Ehrlich II –2nd World Conference on Magic Bullets

Growth Hormone: how to catch the magic bullet of the modern athlete HOLT RIG on behalf of the GH-2004 project team University of Southampton, UK There is widespread anecdotal evidence that growth hormone (GH) is used by athletes for its anabolic and lipolytic properties. There has been considerable debate, however, whether GH has a performance enhancing effect in healthy adults. Although clinical trials are not best placed to address this issue because of the small margins of victory in many athletic events, recent studies have shown that GH can improve physical fitness as well as body composition. Despite GH appearing on the World Anti-Doping Agency list of banned substances, the detection of abuse with GH is challenging because unlike manysubstances of abuse, such as synthetic anabolic steroids, GHis a naturally occurring substance. Demonstration of exogenousadministration must rely on detecting concentrations exceedingnormal physiological circumstances. Two approaches have been developed to detect GH abuse. The first relies on the measurement of pituitary GH isoforms. When rhGH, which contains only 22kD GH, is administered in sufficiently high doses, there is a suppression of endogenous GH secretion and therefore the ratio between 22kD GH and total GH is altered. The second method is based on the measurement of markers of GH action. After evaluation of 25 potential markers, the GH-2000 team proposed a method using insulin like growth factor-I (IGF-I) and type 3 pro-collagen (P-III-P). The administration of rhGH leads to a significant rise in these markers, the magnitude and duration of elevation of which is dependent on the rhGH dose and gender. As a result GH abuse can be detected with reasonable sensitivity and specificity.

The piggy back approach or how to find new lead structures for antiinfective or antiviral drugs HOLZGRABE U, TISCHER M Institute of Pharmacy, University Wuerzburg, Germany Background: Mono- and bisnaphthalimides such as amonafide and elinafide have been developed as cancerostatic drugs by Brana et al. [1]. Recently, naphthalimides could be profiled as potent ligands of the muscarinic receptor. Methods: Within the frame of a broad screening program for drugs a small library of recently synthezised mono- and bisquartary naphthalimides were tested for their activity against Trypanosoma brucei [2], Leishmania major, Candida albicans, Staphylococcus aureus, Plasmodium falciparum, and Measle and Nipah virus as well as for toxicity against macrophages. Results: Depending on the substitution pattern the mono- and bisnaphthalimides were active against either microorganism in the lower micromolar and nanomolar range of concentration combined with almost no cytotoxicity. Conclusion: The bisnaphthalimides studied here are perfect lead compounds for further drug development. Interestingly, the structure-activity relationships were different for each purpose indicating that the piggy-back approach can be considered as a worthwhile method for drug development. 1. Brana et al. Curr. Med. Chem. Anti-Cancer Ag. 1 (2001) 237-55; 2. M. Muth, V. Hoerr, A. Stich, U. Holzgrabe, Bioorg. Med. Chem. Lett. 17 (2007) 1590-1593.

Inhibitors of Serine/Threonine Protein Phosphatases at the Dawn of a Clinical Era. HONKANEN RE Dept. of Biochem and Mol. Biol., University of South Alabama, Mobile, AL USA. The reversible phosphorylation of proteins regulates the biological activity of many diverse cellular processes, including gene transcription, protein-protein interactions, cell-cycle progression and apoptosis. Phosphorylation occurs principally on ser/thr and tyr residues, with the addition and removal of phosphate catalyzed by kinases and phosphatases, respectively. Although protein phosphatases were once viewed as simple house keeping proteins, in the last decade it has become eminently clear that they are actually dynamic and highly regulated enzymes. Therefore, the development of compounds that alter the activity of “key” phosphatases is rapidly emerging as an important area in drug discovery. Because the majority of protein phosphorylation (>90%) occurs on serine and threonine residues, the identification of specific agents that alter the activity of key ser/thr phosphatases seems especially promising for development. For the PPP-family of phosphatases, several lead compounds for drug development have come from studying the biological actions of natural products, such as fostriecin and cantharidin, which was identified as an activate constituent in a Chinese beetle extract developed over 1500 years ago for the treatment of human cancer. The current challenge for further drug development is to identify “key” phosphatases and more selective inhibitors, because non-selective inhibitors, such as calyculin A, microcystins and nodularin are highly toxic to both normal and cancer cells. The high-resolution crystal structures of PP1, PP2A and PP5 have been solved, and structure/function studies with derivatives of highly selective inhibitors (i.e. fostriecin and cytostatin A) provide insight into methods for developing novel inhibitors, as well as challenges that must be overcome for small molecule development. The development of siRNA and antisense oligonucleotides that support RNAase H mediated degradation of the targeted mRNA has resulted in compounds capable of specifically suppressing the expression of each ser/thr phosphatase in human cells. Such compounds have already proven useful for the validation of drug targets, and if difficulties associated with in vivo stability for siRNA or systemic delivery of antisense oligonucleotides can be overcome, these compounds are poised to have a major impact on the clinical management of many human disorders.

