Ehrlich II –2nd World Conference on Magic Bullets

Titanium-Based Magic Bullets Against Renal-Cell Cancer TACKE M UCD School of Chemistry and Chemical Biology, Belfield, Dublin 4, Ireland. Background: Titanocene dichloride (Cp2TiCl2) shows significant activity against a variety of human cancer cell lines, overcomes the resistance against platinum drugs and reached two clinical Phase II studies against renal cell and metastatic breast cancer, where Cp2TiCl2 failed due to a lack in anti-proliferative activity. The aim of our research is to synthesise suitably substituted titanocenes and ansa-titanocenes in order to control the physiological uptake of the titanocenes and to overcome the toxicity effects exhibited by titanocene dichloride. In addition we model the titanocene-DNA interaction leading finally to apoptosis. Methods: Ansa-titanocene dichloride derivatives are obtained through reductive dimerisation of substituted aryl fulvenes with titanium dichloride, while un-bridged titanocene compounds are available from fulvenes through the addition of lithium hydride or aryl lithium [Bioorganometallic Fulvene-Derived Titanocene Anticancer Drugs, K. Strohfeldt, M. Tacke, Chem. Soc. Rev., 2008, 37, 1174-1187]. Results: We investigate the cytotoxic activity of all titanocenes on LLC-PK pig kidney carcinoma cells and that of selected titanocenes on a 36 cell-line panel in vitro. Results show that our most effective titanocene has a significantly higher activity than Cp2TiCl2 itself. Recently, our best titanocenes have successfully finished pre-clinical animal studies (EAT, MCF7, PC3, A431 and CAKI-1 in mice) as well as clinical ex vivo tests involving a variety of explanted human tumors. Conclusions: Our best titanocene derivatives are ready for clinical trials and have the potential to become the first effective chemotherapy against advanced renal-cell cancer in the nearby future. Acknowledgement: Support is acknowledged from CESAR, COST, HEA/CSCB and UCD.	Chemotherapeutic agents for Merkel cell carcinoma (MCC) of skin – past, present and future TAI P^1, YU E^2, ASSOULINE A^3, JOSEPH K^4, KRZISCH C^3 ^1Univ. Saskatchewan, Regina, Canada; ^2Univ. Western Ontario, London, Canada; ^3Centre Hospitalier Universitaire d' Amiens, Amiens, France; ^4Cross Cancer Institute, Edmonton, Canada. Background: In the past, chemotherapy was mostly used for metastatic MCC, although a few authors tried adjuvant chemotherapy as well. At the present time, multi-modality treatments are used. This study reports the multicenter experience on management of MCC. Methods: Patient information was obtained from medical records of 3 Canadian and 1 French institutes. Cox-proportional hazard analysis was used to find significant prognostic factors for cause-specific (CSS) and overall survivals (OS). Results: From 1987 to 2007, 121 cases from 4 institutes were collected. Median follow-up was 21.5 (range:0.5–163.4) months. The overall rate of distant metastases was 36/121 (30%). A total of 25 patients received chemotherapy either as initial treatment (n=10 or later on at time of relapse (n=15). Patients were treated with multiple regimens at recurrence. Chemotherapeutic agents included: Etoposide (E), Cisplatin (P), Carboplatin, Cyclophosphamide (C), Adriamycin (A), Vincristine (V), Epirubicin (Epi), Irinotecan, Taxotere and Topotecan. The regimens were: EP (13 patients), CAV/EP (6 patients), CAV or CEpiV (3 patients), AV/E or CEpiV/E (2 patients), CEP (1 patient), weekly carboplatin (1 patient), irinotecan+carboplatin (1 patient), taxotere and later topotecan (1 patient). The latter were used as third and fourth line chemotherapy in the same patient, with no response. Irinotecan+carboplatin produced a partial response. The patient who received weekly carboplatin as a radiosensitizer had a partial response. Cox-proportional hazard analysis showed that postoperative adjuvant radiotherapy and stage were significant prognostic factors for CSS and OS. Conclusions: The future magic bullet for MCC will likely be radiotherapy in combination with modern chemotherapy. Future studies of MCC should focus on newer agents used for other neuroendocrine tumors, e.g., irinotecan, taxotere and topotecan.
