Ehrlich II –2nd World Conference on Magic Bullets

Interactions Between NE And EAAT ORTIZ JG, BERRIOS-CARTAGENA N, RUBIO-DAVILA M Department of Pharmacology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico Background: Excitatory Amino Acid Transporters (EAAT) are the main mechanism for its removal from the synaptic space. We have reported that alpha-1 antagonism (prazosin) prevents cocaine sensitization and it is accompanied by marked changes in Glutamate transmission (Ortiz et al 2006). We examined possible changes in EAAT acttivity brought about by cocaine exposure. Methods: Rat cortex (CTX) and prefrontal cortex (PFC) slices were incubated with different agents for at least one hour prior to measuring EAAT activity. The slices were then incubated with 50 μM [^3H]Glutamate for 10 minutes. The media was the removed and the slices washed twice. The slices were then solubilized in 50 μL of 0.5 M NaOH overnight. The radioactivity was quantified after acidification with 50 μL of glacial acetic acid and addition of 1 mL Ecolume using a Beckman LS 1800 scintillation counter. Results: Fifty micromolar NE increases EAAT activity in PFC but not in cortical slices. Cocaine increases EAAT activity in PFC slices but not cortical slices. Prazozin also increases EAAT activity, however, propranolol (beta blocker) had no effect, nor did it prevent the effects of NE. Conclusions: 1) Our results show modulation of EAAT activity by NE and 2) suggest non-alfa, non-beta mechanism(s).

Micro RNA As A Novel Drug For Cancer Metastasis: Suppression Of Lung Metastasis Of Osteosarcoma In In Vivo Model OSAKI M^1,2), TAKESHITA F^1), OSHIMURA M^2), OCHIYA T^1) ^1Sec. Meta. Natl. Cancer Ctr. Res. Inst., Div. Mol. Genet. Biofanct. Tottori Univ. Grad Sch Med. Sci. Background: MicroRNA (miRNA) is a small non-coding RNA consisting of 21~25 nucleotides. Recent studies disclosed that many miRNAs show some correlations with cancer progression including metastasis. Methods: To detect candidates of miRNA in human osteosarcoma that should be implicated in lung metastasis, we performed miRNA microarray analyses on a 143B cell, a human osteosarcoma cell line showing high frequent spontaneous metastasis to lung in an in vivo mouse model, and comparing it with that of an HOS cell, which was a parent cell of the 143B cell and showed no spontaneous lung metastasis in mice. The detected miRNA candidates were examined to identify miRNA(s) correlated to cellular invasion by in vitro assay. An identified miRNA was inoculated into mice that showed spontaneous lung metastasis from primary osteosarcoma lesion at right knee joint. Expression of the identified miRNA was also examined in human osteosarcoma tissue samples. Results: We identified nine miRNAs that were significantly down-regulated in 143B compared to HOS (P<0.01). In a matrigel invasion assay, transfection of one of those miRNAs (termed occasionally as miR-X) into 143B decreased their invasiveness. On the other hand, the miR-X poorly affected proliferation of 143B cells in vitro. Intravenous injection of the miR-X, but not negative control miRNA, into the spontaneous metastasis model mice successfully suppressed lung metastasis of osteosarcoma cells. Moreover, expression levels of miR-X in human primary osteosarcoma with lung metastasis after primary tumor resection showed low compared to those without lung metastasis. Conclusion: Our data suggest that the down-regulation of miR-X correlates lung metastasis of human osteosarcoma cells by promoting cellular invasion, and that the miR-X might be a novel drug to inhibit lung metastasis of osteosarcoma cells.

Applied Reverse Vaccinology: A Meningococcal Serogroup B Vaccine For Infants OSTER P^1, BORROW R^3, FINLOW J^3, POLLARD AJ^4, SNAPE M^4, MILLER E^2 ^1Novartis Vaccines, Siena, Italy, ^2Immunisation Department, Health Protection Agency, London, UK, ^3Vaccine Evaluation Unit, Health Protection Agency, Manchester, UK, ^4Department of Paediatrics, University of Oxford, Oxford, UK Background and Aims: Although strains of serogroup B N.meningitidis are the predominant disease causing strains in many regions of the world today, there is no truly global vaccine available to prevent this particular meningococcal infection. In a technique known as “reverse vaccinology,” potential vaccine candidates were predicted by analyzing the entire genome sequence of an invasive MenB strain. Through genetic engineering and from the investigation of 350 potential antigens, surface proteins were identified that best induced an antibody response. Building on this genomics approach, it was possible to develop a vaccine that offered protection against multiple strains of MenB. Phase II trials have demonstrated satisfactory safety, tolerability and immunogenicity. In addition, rMenB is the first recombinant MenB protein vaccine to induce an immune response in infants. Methods: Safety and immunogenicity of the rMenB vaccine was assessed in a 2, 4 and 6 month schedule. The immunogenicity was measured by serum bactericidal assay using human complement (hSBA). Results: The trial demonstrated satisfactory safety, tolerability and immunogenicity. Local and systemic reactions of the vaccine candidate were similar in frequency and intensity to routine infant immunisations with the exception of fever. A moderate, short-lasting temperature rise not exceeding 39.0°C following the first dose was reported more frequently in the rMenB arm than in the control. Preliminary analysis shows 89% (44/76-SL, ST32), 96% (5/99, ST8) and 85% (NZ98/254, ST41/44) hSBA ≥1:4 post 3^rd dose against three serogroup B strains representing the major vaccine antigens. The majority of disease causing strains worldwide express at least one of rMenB antigens. Conclusions: rMenB vaccine is well tolerated and immunogenic against a panel of serogroup B strains in young infants when administered in a three dose schedule two months apart. This vaccine is entering phase 3 clinical trials.

