The objective of this series of investigations was two-fold: (1) to investigate new synthetic routes to the sterically hindered amines and hydrazines, and (2) the preparation of potential hypotensive and anticholinergic agents to be screened for pharmacological activity. A series of N-(substituted amino)-2,5-dimethylpyrroles was synthesized by means of the Knorr-Paal condensation, and the N-alkylamino analogs were found to undergo catalytic hydrogenation to the corresponding pyrrolidines, while the N-arylamino compounds underwent no reaction or yielded to N-N bond cleavage, depending upon severity of the conditions. In applications expanding upon the fundamental ground-work laid by Broadbent and Olsen^1 a series of 4-substituted 1,7-dimethyl-2,6-dioxa-10-azatricyclo[5.2.1.0^4,10]decanes was prepared by the interchange reaction of sterically hindered carboxylic and carbamic acid esters with 1,7-dimethyl- 4-hydroxymethyl-2,6-dioxa-10-azatricyclo[5.2.1.04,10]decane. Other exploratory work in this area resulted in the preparation of the 4-ethoxymethyl analog of the tricyclic nucleus by the Williamson synthesis. A correlation was made between the synthesis of the 1,7-dimethyl-2,6-dioxa-10-azatricyclo[5.2.1.0^4,10]decanes by condensation of 2-amino-1,3-propanediols with 2,5-hexanedione and the more common Knorr-Paal reaction of primary amines with the same diketone. These results indicated that both pathways were functioning simultaneously in the condensation of 2-amino-1,3-propanediols with Υ-diketones or their equivalent, production of the tricyclic system taking preference over pyrrole formation when the effects of steric hindrance become operative. As an outgrowth of studies directed toward the synthesis of analogous heterotricyclic systems a family of 2,2'- ethylenebis(1,3-dioxanes) was prepared. The 5-amino-5- methyl compound of this series was found to form a stable N-bromo derivative and is undergoing further consideration in regard to this property. These investigations also led to the synthesis of the parent compound of the 2,2'-ethylenebis(1,3-dithiane) series. All compounds synthesized during the course of this research have been submitted to private industry for pharmacological screening as well as to the Cancer Chemotherapy National Service Center. Pharmacological data resulting from this work is not currently available for public use.

Evofosfamide, also formerly known as TH-302, is an investigational hypoxia-activated prodrug and is used to target cancerous cells under hypoxic conditions, which is a feature possessed by multiple solid tumors including pancreatic tumors. Gemcitabine, a cytotoxic agent, has for many years been the standard first-line treatment for metastatic pancreatic cancer in patients. In recent years, combination chemotherapeutic therapies have provided a new avenue for molecular targeting by increasing the probability of eliminating the cancer and minimizing the likelihood of resistance. We have evaluated multiple studies in an effort to shed light on an emerging prodrug, evofosfamide, which operates by selectively targeting the tumor hypoxic compartment. A web-based literature search was performed through PubMed and Google Scholar using the keywords 'evofosfamide', 'TH-302,' and 'pancreatic tumor.' Of the available results, 53 relevant studies were reviewed and summarized. Chemotherapeutic agents such as evofosfamide, which targets tumor hypoxia, are new agents against cancer cells. Current experience with these agents is limited as additional and longer prospective studies are needed to further evaluate the clinical efficacy and postmarketing safety profile.

Studies of drug resistance mostly characterize genetic mutations, and we know much less about the phenotypic mechanisms of drug resistance, especially at a quantitative level. p53 is an important mediator that regulates the cellular response to chemotherapy, but even cancer cells with wild-type p53 exhibited variable drug sensitivity for unclear reasons. In this PhD thesis, I investigated the mechanistic basis underlying differential p53 pathway activation in response to two types of chemotherapeutics, i.e., etoposide (a DNA-damaging drug) and 5-fluorouracil (5-FU, an antimetabolites), which led to distinct cell fate outcome in drug sensitive vs. resistant cancer cells. Specifically, I uncovered a new resistance mechanism to etoposide through bimodal modulation of p53 activation dynamics and characterized a four-component regulatory module, involving ATM, p53, Mdm2 and Wip1, which generates bimodal p53 dynamics through coupled feed-forward and feedback loops. Moreover, I found that the inhibitory strength between ATM and Mdm2 determined the differential modular output between drug sensitive and resistant cancer cell lines, and that combinatorial inhibition of Mdm2 and Wip1 was an effective strategy to alter p53 dynamics in resistant cancer cells and sensitize their apoptotic response, pointing to p53 pulsing as a potentially druggable mechanism that mediates resistance to DNA damaging chemotherapy. As for response to 5-FU, preliminary results illustrated that 5-FU activated p53 and differential cell fate outcome via ribosomal stress, rather than DNA damage. Different from dose response to etoposide, 5-FU-induced p53 activity was not only regulated by p53 induction level but also p53 phosphorylation by kinases, such as DNA-PK. Overall, this thesis presented original results that elucidated phenotypic mechanism of chemoresistance and provide new angles towards developing more effective combinatorial anticancer therapy.

