The goal of this research was to design, prepare and study a new class of supermolecules coupling ruthenium and platinum, which would display covalent binding to DNA. Drawing upon the well-established efficacy of cis-diamminedichloroplatinum(II) (cisplatin) and the DNA-binding properties of select ruthenium polyazine complexes, the approach was to bind the cis-PtIICl2 active site of cisplatin to ruthenium light absorbers using the dpq and dpb bridging ligands (where dpq = 2,3-bis(2-pyridyl)quinoxaline, dpb = 2,3-bis(2-pyridyl) benzoquinoxaline). These complexes are potentially bifunctional, capable of DNA intercalation through the bridging ligand and covalent binding to DNA through the cis-PtCl2 site. Synthetic methods were developed to prepare the mixed-metal, bimetallic complexes [(bpy)2Ru(BL)PtCl2](CF3SO3)2 and [(phen)2Ru(BL)PtCl2](CF3SO3)2 (where bpy = 2,2¢-bipyridine, phen = 1,10-phenanthroline) in high purity and good overall yields. The DNA-binding ability of these complexes was probed by reaction with linearized plasmid DNA and subsequent analysis by native and denaturing gel electrophoresis. The known DNA binders, cisplatin and trans-{[PtCl(NH3)2]2(m-H2N(CH2)6NH2)}(NO3)2 (1,1/t,t), were examined under equivalent conditions and used as positive controls. Native gel electrophoresis was used to show that these complexes strongly bind DNA, retarding the migration of DNA through the gel in a fashion inversely proportional to the ratio of DNA base pairs (bp) to metal complex (mc). Analysis by denaturing gel electrophoresis determined that the Ru-Pt complexes bind to DNA in a fashion similar to cisplatin, forming primarily intrastrand adducts. However, these systems also appear to form interstrand adducts at a 10-fold lower metal concentration than cisplatin. In addition to affecting the migration rate, the bimetallic complexes also significantly reduced the fluorescence of DNA-intercalated ethidium bromide for the Ru-Pt reacted samples at low-DNA bp: mc ratios. This was not observed for the cisplatin and 1,1/t,t treated samples. This observation was quantitated by gel densitometry. Precipitation of the DNA by cisplatin, 1,1/t,t and all four Ru-Pt complexes was determined not to be the cause of reduced ethidium bromide fluorescence intensity. Homogenous solution fluorescence quenching studies have revealed that the Ru-Pt complexes quench the emission of ethidium bromide even in the absence of DNA, whereas cisplatin and 1,1/t,t do not. In order to compare the effects on DNA migration produced by cisplatin, 1,1/t,t and the Ru-Pt complexes, Rf values were calculated. This analysis has revealed that all four Ru-Pt complexes retard DNA migration to approximately the same degree. Calculation of theoretical DNA migration distances, based upon the molecular weight change of DNA caused by metal-complex binding, have revealed that the observed affect on DNA migration cannot be accounted for by an increase in molecular weight alone. This indicates that changes in charge and three-dimensional shape of the DNA upon binding of the Ru-Pt complexes may also contribute. / Ph. D.

