Neural Tube Defects (NTDs) are a group of common congenital malformations that result from incomplete neural tube closure leading to abnormalities of the brain and/or spinal cord. Unfortunately, their etiology remains unknown, probably due to complex multifactorial interactions. The protective effect of dietary folates in preventing NTDs is well known, but this beneficial effect is limited to the 60 to 70% of cases; leaving 30% of the population without any known option for improving pregnancy outcomes. The mechanism by which folates rescue NTD-affected embryos is poorly understood, but the ability of folate supplementation to overcome a significant percentage of NTDs and the critical role of 5-methyltetrahydrofolate in the remethylation of homocysteine (Hcy) to methionine in the placenta suggests that folate binding and/or transport might play a critical role during development. We hypothesized that maternal autoantibodies (AB) targeting placental folate receptor alpha (FRα) are blocking the receptor and limiting the ability of the FRα to bind folates, reducing intraembryonic folate levels. Furthermore, we hypothesized that AB binding to other relevant proteins required for trophoblastic growth and placentation can be involved in activating pathologic inflammatory pathways that can result in suboptimal uptake of nutrients and contribute to an abnormal closure of the neural tube. We developed a high throughput ELISA to evaluate whether mothers experiencing pregnancies complicated with NTDs are more likely to have placental AB to FRα than are mothers experiencing normal pregnancies. We optimized and simplified a protocol for AB elution from placental tissues and determined whether these antibodies were blocking the FRα from binding with available folates. Although anti-FRα IgG antibodies were not associated to the blocking activity in this study, we found that the blocking activity was higher in the placentas from NTD-affected pregnancies compared to controls, that FRα IgM antibodies are most likely the type of antibody produced during gestation that is most relevant to the blocking activity and that it is unlikely that autoimmunity against other developmental proteins associated with NTDs is generating the NTDs. / text

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Conventional methods of cancer therapy have been severely limited by inefficient delivery of therapeutic doses without incidence of harsh and toxic side effects in normal tissues. Consequently, countless new methods for early detection and drug delivery have been investigated in the area of nanoparticles and hydrogels. Although many of these methods are promising, the complex nature of cancer increases the difficultly for the development of the perfect system. Cellulose nanocrystals (CNCs) have been studied widely for a variety of applications. Despite their advantages, investigations of their abilities in the biomedical field have not been explored. The goal of this project is to delve into the potential uses of CNCs in detection, targeted drug delivery, and potentiation of irreversible electroporation (IRE)-induced cell death in folate receptor (FR)-positive cancers. To accomplish this task we have prepared stable and reproducible CNCs from wood pulp via sulfuric acid hydrolysis. Furthermore, we have functionalized the surface of these nanoparticles and conjugated them with the targeting ligand folic acid (FA) and the fluorescent imaging agent fluorescein-5\'-isothiocyanate (FITC) to create FITC-CNC-FA; CNCs have also been conjugated with doxorubicin (DOX), a potent chemotherapeutic (DOX-ALAL-CNC-FA). We have determined FITC-CNC-FA's and DOX-ALAL-CNC-FA's ability to specifically target FR-positive cancer cells in vitro; meanwhile non-targeted CNCs (FITC-CNC) were shown unable to bind to these cell types. In addition, we have investigated FITC-CNC-FA's pharmacokinetic activity in vivo. To properly model the CNC conjugate's activity in vivo, a physiologically based pharmacokinetic (PBPK) model has been constructed. We have also examined CNCs' ability to potentiate a new technique for tumor ablation, IRE. Pre-incubation with FA-conjugated CNCs (CNC-FA) have shown an increase in cytotoxicity in FR-positive cancer cells induced by IRE. In addition, CNC-FA did not potentiate IRE-induced cytotoxicity in a FR-negative cancer cell type. For a more comprehensive understanding of CNC-FA's ability to potentiate IRE induced cytotoxicity, we optimized a 3D in vitro hydrogel system. Preliminary data suggest this method of experimentation will be more realistic to in vivo studies to be completed in the future. Together, these studies showcase CNCs as efficient and effective nano-carriers in tumor detection and treatment. / Ph. D.

