Ehrlich II –2nd World Conference on Magic Bullets

ATP and adenosine as biomarkers for drug development YEUNG PKF, DAUPHINEE J, SIMONSON K, BEATON S, GOUZOULES T Dalhousie University, Halifax, Nova Scotia, Canada Background: ATP and adenosine are major regulators of energy metabolism maintaining optimum balance between oxygen supply and demand within the cardiovascular system. In addition to these fundamental roles, they are also potential biomarkers for cardiovascular protection, and as therapeutic targets for treatment of ischemic heart disease (IHD), stroke, inflammatory diseases, cancer, and neurodegenerative diseases. The effectiveness of many cardiovascular drugs could be attributed to their effect on increasing plasma and tissue concentrations of adenosine and ATP. The main objective of our research effort in this program is to determine whether or not adenosine and ATP concentrations in plasma and RBC are useful surrogate biomarkers for development of cardiovascular agents in vivo. Methods: Male New Zealand White rabbits (4 – 6 kg), normotensive SD rats and hypertensive SHR (325 – 400 g) were used for these investigations. They were treated with a known cardiovascular drug or normal saline (control), and kept either in a restrainer or placed on a research treadmill for an exercise test for blood sample collection and hemodynamic recordings. Blood samples were collected via an indwelling catheter from a carotid artery and immediately mixed with a stopping solution to stabilize adenosine and ATP during subsequent processing. Plasma concentrations of adenosine and red blood cell (RBC) concentrations of ATP and their metabolites were measured by a previously validated HPLC. Data between groups were compared by ANOVA and differences considered significant when p < 0.05. Results: We have found that many cardiovascular agents block the uptake of adenosine by RBC, and that some of them enhance the hemodynamic effects of adenosine in the rabbit model. In addition, we have found that SHR have higher plasma concentrations of adenosine and RBC concentrations of ATP than the normotensive SD rats, and that exercise increases the concentrations of these biochemical regulators particularly in rats treated with the cardiovascular agents. Conclusion: Many cardiovascular drugs and perhaps also drugs affecting energy metabolism could modulate the circulatory concentrations and cardiovascular effects of Adenosine and ATP which could be exploited for development of novel drugs and innovative therapies. (Supported in part by a grant-in-aid from CIHR/NSHRF/PEF Regional Partnership Program).	Antineoplastic effects of an aurora B kinase inhibitor against breast cancer YEUNG SJ, GULLY C, YEUNG JA, LEE MH Department of General Internal Medicine, Ambulatory Treatment & Emergency Care, Department of Molecular & Cellular Oncology, The University of Texas M D Anderson Cancer Center, Houston, Texas, USA Aurora B kinase is an important mitotic kinase involved in chromosome segregation and cytokinesis. It is overexpressed in some human breast cancer as well as other cancers, and has been linked to genetic instability, tumorigenesis and invasive disease. Aurora B is an important molecular target for chemotherapeutics, and several small molecule inhibitors of Aurora B are now in clinical trials. AZD1152 is a dihydrogen phosphate prodrug which is converted in the serum to AZD1152-HQPA [hydroxyquinazoline pyrazol anilide]. AZD1152-HQPA is a small molecule ATP binding pocket competitor that selectively inhibits Aurora B [Ki = 0.36 nM] compared with Aurora A [Ki =1369 nM] and 50 other kinases. AZD1152 has been shown to have antineoplastic activity in acute myelogenous leukemia, multiple myeloma and colorectal cancer. To date, AZD1152 has not been evaluated in human breast cancer, the leading cancer among US women. We investigated the effect of AZD1152 on breast cancer cells. AZD1152-HQPA inhibited a panel of human cell lines including Her18, a Her-2 overexpressing breast cancer line. The IC50 for these lines were 14-125 nM and in the same range as reported by others. Time-lapse photomicrography of Her18 cells treated with AZD1152-HQPA showed enlarged multinucleate cells. Micronuclei and chromosome bridges were observed. FACS analysis demonstrated polyploidy consistent with the increase in DNA copy number in multinucleate cells. AZD1152-HQPA treatment resulted in decrease in Aurora B activity (as indicated by decrease in phosphorylated Histone H3), mitotic failure and apoptosis (as indicated by Annexin V-FITC FACS, increases in PARP cleavage and increase in Bax). AZD1152-HQPA inhibited the clonogenic potential of Her18 cells. AZD1152 (62.5 mg/kg/day on days 1 and 2 of a 7-day repeating cycles) also demonstrated significant antineoplastic activity against Her18 xenografts in nude mice. Reduction in phospho histone H3 was demonstrated by immunoblotting in tumor samples from the AZD1152-treated group. Bax was increased in the AZD1152-treated tumors. Immunohistochemistry for Ki-67 and activated caspase 3 showed that AZD1152 reduced proliferation and increased apoptosis in the treated xenografts. In conclusion, these data suggest that AZD1152 may be an effective chemotherapy agent for human breast cancer. Further investigation and possibly a clinical trial in breast cancer patients are warranted