Assay of Therapeutic Effect in Hepatitis Using Pharmacokinetics of Salicylamide: Effects of Sho-saiko-to, a traditional Kampo medicine, and Its Chemical Components in Carbon Tetrachloride Intoxicated Rats TAIRA Z, UEDA Y Tokushima Bunri Univ., Tokushima, Japan Background: Pharmacokinetics (PK) is a powerful tool for detecting physiological changes in vivo resulting from injury, tumors or pregnancy. Aims: The therapeutic effects of Sho-saiko-to and its chemical components, baicalin, baicalein, glycyrrhizin and glycyrrhetic acid, were examined using daily changes in PK behavior of salicylamide (SAM) in rats with carbon tetrachloride (CCl4)-induced hepatitis. Methods: Male Wistar rats were intraperitoneally (i.p.) injected with 0.2 ml/kg of CCl4 in 20% olive oil, and serum ALT value measured daily. A suspension of Sho-saiko-to extract was given orally at 40 mg/kg one hour before CCl4 dose; while each component solution was given i.p. at the same dose. The rats were treated daily with the reagents in the same manner. SAM was injected intravenously into CCl4-intoxicated rats at days 0 to 5 and blood samples collected for 90 min. SAM in blood samples was determined by its fluorescence. The PK of SAM were examined using the parameters; plasma clearance (CL), the mean residence time (MRT), the volume of distribution (Vdss) at steady state, etc. Results: Serum ALT value rose to a peak one day after CCl4 dose and then decreased to the control level after three days. Baicalin (25.3%) and glycyrrhizin (19.9%) effectively suppressed the peak value of CCl4-induced hepatitis, but Sho-saiko-to extract (39.1%), baicalein (36.4%) and glycyrrhetic acid (47.1%) were less protective. However, the PK of SAM showed that at day one the primary decrease of CL resulted in liver damage because of CCl4 intoxication, in which the metabolic enzymes were damaged but the damaged tissue remained. Thus there was delayed MRT and Vdss remained at a control level. Furthermore, the secondary decrease of CL at day three may result in liver regeneration where the active enzymes were eliminated and the damaged tissue was removed. That is, MRT remained at a high level and Vdss decreased. Sho-saiko-to extract or its components protected the liver from damage in the different ways, as seen in the PK parameters; e.g. baicalin kept MRT at a control level but glycyrrhizin decreased it. Conclusions: The PK for SAM showed that liver function recovered in a biphasic manner by five days after CCl4 intoxication. Sho-saiko-to extract or its components effectively protected the liver against damage.	Telephone counseling of athletes abusing anabolic-androgenic steroids and the state of drugabuse in Japanese athletes TAKAHASHI M^1, TATSUG Y^1, KOHNO T^2 International Budo University, Katsuura, Japan Ryotokuji University, Urayasu,Japan Background: Drug abuse, most notably anabolic-androgenic steroid (AAS) use, in athletes is widespread. As a result, athletes and exercise enthusiasts who abuse these drugs are troubled by the side effects of these illicit drugs, especially AAS. Methods: In an attempt to improve this situation, since 1993, we have counseled athletes who abuse drugs and others with questions about AAS via telephone and tabulated the results. Counseling sessions took place by telephone every Monday between 19:00-23:00 hours. The number of cases was tabulated each year and the specific items discussed during each consultation were categorized by key words. Cases consisted of both drug abusers and athletes who did not abuse drugs and were concerned about the side effects or other various problems surrounding the use of AAS. Results: From 1993 to 1996 there were about 50 cases yearly; thereafter, the number of consultations dropped to about 30 to 40 cases each year. In 2002, consultations with drug abusers accounted for 52.2% of all consultations compared with 46% of all consultations from 1993 to 2002. We have found that abusers of endocrine agents exist in Japan, as well as elsewhere. Conclusions: 1) We hope these results will demonstrate the necessity of employing public institutional counseling systems for athletes who are drug abusers in Japan, similar to the successful system instituted by the Swedish National Service. 2) Furthermore some athletes and exercise enthusiasts has been recently using the website that drugabusers control in Japan. They exchange the information about the knowledge of drugabuse each other. We accessed to the website and analyzed the contents of them by key words. We would like to report the result in detail, too