Anticancer therapeutics are often limited to suboptimal doses due to their lack of selectivity for tumor cells and resultant damage to healthy tissue. These limitations motivated researchers to develop tumor-specific delivery systems for improved therapeutic efficacy and reduced unintended cytotoxicity. Polyamidoamine dendrimers offer an ideal platform for designing targeted therapeutics with tunable characteristics that optimize pharmacokinetic behavior and targeting specificity. Ligand conjugation to dendrimer provides the biochemical interaction necessary to activate tumor-specific receptors for receptor-mediated endocytosis and effective internalization of polyplexes. Tumor-specific receptors overexpressed in carcinomas, like folate receptor-alpha (FOLRα), are targeted by ligand-conjugated dendrimer to allow enhanced internalization of dendrimer and its therapeutic cargo. We examined the cellular trafficking dynamics and potential of folate-conjugated dendrimer for nucleic acid delivery in vitro. Results show folate-conjugation to G4 PAMAM dendrimer (G4FA) confers enhanced uptake in FOLRα-positive tumor cells. Cells internalize G4FA in a receptor-dependent manner with specificity for FOLRα-positive tumor cells.

Cancer remains one of the most dreaded diseases due to inevitable suffering and possible fatality. Only cardiac disease has caused more deaths than cancer. Present day cancer treatment involves radiation, surgery or chemotherapy. In chemotherapy, an anti-tumoral drug is used to treat the tumor either by killing or stalling the growth of the tumor cells. In certain types of cancer, for e.g. metastatic breast cancer, the first line of therapy is often chemotherapy. But the inability of current clinically approved drugs to selectively target tumor cells, ultimately results in side effects. To reduce these side effects, prodrug therapies have been developed. A prodrug is defined as a drug molecule inactivated by a temporary cap or carrier, subsequently removed by an external intra or extracellular stimulus. Several prodrug strategies such as ADEPT (Antibody–Directed Enzyme Prodrug Therapy) have been tested in clinical trials but have thus far met with limited success. In the wake of these limitations, development of photo-activatable prodrugs may be particularly desirable for minimizing the adverse side effects associated with current cancer chemotherapeutics. Photodynamic therapy (PDT) is a light dependent tumor treatment modality that has existed for many years. PDT involves a photosensitizer which is administered to the patient and later activated using the light of wavelengths between 650-800 nm. The activated photosensitizer creates singlet oxygen, which acts as cytotoxic agent to the tumor cells. But this approach has several drawbacks including slow uptake of the photosensitizer by the tumor cells and the dependence on molecular oxygen that is not always present at even moderate levels in the tumor tissues. To address these limitations of PDT, we developed a new prodrug concept called ‘Photocaged Permeability’ in our first project, and demonstrated drug delivery using this approach. The basis of this concept is that, by attaching a hydrophilic molecule to the drug via a photosensitive linker, the permeability of the drug could be restrained. But the drug could be released at the site of the tumor after irradiating with UV light. To achieve this goal, we designed and synthesized a photosensitive drug conjugate that was comprised of doxorubicin attached to a negatively charged, cell impermeable molecule, EDANS (5-((2-Aminoethyl) amino) naphthalein-1-sulfonic acid) via a photosensitive nitroveratryl linker. Later, we performed MTT (cell viability) assays using esophageal adenocarcinoma (JH-EsoAd1) cells to determine the efficiency of our drug conjugate to induce cell death. As expected our drug conjugate was able to induce cell death, but only in presence of light. But in the dark, the cells remained unaffected. Also, we did several control studies to substantiate the fact that the cell death was actually due to drug release but not due to light or other entities. Further, we performed FACS (Fluorescence Assisted Cell Sorting) and confocal assays to show that in dark, the drug conjugate did not permeate cells. But upon irradiation with UV light, the drug was released from the conjugate, permeated the cells and induced cell death. A weakness of the above mentioned approach is that the drug is “decaged” or photo-released from the conjugates only under UV light; which cannot be translated to physiological conditions. This is because the UV light cannot penetrate deeper than 5 mm into the human skin. As a result, tumor cells that are deeply embedded in the human body cannot be treated using these approaches. To address this problem, Near Infrared (NIR) light could be used as it penetrates deeper than UV. Recently, several groups have reported using Upconverting Nanoparticles (UCNP) for the purpose of drug activation. The basis of this phenomenon is that the incidence of NIR light on these particles initiates multi-photon processes, eventually emitting UV/VIS wavelengths. The advantage of the NIR is that it deeply penetrates into the human skin. In our latest project, we have designed a drug conjugate that would be attached to UCNPs. We envision that after grafting the drug conjugate onto the nanoparticles and irradiating it with NIR drug release will occur as a result of upconversion. The above two systems describes novel methodologies for controlled release of the drug. To further improve the efficacy of the drug action, we designed new photosensitive systems based on the concept of targeted drug delivery. Targeted drug delivery is a treatment methodology in which the modified chemotherapeutic drug with higher tumor affinity could be concentrated in the tumor tissues. In certain cases, the receptors of tumor cells are targeted for the purpose of therapy. Receptors are cell surface proteins that are expressed on their plasma membrane. A select few of them such as Folic Acid Receptor (FAR) and PSMA (Prostate Specific Membrane Antigen) are overexpressed in malignant cells. In our new designs, we attached folic acid and urea based (DUPA) ligand, which were previously reported to bind to FAR and PSMA receptors respectively. Cell studies are currently underway to determine the specificity of these drug conjugates in targeting tumor cells. Once we demonstrate the above drug delivery strategies in vitro and later in vivo, we will have established novel drug delivery systems that could potentially be applied towards chemotherapeutic treatment.