Metronomic cyclophosphamide (CPA) treatment activates robust anti-tumor immunity and induces regression of implanted tumors in mouse models of brain cancer when administered on an intermittent, every 6-day schedule (CPA/6d), but not on a daily low-dose or a maximum-tolerated dose schedule. Five intermittent metronomic CPA schedules were investigated in GL261 gliomas implanted in scid mice. Metronomic CPA treatments spaced 9 or 12 days apart induced extensive tumor regression, however, tumor-infiltrating natural killer cell responses were not sustained, and tumor growth rapidly resumed after treatment day 24. Increasing the CPA dose prolonged the period of tumor regression on the every 9-day schedule, but natural killer cell activation was markedly decreased. Thus, sustained immune and anti-tumor responses were only achieved on the CPA/6d schedule. Furthermore, CPA/6d treatment eradicated GL261 tumors implanted in immunocompetent C57BL/6 mice by activating anti-tumor CD8-T cell responses and immune memory, which provides proof-of-concept that single agent chemotherapy delivered on an optimized metronomic schedule can cure large established cancers. Transcriptomic profiling, KEGG pathway, and upstream regulator analysis were employed to compare CPA/6d-induced gene expression changes between: immune-responsive GL261 tumors and immune-unresponsive Lewis lung carcinoma (LLC) and B16F10 melanoma tumors; between GL261 tumors implanted in immunocompetent mice versus in scid immunodeficient mice; and between GL261 tumors in scid mice treated with CPA every 6-days or every 9-days. CPA-treated LLC tumors were associated with inhibited VEGFA-targeted genes, down-regulated cell adhesion and transendothelial migration genes, and up-regulated drug metabolism pathways. In B16F10 tumors, CPA activated genes in chemokine signaling and antigen processing and presentation pathways, but no NK cell and T cell effector pathways were activated. GL261 tumors in scid mice were deficient in CPA activation of a subset of cytokine and cytokine receptor genes and T cell receptor signaling genes seen in immunocompetent mice. Cytokine gene expression was lower and drug metabolism gene expression was higher in every 9-day CPA-treated tumors versus CPA/6d-treated tumors. Together, these studies elucidate the dose, schedule, and adaptive immune-dependence of CPA-induced anti-tumor immune responses, giving new insight into the molecular signaling events underlying the deficiencies in immune responses seen in intermittent metronomic CPA-unresponsive tumor models. / 2017-05-31T00:00:00Z

While CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone), the current standard of care for non-Hodgkin lymphoma (NHL), kills tumor cells, the accumulation of tumor cell “debris” can stimulate inflammation and tumor growth. Thus, cytotoxic cancer therapies are a double-edged sword. Previous studies have shown that apoptotic debris stimulates tumor growth. We hypothesize that (1) CHOP-generated tumor cell debris can promote lymphoma progression via release of pro-inflammatory cytokines; (2) blocking phosphatidylserine (PS), which is presented on the surface of apoptotic cells, may inhibit debris-stimulated cancer progression. METHODS: Lymphoma EL4 debris was generated by treating tumor cells with CHOP chemotherapy. EL4 debris was isolated via Ficoll gradient and co-injected with living EL4 tumor cells into immunocompetent C57BL/6 mice. Macrophage-secreted cytokines were measured via array analysis. RESULTS: Flow cytometry confirmed CHOP chemotherapy generated apoptotic/necrotic debris. Vincristine-, mafosfamide-, and prednisolone-generated lymphoma EL4 debris stimulated tumor growth by over 100-fold in a dose-dependent manner. Debris alone did not induce tumors, even at 250 days post-injection. Doxorubicin-generated EL4 debris stimulated tumor growth at low dose (1x105), but inhibited growth at high dose (9x105). Systemic administration of doxorubicin-generated EL4 debris or blocking PS in the cell debris generated by doxorubicin using annexin V or an anti-PS neutralizing antibody inhibited doxorubicin-generated debris-stimulated tumor growth. Therapy-generated debris stimulated macrophage pro-inflammatory cytokine production. CONCLUSIONS: CHOP chemotherapy-generated debris regulates tumor growth via cytokine production. Thus, harnessing the anti-tumor activity of inhibitory debris or neutralizing PS on stimulatory debris may be a novel anti-cancer approach. / 2018-07-11T00:00:00Z