Interferon Gene Therapy against Metastatic Cancer TAKAKURA Y, NISIKAWA M Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Background: The interferon (IFN) is the family of one of the most potent cytokines with various biological activities. Both type I (IFN-arfa and beta) and type II (IFN-gamma) IFNs have been used as therapeutic agents for malignant tumors and hepatitis in clinic. However, the biological half-lives of IFNs are very short. One of the promising approaches to overcome this drawback may be IFN gene therapy. Methods: The therapeutic effect of IFNs was examined using mouse models of liver or lung metastasis following gene transfer by intravenous injection of plasmid DNA vector by the hydrodynamics-based procedure (1). In order to prolong the duration of gene expression, we designed CpG-reduced (about 80%) or completely depleted vector encoding IFNs. Therapeutic effect on allergic diseases was also evaluated using NC/Nga mice, a model for human atopic dermatitis. Results: A significant amount of IFNs was observed in the liver and blood circulation following gene expression. In the liver metastasis experiment, IFN-expressing vector showed a profound reduction of liver metastasis and a prolonged survival (2). Hydrodynamic delivery of CpG-reduced vectors resulted in more sustained production of IFNs and a better therapeutic effect against the lung metastasis (3). Moreover, a single hydrodynamic injection of completely CpG depleted vector resulted in much more prolonged concentration of IFN-gamma over 80 days in NC/Nga mice. The correction of the imbalance of helper T lymphocyte subpopulations was shown in the mice and various symptoms associated with the allergic disease were eliminated. Conclusions: 1) Novel CpG-reduced vectors encoding IFNs showed prolonged gene expression compared with CpG-rich conventional vectors following intravenous injection by the hydrodynamic method. 2) Sustained IFN gene expression by CpG-reduced vectors resulted in enhanced therapeutic effect in lung metastatic model in mice. 3) Prolonged plasma IFN concentration was also effective for the treatment of atopic dermatitis in mice. In conclusion, IFN gene therapy would be possible through optimization of design and delivery of IFN plasmid vector. References: 1. Kobayashi N, Nishikawa M, Takakura Y, Adv. Drug Delivery Rev., 57, 713-731 (2005) 2. Kobayashi N. et al, Mol Ther., 6, 737-744 (2002) 3. Kawano H et al, Int J Cancer., 121:401-406 (2007)	Interaction Between Local Anesthetics, Specially Articaine, and the Sarcoplasmic Reticulum Ca^2+-Adenosine Triphosphatase TAKARA D; SÁNCHEZ GA, ALONSO GL University of Buenos Aires, Buenos Aires, Argentina. Background: The calcium-dependent adenosine triphosphatase (Ca^2+-ATPase) is a major intrinsic protein in the sarcoplasmic reticulum (SR) membranes from skeletal muscles. It actively transports Ca^2+ from the cytoplasm to the SR lumen, reducing the cytoplasmic Ca^2+concentration to promote muscle relaxation. Local anesthetic (LA) may induce sustained muscle contraction by inhibiting Ca^2+-ATPase. Aims: 1) to determine the action mechanism of articaine on the Ca^2+-ATPase cycle, 2) to study the different LA sensitivities on skeletal muscle isoforms of the Ca^2+-ATPase. Methods: SR vesicles from rabbit skeletal muscle were obtained as in Champeil et al (1985). Ca-dependent ATPase activity was determined as in Baginski et al (1967). ATP-dependent calcium uptake and phosphorylation with inorganic phosphate (Pi) were determined with a radioisotopic technique. Transient kinetics of the Ca^2+-ATPase active transport was analyzed by