Fluoroquinolones in the Treatment of Tuberculosis: Current Status and Future Issues YEW WW^1 ^1Grantham Hospital, Hong Kong, China In the 1980s to 1990s, fluoroquinolones were shown to have significant activities, both in vitro and in the murine model, against Mycobacterium tuberculosis. Later on, the clinical activity of fluoroquinolones in treating both drug-resistant and drug-susceptible tuberculosis, was demonstrated in some largely uncontrolled chemotherapy trials. The utility in the former setting became rather important due to the surge in multidrug-resistant tuberculosis globally in the past two decades. In parallel, fluoroquinolones were also found to have a useful role in treating patients with significant intolerance to conventional antituberculosis drugs. Furthermore, in order to improve patient adherence and curtail development of drug resistance, a genuine need arose to discover more efficacious drug regimens to shorten the six-month duration of standard antituberculosis chemotherapy for drug-susceptible disease. This prompted focused research on the sterilizing capacity of fluoroquinolones against M. tuberculosis persisters in disease lesions, especially regarding that of the newer 8-methoxyquinolones. Animal experiments have apparently demonstrated the potential ability of these new agents in shortening the duration of therapy, in comparison to standard short-course treatment, and producing stable cure of disease without relapse. Controlled clinical studies are now ongoing to evaluate these issues in patients with tuberculosis. Some preliminary results appear rather promising. However, the emergence of resistance against fluoroquinolones in M. tuberculosis strains, often in the deadly form of extensively drug-resistant tuberculosis would pose a great concern regarding the effectiveness of these new agents in treatment of the disease. Novel strategies are thus required to circumvent this challenge.	Pharmacokinetic and Drug Metabolism: A Bioanalytical Perspective YIN J PharmaMar USA, Inc., Cambridge, MA, USA Drug development is a complex process that requires technical and scientific expertise in many area and contributive specialist knowledge integration. Evaluation of the pharmacokinetics (PK), drug metabolism, and pharmacodynamics (PD) is essential for understanding the characteristics of a new molecule entity and is necessary for a rational drug development program. Among these processes a key component is the development of bioanalytical methods needed to accurately quantify biological samples of administered drugs. This is an important factor in the study of the relationship of dose, exposure and effect, and reaching reasonable PK/PD conclusions regarding the drug safety and efficacy. A fundamental flaw in drug development program would be the lack of reliable effectiveness and safety profiles and rich database to support pivotal dose regimen design for the clinical trials. Validated bioanalytical assays ensure accurate information regarding PK properties and further the establishment of informative doseexposure (PK)-response (PD) relationship throughout the drug development program, and eventually provide the best possible foundation relevant to more efficacious and safer therapeutic use of the drug in man.
Inhibitory balance of γ-aminobutyric acid in cerebellar circuitry in autism: a circuit-centered approach to drug target design for developmental neurobiological disorders YIP J, SOGHOMONIAN JJ, BLATT GJ Department of Anatomy and Neurobiology, Boston University School of Medicine, Boston MA 02118 The maintenance of the balance between excitation and inhibition in the brain is essential to avoid pathological consequences. γ-aminobutyric acid (GABA) is the principal source of inhibition, it uses glutamate (the principal excitatory transmitter) to block over-excitation in the synapses of the brain and thus preventing seizures. GABAergic agents that enhance inhibition in the brain are anticonvulsive and anxiolytic. A dilemma arises during brain development when a mismatch between the strength of GABA and glutamatergic synapses which may prevent growth and synapse formation and perturb the sculpting of neuronal circuit connections, If this condition prevails, there will be a failure of neuronal communication and may contribute to disordered information processing in autism. Therapeutic drug design based on drug-receptor interaction becomes more challenging in disorders of developmental neurobiological origin involving multiple signalling processes such as autism. The discovery of drug development program in autism will require the application of basic science research with therapeutic modalities that can address malfunctions in the homeostatic mechanisms of the disorder. A circuit-approach study of inhibitory neuromodulation, via post mortem brain studies from autistic compared to control individuals, represents one of our efforts to identify key neurosubstrates in autism as a prelude to future rational drug design.	