The goal of this research was to develop an understanding of the impact of component modifications on spectroscopic properties, DNA interaction, and bioactivity of tridentate, terpyridine containing ruthenium-based chromophores and chromophore coupled cisplatin analogs. The coupling of a light absorbing unit to a bioactive site offers the potential for developing supramolecules with multifunctional interactions with DNA and other biomolecules. A series of supramolecular complexes of the form [(TL)RuCl(dpp)](PF₆) and [(TL)RuCl(BL)PtCl₂](PF₆) with the BL (bridging ligand) = 2,3-bis(2-pyridyl)pyrazine (dpp) and varying TL (terminal ligand) (tpy = 2,2'':6'',2''-terpyridine, MePhtpy = 4''-(4-methylphenyl)- 2,2'':6'',2''- terpyridine, or tBu3tpy = 4,4'',4''-tri-tert-butyl-2,2'':6'',2''-terpyridine) have been designed and developed. The investigations described in this thesis were focused on the design and development of multifunctional supramolecules with improved DNA interaction and antibacterial properties. The impact of component modifications on photophysical and biological properties of the designed the supramolecular complexes was investigated. A series of supramolecular complexes of the type, [(TL)RuCl(dpp)](PF₆) and [(TL)RuCl(dpp)PtCl₂](PF₆), have been synthesized using a building block approach. Electronic absorption spectroscopy of these types of complexes displayed intense ligand-based transitions in the UV region and metal to ligand charge transfer (MLCT) transitions in the visible region. The Ru to dpp MLCT transitions in RuIIPtII bimetallic complexes were found to be red-shifted relative to the monometallic synthons. The MLCT transitions for [(TL)RuCl(dpp)](PF₆) and [(TL)RuCl(dpp)PtCl₂](PF₆) were centered at ca. 520 and 545 nm, in CH₃CN respectively. The RuIIPtII bimetallic complexes with (TL = tpy, MePhtpy, and tBu3tpy) displayed reversible RuII/III couples at 1.10, 1.10, and 1.01 V vs. Ag/AgCl, respectively. The tpy0/- reductions occurred for TL = tpy, MePhtpy, and tBu3tpy at -1.43, -1.44, and -1.59 V vs. Ag/AgCl, respectively. The RuIIPtII complexes displayed a more positive potential for the dpp0/- couples (-0.50 -0.55, -0.59 V for tpy, MePhtpy, and tBu3tpy, repectively) relative to their monometallic synthons (-1.15, -1.16, and -1.22 V), consistent with the coordination of electron deficient Pt(II) metal center. This research also presents first extensive DNA photocleavage studies of these relatively unexplored tridentate, tpy-containing chromophores. The DNA binding and photocleavage properties of a series of homoleptic and heteroleptic chromophores and RuIIPtII bimetallic complexes were investigated using agarose gel electrophoresis and equilibrium dialysis experiments. The heteroleptic complexes, [(MePhtpy)RuCl(dpp)](PF₆), [(tpy)RuCl(dpp)](PF6), and [(tBu3tpy)RuCl(dpp)](PF6), were found to photocleave DNA more efficiently than homoleptic complexes, [Ru(MePhtpy)2]2+, [Ru(tpy)2]2+, and [Ru(tBu₃tpy)2]2+, in the presence of oxygen. Coupling of [(TL)RuCl(BL)] subunit to a cis-PtIICl2 site provides for the application of typically shorter lived RuII(tpy) based chromophores in DNA photocleavage. The [(TL)RuCl(dpp)PtCl₂]+, complexes displayed covalent binding to DNA and photocleavage upon irradiation with visible light modulated by TL identity. The impact of component modifications on antibacterial properties of the designed molecules was explored for the first time. Both the RuIIPtII bimetallic complexes and their monometallic analogs displayed antibacterial properties. [(MePhtpy)RuCl(dpp)](PF₆) was found to be the most efficient antibacterial agent in the series of monometallic and RuIIPtII bimetallic complexes, displaying cell growth inhibition at 0.05 mM concentration compared to 0.1 mM concentration of [(MePhtpy)RuCl(dpp)PtCl₂](PF₆) needed to display the similar effect. A direct correlation was found to exist between the DNA interaction and bactericidal properties of the designed supramolecules. The effects of light on antibacterial properties of [(MePhtpy)RuCl(dpp)](PF₆) were also briefly examined. This complex represents the first inorganic chromophore-based photodynamic antibacterial agent. / Ph. D.