Reduction Of Flubiprofen-Induced Gastric Toxicity By Prodrug Formation And Enhancement Of Oral Bioavailability Of Flubiprofen By Chitosan Complexation OTAGIRI M, IMAI T Graguate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan Background: Acidic nonsteroidal anti-inflammatory drugs (NSAID), including flubiprofen (FP), are poorly soluble in water and cause gastric irritation. Aims: 1) To develop a NSAID with reduced gastric toxicity by combining it with a clinically potent histamine H2 antagonist; and, 2) To improve the dissolution characteristics for rapidly absorbed solid drug formulation using low molecular (LM) chitosan. Methods: A conjugate of FP with N-[3-{3-(1-piperidenylmethyl)phenoxy} propyl] 2-(2-hydroxyethyl-thio) acetamide (PPA) histamine antagonist was synthesized. A complex of FP and LM chitosan (MW: approximately 750) was prepared using a kneading method. The dissolution of FP from the samples was measured using a padded method. In vivo absorption studies were carried out using male wistar rats, and FP was analyzed using HPLC. Gastric mucosal irritation was evaluated from the total sum of the products of length and width of each affected mucosal part from each rat. Results: The prodrug FP-PPA was partly hydrolyzed in vitro in buffer (pH 1.2-7.4) in the presence or absence of pepsin and trypsin, then it was slowly hydrolyzed in gastric mucosal homogenate and quickly hydrolyzed in 10% rat plasma. The prodrug inhibited carrageenan-induced paw edema to the same extent as FP alone. The plasma concentrations of FP after oral administration of the prodrug were similar to FP alone. The prodrug formation significantly reduced gastrotoxicity in comparison with an equivalent dose of FP, whereas the coadministration of FP and PPA did not affect the gastrotoxicity of FP. The gastrotoxicity of the FP methyl ester was dependent on the drug concentration in the gastric mucosa. The dispersion of FP in LM chitosan causes a decrease of crystallinity and microcrystal size and a change of crystal lattice and microcrystal shape. The dissolution rates of FP from the LM chitosan complex were enhanced with increasing amounts of LM chitosan. The oral bioavailability of FP was improved through complex formation with LM chitosan. Conclusions: 1) The prodrug of FP with histamine H2 antagonist, PPA, causes less gastric damage than ester prodrugs like the methyl ester of the free drug FP. 2) The complex of FP with LM chitosan induced a faster absorption rate of FP, owing to the rapid dissolution into the GI fluid.

Complex Carbohydrate-Based Cancer Vaccines: Magic Bullets In The Making? Yang G^1, Spassova MK^1, Qian W^2, Zhu J^2, Lee D^2, Cao C^1, Ragupathi G^3, Livingston PO^3, Danishefsky SJ^2,4, Ouerfelli O^1 ^1Organic Synthesis Core Laboratory, ^2The Laboratory for Bioorganic Chemistry, Molecular Pharmacology and Chemistry Program, ^3Laboratory of Tumor Vaccinology, Clinical Immunology Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, ^4Department of Chemistry, Columbia University, Havemeyer Hall, New York Background: A hallmark of cancer establishment is heterogeneity within a single patient and even within a single organ. Heterogeneity of cancer manifests itself in many ways. In addition to all disrupted signaling pathways, the complex carbohydrates that are displayed at the cell membrane are also severely disrupted. Some are overexpressed in certain cancers more than others, while others are not properly processed. Challenging the immune system with these aberrant carbohydrate motifs seems to be a promising strategy. Vaccination against cancer though deprived of glory to date, has provided more than was originally expected of it. Methods: Over the past decade, several major developments in chemical synthesis and manipulation of complex carbohydrates have made these complex structures available in pure single forms. This has re-opened the door to the systematic study and improvement of carbohydrate based cancer vaccines. So far, vaccination against cancer was tested in late stages of disease when everything else failed, and when patient’s immune system is equally compromised. In the many vaccination trials as a means of “preventive measure against cancer relapse” complex carbohydrates have proven efficacious in prolonging patients’ life compared to untreated patients. Results: Challenging the immune system with a unimolecular multiepitopic synthesized carbohydrate-based cancer vaccine produced an antibody count that is superior to the sum of its epitopes used together as a mixture. Conclusions: In the quest to harness the immune system to recognise and kill cancer cells using a mulitivalency strategy, made possible through major advances in organic synthesis, complex carbohydrates-based cancer vaccine strategies might be the sought after magic bullet in cancer treatement and prevention. Our synthetic efforts as well as an overview of some of our recent results will be presented.