Ovarian cancer is the second most common, and first most lethal gynecological cancer. It will affect one in seventy-eight women, and is commonly diagnosed in the later stages of the disease. The majority of the cancer’s lifespan is spent within the peritoneal cavity. Hyperthermic intraperitoneal chemotherapy (HIPEC) is an innovative new treatment that has been proven as an effective treatment in other peritoneal cancers. There is strong scientific evidence to support HIPEC as an ideal treatment for advanced ovarian cancer. Over the past two decades, there has been an increase in the number of studies focused on the efficacy of HIPEC with regards to advanced ovarian cancer. These studies have shown great promise, with two very recent phase III studies showing resounding results. It is also clear that there is a need for standardization throughout these scientific studies in order to reasonably introduce HIPEC as a standard of treatment.

Current chemotherapeutic treatment schedule prediction methods rely heavily on PK/PD-based models and overlook the important contribution of tissue-level transport and binding. Tissue-level transport and binding phenomena are essential to understanding drug delivery and efficacy in tumors. Drugs with desirable PK/PD properties often fail in vivo due to poor tissue-level transport. We developed an in silico method to predict the effect of treatment schedule on efficacy that couples PK/PD with tissue-level transport. Treatment schedules were implemented on theoretical drugs with different PK/PD and transport properties. For each drug with a given clearance rate, diffusivity, and binding, treatment schedules consisting of one to 20 doses were simulated. Results show that at binding constants around one, high diffusivities, and high clearance rates, implementation of a treatment schedule becomes more significant. At low clearance rates, regardless of tissue-level transport and binding, one dose was predicted to be most efficacious. Tissue Drug Exposure (TDE) was shown to be to a crucial factor for treatment schedule efficacy. Efficacy was improved by increasing TDE. Implementation of a treatment schedule with more doses than one curbed the effect of poor retention with drugs. This model investigates the effect of treatment schedule on a tissue transport model and shows implementation of a proper dosing regimen is crucial to maximize TDE and chemotherapeutic efficacy.