What Sort of Light Is At the End Of the Tunnel In Anti-Allergic Drugs? HORAK F Medical University of Vienna, ENT Dptm., Vienna, Austria Background: Studies on the efficacy of anti-allergic drugs in the treatment of allergic rhino-conjunctivitis have yielded inconsistent and unsatisfying results, notwithstanding their approval for this indication. Objective: The objective of this study was to evaluate and compare the therapeutic effect of approved anti-allergic drugs as well as compounds under development to placebo in subjects with allergic rhino-conjunctivitis. Methods: In consecutive single-centre, double-blind, placebo controlled, cross over studies, more than 500 subjects with confirmed allergic rhinitis due to grass pollen or house dust mite were randomised to receive active or placebo drugs and exposed to the accordant allergen (grass or mite) in the Vienna Challenge Chamber (VCC) for several hours. Patients recorded symptoms at 15-minute intervals, nasal secretion and nasal resistance were evaluated every 30-minutes. The primary endpoint was the mean change from placebo in total symptom score. Results: All of the investigated compounds like H1-receptor antagonists, topically used steroids and other mediator antagonists were significantly different from placebo in the primary endpoint but not very different in their therapeutic power. However, all of the approved drugs did not reach a therapeutic potency of more than 35% over placebo reflecting the frustration of many patients suffering from allergic rhinitis. Never the less, some compounds, out of the pipeline, have overcome this threshold, whereas others reaching the target were discontinued in their development, due to the risk/benefit viewpoint. Conclusion: The standardised and validates model of an allergen challenge chamber like the VCC enables us to compare results from different trials on a historical basis. Albeit approved anti-allergic drugs do not fully satisfy suffering individuals, several new compounds are under investigation and hopefully reaching a risk/benefit ration which will be acceptable for the patients.

Antitumor Effect of a Novel NF-B Targeting Therapy in Bladder Cancers HORIGUCHI Y^1, KIKUCHI E^2, OZU C^1, YOSHIOKA K^1, NAKASHIMA J^2, UMEZAWA K^3, TACHIBANA M^1, ^1Tokyo Medical Univ., Tokyo, Japan; ^2Keio Univ., Schl. of Med., Tokyo, Japan; ^3Faculty of Science and Technology, Keio Univ., Yokohama, Japan. Background: Nuclear factor (NF)- B is a transcription factor not only induces and controls various genes including inflammatory cytokines, but also activates genes with antiapoptosis, tumor proliferation and invasion. It has been clearly demonstrated that certain advanced human bladder cancer cells constitutively acquire the ability to activate NF-B, which not only protects cancer cells from apoptotic cell death, but also up-regulates the production of various cytokines that might induce malignant potential of the disease. NF-B activation inhibitor, therefore, might be useful as novel anticancer agents. We have designed and synthesized a NF-B inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) and investigated the effectiveness of DHMEQ against advanced human bladder cancer cells, KU-19-19 in which NF-B is constitutively activated. Methods: KU19-19 was implanted s.c. in the flank of nude mice. Daily i.p. administration of 2 mg/kg DHMEQ was started from 7 days after tumor implantation. After 28 days, mice were sacrificed and all the tumors were evaluated. Microvessels in tumor specimens were counted after immunostaining with an anti-CD34 monoclonal antibody. Apoptosis was measured by TUNEL assay using Apoptosis in situ Detection Kit. The average number of positively stained cells was counted and apoptosis index was calculated. Results: Inhibition of NF-B by transfection of adenovirus vectors expressing stable form of NF-B inhibitor, IB, inhibited KU19-19 cell growth and induced apoptosis. DNA binding activity of NF-B was completely inhibited by DHMEQ. Marked levels of apoptosis were observed after DHMEQ administration. DHMEQ treatment in vivo inhibited KU19-19 tumor growth. Tumor volume after DHMEQ treatment was 3110945 cmm vs 60192309 cmm of control mice (P<0.05). A statistically significant decrease in MVD in DHMEQ-treated tumors was observed. Blood vessels in the tumors derived from control mice showed well-developed vascular networks. In contrast, the vessels in the tumors of DHMEQ-treated mice consisted of poorly developed networks. The apoptotic index was increased 2.3-fold in DHMEQ-treated tumors than control. Conclusions: Targeting NF-B could be a new strategy of treatment against advanced bladder cancer.