The Ocular Penetration Of Antibiotics Using A Rabbit Model That Emulates Human Topical Dosing. OWEN GR, BROOKS AC, BERNAL-PEREZ LF, CAMPBELL-FURTICK MB Alcon Research Ltd., Fort Worth, TX, USA Background: Various antibiotics are being used topically to treat ocular infections. We have developed a new rabbit model that emulates human topical ocular dosing. The model gives a level of precision that is superior to both human and animal pharmacokinetic studies, and the results correlate well with human aqueous humor levels obtained during cataract surgery. The model has been used to explore the ocular penetration and distribution of a number of commercial ophthalmic antibiotics. Methods: Anesthetized rabbits were given a single topical dose (50 µL) of the various commercial antibiotics followed by a 30 minute controlled period that emulates the human eye with manual blinking (4 blinks/min) and a supplementary tear flow (2 μL/min). Tear samples (1 μL) were collected at 2 minute intervals throughout the dosing. The rabbits were euthanized after 60 minutes and ocular tissue samples were collected. In another study, a more frequent dosing regimen was explored. All tissue samples were extracted by sonication in water, and antibiotic concentrations were quantified using HPLC. Results: The tear concentrations of the antibiotics decreased at a first-order rate and generally were below detectable levels after 20 minutes. The tissue levels for some of the antibiotics evaluated, 60 minutes after dosing, are shown in the table: Antibiotic Moxifloxacin Levofloxacin Gatifloxacin Tobramycin Dosing Single 4 times Single Tissue Concentration (µg/g or µg/mL) Mean ± se, n = 4 Aqueous humor 2.2 ± 0.4 7 ± 1 0.60 ± 0.07 0.7 ± 0.1 0.03 ± 0.01 Conjunctiva 2.6 ± 0.1 16 ± 3 0.3 ± 0.1 0.4 ± 0.1 1.2 ± 0.2 Cornea 7 ± 3 32 ± 1 3.7 ± 0.6 7 ± 1 0.31 ± 0.03 Iris-ciliary body 1.4 ± 0.3 6 ± 1 0.37 ± 0.02 0.5 ± 0.1 < 0.1 Sclera 0.7 ± 0.2 3 ± 2 0.8 ± 0.7 1.0 ± 0.3 < 0.4 Conclusions: Fluoroquinolones provide higher levels of ocular antibiotic penetration, with moxifloxacin providing the highest levels in the aqueous humor (p<0.01). Dosing moxifloxacin every 15 minutes for one hour prior to collection results in aqueous humor concentrations more than 3x the MIC of fluoroquinolone resistant staphylococci (2.0 μg/mL) and more than 100x the MIC of fluoroquinolone sensitive staphylococci (0.05 μg/mL).

Racial Disparity In Stroke Risk Factors: The Berlin–Ibadan Experience; A Retrospective Study OWOLABI MO^1, PLATZ T^2 ^1Department of Medicine, University College Hospital, Ibadan, Nigeria; ^2Ernst-Moritz-Arndt Universität, Greifswald, Germany Background: Different workers have reported racial disparities in the distribution of risk factors for stroke and stroke subtype (ischemic vs hemorrhagic). No transcultural transnational studies have been conducted to confirm and relate these disparities to one another. Our objective was to identify differences in the distribution of risk factors for stroke and stroke subtypes among urban-dwelling stroke patients in Nigeria, a developing country, and Germany, an industrialized country. Methods: Consecutive stroke patients in Ibadan (100) and Berlin (103) were studied. Their hospital records were screened to identify documented vascular risk factors and stroke subtype. Results: The stroke patients in Ibadan were younger than those in Berlin (t = 4.940, P = 0.000). Hypertension was significantly more common in Ibadan while cigarette smoking, dyslipidemia, atherosclerosis, and cardiac factors were significantly more frequent in Berlin. Cerebral infarction was more common in Berlin (80%) than in Ibadan (63%). Conclusion: The risk factors associated with cerebral infarction were more frequent in Berlin. We suspect that racial disparity in risk factors for stroke may account for the difference in proportions of stroke subtype in black and white populations. Larger prospective community-based multinational multiracial studies are required to confirm these disparities and identify possible underlying genetic, dietary, and socio-economic factors.