For many years hexamitids, Hexamita spp. and Spironucleus spp., have frequently been reported in vertebrates, particularly in fish. This suggests a potentially important role of these parasites in the fish culture industry. Though the majority of hexamitids are not known to cause disease in their vertebrate host, those that have been documented as associated with disease are still in need of further investigation into their geographical distribution, host range, life cycle, host-parasite relationship, pathogenicity, diagnosis, prevention, treatment, and control. Spironucleus vortens is a hexamitid recently described from angelfish (Pterophyllum scalare). Although the structure of this parasite has been investigated using the electron microscope (Poynton et al., 1995), other information on this organism is poorly understood. Thus, the purpose of this research was to study the nature of S. vortens in TYI-S-33 culture medium and in the angelfish host. The optimal environmental conditions for S. vortens growth were investigated using variations of temperature, pH, and bile concentrations. This study is useful in helping to understand the locations and environmental conditions in the host that are suitable for the growth of S. vortens. Treatment of S. vortens, using seven chemotherapeutic agents; dimetridazole, metronidazole, pyrimethamine, albendazole, fenbendazole, mebendazole, and magnesium sulfate was evaluated. The pathogenicity of S. vortens in angelfish was investigated in fish experimentally inoculated with trophozoites. This study provided information to help understand the pathogenesis of the parasites in the host. Finally, to examine the protective defense mechanisms, the presence of anti-S. vortens antibodies in angelfish serum were evaluated along with the presence of immune cells (lymphocytes, macrophages, eosinophilic granular cells, neutrophils, and plasma cells) at invaded sites of the intestine and other internal organs in response to an experimental Spironucleus vortens infection. The results of this research provide information on this parasite's effect on the fish host which may be useful in understanding the nature of other hexamitids. A few published reports have suggested the in vitro growth requirement of fish Spironucleus (Poynton et al., 1995; Sterud, 1998), but none have examined the optimal conditions required for growth and the pathogenicity of S. vortens. The first study was to examine the optimal requirements for the in vitro growth of the parasite. The organisms were cultivated in either an artificial medium (TYI-S-33) at different temperatures or various pH conditions, or in medium supplemented with different bile concentrations at 25°C. Criteria used to justify the optimal conditions were average cell number ml-1, growth rate, survival time, and cell conditions (motility and morphology). The organisms survived longest at 22°C, and had the highest average cell number ml-1 at 25°, 28° and 31°C. At 25°C the parasites were highly active and survived up to 6 days. The organisms cultivated at pH 6.5, 7.0 and 7.5 yielded the highest average cell number ml-1 with survival periods up to 13-14 days. Most of the organisms cultivated at a pH lower than 6.0 or a pH higher than 7.5 were suppressed and killed within 5-6 days of cultivation. All cultures supplemented with bovine or fish bile yielded lower maximal numbers of parasites than cultures with no bile. These results indicate that the optimal condition for the in vitro cultivation of S. vortens is 25°C and pH 6.5 to 7.5 without supplementation with bile. In order to treat spironucleosis, the efficacy of various chemotherapeutic agents on the growth of S. vortens was examined in vitro. In this study nitroimidazoles and benzimidazoles, formerly reported as drugs of choice for the treatment of diplomonads, pyrimethamine and magnesium sulfate (Epsom salt) were evaluated at different concentrations on the growth of S. vortens. Dimetridazole and metronidazole were effective in inhibiting the parasite's growth at concentrations of 1 μg ml-1 or higher. Albendazole and fenbendazole suppressed the growth of parasites at concentrations of 1.0 μg ml-1 or higher after 24 h exposure. Mebendazole was the most effective agent of the benzimidazole group; and inhibited the parasite's growth at concentrations of 0.5 μg ml-1 or higher. Pyrimethamine at concentrations of 1-10 μg ml-1 failed to inhibit the parasite's growth. Magnesium sulfate inhibited the growth of the parasites only at high concentrations (70 mg ml-1 or higher) . This study indicates that dimetridazole, metronidazole and mebendazole are the most effective chemotherapeutic agents in vitro at inhibiting the growth of S. vortens. To investigate the pathogenesis of spironucleosis, angelfish were orally (PO) or intraperitoneally (IP) inoculated with S. vortens. Control angelfish which were orally gavaged or intraperitoneally injected with PBS were in normal body condition and had no morbidity or mortality. Compared to the control angelfish, PO-infected angelfish were inappetent with no other clinical signs, while IP-infected angelfish showed clinical signs of inappetite, weakness, respiratory distress, and laying on their sides. Twenty percent of the IP-infected angelfish died within the first three weeks after infection. In PO-infected angelfish, the organisms were located only in the intestinal lumen. In IP-infected angelfish, S. vortens were found in the blood, stomach, intestine, and other internal organs (spleen, gall bladder, and ovary). However, no parasites were observed within the intestinal mucosa of either PO- or IP-infected fish. Histopathologic examination of the intestines revealed mild to moderate multifocal enteritis in both PO- and IP-infected angelfish. The mucosal epithelium appeared undamaged although the parasite was closely located and appeared attached to the intestinal mucosa. The results suggest that S. vortens normally causes mild to moderate multifocal enteritis with no morbidity. However, the parasites can cause granulomatous inflammation in a wide variety of host tissues, and may be lethal if they enter the abdominal cavity and disseminate to other organs via the blood circulation. Immunity, both cell mediated and humoral, against S. vortens was investigated in this study. Histopathologic examination revealed a response from inflammatory cells infiltrated and localized in the affected tissues. Macrophages, lymphocytes, and plasma cells were the most common cell types found in the internal organs. Macrophages were active in the affected tissues where the parasites lived in situ. However, in vitro studies indicated that there were no differences in a production of H₂O₂ or in phagocytosis between macrophages of control and infected angelfish regardless of inoculum dosage and administration route. A preliminary study of humoral antibody indicated that angelfish did not develop anti-S. vortens antibody after they were orally or intraperitoneally infected with either a low or a high number of the organisms. It is suggested that localized leucocyte response may be an important mechanism against Spironucleus vortens infection in angelfish. This research has indicated some of the important environmental factors affecting the parasite's growth, and has provided some initial information on the pathogenicity of S. vortens. In addition, preliminary information on the host's protective immune systems, humoral and cell-mediated immunity, against the parasite have been documented. The results from this research will be useful for aquaculture, particularly of tropical freshwater angelfish, and may help to provide an understanding of the biological roles of other hexamitids. / Ph. D.

No description available.

No description available.