CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Oxaliplatina (OXL) à um composto de platina de terceira geraÃÃo com potente atividade citotÃxica contra vÃrios tipos de cÃncer, possuindo um efeito colateral de difÃcil tratamento, uma severa neuropatia perifÃrica. Estudos sugerem que o oxalato, metabÃlito da OXL, està envolvido no desenvolvimento dessa neuropatia sensitiva perifÃrica (NSP); de modo que foi publicada a sÃntese de um anÃlogo (LLC-1402) da OXL, livre de oxalato, tendo propriedades antitumorais (LIU et al., 2013). O objetivo do trabalho à estudar o papel do oxalato na neuropatia induzida por OXL em camundongos, comparando a OXL com LLC-1402. A NSP foi induzida por 2 injeÃÃes (iv.) por semana de OXL (2 mg/kg), em camundongos machos Swiss, durante 4  semanas, totalizando 9 injeÃÃes. LLC-1402 (7, 14 e 28 mg/kg) foi administrado (iv.) seguindo o mesmo esquema. Foram realizados testes nociceptivos (Von Frey eletrÃnico e TIC), semanalmente. Depois, foi escolhida a dose de 14 mg/kg do LLC-1402 para fazer um outro experimento, no qual foi acrescentada a injeÃÃo ip. de oxalato (1,7 mg/kg). No 28 dia, foi feita coleta de sangue para contagem total de leucÃcitos e dosagens bioquÃmicas. Nos 28 e 56 dias, foi feita a coleta de medula espinhal e GRD para imunofluorescÃncia para ATF-3, c-FOS, iNOS e NeuN. Os resultados mostraram que a OXL e o LLC-1402 foram capazes de diminuir o limiar de retirada da pata e o tempo de retirada da cauda significativamente (p<0,05), comparado ao grupo controle. A injeÃÃo de LLC-1402 junto com oxalato e somente oxalato tambÃm foi capaz de reduzir o limiar de retirada da pata e o tempo de retirada da cauda. Os grupos tratados com OXL, LLC-1402 e oxalato mostraram uma reduÃÃo significativa da contagem total de leucÃcitos. Para as dosagens bioquÃmicas (TGO, TGP, ureia, creatinina), nÃo houve diferenÃa estatÃstica entre os grupos. Houve um aumento da imunoexpressÃo de c-Fos no GRD nos grupos tratados com OXL, LLC-1402 e LLC-1402 junto com oxalato nos 28 e 56 dias, e somente oxalato no 28 dia. Foi observado esse aumento no corno dorsal da medula espinhal no 28 dia em todos os grupos tratados. Foi observado um aumento da imunoexpressÃo de ATF-3, no GRD e corno dorsal da medula espinhal, em todos os grupos tratados, nos 28 e 56 dias. NÃo houve diferenÃa significativa entre os grupos no GRD nem medula espinhal na imunoexpressÃo de iNOS no 56 dia; jÃ, no 28 dia, houve um aumento da imunoexpressÃo de iNOS no GRD. Os resultados mostraram que o oxalato pode estar envolvido parcialmente na NSP induzida por OXL. / Oxaliplatin (OXL) is a third generation platinum compound with potent cytotoxic activity against several types of cancers, having a side effect is difficult to treat, a severe peripheral neuropathy. Studies suggest that oxalate, OXL metabolite, is involved in the development of peripheral sensory neuropathy (PSN); so that was published the synthesis of an OXL analogue (LLC-1402), oxalate free, which has antitumor properties (LIU et al., 2013). The objective of this research is to study the role of oxalate in OXL induced neuropathy in mice, comparing OXL with LLC-1402. The PSN was induced by two injections (iv.) OXL (2 mg/ kg) per week, in male Swiss mice, for 4  weeks, totaling 9 injections. LLC-1402 (7, 14 and 28 mg / kg) was administered (iv.) following the same scheme. Nociceptive tests were performed (electronic von Frey and tail immersion test) weekly. After, it was chosen dose of 14 mg / kg of the LLC-1402 to perform another experiment, in which was added oxalate injection (1.7 mg/kg). In the 28th day, it was made blood collection for total leukocyte count and biochemical measurement. In the 28th and 56th days, spinal cord and DRG were removed for immunofluorescence for ATF-3, c-FOS, iNOS and NeuN. The results showed that both OXL and LLC-1402 were able to decrease the paw withdrawal threshold and tail withdrawal time significantly (p<0.05) compared to the control group. The injection of LLC-1402 together with oxalate and only oxalate was also able to reduce the paw withdrawal threshold and the tail withdrawal time. Moreover, all groups treated with