Cytokine Profiles Of Patients With Cutaneous Leishmaniasis OZBILGE H, KAYA E University of Erciyes, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Kayseri, Turkey. Background: The Leishmania spp. are obligate intracellular protozoa thatcause a spectrum of diseases, including cutaneous, mucocutaneous,and visceral leishmaniasis, in tropical or subtropicalcountries. Sanliurfa is an area highly endemic for cutaneous leishmaniasis caused bythe protozoan Leishmania tropica and it has been for many years an important focus in Southern Anatolia of Turkey. This research was planned to detect the cytokines in sera of patients with active cutaneous leishmaniasis in Sanliurfa. Methods: In this study, the cytokine measurements were made in sera of 25 patients with active cutaneous leishmaniasis, before and after the sodium stibogluconate therapy and 25 healty control people. Cytokines such as IFN- g, TNF-α, IL-2, IL-4, IL-6, IL-10 and IL-12 were measured in groups and the results were compared. Results: IL-2, IL-4, IL-6, IL-10, IFN- γ levels were found higher in active cutaneous leishmaniasis than control group, while no differences in IL-12 levels were found. IL-4, IL-6, IL-10, IFN- γ levels were found lower at post treatment compared to pretreated patients with cutaneous leishmaniasis. Conclusion: Following up the cytokine levels of these patients with cutaneous leishmaniasis can give an idea on the course of the disease and can contribute to schematize cytokine treatment

A Molecular Perspective On Integron-Associated And Transferable Antibiotic Resistance In Clinical And Aquatic Isolates Of Gram-Negative Bacteria In Northern Region Of Turkey OZGUMUS OB^1, SANDALLI C^1, CAYLAN R^2, TOSUN I^3, AYDIN K^2, KOKSAL I^2, SEVIM A^1, SEVIM-CELIK E^1, ALPAY-KARAOGLU S^1, COLAKOGLU F^1, SIVRI N^4, KOLAYLI F^5, YESILGIL-ATASOY P^1 ^1Microbiology & Molecular Biology Research Laboratory, Department of Biology, Faculty of Arts & Sciences, Rize University, Rize, ^2Department of Clinical Microbiology & Infectious Diseases, Faculty of Medicine, Karadeniz Technical University, Trabzon, ^3Department of Microbiology & Clinical Microbiology, Faculty of Medicine, Karadeniz Technical University, Trabzon, ^4Department of Environmental Engineering, Faculty of Engineering, Istanbul University, Istanbul, ^5Department of Medical Microbiology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey Background: We analyzed the data obtained from our earlier studies concerning the mobile genetic elements such as conjugative resistance plasmids and class 1 and class 2 integrons in multi-drug resistant Gram-negative pathogens isolated from different aquatic or clinical environments in the northern region of Turkey. Methods: Disk diffusion and agar dilution methods were used in antibiotic susceptibility testing. Transferable antibiotic resistance was detected by conjugation assays. Class 1 and class 2 integron gene cassettes were screened and then described by sequencing. Sequences were compared to the current GenBank databases using the BLAST suite of programs. CLUSTALW amino acid sequence alignments were produced for comparison. Results: Resistance to ampicillin, tetracycline, streptomycin, trimethoprim and sulphamethoxazole was commonly high in multi-drug resistant organisms from both aquatic and clinical isolates, and much of them were transferable traits carrying on conjugative plasmids. The gene cassettes such as the families of aadA and dfrA inserted in integrons were commonly shared between aquatic and clinical isolates, and some of them were novel gene cassette arrays and new alleles. Conclusions: We conclude that further molecular epidemiological investigations should be committed to monitor for these antibiotic resistance genes in various environments in the other regions of Turkey to take precautions for slowing down the progression of antibiotic resistance evolution country-wide.

