Studies of submillimeter gels composed of covalently cross-linked poly(acrylic acid)-g-poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic-PAA) networks are reviewed in light of potential applications of the microgels as drug carriers in oral delivery. The microgels are capable of volumetric transitions in response to environmental stimulae such as pH and temperature. It is shown that the type of Pluronic used in the microgel synthesis changes the structure of the resulting microgels, with the more hydrophobic Pluronic imparting porosity. Microgels based on Pluronic L92 (L92-PAA-EGDMA) possess higher ion-exchange capacity than microgels based on Pluronic F127 (F127-PAA-EGDMA), albeit the former are more hydrophobic. Analogously, more hydrophobic but heterogeneous L92-PAA-EGDMA exhibit superior capacity for equilibrium loading of hydrophobic drugs such as taxol, camptothecin and steroid hormones, as well as higher capacity for weakly basic drugs such as doxorubicin, mitomycin C, and mitoxantrone. / Singapore-MIT Alliance (SMA)

Cardiotoxicity associated with many cancer drugs is a critical issue facing physicians these days and a huge hurdle that must be overcome for a side effects-free cancer therapy. Survival of cardiac myocytes is compromised upon the exposure to certain chemotherapeutic drugs. Unfortunately, the mechanisms implicated in cardiac toxicity and the pathways governing myocyte survival are poorly understood. The following thesis addresses the mechanisms underlying the cardiotoxicity of two anticancer drugs, doxorubicin (DOX) and Imatinib mesylate (Gleevec). Transcription factor GATA-4, has recently emerged as an indispensable factor in the adult heart adaptive response and cardiomyocyte survival. Therefore, the specific aim of this project was to determine the role of GATA-4, its upstream regulators, as well as partners in survival. A combination of cell and molecular techniques done on in vivo, and ex vivo models were utilized to tackle these issues. In this study, we confirmed the cardiotoxicity of the anticancer drug, Imatinib mesylate and found to be age dependent. GATA-4, already known to be implicated in DOX-induced toxicity, was confirmed as an Imatinib target. At the molecular level, we identified IGF-1 and AKT as upstream regulators of GATA-4. Moreover, we confirmed ZFP260 (PEX-1), a key regulator of the cardiac hypertrophic response, as a GATA-4 collaborator in common prosurvival pathways. Collectively, these results provide new insights on the mechanisms underlying drug-induced cardiotoxicity and raise the exciting possibility that cancer drugs are negatively affecting the same prosurvival pathway(s), in which GATA-4 is a critical component. Therapeutic interventions aimed at enhancing GATA-4 activity may be interesting to consider in the context of treatments with anticancer drugs.

1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes containing ptrifluoromethyl (DIM-C-pPhCF3), p-t-butyl (DIM-C-pPhtBu), and phenyl (DIM-CpPhC6H5) substituents have been identified as a new class of peroxisome proliferatoractivated receptor γ (PPARγ) agonists that exhibit antitumorigenic activity. In this study, the PPARγ-active compounds decreased HT-29, HCT-15, RKO, HCT116 and SW480 colon cancer cell survival and KU7 and 253JB-V33 bladder cancer cell survival. In HT- 29, HCT-15, SW480 and KU7 cells, the PPARγ agonists induced caveolin-1 expression and this induction was significantly downregulated after cotreatment with the PPARγ antagonist GW9662. Since overexpression of caveolin-1 is known to suppress cancer cell and tumor growth, the growth inhibitory effects of the DIM compounds in these cell lines are associated with PPARγ-dependent induction of caveolins. These PPARγ-active compounds did not induce caveolin-1 in HCT-116 cells. However, these compounds induced NSAID-activated gene-1 (NAG-1) and apoptosis in this cell line. This represents a novel receptor-independent pathway for C-DIM-induced growth inhibition and apoptosis in colon cancer cells. In SW480 colon cancer cells 2.5-7.5 μM C-DIMs induced caveolin-1 whereas high concentrations (10 μM) induced pro-apoptotic NAG-1 expression. In athymic nude mice bearing SW480 cell xenografts DIM-C-pPhC6H5 inhibited tumor growth and immunohistochemical staining of the tumors show induction of apoptosis and NAG-1 expression. Thus, the PPARγ-active compounds induce both receptor-dependent and-independent responses in SW480 cells which are separable over a narrow range of concentrations and this dual mechanism of action enhances their antiproliferative and anticancer activities. Similar results were obtained for another structural class of PPARγ agonists namely 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and the corresponding methyl (CDDO-Me) and imidazole (CDDO-Im) esters. Structure-activity studies show that 1,1-bis(3'-indolyl)-1-(psubstitutedphenyl) methanes containing p-trifluoromethyl (DIM-C-pPhCF3), hydrogen (DIM-C-pPh) and p-methoxy (DIM-C-pPhOCH3) substituents activate Nur77 and induce apoptosis in pancreatic, prostate, and breast cancer cell lines. Nur77 agonists activate the nuclear receptor, and downstream responses include decreased cell survival, induction of cell death pathways including tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and PARP cleavage. Nur77 agonists also inhibit tumor growth in vivo in athymic nude mice bearing Panc-28 cell xenografts.