The Clinical Pipeline of a Candidate Malaria Vaccine that Targets the Achilles’ Heel Antigen of Plasmodium falciparum HORII T. Res. Inst. for Microbial Dis., Osaka University, Japan Background: Malaria remains as one of the most infectious disease globally. Failure of existing control strategies necessitates the need for vaccine development. We focused on the development of an effective vaccine using a recombinant SE36 protein based from the N-terminal domain of Serine Repeat Antigen (SERA5) of Plasmodium falciparum. Methods: In vitro studies have elucidated the transcription profile, processing and localization of SERA5. A recombinant version of the 47 KDa N-terminal domain, and a modified version with high hydrophilicity (SE36), has been produced in E. coli. Molecular and immunology based epidemiological studies have been conducted in Uganda and Solomon Islands. An efficient fermentation and purification process has also been developed for the production of this antigen in a scale comparable with industrial manufacture and this has been used for preclinical and toxicology studies in animals to demonstrate safety and immunogenicity. The candidate vaccine, BK-SE36, was formulated adsorbed to aluminum hydroxide gel and is manufactured as a lyophilized product under Good Manufacturing Practices (GMP). A phase 1a clinical trial has been conducted in Japan. Results: In vitro, several lines of evidence suggest that SERA5, which belongs to a multigene family unique to Plasmodium, plays an essential role in parasite development and merozoite egress. Immunoepidemiological data underscores the uniqueness of SERA: naturally induced antibody response to SE36 protein correlated with increased protective immunity in adults and children. Higher levels of anti-SE36 IgG3 titer were associated with the absence of fever and lower parasitemia in children under 15 years; and were associated with protection against severe malaria in children under 5. Sero-conversion rates were 50% or less in >16 yr-old; and less than 10% in <10 yr-old individuals. Preclinical studies in animals showed that BK-SE36 was safe and highly immunogenic. Immunological test using squirrel monkeys provided significant protection after P. falciparum challenge infection; and antibody titers were significantly boosted. BK-SE36 was, likewise, safe and highly immunogenic in chimpanzees. No significant safety issues have been identified in healthy, malaria-unexposed adults in a Phase Ia clinical trial in Japan. Cumulative data confirms the potential of this candidate vaccine.

The success story of Japanese DTaP control and a further development of vaccine evaluation HORIUCHI Y^1^,2, KATAOKA M^1, YAMAMOTO A^1, OCHIAI M^1, HARASHIMA A^1, NAGATA N^1, HASEGAWA H^1, KURATA T^1 ^1 National Institute of Infectious Diseases, ^2Pharmaceuticals and Medical Devices Agency, Tokyo, Japan. Background: Different from therapeutic drugs, vaccines for controlling infectious diseases are given to majority of healthy population and, in most cases, need booster immunizations. However, many procedures applied to vaccine evaluation particularly for licensure are specific for therapeutic drugs and need to be revised. Methods: Adverse Event Following Immunization (AEFI) cases before and after implementation of diphtheria tetanus acellular pertussis vaccine (DTaP) were compared. Acellular pertussis based combination vaccnes imported from foreign markets were compared with Japanese DTaPs for injection site injuring effects in mouse footpad swelling, rabbit skin swelling and mouse quardriceps muscle injection models. Regarding local reaction to a booster dose, mice were intramuscularly immunized with a vaccine sample twice at one month interval and challenged at footpad two weeks later with 50 microlitre of diphtheria toxoid to measure swelling reaction. Results: Annually two mortality and three encephalopathy cases, in average, were reported among five million doses of diphtheria tetanus whole cell pertussis vaccine (DTwP). However, report of such severe AEFI has become quite rare after 1991 when minimum requirements of biological products (MRBP) for DTaP was revised to strengthen detoxification of pertussis toxin (Table 1) to suggest relevance of DTwP with the rare but severe AEFI. Regarding local reaction, all imported vaccines induced very strong inflammation and tissue injury at injection sites of mouse footpad, rabbit skin and mouse quardriceps muscle while Japanese DTaP induced no such reaction to suggest difference in tissue damaging effect of the vaccines. We evaluated enhancing effect of DTaP with varied levels of residual PT activity on local reaction to a booster dose using mouse footpad swelling model. A significant correlation was seen between mouse footpad swelling and residual PT activity of immunized vaccine batches. Conclusions: 1) Reduced toxicity in laboratory tests seemed to be relevant to safety. 2) Clinical observation could not evaluate injection site injury. 3) DTaP may enhance local reaction to booster doses. 4) Not only clinical evaluations, laboratory models need to be focused on in vaccine evaluation.