Circulating epithelial tumor cells (CETC) allow gene analysis of the residual tumor burden individual chemosensitivity testing and monitoring of the adjuvant setting in a curative PACHMANN K^1,2 and PACHMANN U^2 ^1Clinic for Internal Medicine II University Hospital Friedrich Schiller University Jena Germany and ^2Center for Transfusion Medicine Bayreuth Germany Background: Although most malignant tumors have no detectable metastases at diagnosis, most patients do not die from the primary tumor but from metastases during the subsequent course of disease. Circulating epithelial tumor cells (CETC) emanating from the primary tumor are assumed to be the source of such metasetases. Understanding the composition and the genetic variability of CETCs, chemosensitivity of remnant CETCs before and after specific therapies and monitoring during systemic therapy will further our understanding of therapy reponse and resistance. Methods: Monitoring of neoadjuvant systemic therapies included 50 lung cancer patients and 75 breast cancer patients, of adjuvant therapies 200 breast cancer patients, and chemosensitivity testing has been performed in over 100 patients. CETCs were detected and monitored from peripheral blood of cancer patients requiring not more than 1ml of blood by staining with fluorochrome labeled antibodies to EpCAM and CD45 antigen by laser scanning cytometry or automated image analysis. Reanalysis for genetic aberrantions was performed by fluorescence in situ hybridization and single cell PCR and in vitro cytotoxicity testing by analysis of specific tumor cell killing by chemotherapeutic agents. Results: Viable epithelial cells, suspect of tumor origin, were detectable in more than 90% of tumor patients with a good correlation between tumor size and the number of CETCs before therapy indicating that such cells are released during tumor growth and can be disseminated during diagnostic and therapeutic interventions. CETCs from individual patients showed a wide genetic heterogeneity of e.g. the her2/neu amplification and this may in the future be used for optimizing therapy. Longitudinal monitoring allowed supervision of the action of therapeutic agents on CETCs in vivo. Neoadjuvant therapy (with the primary tumor still present) first eliminated CETCs by but with continuing tumor disintegration cells were again released into circulation. Numbers could vary up to 10 thousand fold. Monitoring of the adjuvant therapy success revealed that an increase in CETC numbers more than tenfold towards the end of therapy was a strong predictor of early metastasis formation. The correlation between in vitro sensitivity of CETCs and therapy response is now under investigation. Conclusions: Analysis of CETCs responsible for fatal metastasis formation, of their gene endowment, their sensitivity to chemotherapeutic agents and their in vivo response to therapy allows, insight into the mechanisms of tumor cell killing and resistance and will greatly contribute to individualized therapeutic approaches

A biomarker panel for acute graft versus host disease PACZESNY S^1,2, KRIJANOVSKI O^1, BRAUN TM^1, CHOI S^1, CLOUTHIER SG^1, KUICK R^1, MISEK DE^1, COOKE KR^1, KITKO CL^1, WEYAND A^1, BICKLEY D^1, JONES D^1, WHITFIELD J^1, REDDY P^1, LEVINE JE^1, HANASH SM^2,AND FERRARA JLM^1. ^1University of Michigan, Ann Arbor, MI, 48109, USA and ^2Fred Hutchinson Cancer Center, Seattle, WA, 98103, USA. Background: No validated biomarkers exist for acute graft versus host disease (GVHD), the major complication of allogeneic hematopoietic cell transplantation. Methods: We screened plasma with antibody microarrays for 120 proteins in a discovery set of 42 transplant patients. We then measured the levels of the best biomarkers from the discovery set by sequential ELISA to create a composite biomarker panel that we tested in samples from 424 transplant patients randomly divided into training (n = 282) and validation (n = 142) sets. Results: Analysis of 23 proteins in the discovery set revealed eight potential biomarkers. Logistic regression analysis of these eight proteins in the samples of the training set determined a composite biomarker panel of four proteins (interleukin-2-receptor–alpha, tumor-necrosis-factor-receptor-1, interleukin-8, and hepatocyte growth factor) that optimally discriminated patients with and without GVHD. The area under the receiver operating characteristic curve distinguishing these two groups in the training set was 0.91 [95% confidence interval, 0.87 - 0.94] and 0.86 [95% confidence interval, 0.79 - 0.92] in the validation set. A model utilizing protein levels with 95% specificity for GVHD in the training set provided 94% specificity in the validation set. In patients with GVHD, Cox regression analysis revealed that the biomarker panel independently predicted survival independently of GVHD severity (p < 0.001, Table 1). Table 1: Association of Maximum GVHD Grade and Biomarker Panel with Overall Survival Univariate Multivariate Hazard Ratio p value Hazard Ratio p value Maximum GVHD Grade (1/2 vs 3/4) 2.35 <0.001 2.11 0.001 Biomarker Panel^# 2.46 <0.001 2.43 <0.001 ^#For subject with levels of each biomarker in the panel that are 1.5 times the median versus a subject with levels of each biomarker at the median. Conclusions: A panel of four biomarkers can confirm the diagnosis of GVHD in patients at onset of clinical symptoms of GVHD and provide prognostic information independent of GVHD severity.