Cisplatin is an antineoplastic drug used to treat malignant tumors in human beings and dogs. Nephrotoxicosis was initially considered dose limiting. The use of saline loading and hypertonic saline administration protocols allowed dose escalation, reduced nephrotoxicosis, and increased remission rates in the treatment previously poorly responsive malignant tumors in human beings. A pilot study was performed to determine efficacy of 4-hour saline loading in providing renal protection for dogs receiving high-dose cisplatin (150 mg/m² IV). Two beagles were saline loaded (25 ml/kg/hr of 0.9% NaCI, IV) for 4 hours and infused with cisplatin (150 mg/m²). We demonstrated that high-dose cisplatin (150 mg/m² IV) can be administered to dogs without biochemical evidence of acute nephrotoxicosis; however gastrointestinal toxicoses (fibrinonecrotic enteritis) and severe myelosuppression (leukopenia) were incompatible with patient survival and therefore, dose limiting. In another study we compared efficacy of hypertonic saline with normal saline in preventing nephrotoxicosis associated with administration of high-dose cisplatin (90 mg/m² IV) to dogs. In this study we demonstrated that a single IV dose of cisplatin (90mg/m²) can be administered to dogs in normal saline (0.9%) or hypertonic saline (7%) in combination with 4 hour saline loading (25 ml/kg/hr) without evidence of reduced renal function as measured by exogenous creatinine clearance. Platelet numbers were significantly increased in dogs that received cisplatin in hypertonic saline. Nephrotoxicosis was not dose limiting in either study. Future studies should attempt to determine the efficacy and toxicoses of multiple doses of cisplatin (90 mg/M² administered in hypertonic saline to tumor bearing dogs. / Master of Science

A series of bimetallic (Ru^II, Pt<sup>II</sup) complexes were synthesized with the general formula [(tpy)RuCl(BL)PtCl₂](PF₆) (tpy = 2,2':6',2"-terpyridine and BL = bridging ligand) and their spectroscopic, electrochemical, and DNA binding properties studied. The bridging ligands used in these complexes were 2,3-bis(2'-pyridyl)pyrazine (dpp), 2,3-bis(2'-pyridyl)quinoxaline (dpq) and 2,3-bis(2'-pyridyl)benzoquinoxaline (dpb). These complexes combine light-absorbing Ru^II-polypyridyl chromophores and a cis-PtCl₂ structural motif known to bind DNA. The Ru-bound chloride may be substituted, enabling further modification of the spectroscopic properties. The synthesis of [(tpy)RuCl(BL)PtCl₂](PF₆) utilizes a building block approach that allows modifications to the series of complexes within the general synthetic scheme. This illustrates the applicability of this scheme to the development of new series of complexes. The lowest-energy absorption for the three complexes is assigned to a Ru(dπ) → BL(π*) charge transfer transition. This transition shifts to lower energy as the ligand is varied from dpp to dpq to dpb. The first and second reductions are BL^0/- and BL^-/2- based and shift to more positive potentials from dpp to dpq to dpb. The Ru^II/III redox couple remains at a nearly constant potential for the series. All three compounds show DNA binding when incubated with linearized plasmid DNA. Adduct formation was assessed by agarose gel electrophoresis as a retardation of band migration. / Master of Science

Yes / Malignant pleural mesothelioma (MPM) is associated with an extremely poor prognosis, and most patients initially are or rapidly become unresponsive to platinum-based chemotherapy. MicroRNA-31 (miR-31) is encoded on a genomic fragile site, 9p21.3, which is reportedly lost in many MPM tumors. Based on previous findings in a variety of other cancers, we hypothesized that miR-31 alters chemosensitivity and that miR-31 reconstitution may influence sensitivity to chemotherapeutics in MPM. Reintroduction of miR-31 into miR-31 null NCI-H2452 cells significantly enhanced clonogenic resistance to cisplatin and carboplatin. Although miR-31 re-expression increased chemoresistance, paradoxically, a higher relative intracellular accumulation of platinum was detected. This was coupled to a significantly decreased intranuclear concentration of platinum. Linked with a downregulation of OCT1, a bipotential transcriptional regulator with multiple miR-31 target binding sites, we subsequently identified an indirect miR-31-mediated upregulation of ABCB9, a transporter associated with drug accumulation in lysosomes, and increased uptake of platinum to lysosomes. However, when overexpressed directly, ABCB9 promoted cellular chemosensitivity, suggesting that miR-31 promotes chemoresistance largely via an ABCB9-independent mechanism. Overall, our data suggest that miR-31 loss from MPM tumors promotes chemosensitivity and may be prognostically beneficial in the context of therapeutic sensitivity.

