl cellulose was able to retard the precipitation and enhanced in vitro permeation due to the increased thermodynamic activity. The significance of this work was the systematic approach to understand the mechanism of nanoemulsion absorption and to utilize nanoemulsion for permeation enhancement. The knowledge gained in this work will help rationally design the formulation in the future.

Depression is a common mood disorder. Although major ethical challenges make it nearly impossible to invasively and directly measure serotonin (5-hydroxytryptamine, 5-HT) levels in human brains, neuroimaging technologies have shown macroscopic structural and functional abnormalities in the prefrontal cortex (PFC) of depressed patients. The monoamine hypothesis of depression is based on the neurotransmitter imbalance, such as deceased serotonin brain levels are implicated in the cause of depression. Research has focused on the control mechanisms involved in the dorsal raphé nucleus (DRN) which is the serotonergic control center located in the midbrain. We hypothesized that activation 5-HT 2A receptor in PFC would increase serotonin levels by an AMPA-dependent mechanism in both DRN and PFC. Enhancement of the 5-HT in DRN may inhibit 5-HT level in PFC by 5-HT 1A receptor. This becomes the full feedback loop system. While 5-HT levels in the PFC have been well studied, pathway that modulate this DRN pool through upstream cascade interactions leading to a downstream feedback loop have been difficult to elucidate. Developing a mechanism-based pharmacokinetics (PK) and pharmacodynamics (PD) model to quantitatively describe the effect of 5-HT 2A receptors regulation to serotonin in the DRN and PFC would help us to better understand the complex brain. 5-HT 2A receptor agonist and AMPA receptor agonist and antagonist were used to activate or block the related receptor. Male Wistar rats underwent neurosurgery for implantation of microdialysis (MD) probes. Three to five rats were randomly assigned to experimental arms. Using the MD method, the drug combination was examined to explore the drug effect on time course of 5-HT release in DRN and PFC. Based on the experiment results, a mechanism-based PD model was developed. Phoenix WinNonlin ® and Berkeley Madonna™ were used for model estimation, external validation with secondary data set, and simulation. The result supports the possibility of a 5-HT 2A /AMPA feedback control circuit that originates in the PFC and modulates DRN and PFC 5-HT levels through feedback coupling of 5-HT. The time-course profiles of 5-HT in both DRN and PFC was well modeled and model parameters were estimated with good precision (CV% ranged from 1.37% to 35.03%). The mechanism model was developed to characterize and better understand the neurotransmitter mechanisms, providing estimations of various parameters of the disease related receptor system.

The DART ion source was introduced in 2005 at the ASMS Sanibel Conference and immediately afterward Professor Sparkman was contemplating of a way to get our lab this revolutionary mass spectrometry ionization technique. It did not take long because it was delivered to the Pacific Mass Spectrometry Facility in August 2006 and I was able to being using and learning the technique. The ion source creates excited state helium metastables (2 3 S) with an ionization potential of 19.8 eV are created by a glow discharge at atmospheric pressure. The metastables are sent through an optional heater, to aid in desorption, enter the open-air to directly ionize your sample or ionize reagent species to react with the analyte molecules. The most observed ionization mechanism is the formation of protonated molecules from a proton-transfer reaction between the analyte and protonated water clusters. The limited to no sample preparation with the "soft" ionization provide very quick identification of intact organic ions in or on various types of matrices. When the DART is coupled to a high resolving power instrument, such as the JEOL AccuTOF, accurate masses and accurate isotope ratios are assigned to aid in the determination of unknown elemental compositions. This research discusses the formation of the metastable species and how they are used to produce analyte and reagent ions within the open-air sample gap of the DART-mass spectrometer interface. A description of the fundamentals on the operation including real time visualization of the fluid dynamics and confirmation of the formation of a hydroxyl radical in the proposed formation of the protonated water clusters, along with applications developed in the Pacific Mass Spectrometry Facility will also be discussed. These include cleavage, desorption, and ionization of solid-phase peptides, desorption of aqueous metal ions using a heated wire filament and the increased ion transmission with the Vapur interface using metal coated glass tube for the transfer tube.

Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Second we developed lipids with imidazole headgroups. Such lipids can protonate to provide positive charges on liposome surface at lowered pH. Additionally, negatively charged PEGylated phospholipids can cluster with the protonated imidazole lipids to display excess positive charges on the surface of the liposomes, thus enhancing their interaction with negatively charged cancer cells. We prepared convertible liposome formulations I, II and III consisting of one of the three imidazole-based lipids DHI, DHMI and DHDMI with estimated pKa values of 5.53, 6.2 and 6.75, respectively. Zeta potential measurement confirmed the increase of positive surface charge of such liposomes at lowered pHs. DSC studies showed that at pH 6.0 formulation I formed two lipid phases, whereas the control liposome IV remained a one-phase system at pHs 7.4 and 6.0. The interaction of such convertible liposomes with negatively charged model liposomes mimicking biomembranes at lowered pH was substantiated by 3-4 times increase in average sizes of the mixture of the convertible liposomes and the model liposomes at pH 6.0 compared to pH 7.4. The doxorubicin-loaded convertible liposomes show increased cytotoxicity in B16F10 (murine melanoma) and Hela cells at pH 6.0 as compared to pH 7.4. Liposome III shows the highest cell kill at pH 6.0 for both the cells. The control formulation IV showed no difference in cytotoxicity at pH 7.4 and 6.0. Uptake of convertible liposome II by B16F10 cells increased by 57 % as the pH was lowered from 7.4 to 6.0.

It has long been known that the efficiency of anticancer drugs is limited by the emergence of resistance due to the evolving repair of such DNA lesions in malignant cells. Therefore, development of pharmaceutical agents, which can interfere with the DNA repair pathways, may represent a novel approach to enhance the cytotoxic effects of chemotherapy by reducing drug resistance. Abasic sites (AP sites) are the key intermediates in the BER pathway and promising targets for BER inhibition. In chapter 2, we report the synthesis of two small molecules specifically targeting at AP sites and the evaluation of their activity in terms of interstrand crosslinking formation. Our results show no covalent adduct is induced, which is due to the weak DNA binding affinity. In chapter 3, we try to use TFOs to deliver the interstrand crosslinking moiety to the AP site in a sequence specific manner. Two modified phosphoramidites were synthesized and incorporated into the 5' end of TFOs. The activity was evaluated by using various biophysical and biochemical experiments. The work reported in chapter 4 is focused on the G-quadruplex structure formed in the guanine rich telomeric sequence. Many studies have shown G4 ligands can induce and stabilize G-quadruplex within telomere region and inhibit the activity of telomerase that is overexpressed in 80-90% of cancer cells. Our results indicate that phenanthroline based metal complexes, Ni(Phen) 2 , have strong binding affinity and selectivity towards G-quadruplex over duplex DNA. The effect of Ni(Phen) 2 on telomerase activity and cytotoxicity towards cancer cells was also investigated. Calixarenes containing DNA building units such as nucleotides, nucleosides, and nucleobases have recently aroused much interest because of their versatile applications. In chapter 5, we report the synthesis of calix[4]arenes ( 5.11-5.14 ) functionalized with a single nucleobase (thymine, adenine, guanine, or cytosine) at the upper rim via click chemistry. Their complexation with alkali metal ions was examined using MALDI-TOF mass spectrometry and their molecular interactions were determined using 1 H NMR. All calix[4]arene derivatives show good complexation with alkali metal ions with apparent selectivity. The results also reveal that nucleobase-calix[4]arenes are capable of self-association in CDC1 3 and calix[4]arenes bearing complementary nucleobases can bind to each other via base pairing.

Trans-2-Aminocyclohexanol (TACH) is a promising model for pH-triggerable molecular switches with a variety of potential applications. In particular, such a switch, when incorporated into cationic liposomes, provides a novel design of the pH-sensitive helper lipids for gene delivery. Protonation of TACH molecules results in a strong intramolecular hydrogen bond between the amino and its neighboring hydroxyl groups, which triggers a conformational flip, and forces changes of the relative position of other substituents on the ring. In this work, a library of TACH-lipids has been designed and built based on structural modifications of both hydrophilic headgroups and hydrophobic tails, and their conformational behavior has been studied by 1 H NMR. NMR-titration has been done to quantitatively monitor the conformational switch for TACH derivatives. It was discovered that conformational behavior of TACH-lipids is independent from the length or shape of their hydrophobic tails. Therefore, a simplified model was suggested based on TACH with diethyl groups instead of hydrocarbon tails. Conformational study of these models has demonstrated that the position of equilibrium shift A [special characters omitted] BH + can be effectively changed by altering structure of NR 2 R 3 group. Furthermore, the pH-induced conformational flip occurs in a certain pH range that mostly depends on the basicity of group NR 2 R 3 , allowing a broad tuning of the pH-sensitivity of TACH-based conformational switches in a wide range of acidity. The hydrophilic OH group was also modified to influence the conformational equilibrium. External stimuli including addition of acid, change of solvent and of the solution ionic strength also showed impact on conformation equilibrium to different extents. To explore the potential to serve as pH-sensitive helper lipids in gene delivery, a variety of TACH-lipids were incorporated into lipoplexes together with the cationic lipid DOTAP to mediate DNA transfection in Bl6F1 and HeLa cancer cell lines. The lipoplex comprising TACH-lipid 3o (R 1 = C 19 H 37 ; R 2 R 3 = CF 3 CH 2 NH) exhibited one to two orders of magnitude better transfection efficiency than the one with the conventional helper lipid DOPE while only inducing slight higher cytotoxicity. Thus, the lipid can be suggested as a novel helper lipid for efficient gene transfection with low cytotoxicity.