Predicting the Impact of Hepatic Transporters on the Pharmacokinetics of Statins in the Liver PAINE SW, SOARS M, WEBBORN P, GRIME K, RILEY RJ AstraZeneca, Loughborough, UK Background: The statins form a class of hypolipidemic drugs used to lower cholesterol levels in people with or at risk of cardiovascular disease by inhibition of the HMG-CoA reductase enzyme. The enzyme is found in the smooth endoplasmic reticulum within the hepatocytes of the liver. Statins are known to be transported into hepatocytes thus raising their intracellular concentration. The ability to predict the free concentration of a drug within the hepatocytes of the liver will lead to a better understanding of the pharmacokinetics, pharmacodynamics and potential drug-drug interactions of statins. Methods: This study included the drugs atorvastatin, cerivastatiin and indomethacin as a control. Disposition of these compounds was measured in isolated rat hepatocytes and the data fitted to a kinetic model incorporating active uptake, permeation, binding and metabolism. Parameters from the hepatocyte kinetic model were transposed to an in vivo kinetic model and used to predict the pharmacokinetics of the drugs including the all-important free concentration within the hepatocytes of the liver. These predictions were compared to measured in vivo pharmacokinetics (plasma, liver and muscle) from rat bile duct cannulated animals. Results: Use of the hepatocyte model to estimate the ratio of intracellular to extracellular steady-state free-drug concentrations demonstrated the strong influence of active uptake on the kinetics of atorvastatin (18:1) and cerivastatin (8:1), in comparison with indomethacin (3.5:1). Indomethacin, however, was shown to have a higher uptake clearance (599 ± 101 µl/min/106 cells) than atorvastatin (375 ± 45 µl/min/106 cells) and cerivastatin (413 ± 47 µl/min/106 cells). The high passive permeability of indomethacin (237 ± 63 µl/min/106 cells) clearly negated the effect of the active transport on the overall disposition. Hepatic clearance was well predicted by the analogous in vivo model, in contrast to predictions based on standard methods. Volume of distribution was well predicted for indomethacin and predicted reasonably for atorvastatin and cerivastatin and higher than might be expected for an acid compound. Furthermore, the terminal half-life predictions for all 3 compounds were within two-fold of the observed values. Conclusions: 1) The disposition of atorvastatin and cerivastatin in isolated rat hepatocytes can be fitted to an hepatocyte model that incorporates active uptake, permeation, binding and metabolism. 2) In vivo pharmacokinetics in plasma, liver and muscle can be predicted from disposition in isolated hepatocytes.

HPLC and MALDI TOF MS analysis of novel antileishmanial compounds from Quassia amara PAL A^1, CHAKRABORTY D^1, BHATTACHARJEE S^2, MAJUMDAR S^2 ^1 Plant Molecular & Cellular Genetics, ^2Molecular Medicine, Bose Institute, Kolkata-700054, India. Background: Quassinoids are used in folk medicine for centuries and have antileukemic and antimalerial activities. Aims: 1) To charecterise and quantify quassinoids from Quassia amara bark tissue using HPLC and MALDI TOF 2) To assess antileishmanial activity of purified quassinoid and neoquassinoid. Methods: Lypholysed methanolic extract of dried, powdered 1.0g bark tissue was dissolved in 1ml methanol and filtered with 22µ filters. A HPLC DAD (LC-20 AT Liquid Chromatogram) was performed on C-18 column with a linear gradient mobile phase of methanol: water; for detection of quassinoids. HPLC elutes were collected, pooled, lypholysed and stored. MALDI TOF analysis of elutes dissolved in 100µl of 5% formic acid: acetonitrile buffer (1:2) was done on a Bruker Auto Flex II TOF/ TOF MALDI mass spectrometer in positive ion reflector mode using delayed extraction and nitrogen laser (337nm). Cytotoxicity test was performed by MTT assay of the elutes at 1-500ng^-mland 1-250mg^/ml. The effects of two quassinoids on viability of Leishmania donovani promastigotes were assessed by monitoring MTT metabolism after 96h cultured in presence of 1-100ng^/ml compound. Intracellular parasitic load of amastigotes/ 100 macrophages was determined by Giemsa staining of L. donovani infected peritoneal macrophage cells incubated with the extracts at 1-100µg concentration. Results: 8.28µg^-mg Quaasin & 7.66µg^-mg neoquassin were detected at 14min and 21-22min, respectively from Q. amara bark as confirmed by retention time (HPLC) and UV-vis spectral (DAD) analysis with authentic compound. On MALDI TOF analysis showed protonated quassin at m/z 389.249 and that of neoquassin at m/z 391.316. For antilieshmanial assay, quassin and neoquassin was found to be non–cytotoxic to 25µg^-ml. The LD50 value of quassin and neoquassin for L. donovani promastigotes was 62µg^-ml & 0.098µg^-ml, respectively. The ED50 value of quassin and neoquassin in reducing the intracellular parasitic load was 12.63µg^-ml & 8.27µg ^-ml, respectively. Conclusions: 1) Significant amount of quassin and neoquassin was detected from Quassia amara bark tissue on HPLC analysis. 2) This is the first report on MALDI TOF analysis of quassinoids, a soft ionisation technique that is increasingly being used for the detection of complex organic molecules. 3) Neoquassin was found to be more effective than quassin as an antileishmanial agent.