numerical simulation (Hecht et al, 1990). Results: Articaine inhibited Ca^2+-ATase activity, with Ki depending on Ca^2+concentration: it increased from approximately 6 mM for 0.1 μM Ca^2+ up to a constant value around 40 mM at [Ca^2+] higher than 20 μM. Anesthetic also inhibited the Ca^2+ uptake by isolated SR vesicles (Ki=30.533.4mM, n=5). Articaine increased the permeability of the membrane to Ca^2+ and was prevented by Ca^2+ and Mg^2+. Ca^2+, Mg^2+ and H^+ affected the inhibitory action of articaine on the Ca pump protein. In addition, we studied the properties and inhibitory effect of several LA (unplublished results) on Ca^2+-ATPase from masticatory muscles (Ki(lidocaine)= 19.311.87, n= 6 for ATPase activity, Ki(lidocaine)= 25.102.95,n=5 for Ca^2+ uptake. Ki(bupivacaine)= 19.301.12, n= 5 for Ca^2+-ATase activity and Ki(bupivacaine)= 8.122.81, n=5 for Ca^2+ uptake). Conclusions: 1) The activating effect of Ca^2+ on the ATPase activity was competitively inhibited by articaine. 2) The activating effect of Mg^2+ on the phosphorylation of Ca^2+-ATPase by Pi was also inhibited by articaine. 3) Decreasing pH increased Ki for articaine to inhibit the Ca^2+-ATase activity. 4) Articaine did not affect the reaction mechanism of the cations acting as cofactors of ATP in the catalytic site. 5) Ki values for lidocaine and bupivacaine in masticatory muscles were lower than those reported for these LA in white fast muscle.
Paclitaxel works nicely with molecular targeting cancer therapy via RNA interference TAKEI YOSHIFUMI Department of Biochemistry, Nagoya University Graduate School of Medicine, Nagoya, JAPAN BACKGROUND: Paclitaxel (PTX; Taxol^®) is used to treat patients with lung, ovarian, breast, head, and neck cancer or advanced Kaposi’s sarcoma. PTX works by interfering with normal microtubule breakdown during cell division. Although it acts as a potent anti-cancer reagent, it has a major dose-limiting toxicity in that it causes bone marrow suppression, especially neutropenia. To reduce the dosage of PTX, and to minimize hematologic side effects, we designed a combination therapy based on molecular targeting (in which the target is VEGF) via RNA interference (RNAi). VEGF (vascular endothelial growth factor) is a potent angiogenic factor in cancers. METHODS AND RESULTS: We established a human VEGF blockade system via RNAi. The synthetic short interfering RNA (siRNA) targeting human VEGF almost completely inhibited VEGF secretion by a human prostate cancer cell line (PC-3). The VEGF siRNA, together with atelocollagen, significantly suppressed the growth of PC-3 tumors that had been subcutaneously xenografted in nude mice. Atelocollagen, which is derived from bovine collagen digested with pepsin to reduce the antigenicity, is used as a delivery carrier of siRNAs. Atelocollagen contributes to the stabilization of the siRNA in tumors and delivers the siRNA to tumor tissues. These inhibitory effects of VEGF siRNA were dramatically augmented by combined treatment with PTX. The combinatory effect was cytocidal, thus reducing the initial volume of the tumor. The PTX dose (12 mg/kg) used for the combinatorial treatment did not cause any neutropenia or liver damage. VEGF siRNA suppressed VEGF contents in tumors, leading to the inhibition of tumor angiogenesis. Surprisingly, we also observed, for the first time, that PTX itself significantly reduced VEGF expression in the tumor. The evidence supports the phenomenon that PTX inhibits tumor angiogenesis even without any evidence of the molecules involved. DISCUSSION AND CONCLUSION: We made it possible to reduce the dosage of PTX, a chemotherapeutic agent, in combination with a molecular targeting therapy consisting of nucleic acids, especially those based on RNAi. We also found that PTX works nicely in combination with molecular targeting cancer therapy, i.e., anti-angiogenesis (ant-VEGF), via RNAi.	