Le pronostic des cancers ovariens reste sombre, en particulier en raison du retard diagnostic. Les traitements actuels associent une chirurgie de cytoréduction complète à l'administration de chimiothérapie à base de sels de platine. Les métastases viscérales sont rares dans cette pathologie, et la maladie est longtemps localisée à la cavité péritonéale. Pour cette raison, une attention particulière est portée au traitement des métastases péritonéales. Il est admis en effet que le facteur principal de réduction des récidives est l'absence de résidu tumoral en fin d'intervention. Malgré les progrès et la standardisation des techniques chirurgicales, la chirurgie de cytoréduction macroscopiquement complète, associée à une chimiothérapie efficace, ne prévient pas la survenue des récidives qui concerneront 60% des femmes en rémission à l’issu de ce traitement.Parmi les hypothèses expliquant ce taux élevé de récidive, l’existence d’une maladie microscopique résiduelle à l’issu de la chirurgie est évoquée. Le traitement de cette maladie microscopique représente un nouveau défi à relever pour les oncologues médicaux et les chirurgiens, et de nouvelles stratégies sont à développer dans ce domaine.Notre objectif est de réaliser la destruction ciblée par thérapie photodynamique (PDT) des métastases péritonéales microscopiques qui sont ignorées lors de la chirurgie. Nous espérons ainsi diminuer l’incidence des récidives locales qui concernent la majorité des patientes. Pour apporter la preuve de l’efficacité de cette stratégie innovante, un ciblage thérapeutique est indispensable car le développement de la PDT dans cette indication est limité par la mauvaise tolérance des tissus sains.Nous présentons ici les résultats précliniques obtenus in vitro et in vivo pour l’évaluation de photosensibilisateurs couplés à l’acide folique (PS1 et PS2) et ainsi dirigés vers le récepteur au folate, récepteur membranaire spécifique des cancers épithéliaux de l’ovaire (CEO).Nous avons travaillé sur des lignées cellulaires murine (NuTu-19) et humaines (SKOV-3, OVCAR-3) de CEO et sur un modèle animal de carcinose péritonéale. Après validation du modèle animal pour l'évaluation de molécule couplée à l'acide folique, nous avons montré la bonne spécificité du PS1 pour sa cible tumorale, meilleure que celle rapportée pour les autres photosensibilisateurs utilisés dans cette indication. Les lignées cellulaires émettent une fluorescence détectable après mise en culture dans un milieu enrichi en PS ce qui indique leur capacité à incorporer la molécule d’intérêt. Cette fluorescence a été détectée par spectrofluorimétrie (PS1 et PS2) et en photodiagnostic (PS2) in vivo au niveau des métastases péritonéales. La PDT permet d'obtenir la mort cellulaire des cellules humaines in vitro avec une excellente efficacité. Les premières données précliniques obtenues in vitro sur lignées humaines indiquent que la PDT utilisant un photosensibilisateur couplé à l'acide folique pourrait avoir des applications en immunothérapie.Un photosensibilisateur spécifique pourrait autoriser le développement d'une technique de PDT sure et efficace et jouer ainsi un rôle dans le traitement et la prévention des récidives péritonéales des cancers épithéliaux de l'ovaire. / Ovarian cancer’s prognosis remains dire after primary therapy. The standard of care remains debulking surgery in combination with platinum-based chemotherapy. This consists of either primary debulking surgery and adjuvant chemotherapy or neoadjuvant chemotherapy followed by interval debulking surgery, depending on FIGO stage and predictive factors concerning residual macroscopic disease after surgery. Recurrence rate is disappointingly high as 60-80% of women with epithelial ovarian cancer (EOC) considered in remission will develop recurrent disease within five years. Special attention to undetected peritoneal metastases and residual tumorous cells during surgery is necessary as they are the main predictive factors of recurrences.An option to improve the completion of cytoreductive surgery is using photodynamic therapy (PDT) to induce necrosis of micrometastases. A limit of this technique is the toxicity induced by the low photosensitizer (PS) specificity for tumor tissue if the light cannot be specifically applied. This would be the case in advanced ovarian cancer. To solve this problem, a solution is the design of selective PS, that is to say PS coupled to a unit that target over-expressed receptors on tumor cells. Approximately, 72-100% of ovarian carcinoma overexpress Folate Receptor α (FRα) in particular the