OXL, LLC-1402 and oxalate showed a significant reduction in the total leukocyte count. The biochemical measurement (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, urea, creatinine) showed no statistical difference between the groups. The results also showed increased c-Fos immunoexpression in GRD in groups treated with OXL, LLC-1402 and LLC-1402 together with oxalate in the 28 and 56 days, and only oxalate the 28th day. This increase was observed in the dorsal horn of the spinal cord at 28th day in all the treated groups. It was not observed this increase in the dorsal horn of the spinal cord in the 56th day. It was observed an increase in immunoexpression of ATF3 in DRG and spinal cord of dorsal horn in all treated groups in the 28th and 56th days. iNOS immunofluorescence showed no significant difference between groups in the DRG nor spinal cord, already in the 28th day, there was an increase in immunoexpression of iNOS in GRD. Thus, the results showed that the oxalate may be partially involved in PSN induced OXL.

M.Sc. (Chemistry) / Please refer to full text to view abstract

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This thesis mainly deals with some biochemical aspects regarding the efficacy of novel platinum anticancer compounds alone and in combination with mangiferin, as part of a broader study in which both chemistry and biochemistry are involved. Various novel diamine and N-S donor chelate compounds of platinum II and IV have been developed in which factors such as stereochemistry, ligand exchange rate and biocompatibility were considered as additional parameters. In the first order testing, each of these compounds was tested with reference to their “killing” potential by comparing their rate of killing, over a period of 48 hours with those of cisplatin and oxaliplatin. Numerous novel compounds were tested in this way, using the MTT cell viability assay and the three cancer cell lines MCF7, HT29 and HeLa. Although only a few could be regarded as equal to or even better than cisplatin, CPA7 and oxaliplatin, the testing of these compounds on cancer cells provided useful knowledge for the further development of novel compounds. Three of the better compounds, namely Yol 25, Yol 29.1 and Mar 4.1.4 were selected for further studies, together with oxaliplatin and CPA7 as positive controls, to obtain more detailed knowledge of their anticancer action, both alone and when applied in combination with mangiferin. In addition to the above, resistant cells were produced for each of the three different cell lines tested and all the selected compounds, both in the presence and absence of mangiferin. The effects of these treatments on the activation of NFĸB when applied to normal and resistant cell lines were also investigated. All the compounds induced apoptosis in the cell lines tested as well as alter the DNA cycle at one or more phase. Additionally, combination of these compounds with mangiferin enhanced the above-mentioned effects. Mangiferin decreases the IC50 values of the platinum drugs by up to 3.4 times and, although mangiferin alone did not induce cell cycle arrest, the presence of mangiferin in combination with oxaliplatin and Yol 25 shows an earlier and greatly enhanced delay in the S-phase, while cells treated with CPA7, Yol 29.1 and Mar 4.1.4 in combination with mangiferin showed a later, but greatly enhanced delay in the S-phase. It was also found that mangiferin acts as an NFĸB inhibitor when applied in combination with these drugs, which, in turn, reduces the occurrence of resistance in the cell lines. Resistance to oxaliplatin was counteracted by the combination with mangiferin in HeLa and HT29, but not in MCF7 cells, while resistance to CPA7 was only counteracted in the MCF7 cell line. Yol 25 and Mar 4.1.4 did not seem to induce resistance in HeLa and MCF7 cells, but did in HT29 cells, whereas Yol 29.1 caused resistance in HeLa and HT29 cells, but not in MCF7 cells. Finally, an effort was made to evaluate the different compounds by comparing them with respect to their properties relating to anticancer action with and without the addition of mangiferin.