Crosstalk Between TNF and NGF: Potential Implications for Alzheimer's Disease and Neuroblastoma TAKEI YOSHINORI, LASKEY R MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road Cambridge CB2 0XZ, UK Background: Neuroblastoma is a pediatric tumour of the sympathetic nervous system. While early stage neuroblastoma sometimes shows spontaneous regression, advanced neuroblastoma has poor outcome and new therapeutic strategies are needed. Nerve growth factor (NGF) can induce differentiation of neuroblastoma cells expressing high levels of an NGF-specific receptor p140^TrkA. However, many neuroblastoma cells in advanced stages express p140^TrkA poorly and they cannot differentiate in response to NGF, though overexpression of p140^TrkA allows NGF to induce their differentiation. Thus, inadequate expression of p140^TrkA is supposed to cause the insensitivity of neuroblastoma cells to NGF-dependent differentiation. Results: we have reported recently that overexpression of p140^TrkA is not necessary for NGF-dependent differentiation of neuroblastoma cells. NGF can induce synthesis of tumour necrosis factor  (TNF) in neuroblastoma cells. Although TNF is a cytokine that can promote cell death through TNFα receptor 1, TNF induced by NGF activates another TNFα receptor, TNFR2. When signalling through TNFR2 was inhibited, NGF induced differentiation of neuroblastoma cells without overexpression of p140^TrkA. Thus, signalling through TNFR2 appears to cause the insensitivity of neuroblastoma cells to NGF-dependent differentiation, rather than insufficient expression of p140^TrkA. Conclusion: Our findings could provide new therapeutic strategies for neuroblastomas. Furthermore, induction of TNF in neural cells by NGF suggests a positive feedback loop of TNF and NGF expression between neurons and glial cells, since glial cells can produce NGF in response to TNF. This loop could promote survival of neural cells in inflammation. However, once their balance is disturbed, this loop could contribute to either excessive death of neural cells or excessive proliferation of immature neural cells, i.e. neurodegenerative or cancerous conditions. Takei, Y. & Laskey, R. (2008) Tumor Necrosis Factor α Regulates Responses to Nerve Growth Factor, Promoting Neural Cell Survival but Suppressing Differentiation of Neuroblastoma Cells. Mol Biol Cell, 19, 855-864. Takei, Y. & Laskey, R. (2008) Interpreting crosstalk between TNF and NGF: potential implications for disease, Trends in Molecular Medicine, 14, in press.
Mechanism of Fluoroquinolone resistance in Shigella and Salmonella species TALUKDER KA, AZMI IJ, KHAJANCHI BK, ISLAM Z, MONDOL AS, ASLAM M, DUTTA DK, FARUQUE ASG, AHMED D, HOSSAIN MA, WATANABE H, SACK DA, ENDTZ HP, CRAVIOTO A ICDDR, B, GPO Box-128, Dhaka-1000, Bangladesh Background: Enteric fever and shigellosis remain a major cause of morbidity and mortality in worldwide. Ciprofloxacin, a fluoroquinolone antimicrobial agent is generally highly effective in treating typhoid fever and shigellosis patients. We report the emergence of complete ciprofloxacin resistance in Bangladesh. Objectives: The aim of the present study was to characterize and understand the molecular mechanism of fluoroquinolone resistance in Shigella flexneri and Salmonella Typhi strains recently isolated in Bangladesh. Methods: A total of 1728 Shigella species isolated between 2004 and 2008 and 765 Salmonella Typhi strains isolated between 2006 and 2007 in the Clinical Microbiology Laboratory from diarrhoeal patients at ICDDR,B hospital were tested for MIC by E-test and agar dilution method in addition to antibiotic susceptibility testing following the recommendations of CLSI. Plasmid profiling, PFGE and sequencing analysis were performed in order to determine the clonal relationships and mutation analysis of QRDR of gyrA, gyrB and parC. Results: Of 1728 Shigella species isolated between 2004 and 2008, 104 (6%) isolates were resistance to ciprofloxacin. Most of ciprofloxacin resistant isolates were S. flexneri 2a (90%). Resistance to ciprofloxacin of S. flexneri 2a increased from 0% in 2004 to 95% in June 2008. The MIC for ciprofloxacin, norfloxacin, ofloxacin, were 6-8 μg/ml, 8-32 μg/ml, and 8-24 μg/ml, respectively. Of 765 Salmonella Typhi strains, 474 (62%) strains were resistant