GAD67 mRNA decrease in cerebellar Purkinje cells in autism: an in situ hybridization study YIP J, SOGHOMONIAN JJ, NGUYEN L, BLATT G Boston Univ, Boston, MA, USA Reduced levels of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD) isoforms have been reported by Fatemi et al (2002; Soc Biol Psych, 52: 805-10) in the cerebellum of adult autistics but the specific cerebellar regions or cell types were not identified. A likely candidate is Purkinje cells in the posterolateral cerebellar hemisphere, an area previously identified as having moderate to severe decrease of Purkinje cell number. To investigate the role of GAD in Purkinje cells, the current study measures GAD67 mRNA levels in the posterolateral cerebellar cortex. Fresh frozen brains for the Harvard Brain Tissue Resource Center and Autism Tissue Program consisted of 8 autistics, and 8 controls matched for age, PMI, gender and pH. In situ hybridization procedure was described in Nielsen and Soghomonian (2004; Neurosci, 123: 31-42). Each section covered with 2-8 ng of radiolabeled cRNA was hybridized for 3.5 hr at 50^oC. Brain sections radiolabeld with ^35S-GAD67 probe were processed for film radioautograph. Area of occupancy of reduced silver grains (μm2) corresponding to GAD67 mRNA in individual neurons was quantified on emulsion radioautographs using NIH image software. GAD67 mRNA levels were reduced by 40% (p = 0.002, student t-test) in autistic compared to control (mean ± SEM for controls 1.54 ± 0.08, autistics 0.92 ± 0.09) brains, suggesting downregulation of GAD67 mRNA in Purkinje cells in autism. Whereas GAD67 mRNA levels were reduced, the effects were not paralleled by a significant loss of Purkinje cells (mean ± SEM for controls 4.0 ± 0.2, autistics 3.7 ± 0.2). The reduction in GAD67 mRNA level may result in reduced GABA input to deep cerebellar nuclei disrupting cerebellar output. This may also contribute to autism neuropathology seen in the cerebellar nuclei such as previously demonstrated by Bauman and Kemper (1985; Neurol, 35: 866-74). Support by: NIH NICHD #HD39459-04
The fecundity ofSchistosoma japonicum was impaired by administration of low dose cyclophosphamide CHUANXIN YU^1, QI G^1, XUREN Y^1, KIKUCHI M^2, KENJI H^2 1 Jiangsu Institute of Parasitic Diseases, Wuxi city, Jiangsu Province, P.R. China; 2 Institute of Tropical Medicine, Nagasaki University, Nagasaki city, Japan. Background: Development of female schistosomes from infectious cercariae to mature egg producing adult is the key pathogenic step of schistosome infection. To explore the factors promoting the development of schistosome will helpful for developing some new methods of anti-pathological process. Aim: To explore the impact of administrating low-dose cyclophosphamide (CTX) on the level of CD4+CD25+ Treg cells of mice and on the fecundity development and egg production of S. japonicum. Methods: This study included 72 C57BL/6J female mice, which were divided into control group, single treating group and consecutive treating group (24 mice per group, weight: 20±3g). The mice in single treating group were given a single low dose of CTX (50mg/Kg) by I.P., mice of consecutive treating group were given a dose CTX per week by I.P., and mice of control group were given placebo of normal salt solution (NS). Mice were infected with 30±1 cercaria of S. japonicum at seventh day after first injection. The level of CD4+CD25+ Treg cell in peripheral blood and spleen of three mice from each group were examined by FACS per week. All the remained mice were sacrificed at 42^th day post infection，and the worms recovered from portal vein by perfusion and eggs in liver were counted. The size of egg granuloma was checked by histological observation. This experiment repeated twice. Results: The percentage of CD4+CD25+ Treg cell in peripheral blood and spleen of mouse was declined and reached to the lowest level at seventh day after received single dose CTX, then rose gradually. The percentage of CD4+CD25+ Treg cell of mice received CTX treatment consecutively maintained a low level constantly. The egg reduction rate was 31.2 to 36.2 % in single treating group, and was 46.1 to 50.4 % in consecutive treating group. The egg granuloma size was reduced significantly both in single treating group and in consecutive treating group than the one in control group, the reduction rate of average equivalent area of single granuloma was 51.5% in consecutive treating group and 19.2% in single treating group respectively. Conclusion: CD4+CD25+ Treg cell play an important role during the fecundity development of S. japonicum.	