serous carcinoma. FRα is absent in most of the healthy tissues; thus, representing a promising target for EOC targeted therapy.We present preclinical results of in vitro and in vivo studies concerning properties of folic-acid targeted photosensitizers (PS1 and PS2). Those studies have been performed on murine and human cell lines of EOC and on a preclinical model of peritoneal carcinomatosis (Fisher F344 rat / NuTu-19 cell line). Results suggest that specificity for ovarian cancer metastases is better than previously reported with other photosensitizer. Fluorescence emission was higher in peritoneal metastases than in liver and healthy peritoneum. Tissue quantification of the PS showed specific incorporation of the folate-targeted PS within tumor tissue. Folic acid targeted PDT induced cellular death on EOC human cell lines.Specific PS may allow the development of efficient and safe intraperitoneal PDT procedure which could play a role in the prevention of EOC peritoneal recurrences.

This dissertation comprises an introduction, background and current research progress in the area of classical antifolates as the targeted anticancer therapies.<br>In this study, twelve series of classical 5-, 6- and 7-substituted pyrrolo[2,3-d]pyrimidines were designed and synthesized. Extensive structure modifications of the pyrrolo[2,3-d] pyrimidine scaffold were investigated to determine selective transport via FR or/and PCFT and tumor targeted antifolates with GARFTase or multiple folate metabolizing enzyme inhibition.<br>The design strategies employed include: variation of the side chain substitution position (5-,6- and 7-substituted); variation of the side chain length (n=1-6); isosteric replacement of the 1,4-disubstituted phenyl ring with 1,2- and 1,3- disubstituted phenyl ring and 2,5- disubstituted thiophenyl ring; replacement the L-glutamate with variation at the á and ã carboxylic acids.<br>As a part of this study, a total of one hundred and fifty six new compounds (including new intermediates) were synthesized and separated. Of these, twelve series consisting of forty two classical antifolate final compounds were submitted for biological evaluation. In addition, bulk synthesis of some potent final compounds (2, 2.0 g; 161, 500 mg; 175, 1.0 g; 166, 500 mg; 194, 500 mg) was carried out to facilitate in vivo evaluation.<br>More importantly, a new Heck coupling of the thiophene iodide 301 and allyl alcohols to synthesize aldehydes in one step was discovered. Due to its potential use in analog synthesis of clinically used antifolates such as RTX and PMX, this mild conditioned and easy to handle Heck coupling reaction is highly attractive.<br>During this study, the SAR of folate transporters (RFC, FR and PCFT) and GARFTase inhibitors were extensively explored. The 6-substituted straight chain compound 166 (n=7) was extremely potent against KB tumor cells (IC50=1.3 nM, about 80-fold more potent than clinically used PMX) without any RFC activity. The 5- substituted phenyl compound 175 (n=4) showed AICARFTase as the primary target with potent KB tumor cell inhibition (IC50=7.9 nM, about 8-fold more potent than PMX) and also indirectly inhibited the mTOR pathway leading to tumor cell apoptosis. Due to their potent antitumor activities, these two compounds serve as leads for future structural optimization. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Medicinal Chemistry / PhD; / Dissertation;

Elevated total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease and neurocognitive disease such as dementia. The B vitamins folate and B12 are the main de terminants of tHcy. tHcy concentration can also be affected by mutations in genes coding for receptors, enzymes and transporters important in the metabolism of Hcy. This thesis focuses on mutations in the genes for folate receptor-alpha and methylenetetrahydrofolate reductase (MTHFR) and the effect they have on tHcy concentrations. Six novel mutations in the gene for folate receptor-alpha were described in Paper I. Taken together they exist in a population with a prevalence of approximately 1% and thus are not unusual. There may be an association of –69dupA and –18C>T to tHcy but for the 25-bp deletion, –856C>T, –921T>C and –1043G>A there is probably no association to tHcy. Mutation screening was continued and four additional mutations, 1314G>A, 1816delC, 1841G>A and 1928C>T, were described in Paper II. The prevalences for the heterozygotes were between 0.5% and 13% in an elderly population. There was no significant difference in prevalence between the elderly subjects and