This dissertation mainly deals with some biochemical aspects regarding the efficacy of novel platinum anticancer compounds, as part of a broader study in which both chemistry and biochemistry are involved. Various novel diamine and N-S donor chelate compounds of platinum II and IV have been developed in which factors such as stereochemistry, ligand exchange rate and biocompatibility were considered as additional parameters. In the first order testing, each of these compounds was tested with reference to their “killing” potential by comparing their rate of killing, over a period of 48 hours with those of cisplatin and oxaliplatin. Some 80 compounds were tested in this way. Although only a few could be regarded as equal to or even better than cisplatin and oxaliplatin, the testing of these compounds on cancer cells provided useful knowledge for the further development of novel compounds. Four of the better compounds, namely Y9, Y14, Y16 and Lt16.2 were selected for further studies to obtain more detailed knowledge of their anticancer action, including some flow cytometric studies. In addition to the above, cisplatin resistant cells were produced for each of the three different cell lines tested, namely, HeLa, HT29 and MCF7 cancer cell lines, by intermittent and incremental exposure to cisplatin (all the cell lines tested became resistant to cisplatin). Each of the selected compounds were exposed to the cells in the same manner, in order to attempt the induction of resistance against these compounds in the three cell lines tested (i.e. whether these cells will become resistant to the various compounds). Each of these selected platinum containing compounds were subsequently tested against the “cisplatin resistant” cell lines in order to determine their efficacy against such cells. One such compound could be singled out, since cervical cancer cells (HeLa cells) do not become resistant to it. This behaviour is similar to that of oxaliplatin against cervical cancer and colon cancer (HT29) cells (oxaliplatin is the number one treatment for colon cancer at present). This compound also proved to be more active against cisplatin resistant cell lines. It was found that all the compounds induced apoptosis in the cell lines tested as well as inhibit the DNA cycle at one or more phase. Finally, an effort was made to evaluate the different compounds by comparing them with respect to their properties relating to anticancer action.

Please read the abstract in the section front of this document / Thesis (PhD (Chemistry))--University of Pretoria, 2006. / Chemistry / unrestricted

Development of Non-Traditional Platinum Anticancer Agents: trans-Platinum Planar Amine Compounds and Polynuclear Platinum Compounds By Daniel E. Lee, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2015 Major Director: Nicholas P Farrell, Ph.D., Professor, Department of Chemistry Platinum anticancer compounds with cis geometry, similar to cisplatin, have been explored to circumvent the cisplatin resistance; however, they were not considered broadly active in cisplatin cells due to exhibiting similar or same cell death mechanism as cisplatin. Platinum compounds with trans geometry were less studied due to transplatin being clinically inactive; but with few structural modifications, they resulted in unaffected cytotoxic activities in cisplatin resistant cells with structural modification by exhibiting different modes of DNA binding. This research focused on further exploring and establishing structure-activity relationship of two promising non-classical series of platinum compounds with trans-geometry: trans-platinum planar amine (TPA) compounds and noncovalently binding polynuclear platinum compounds (PPC-NC). During this research, further optimizations of the reactivity of TPA compounds were accomplished by modifying the leaving carboxylate groups. The effects of modified reactivity were probed by a systematic combination of chemical and biophysical assays, followed by evaluating their biological effects in cells. To establish the structural-activity relationship of PPC-NCs, Mono-, Di-, Tri-, and Tetraplatin NC with charge of 4+, 6+, 8+, and 10+ were synthesized and evaluated by utilizing biophysical and biological assays. Lastly, a new class of polynuclear platinum compounds, Hybrid-PPCs, were synthesized and evaluated to overcome the pharmacokinetic problems of BBR3464, phase II clinical trial anticancer drug developed previously in our laboratory.