No description available.

Doctor of Philosophy(PhD) / The present study deals with the synthesis and characterization of six tri-palladium complexes code named MH3, MH4, MH5, MH6, MH7 and MH8 that contained two planaramine ligands bound to the central or each of the terminal metal ions. The activity of the compounds against human cancer cell lines: A2780, A2780cisR and A2780ZD0473R, cell uptake, levels of DNA-binding and nature of interaction with salmon sperm and pBR322 plasmid DNA have also been determined. Whereas cisplatin binds with DNA forming mainly intrastrand GG adduct that causes local bending of a DNA strand, the tri-palladium complexes are expected to bind with DNA forming a number of long-range interstrand GG adducts that would cause a global change in DNA conformation. Among the designed complexes, MH6 that has two 2-hydroxypyridine ligands bound to each of the two terminal palladium ions is found to be most active. The compound also has the highest cell uptake and Pd-DNA binding levels. In contrast, MH8 which has two 4-hydroxypyridine ligands bound to each of the two terminal palladium ions is found to be least active. The results indicate that, as applied to the terminal metal centres, 2-hydroxypyridine would be more activating than 4-hydroxypyridine perhaps because of greater protection provided to the terminal centres from coming in contact with the solvent molecules. In contrast, when bound to the central metal centre, 4-hydroxypyridine appears to play a slightly greater activating role than 2-hydroxypyridine or 3-hydroxypyridine, suggesting that non-covalent interactions such as hydrogen bonding associated with the ligand rather than its steric effect may be a more important determinant of antitumour property. The results illustrate structure-activity relationships and suggest that the tri-palladium complex containing two 2-hydroxypyridine ligands bound to each of the three metal centres or the compound that contains two 2-hydroxypyridine ligands bound to each of the two terminal metal centres and two 4-hydroxypyridine ligands bound to the central metal centre, may be much more active than any of the designed complexes.

Cardiotoxicity associated with many cancer drugs is a critical issue facing physicians these days and a huge hurdle that must be overcome for a side effects-free cancer therapy. Survival of cardiac myocytes is compromised upon the exposure to certain chemotherapeutic drugs. Unfortunately, the mechanisms implicated in cardiac toxicity and the pathways governing myocyte survival are poorly understood. The following thesis addresses the mechanisms underlying the cardiotoxicity of two anticancer drugs, doxorubicin (DOX) and Imatinib mesylate (Gleevec). Transcription factor GATA-4, has recently emerged as an indispensable factor in the adult heart adaptive response and cardiomyocyte survival. Therefore, the specific aim of this project was to determine the role of GATA-4, its upstream regulators, as well as partners in survival. A combination of cell and molecular techniques done on in vivo, and ex vivo models were utilized to tackle these issues. In this study, we confirmed the cardiotoxicity of the anticancer drug, Imatinib mesylate and found to be age dependent. GATA-4, already known to be implicated in DOX-induced toxicity, was confirmed as an Imatinib target. At the molecular level, we identified IGF-1 and AKT as upstream regulators of GATA-4. Moreover, we confirmed ZFP260 (PEX-1), a key regulator of the cardiac hypertrophic response, as a GATA-4 collaborator in common prosurvival pathways. Collectively, these results provide new insights on the mechanisms underlying drug-induced cardiotoxicity and raise the exciting possibility that cancer drugs are negatively affecting the same prosurvival pathway(s), in which GATA-4 is a critical component. Therapeutic interventions aimed at enhancing GATA-4 activity may be interesting to consider in the context of treatments with anticancer drugs.