to nalidixic acid. Of the 474 nalidixic acid resistance strains, 402 (85%) showed reduced susceptibility to ciprofloxacin. The complete resistance to ciprofloxacin increased from 18 (2.8%) in 2006 to 11 (4.5%) in July 2007. The isolates showed reduced susceptibility to ciprofloxacin had the MIC ranged from 0.064-0.25 μg/ml and the complete ciprofloxacin resistance strain showed the MIC ranged from 6-32 μg/ml. Sequence analysis of QRDR of ciprofloxacin resistant strains of S. flexneri and S. Typhi revealed that all had mutations in gyrA (Ser^83 ® Phe) and/or (Asp^87 ® Asn or Gly) and a single mutation in parC (Ser^80 ® Ile) whereas none of the susceptible strain had the mutation in their QRDR region. In addition, a novel mutation point was detected at outside the QRDR at position 211 (His Tyr) in gyrA gene of fluoroquinolone resistant strains of S. flexneri 2a. None of the strains of S. flexneri 2a and S. Typhi had mutations in gyrB and parE genes. Qnr gene was not detected in any of strains which exclude the possibility of plasmid mediated quinolone resistance in S. flexneri and S. Typhi in this region. A single PFGE type A, subdivided into four subtypes A1 to A4, were found in S. flexneri 2a resistant strains suggesting their genetic relatedness. Similar type of findings was observed in case S. Typhi 2a resistant strains. Conclusions: The present study reported the mutation in fluroquinolone resistant S. flexneri 2a and S. Typhi strains in Bangladesh. Increase isolation of ciprofloxacin resistance strains suggests that S. flexneri and S. Typhi are becoming difficult to treat in Bangladesh.	Targeted Delivery of Magic Bullets by the Use of Mechanized Nanoparticles TAMANOI F California NanoSystems Institute, University of California, Los Angeles, CA, USA Background: Recent development in Nanotechnology led to the generation of a variety of nanomaterials including nanoparticles, nanosheets, nanofibers and nanotubes. Nanoparticles with diameters in the range of 100-200 nm are of particular interest, as they can serve as efficient vehicles to store and deliver magic bullets. In addition, nanoparticles provide protection of stored drugs. Targeting ligands can be attached to the nanoparticles to achieve cancer targeting. Finally, nanomachines can be attached to accomplish controlled release of magic bullets. We are developing mesoporous silica nanoparticles to achieve delivery of anticancer drugs. Methods: Camptothecin was chosen to test the ability of mesoporous silica nanoparticles to deliver poorly water soluble anticancer drugs to cancer cells. The nanoparticles were fluorescently labeled to follow their cellular localization. Camptothecin was loaded onto the nanoparticles, added to human cancer cells and cellular effects were investigated by examining apoptosis induction caused by camptothecin. In the second experiment, we coated the pore interior with azobenzene to generate nanoimpellers that release the stored drug in response to light exposure. Operation of nanoimpellers was examined by the release of a dye, propidium iodide, and following staining of nuclei in response to light exposure. Light dependent release of anticancer drugs using nanoimpeller was examined by using camptothecin. Results: Camptothecin was efficiently stored in fluorescent mesoporous silica nanoparticles and was released in human cancer cells as detected by the induction of apoptosis. Uptake of the nanoparticles and their lysosomal localization were established. Attaching targeting moiety on the surface of nanoparticles enabled preferential effects on cancer cells. Nanoimpeller was shown to operate in human cancer cells. Conclusions: Mesoporous silica nanoparticles provide promising vehicles to deliver anticancer drugs. This is particularly important, as a significant percentage of anticancer drugs being developed is poorly water soluble and has problems in their clinical use. Nanoimpellers provide vehicles that can accomplish controlled release of anticancer drugs in response to light exposure. Our studies open up new approaches to deliver magic bullets.