Metalloporphyrn-Loaded Liposomes as Anticancer Drug Delivery System. Novel Drug Bearing SOD-like and Fenton-reaction Activities YUASA M, MURATA H, YUKI R, TATEISHI T Tokyo Sci. Univ., Noda 278-8510, Japan Background: A novel design of anticancer drug delivery system of ironporphyrin-loaded liposome (FeP/liposome) is reported. A lack of cytotoxicity of FeP/liposome and an efficient generation of toxic OH∙ from O2^-∙ through the iron-catalyzed dismutation and the Fenton reaction allow for targeted necrosis of tumor cells where the O2^-∙ concentration is locally increased as a result of reduced activity of SOD and catalase in the cells. Methods: Lewis lung carcinoma tumor cells were treated with some drugs (0 to 100 M). Cytotoxicity was determined by staining the cells with Alamar blue to obtain the effective concentration (EC50) of drug required to produce 50% lethal dose against cell. Rate constant (kcat) of iron-catalyzed O2^-∙ dismutation was analyzed by stopped-flow method. Particle size () of liposome was measured by DLS and TEM. The relative hydrogen peroxide resistance against SOD (H2O2 resist) of FeP was determined by UV-vis. Results: Significant damage to such impregnated tumor cells is observed in comparison with cisplatin and mitmycin c. The lack of cytotoxicity is shown in control experiments using SOD and manganese-porphyrin (MnP) in place of FeP, which suggests that the cell damage is induced by OH, considering that SOD and MnP only show O2^-∙ dismutation but do not participate in Fenton reaction. The kcat of FeP/liposome is analogous to that of SOD, which also indicates that has a potential to act as catalyst of O2^-∙ dismutation. The FeP/liposome is porphyrin-doped nanoparticle and has a strong H2O2-resistance. The properties of the liposome will permit its continuous circulation in blood, porphyrin-based tumor targeting, and accumulation in a pathological site such as tumor tissue. drugs EC50 (M) kcat (M^-1s^-1) (nm) H2O2 resist FeP/pH-sens liposome 6.5-7.0 1.2-3.1x10^7 33-54 280 FeP/liposome 13-14 1.0-3.1x10^7 23-60 350 cisplatin 31-35 - - - mitomycin c 26 - - - SOD > 100 2.3x10^9 - 1 Conclusions: We have demonstrated that FeP/liposome bearing SOD-like and Fenton reaction activities present a potential target for design and development of novel anticancer drug. Our approach opens the door for liposome-loading and tumor-targeting of a much expanded selection of metalloporphyrin.
Incorporation of Paclitaxel into Well-Defined Amphiphilic Block Copolymer Micelle Having Phospholipid Polymer Sequence YUSA S^1, FUKUDA K^1, ISHIHARA K^2, YOTARO M^3 ^1Univ. of Hyogo, Himeji, Japan; ^2The Univ. of Tokyo, Bunkyo-ku, Japan; ^3Fukui Univ. of Tech., Fukui, Japan Background: Paclitaxel (PTX) is one of the most effective and commonly used drug for the treatment of various cancers. Due to the poor solubility of PTX in water and in many other acceptable pharmaceutical solvents, a specific solubilizing agent such as Cremophor EL is used to formulate PTX in a commercial injection solution. However, serious side effects have been reported. Therefore, a much safer solubilizing reagent has been needed. To realize safer and effective drug administration, novel well-defined and biocompatible micelles of amphiphilic diblock copolymers containing Phospholipid polymer sequences were synthesized. Methods: At first, the homopolymer of 2-methacryloyloxyethylphosphorylcholine (MPC) was synthesized in water by reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization. Using this MPC homopolymer, AB type diblock copolymers of n-butyl methacrylate (BMA) were synthesized. Association behaviors of the amphiphilic diblock copolymer (pMPCm-pBMAn) with varying pBMA block lengths were investigated by nuclear magnetic resonance (NMR), fluorescence probe, static light scattering and quasi-elastic light scattering measurements. A given amount of PTX was dissolved in ethanol, and the PTX solution was added to an aqueous solution containing various amounts of the block copolymers. The solubility was evaluated by the transparency of the solution. Results: Fluorescence spectra of the probe indicated that the probe was solubilized in polymer micelles in water. The formation of polymer micelles comprising a core with pBMA blocks and shell with hydrophilic pMPC block was confirmed by NMR and light scattering data. The size and mass of the micelle increased with increasing pBMA block length. PTX dissolved well in aqueous solutions of the block copolymer as compared with pure water, implying that PTX is incorporated into the hydrophobic core of the polymer micelles. Conclusions: Well-defined amphiphilic block copolymers composed of pMPC and pBMA blocks were prepared via RAFT controlled radical polymerization. The amphiphilic block copolymer formed polymer micelles in aqueous solutions because of hydrophobic associations among pBMA block. By use of the diblock copolymer, the water solubility of PTX can be improved.