The bis-naphthalimidopropyl drugs DMP8408, BNIPDaoct and BNIPDanon, have been developed as novel synthetic lead compounds in cancer therapy. Biochemical assays of these compounds have demonstrated high inhibitor activity (IC50=0.8–1.8μM) against cancer cells, which makes them therapeutically significant. However, knowledge on the structure of these compounds, their target DNA and the drug-DNA complexes is seriously lacking. This justifies the detailed structural and DNA binding studies of these drugs. Determination of the NMR structures of these drugs and their complexes should assist the screening process and enable the discovery of more target specific drugs in the near future. Towards the above goal, the detailed NMR study of DMP8408, BNIPDaoct and BNIPDanon drugs and their interaction with DNA was carried out. The high resolution NMR data and their in depth analysis helped to generate the first NMR structures of DMP8408, BNIPDaoct and BNIPDanon in solution state with a high degree of precision and low RMSD. The results show that the length of the linker chain plays a vital role in the structural and binding properties of these drug compounds, establishing π- π interactions between the two aromatic ends of the drugs. This in return facilitates specific binding to the DNA via intercalation. The classical, self-complementary DNA dodecamer sequence was chosen for drug binding studies. A high resolution NMR structure of the DNA was generated with a low RMSD (Å) and detailed conformational analysis. NMR titrations of DMP8408, BNIPDaoct and BNIPDanon drugs with the 12mer DNA were carried out at 2 ̊C and 25 ̊C, and the effects induced have been monitored by measuring changes to the chemical shifts (1H and 31P) and patterns of intra and intermolecular NOEs. The results show that DNA binding was much stronger in the case of BNIPDaoct and BNIPDanon than DMP8408. Due to a shorter and rigid linker chain, DMP8408 binds to the DNA via the mono intercalation principle, whereas BNIPDaoct and BNIPDanon have a longer and flexible linker chain which facilitates their binding to the DNA via the neighbour exclusion bis-intercalation principle. In addition to the above, similar NMR titration spectra of selectively fluorinated DNA analogues were also measured to probe and provide additional support to the above drug binding results. Based on the above NMR investigation we conclude that BNIPDaoct shows high affinity towards the DNA followed by BNIPDanon and DMP8408 with preference for the major groove of the DNA. The DNA is significantly deformed upon binding to the drugs and this is manifested in the increased line width of the 1H and 31P resonances. It was also shown that the fluorinated DNA is able to distinguish between both the BNIPDaoct and BNIPDanon in its labelled and the unlabelled forms. Similarly, the drugs were able to bind to the DNA and show the same affinity and other effects despite the incorporation of the fluorine label on the DNA. The above mentioned NMR results support the synthetic and biophysical data in the literature and therefore provide new avenues for further research into anticancer drug discovery.