Im Mittelpunkt dieser Arbeit stand die Analytik eines monoklonalen Antikörpers, der spezifisch auf Cisplatin-behandelte DNA reagiert und bereits routinemäßig zu diagnostischen Zwecken bei der Krebstherapie mit ebendiesem Zytostatikum eingesetzt wird. Zu diesem Zweck wurden vor allem massenspektrometrische Methoden eingesetzt, um einerseits den genauen Aufbau sowie die Zusammensetzung des Antikörpers zu untersuchen und andererseits die Antigenspezifität einer genaueren Betrachtung zu unterziehen. Mit Hilfe von nano-ESI-MS konnte der Antikörper unter Erhalt der nativen Struktur in der Gasphase analysiert werden. Tandem-MS-Experimente lieferten nicht nur einen Einblick in typische Fragmentierungs-muster, sondern generierten z. T. auch Informationen über die Primärsequenz einiger Bereiche des Antikörpers. Die im Fc-Teil gebundenen Glycanstrukturen, die sowohl für die Struktur als auch die Funktionalität des Antikörpers eine entscheidende Rolle spielen, wurden nach enzymatischer Abspaltung separat mit Hilfe von MALDI-MS analysiert. Die vollständige Antikörpersequenz wurde durch proteolytische Spaltung des Antikörpers, Analytik mittels hochaufgelöster Tandem-MS und anschließendem Vergleich mit Daten aus der Sequenzierung auf DNA-Ebene bzw. bekannten Antikörpersequenzen aus Proteindatenbanken aufgeklärt. Im selben Zug konnten auf Peptidebene eine Vielzahl verschiedener posttranslationaler Modifikationen nachgewiesen werden. Weiterhin konnte gezeigt werden, dass der Antikörper unter Erhalt seiner Struktur und der antigenbindenden Eigenschaften an den Zuckerstrukturen mit einem eigens synthetisierten Metallkomplex markiert werden kann. Die für die Spezifitätsstudie des Antikörpers notwendigen Antigene wurden durch Inkubation synthetischer einzel- und doppelsträngiger DNA-Oligomere mit Cisplatin erzeugt und analysiert. Anschließend wurden die verschiedenen Antikörper-Antigen-Komplexe unter nativen Bedingungen hergestellt und massenspektrometrisch charakterisiert. Um einen Eindruck von der Komplexstabilität zu erhalten wurden zusätzlich MS/MS-Untersuchungen des Komplexes mit der platinierten doppelsträngigen DNA durchgeführt. Wie bei der Analytik des freien Antikörpers konnten auch hier spezifische Fragmente detektiert werden. / The focus of the thesis was the characterization of a monoclonal antibody, which binds specifically to cisplatin-treated DNA and is already used for diagnostic purposes in cancer therapy. The structure and composition of the antibody, as well, as the antigen specificity were investigated by mass spectrometry. Using nano-ESI-MS it was possible to analyse the antibody in its native state. Tandem-MS experiments revealed not only specific antibody fragments but also gave information on the primary structure of the antibody. The Fc-bound glycans that are responsible for antibody structure and functionality, were cleaved enzymatically and studied with MALDI-MS. For a detailed determination of the primary structure, the antibody was proteolysed with different enzymes and analyzed via high resolution Tandem-MS. The data was evaluated by comparism with results from sequence analysis on DNA level and known antibody sequences from protein databases. Simultaneously many different posttranslational modifications were identified. Furthermore, the antibody was successfully labelled with a metal complex at its sugar structures without loss of its structure and function. For investigation of the antibody specificity antigens were generated by incubation of well-defined single and double stranded DNA oligomers with cisplatin. Antibody-antigen-complexes were synthesized and analyzed by mass spectrometry under native conditions. The stability of the complex with double stranded DNA was also investigated by MS/MS-experiments. Thus, it was also possible to determine the specific fragmentation behavior for the complex just as for the free antibody.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Introduction: This phase II trial evaluated the efficacy and safety of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib, in combination with first-line chemotherapy in advanced urothelial cancer. Methods: Chemotherapy-naïve patients with advanced or metastatic urothelial carcinoma were randomized 1:1:1 to receive six cycles of chemotherapy (gemcitabine 1,250 mg/m 2 on days 1 and 8, and cisplatin 70 mg/m 2 on day 1 of every cycle) concomitantly with gefitinib 250 mg/day (arm A); or with sequential gefitinib (arm B); or alone (arm C). The primary endpoint was the time to progression (TTP). Results: A total of 105 patients received study treatment. Median TTP for arms A, B, and C were 6.1, 6.3, and 7.8 months, respectively. There were no significant differences between treatment arms for any outcomes measured. The most common adverse events were nausea and vomiting. Conclusion: Gefitinib in combination with chemotherapy did not improve efficacy in advanced urothelial cancer.