Aim. The aim of this study was to synthesize and characterize novel analogues of [DACH-Pt-DMC] by using different stereoisomers of DACH; and to investigate any differences in in vitro activity of these complexes in human hepatocellular carcinoma (HCC), colorectal carcinoma (CRC) cell lines and acquired cisplatin or oxaliplatin resistant sub-lines, and to compare that of oxaliplatin and other established Pt-based anticancer agents. Mechanistic roles of DACH-Pt- and DMC components of the TCM-Pt complexes on affecting HCT 116 human CRC cell line were investigated by flow cytometry, COMET assay and cDNA microarray analysis. / Background. Demethylcantharidin (DMC), a modified component of the traditional Chinese medicine (TCM), integrated with a platinum (Pt) moiety created a series of TCM-Pt complexes [Pt(C8H8O 5)(NH2R)2] 1-5 which demonstrated superior antitumor activity and circumvention of cisplatin resistance in vitro. Compound 5, derived from the 1,2-diaminocyclohexane (DACH) ligand (where R=trans-C6H10) had the most potent antitumor activity and closest structural resemblance to oxaliplatin (R,R-DACH-Pt complex) which is the first Pt-based anticancer drug to demonstrate convincing clinical activity against colorectal cancer and has a mechanism of action and resistance that is clearly different from that of cisplatin and carboplatin. / Conclusion. This study is the first to examine the mechanism of anticancer activity of new complexes that integrate DMC with different isomers of DACH. It has shown that both DACH-Pt- and DMC components contribute significantly to the compounds' potent anticancer activity, but likely with different mechanisms of action. The DACH-Pt- component appears to dictate the cell cycle distribution, whereas the DMC component appears to enhance cytotoxicity by inducing more DNA damage in HCT 116 colorectal cancer cells. / Methods. DMC was reacted with appropriate DACH-Pt-(NO3) 2 intermediates, which were prepared from treatment of K2PtCl 4 with stereoisomeric DACH (RR-, SS- & cis-), followed by reaction with silver nitrate. Proton NMR, high-resolution MS, polarimetry and circular dichroism (CD) spectroscopy were used to characterize their chemical structures and optical activities. In vitro antitumor activity (IC50 of 72hr drug exposure time) were assessed by a standard MTT assay. Cell cycle analysis by flow cytometry was determined at 0, 6, 12, 18, 24, 48 and 72 h after drug treatment (cisplatin, carboplatin, oxaliplatin, DMC, compound 1 or trans-DACH-Pt-DMC analogues) at IC50 and 5 x IC50 concentrations with three to four replicates. Comet assay was performed with a fluorescent microscope and used to examine DNA damage after drug treatments (50muM of cisplatin, carboplatin, oxaliplatin, DMC, compound 1 or R,R-DACH-Pt-DMC) for 3hr. cDNA microarray was performed on Affymetrix Human Genome U133A Set and used to analyze gene expression profiles in HCT 116 exposed to trans-(+/-)-DACH-Pt-DMC or oxaliplatin at their IC50 for 72hr. / Results. The in vitro results showed that the trans-analogues were consistently the most potent amongst all the compounds tested in both HCC and CRC cell lines: the trans-(+)(1R,2R)-DACH-Pt-DMC complex, in particular, was the most effective stereoisomer. All of the stereoisomeric DACH-Pt-DMC complexes and oxaliplatin were apparently able to circumvent cisplatin resistance in Huh-7 and SK-Hep1 sub-lines, but cross resistant with oxaliplatin in HCT 116 oxaliplatin resistant sub-line. Flow cytometric analysis revealed the novel trans-DACH-Pt-DMC analogues and oxaliplatin behaved similarly: that is, the compounds at 5 x IC50 concentrations all caused a significant decrease in the S-phase population within 18h and at the same time induced G2/M arrest, and without obvious sub-G 1 phase accumulation, but distinct from that of cisplatin, carboplatin or DMC. Comet assay showed that trans-(+)-(1R,2 R)-DACH-Pt-DMC caused the most significant DNA damage at an equivalent molar concentration. Microarray analysis suggested that the mechanistic role of the DMC ligand can induce the cell cycle to accelerate from the G 1 to S-phase and cause M-phase arrest. / Yu Chun Wing. / "July 2006." / Advisers: Yee-ping Ho; Chik Fun Steve Au-Yeung. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1586. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 191-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Aim: Cisplatin is the first platinum drug that shows promising anti-tumor effect clinically. Oxaliplatin, a third-generation platinum drug that incorporates a diaminocyclohexane (DACH) structural entity, can overcome cisplatin resistance. R,R-5, a novel platinum compound that integrates the DACH entity with a demethylcantharidin (DMC) component that is derived from a traditional Chinese medicine (TCM) , can also overcome cisplatin resistance. The principal objectives of this study was to investigate in detail, the effect of these compounds at the antephase and G2 checkpoints of the cell cycle, and to establish the relationship (if any) between different structural entities with checkpoint activation. The ultimate aim of the study was to ascertain the potential for the development of novel checkpoint abrogators as anti-tumor agents. / Background: A common procedure in current cancer chemotherapy is to induce genomic stress in cancer cells, leading to irreparable DNA damage and eventually cell death. However, there are several DNA repair mechanisms in cancer cells to maintain genomic stability, which require cell cycle checkpoints to stop cell proliferation for DNA damage repair, thereby avoiding errors in cellular events like DNA replication, transcription and mitosis. Among these cell cycle checkpoints, antephase and G2 checkpoints are two gate checkpoints for mitosis. Abrogation of G2 checkpoint has been reported to give rise to synergistic cytotoxic effect with DNA damaging agents, representing a means of circumventing drug resistance in chemotherapy. / Conclusions: Acute stress to cisplatin can activate the MMR/c-Abl/MEKK1/p38MAPK pathway, leading to the activation of antephase checkpoint, and stop cells from entering mitosis immediately. DACH-containing platinum compound oxaliplatin fails to activate this antephase checkpoint. However, both cisplatin and oxaliplatin can activate the G2 checkpoint, which can be abrogated by DMC. In contrast, RR-5 can bypass both the antephase and G2 checkpoints. In summary, novel TCM-platinum compound R,R-5 can bypass mitotic DNA damage checkpoints in cancer cells and thus has the potential for further development as an anti-cancer drug. / Methods: Microarray analysis was used to detect gene transcription profiles after drug treatments. The activation of mitotic checkpoints was inspected by counting mitotic cells and utilizing flow cytometry. Using Western blotting, the activation of certain key players in the antephase and G2 checkpoint was revealed. MTT assays were performed to show the outcome of checkpoint activation. / Results: In HCT116 cells, 35 genes that facilitate G2/M transition were found to be up-regulated after R,R-5 treatment compared with oxaliplatin in the microarray analysis, implying the bypass of mitotic checkpoints by R,R-5 rather than oxaliplatin. Acute stress (2 hour) of cisplatin activated the antephase checkpoint, resulting in a rapid decrease in mitotic index and phosphorylation of histone H1, which avoided mitotic catastrophe and promoted cell survival in HeLa cells. Further experiments demonstrated that this antephase checkpoint could be abrogated by c-Abl and p38MAPK inhibitors, or siRNAs against c-Abl or MEKK1, suggesting that this checkpoint may be controlled by an MMR/c-Abl/MEKK1/p38MAPK pathway. In contrast, oxaliplatin and R,R-5 did not activate this antephase checkpoint. Moreover, after 24 hour oxaliplatin treatment in HeLa cells, the mitotic index and CDK1 activity were decreased, which could be restored by concomitant treatment with ATM/ATR inhibitor and DMC. This indicated the activation of G2 checkpoint by oxaliplatin and implied that DMC can abrogate oxaliplatin-activated G2 checkpoint by restoring CDK1 activity. Cisplatin could also activate G2 checkpoint, whereas R,R-5 apparently bypassed this G2 checkpoint. / Guan, Huaji. / Adviser: Vincent Hon Leung Lee. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 212-249). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.