No / A novel series of 1,4-disubstituted chloroethylaminoanthraquinones, containing alkylating chloroethylamino functionalities as part of a rigid piperidinyl or pyrrolidinyl ring-system, have been prepared. The target compounds were prepared by ipso-displacement of halides of various anthraquinone chromophores by either hydroxylated or chlorinated piperidinyl- or pyrrolidinyl-alkylamino side chains. The chloroethylaminoanthraquinones were shown to alkylate guanine residues of linearized pBR322 (1¿20 ¿M), and two symmetrically 1,4-disubstituted anthraquinones (compounds 14 and 15) were shown to interstrand cross-link DNA in the low nM range. Several 1,4-disubstituted chloroethylaminoanthraquinones were potently cytotoxic (IC50 values: ¿40 nM) in human ovarian cancer A2780 cells. Two agents (compounds 18 and 19) exhibited mean GI50 values of 96 nM and 182 nM, respectively, in the NCI human tumor cell line panel. Derivatization of the potent DNA cross-linking agent 15 to an N-oxide resulted in loss of the DNA unwinding, DNA interstrand cross-linking and cytotoxic activity of the parent molecule.

Yes / Platinum complexes are the most widely used anticancer drugs; however, new generations of agents are needed. The organoiridium(III) complex [(η5-Cpxbiph)Ir(phpy)(Cl)] (1-Cl), which contains π-bonded biphenyltetramethylcyclopentadienyl (Cpxbiph) and C^N-chelated phenylpyridine (phpy) ligands, undergoes rapid hydrolysis of the chlorido ligand. In contrast, the pyridine complex [(η5-Cpxbiph)Ir(phpy)(py)]+ (1-py) aquates slowly, and is more potent (in nanomolar amounts) than both 1-Cl and cisplatin towards a wide range of cancer cells. The pyridine ligand protects 1-py from rapid reaction with intracellular glutathione. The high potency of 1-py correlates with its ability to increase substantially the level of reactive oxygen species (ROS) in cancer cells. The unprecedented ability of these iridium complexes to generate H2O2 by catalytic hydride transfer from the coenzyme NADH to oxygen is demonstrated. Such organoiridium complexes are promising as a new generation of anticancer drugs for effective oxidant therapy. / We thank the ERC (247450), SNSF (PA00P2_145308 for N.P.E.B.), IAS (for I.R.C.), BBSRC (for J.M.H.), Science City (AWM and ERDF), and the EPSRC for support, and Prof. Timothy Bugg and members of EC COST Action CM1105 for stimulating discussions. We also thank Professor Pat Unwin, Mike Snowden, and Rob Lazenby for their help with the electrochemical experiments and the National Cancer Institute for NCI-60 human tumor cell panel screening.

The novel trinuclear complex, BBR3464 has undergone Phase II clinical trials and been shown to have greater cytotoxicity and cellular uptake than clinical anticancer platinum drugs such as cisplatin, oxaliplatin and carboplatin. The clinical efficacy of cisplatin, oxaliplatin and carboplatin is limited due to acquired resistance and dose limiting side effects. The three major pharmacological factors contributing to the intrinsic cytotoxicity of, and cellular resistance to, platinum drugs are (i) cellular uptake and efflux of platinum; (ii) the frequency and nature of Pt-DNA adducts; and (iii) deactivating metabolic reactions with sulfur-containing nucleophiles. Since decreased cellular uptake of platinum drugs is a common feature of resistant cells, investigating mechanisms of cellular uptake and efflux is of a great importance in the field of cancer biology. The mechanisms of uptake of Platinum drugs are diverse and complex. Similar to cisplatin, BBR3464 v as shown to use copper transporter hCTR1 and ATP7B for influx and efflux respectively. Organic cation transporters (OCT) did not play an important role in BBR3464 cellular uptake, however, desipramine, an OCT inhibitor had synergistic effects on platinun drugs-induced cytotoxicity. This effect is of high clinical relevance since desipramine, an antidepressant, is being used in prostate cancer patients for the treatment of neuropathic pain. The mechanism of this interaction was further addressed.Due to the high charge of BBR3464, studies have shown that its DNA binding has a non-covalent component. To examine the non covalent component, labile chloride leaving groups were replaced by non labile ammonia groups. Besides having higher cellular accumulation than BBR3464, the non covalent analogue, AH78, had a different mechanism of action in cells and showed promising results in vivo. These data confirm the validity of searching for new chemotypes outside the cisplatin structural class to aid in the treatment of recurrent, cisplatin-resistant cancers.