Indiana University-Purdue University Indianapolis (IUPUI) / Targeting uncontrolled cell proliferation and resistance to DNA damaging chemotherapeutics using small molecule inhibitors of proteins involved in these pathways has significant potential in cancer treatment. Several proteins involved in genomic maintenance and stability have been implicated both in the development of cancer and the response to chemotherapeutic treatment. Replication Protein A, RPA, the eukaryotic single-strand DNA binding protein, is essential for genomic maintenance and stability via roles in both DNA replication and repair. Xeroderma Pigmentosum Group A, XPA, is required for nucleotide excision repair, the main pathway cells employ to repair bulky DNA adducts. Both of these proteins have been implicated in tumor progression and chemotherapeutic response. We have identified a novel small molecule that inhibits the in vitro and cellular ssDNA binding activity of RPA, prevents cell cycle progression, induces cytotoxicity and increases the efficacy of chemotherapeutic DNA damaging agents. These results provide new insight into the mechanism of RPA-ssDNA interactions in chromosome maintenance and stability. We have also identified small molecules that prevent the XPA-DNA interaction, which are being investigated for cellular and tumor activity. These results demonstrate the first molecularly targeted eukaryotic DNA binding inhibitors and reveal the utility of targeting a protein-DNA interaction as a therapeutic strategy for cancer treatment.

Purpose: Platinum chemotherapy can be considered to treat metastatic castration-resistant prostate cancer (mCRPC) with features of neuroendocrine differentiation. However, platinum compounds are generally only applied after the failure of multiple prior-line treatment options. This study investigated whether acquired resistance against ionizing radiation or docetaxel chemotherapy—two commonly applied treatment modalities in prostate cancer—influences the cisplatin (CDDP) tolerance in mCRPC cell line models. Methods: Age-matched parental as well as radio- or docetaxel-resistant DU145 and PC-3 cell lines were treated with CDDP and their sensitivity was assessed by measurements of growth rates, viability, apoptosis, metabolic activity and colony formation ability. Results: The data suggest that docetaxel resistance does not influence CDDP tolerance in all tested docetaxel-resistant cell lines. Radio-resistance was associated with sensitization to CDDP in PC-3, but not in DU145 cells. In general, DU145 cells tolerated higher CDDP concentrations than PC-3 cells regardless of acquired resistances. Furthermore, non-age-matched treatment-naïve PC-3 cells exhibited significantly different CDDP tolerances. Conclusion: Like patients, different mCRPC cell lines exhibit significant variability regarding CDDP tolerance. The presented in vitro data suggest that previous radiation treatment may be associated with a moderate sensitization to CDDP in an isogenic and age-matched setting. Therefore, previous radiotherapy or docetaxel chemotherapy might be no contraindication against initiation of platinum chemotherapy in selected mCRPC patients.