Surface-modified polyamidoamine (PAMAM) dendrimers for site-specific gene delivery KUMAR A, YELLEPEDDI VK, PALAKURTHI S Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, TX, USA Background: Major limitations of using the PAMAM dendrimers for in vivo gene delivery application are low transfection efficiency, lack of target specificity, and limited transport into the nucleus of the target cells. Herein we report preparation and evaluation of ornithine-conjugated PAMAM dendrimers and their potential for cancer cell-specific gene delivery. Methods: Ornithine-conjugated PAMAM dendrimers were prepared by Fmoc synthesis. A comparative gene transfection study between PAMAM dendrimers and the surface modified dendrimers was conducted in HEK 293T, GM7373, NCI H157G cell lines. Cytotoxicity of the dendriplexes was tested in HEK 293T cells by MTT assay. Effect of excess of ornithine (100 μM) on transfection efficiency of the ornithineconjugated PAMAM dendrimers was investigated. A comparative transfection study in polyamine transport deficient (NCI H157R) and polyamine transport efficient (NCI H157G) cell lines was performed to confirm the role of the polyamine transporter system (PAT) in the dendriplex uptake. Results: 1H NMR and MALDI-TOF spectral analysis showed that about 60 molecules of ornithine (PAMAM-ORN60) were coupled to a PAMAM dendrimer. Comparative study between cancer cell (NCI H157G) and HEK 293T cells showed higher transfection efficiency of PAMAM-ORN60 dendriplexes in cancer cells than normal cells. Cytotoxicity assay has shown that dendriplexes prepared at N/P of 10 were safe at concentrations below 50 μg/mL. Transfection efficiency significantly increased with increase in generation number and degree of ornithine conjugation. Transfection efficiency of the PAMAMG4-ORN60 dendrimers decreased in presence of excess of ornithine while there was no effect on the parent PAMAMG4 dendrimers. Transfection efficiency of PAMAMG4-ORN60 was significantly low in NCI H157R (31.6±12.5%) as compared to NCI H157G cell line (63.1±8.6%). Conclusions: Conjugation of ornithine significantly increased the transfection efficiency of PAMAM dendrimers. PAMAMG4-ORN60 dendriplex uptake was higher in cancer cells as compared to HEK 293T cells. Many tumor types have been shown to contain elevated polyamine levels and an activated PAT system. As the results demonstrated the role of PAT in the uptake of PAMAMG4-ORN60, they may serve as potential cancer cell-specific gene carriers.

Targeted and Multifunctional Dendritic Polymers: Magic Bullets for Drug and Gene Delivery PALEOS CM^1,2, TSIOURVAS D^2, SIDERATOU Z^2, TZIVELEKA L-A^2 ^1Dendrigen SA, Athens, Greece; ^2NCSR “Demokritos”, Aghia Paraskevi, Greece. Background: A significant number of molecules fail to be commercialized due to inability to be delivered to the appropriate tissues. Such bioactive molecules lack blood solubility, stability, tissue specificity and transport ability through cell membranes. For addressing these problems drug carriers based on dendritic polymers have been developed. Methods: Consecutive functionalization of these polymers leads to the preparation of multifunctional dendritic polymers which share the properties of drug delivery and controlled release. Thus, dendritic polymers have been developed bearing targeting ligands, which in addition to exhibiting the property of multivalency they are also complementary to cell receptors. Protective groups have also been introduced which prolong their circulation in biological fluids. Another crucial parameter for effective drug delivery systems is their transport through cell membranes. Finally, nanocavities have been tailored in such a way that drug release is triggered by changes in the biological environment. Results: A schematic representation of a multifunctional dendrimer is shown below. Each group plays a specific role. Thus, specificity has been achieved by targeting ligands while transport through cell membranes has been achieved by molecular transporting moieties. Enhanced water solubility, decreased toxicity, biocompatibility, stability and protection has been achieved by functionalizing dendritic polymers with poly(ethylene glycol) chains. Finally, cationization of dendrimers induces the interaction with genetic material for the formation of complexes employed in gene therapy. Conclusions: Designed functionalization of dendritic polymers scaffolds results in the preparation of non-toxic nanocarriers of significant encapsulating capacity, specificity to certain biological cells and transport ability through their membranes.