Our research involved two projects: Crown Aza and Crown ether. Crown Aza are compounds that have nitrogens as the hetero-atoms in the macrocycle. In the first aim, the 1 H NMR spectrum of strongly-coupled di-iron complexes shows 5 different conformations, but is unable to identify these conformers. Calculation using Density Functional Theory (DFT) were performed to attempt to quantify these conformers to correlate with the experimental NMR data. The second aim of this study was to utilize the carbonyl functional group of the macrocycles to couple the iron complexes to obtain tetramers or higher oligomers. Visible spectra of the selective reduction of iron mono-keto-macrocycles with boron trifluoride and triethylsilane showed a coupling reaction to form a di-iron complex. A mixture of iron mono-keto-macrocycic with iron diketo-dimacrocycle showed a new species with an intense NIR absorption at 1010 nm. This intense band at 1010 nm is extremely rare in transition metal compounds and is of potential interest in photodynamic therapy. This indicates a new species is formed with a very low energy gap between the ground and excited states. Our second interest is in ionizable crown ethers that have many potential applications from environmental to medical. These macrocyclic poly-ethers contain phosphorous and oxygen atoms which produce a charged moiety in the ring to form a neutral complex with our targeted ions. They are significantly different from all ionizable cyclic poly-ethers in the literature. They vary in ring size from 16-crown-6 to 22-crown-8 using ethylene or propylene linkages between the oxygen atoms. Theoretical calculations revealed these ionizable crown ethers can accommodate ions from 2.6 angstroms to 4.2 angstroms in diameter. Strontium 90 ( 90 Sr 2+ ) is a radioactive product ion from nuclear fission reactions. The Phosphorous containing macrocyclic poly-ether with 2 charges could form complexes with Strontium and are possible candidates for clean-up agents of radioactive materials. The synthesis of ionizable crown ethers is discussed.

The current dissertation describes a multidisciplinary research project centered on the discovery and investigation of the anticancer activities exhibited by novel piperazinylpyrimidine derivatives designed to target kinases protein family. Primary screening of the antiproliferative effects implemented by these successfully synthesized new agents has resulted in the candidacy of 4 , 15 , and 16 as not only prototype representatives of the class, but surprisingly also as optimized agents for either globally cytotoxic, 16 , or selectively cytostatic, 4 and 15 , agents. Subjecting 4 , 15 , and 16 to screening tests aiming at measuring their binding to or their functional inhibition of selected sets of kinases has revealed the tendency of 4 to target PDGFR subfamily and the ability of 4 , 15 , and 16 to recognize CSNK1D. Docking as well as binding profiles comparative studies hypothesize 4 , 15 , and 16 as type-I kinase inhibitors. Further preclinical investigation of 15 against MDA-MB-468 triple negative breast cancer cell line revealed that 15 exhibits a time as well as dose-dependent antiproliferative activity mediated by the induction of both time and dose independent G2/M arrest and dose dependent apoptosis. Globally studying the molecular events accompanied with the 15 /MDA-MB-468 incidence has revealed the phosphorylation of TP53 and the consequent activation of its transcriptional activity as a hallmark molecular event relevant to the above observed effects on the cellular, cell cycle, and programmed cell death levels. Apart from the above experimentally oriented investigation, another theoretically driven inquiry was pursued aiming at studying the inherent ability of certain kinases to be more promiscuous towards binding to small molecules than others. Throughout the analysis of a reported dataset, dephosphorylated members of PDGFR subfamily were found to more potently bind to structurally diverse kinase inhibitors compared to INSR subfamily. A molecular dynamics study was performed to compare between the topological, energetic, and dynamic properties of the binding area usually targeted by kinase inhibitors in both KIT, as a representative of the more promiscuous PDGFR subfamily, and INSR, as a representative of the less promiscuous INSR subfamily. Interestingly enough, the binding area in both kinases showed significantly different properties which, to a large extent, can explain their different overall attitudes towards binding small molecules. As a representative example, the binding area of INSR tends to be more energetically self-stabilizing than that in KIT. Additionally, the topological analysis revealed that the binding are in KIT tends to be more rigid and to have bigger size than that of INSR. The current work has successfully cross-implemented experimental, theoretical, and computational studies aiming at the development of novel kinase inhibitors and/or promising anticancer preclinical candidates.