patients with dementia. The 1816(–)-allele and the 1841A-allele were in complete linkage and the haplotype 1816(–)-1841A may possibly have a tHcy raising effect. The 1314G>A and 1928C>T mutations had no association to tHcy. The genotype prevalences and haplotype frequencies of the MTHFR 677C>T, 1298A>C and 1793G>A polymorphisms were determined in a population sample of Swedish children and adolescents (Paper III). The MTHFR 677T-allele was associated with increased tHcy concentrations in both children and adolescents. A small elevating effect of the 1298C-allele and a small lowering effect of the 1793A-allele could be shown. In an epidemiological sample of adults from the Canary Islands, Spain, data for serum folate and vitamin B12 were used for a broader study of the nutrigenetic impact on tHcy (Paper IV). The 677T-allele had a significant tHcy increasing effect in men but not in women. The 1298C-allele had a minor elevating effect on tHcy in men with the 677CT genotype. It was not possible to document any effect of the 1793A-allele on tHcy due to its low prevalence. A slightly superior explanatory power for the genetic impact was obtained using the MTHFR haplotypes in the analysis compared to the MTHFR 677C>T genotype-based approach in both the Swedish children and adolescents and in the Spanish adults. Therefore MTHFR haplotypes should be considered when analysing the impact of the MTHFR 677C>T, 1298A>C and 1793G>A polymorphisms on tHcy. Notwithstanding the large geographical distance between our study populations the haplotype composition is quite similar. The MTHFR 677T-allele is slightly more prevalent in Spain compared to Sweden but it has only an effect on tHcy in the Spanish men. Age, gender and factors linked to the ethnicity of the studied subjects, seem to be able to override the nutrigenetic impact of tHcy-raising genotypes or haplotypes in particular settings, such as in the Spanish women in our study. Gene-nutrient interactions on plasma tHcy levels thus may or may not exist in a certain population. The transferability of nutrigenetic findings may therefore be limited, and must be re-evaluated for each particular setting of age-gender-ethnicity.

Despite the recent advances in cancer therapy, the successful detections and treatments of cancer still remains a challenge. The existing strategies for early cancer detection are often limited due to their poor sensitivity and specificity. Also, the non-selective action of therapeutic interventions hinders treatment success. Our research was therefore directed towards the engineering of excellent bi-functionalized nanoparticles (NPs) based on carbon dots (CDs) that would improve early cancer detection and overcome the limitations of chemotherapy. With the actively targeting agent, these new NPs are expected to effectively deliver pharmacological agents directly to cancer cells. CDs are carbon-based NPs that are utilized as bioimaging agents and drug delivery systems (DDS) due to their excellent biocompatibility, non-toxicity, unique imaging, and facile surface modification. Using folic acid (FA) as targeting agent, the prepared novel CDs will carry doxorubicin (DOX) covalently and non-covalently to the cancer cells with overexpressed folate receptors. The CDs were first synthesized via the hydrothermal bottom-up approach using citric acid and ethylenediamine as precursors. The prepared CDs were then functionalized by FA via a non-cleavable peptide bond followed by complexation with DOX covalently or non-covalently to obtain the desired FA-CD-DOX NPs. All the NPs and intermediates were characterized using ultraviolet-visible spectroscopy (UV-vis), fluorescence spectroscopy (FL), and Fourier transform infrared spectroscopy (FTIR). Assessment of the drug loading capacity (DLC) and drug loading efficiency (DLE) with UV-vis indicated that the non-covalent NPs have low DLC but high DLE compared to the relatively low DLE and high DLC of covalent NPs. In vitro drug release studies were also carried out in phosphate buffered saline (PBS) systems with various pH. It was found that even though both non-covalent and covalent complexes released more DOX at pH 5.0 than at pH 7.0, the DOX release rate was faster in the non-covalent FA-CD-DOX NPs compared to the covalent FA-CD-DOX. Based on these results, we project increased accumulation of drugs in the more acidic (pH 4.5-5.0) microenvironment of cancer cells compared to that of normal healthy cells under physiological pH (7.4). This new FA-CD-DOX NPs could work as efficient theranostic systems to detect and treat cancer.