Potential Reproductive and Developmental Damage Induced by Metronidazole PALERMO AM^1, MERANI MS^2, MUDRY MD^3 ^1CITEFA, Buenos Aires; ^2FMed-UBA; ^3FCEyN-UBA, Universidad de Buenos Aires. Buenos Aires, Argentina. Background: Parasitic illnesses increase all over the world and metronidazole (MTZ) is the well-established antiprotozoal and antibacterial agent usually administered to children and adults at the reproductive age. Aim: To evaluate MTZ 1) as inductor of reproductive damage in Rattus norvegicus females and CFW male mice. 2) as teratogenic in Drosophila melanogaster, since the fly and vertebrates show similar developmental mechanisms. Methods: Reproductive effects in rats were evaluated by scoring 70 adult females (450 g/bw) treated with 250, 500 and 1000mg/kg/bw/day for 7 days. They were mated after treatments and sacrificed at 21 days of gestation. To study the stages of the seminiferous epithelial cycle and spermatozoa morphology, 60 days old male mice received an MTZ dose of v.ip 130 mg/kg/bw. Pachytene spermatocytes, spermatids stages and spermatozoa were analyzed. Developmental effects were studied by allowing wild-type female flies to lay eggs for 24 hr in media with MTZ at 0, 500, 1000, and 2000 µg/ml. Emerging flies (400 to1000 for each concentration) were examined. Control series (C) kept in standard conditions were always run. Results: In female rats MTZ did not affect pre-implantation deaths but increased the frequency of post-implantation deaths (C=3.2%, T=14.9, 16.1, 20.5%) and of dominant lethals (C=3.9, T=12.0, 13.2, 17.8%; P<0.05; Mann-Withney U test) In exposed mice cellular composition and number of stages in the seminiferous tubules were not altered, but the spermatozoa morphology was severely affected (C=39.7±1.0, T=122.9±3.6; P<0.0000; ANOVA Test). In flies, 1000 and 2000µg/ml MTZ-treated series showed higher frequencies of total abnormalities (C=1.6%, T=2.8, 3.9, 3.2%; P<0.05; χ^2 Test) Conclusions: 1) In female rats treatments affected post-implantation death and induced dominant lethals, but abnormal offspring was not increased, probably because conceptions with aberrations are eliminated during post-implantation period. 2) The alteration of spermatozoa morphology by MTZ could represent a potentially serious threat to the normal fertilization process. 3) The morphogenetic alterations induced in Drosophila could indicate a potential developmental toxicity of the drug. 4) side effects of MTZ have to be considered since they represent a conceivable thread regarding fertility or development.

Structure Based Design of Second Generation PDE5 Inhibitors PALMER MJ, BELL AS, FOX DNA, BROWN DG Sandwich Discovery Chemistry, Pfizer Global Research and Development, Sandwich, Kent, U.K. Background: The clinical knowledge gained from the pioneering PDE5 inhibitor sildenafil and subsequent agents, has highlighted the potential of PDE5 inhibition for treatment of additional indications beyond male erectile dysfunction. Such indications would be best treated by highly selective agents suitable for chronic once daily oral dosing. This talk details the discovery and progression of a pyrazolopyrimidine PDE5 inhibitor series designed to display inherently good physicochemistry, and targeting a chronic once daily treatment goal. Methods: Despite a wealth of literature in the PDE5 field, at the outset of the programme we felt that no single chemotype had the profile we were looking for. Thus, at the point of initiation of our chemistry program, we proposed a set of criteria that would enable us to be confident a chemical lead would be likely to result in a clinical candidate with the desired profile. These criteria included measures of potency/selectivity (PDE5 <50nM & >10-fold selective over all other PDEs), physicochemistry (MW<400, LogD 1-2) and adsorption/metabolism (well fluxed, predicted human half-life >12 hours), which were used to track progress at each stage of the project. In targeting these objectives, we set out to harness three technologies (PDE5 structural information, high throughout screening (HTS) and parallel synthesis) to speed our progress. Ligand efficency (LE, log of potency divided by heavy atom count) together with cLogP were used as key measures of compound assessment rather than potency alone. Results: Under the guidance of co-crystal structural information, a non-selective HTS hit (1) with poor physicochemistry was initially modified using parallel chemistry to give a lead compound (2) that established a new pyrazolopyrimidine PDE5 inhibitor series. Notably, (2) displayed physicochemistry compatible with a long plasma half-life, and wide chemical scope. Subsequent optimisation of (2) using crystal structure information to guide design, led rapidly to a highly potent and selective PDE5 inhibitor (3). Optimisation of (3) will be described, and continued focus on physical properties led to a selective series with good pharmacokinetics. Conclusions: In summary, by applying a combination of structure based drug design and rigid physicochemical criteria, we were able to identify a new PDE5 inhibitor series with wide chemical scope. Utilisation of co-crystal structural information and the wide scope enabled rapid transformation into potent and selective leads. Focus on retention of inherently good physicochemistry throughout has enabled the identification of a range of potent and selective PDE5 inhibitors with good physical properties. These leads led to a physicochemically optimised amide with the potential for once daily pharmacokinetics in man.