Characterization of an Active Pharmaceutical Ingredient by Its Dissolution Properties: Amoxicillin Trihydrate as a Model Drug HORKOVICS-KOVATS S^1 ^1Sandoz GmbH, Kundl, Austria. Background: An ordinary powder sample of an active pharmaceutical ingredient (API) is composed of particles having various properties with respect to their shapes, dimensions, etc. When taking into account such differences in particles, the dissolution of a sample can be described using a dissolution theory of heterogeneous particle populations, where every population is characterized by a rate coefficient (), consisting of a geometric factor and a material constant. Aims: 1) To develop a requested theory as a system of differential equations (SDE’s). 2) To prove the applicability of the theory for sieved fractions of API. 3) To predict dissolution profiles for samples of known particle size distribution. 4) To find a coherence between the developed and published theories. Methods: The introduced SDE’s was used for 120 dissolution profiles of sieved amoxicillin trihydrate samples, which were assumed to be two-population systems, by numerical solutions of the SDE’s and the “Solver” function of Microsoft® Excel. The calculated and measured curves were compared by factors of difference (f1) and similarity (f2). The dependence of the dissolution rate coefficients on dose, particle size and rotation speed of the paddles of the dissolution device was established by factorial analysis. Results 1959 data pairs of 120 measured and calculated profiles highly correlated (R^2 = 0.9997), while the average (minimum, maximum) of f1 and f2 is 0.76 (0.09, 3.42) and 96.91 (80.33, 99.91) respectively. The factor analysis of dissolution rate coefficients indicated that the dissolution rate 1 depends only on rotation speed (p < 0.0001), while 2 depends solely on the dose (p = 0.0058). Hence, 1 is a dissolution rate constant of real particles, whereas 2 expresses a rapid disintegration of aggregates of smaller particles. The dissolution profile of a mixed API sample (18 differential equations) was successfully predicted (f1 = 2.8; f2 = 86.0). Conclusions: 1) The developed theory is able to describe the dissolution of heterogonous particle populations. 2) Based on a known sample of particle size distributions, a dissolution profile can be predicted. 3) By solving the introduced SDE’s under certain conditions, the theories developed by Hixon and Crowell, Noyes and Whitney, Niedergall and Goyan, and Pothisiri and Carstensen can be derived, indicating a coherence with the known theories.

Estradiol as Membrane Targeting Modulator of Neuronal Cell Functions HORVAT A, PETROVIC S Institute of Nuclear Sciences "Vinca", Belgrade, Serbia Background: Accumulating evidence from basic science studies demonstrates that estrogens exert profound protective actions against various forms of neurodegenerative diseases and injury. Recently, evidence has accumulated in favour ofmembrane effects of estrogens dueto either a change in some membrane properties or to specificbinding to membrane targets. One examples of estrogen modulation of membrane propertiesinvolve the modulation of intracellular calcium homeostasis. In our earlier as well as recent-nonpublished study both modulation of intracellular calciumstores and regulation of plasma membrane calcium channels havebeen proposed. We examined in vitro effects of 17-estradiol (E2) on intracellular calcium ([Ca^2+]i) concentration in isolated presynaptic nerve terminals (synaptosomes) from whole female rat brain and discrete brain regions by measuring dose-dependent effects of E2 on 1) Ca^2+ influx in synaptosomes through voltage-gated channels and extrusion of Ca^2+ by sodium/calcium (Na/Ca) exchanger, 2) effects of E2 on Ca^2+ influx in synaptosomal mitochondria through uniporter and release of Ca^2+ through mitochondrial Na/Ca exchanger. To evaluate the mechanisms of E2 action we measured its specific binding to isolated membrane of synaptosomes (SPM) and synaptosomal mitochondria, and defined binding characteristics. Methods: Synaptosomes were isolated from whole brain (WB), brain stem (BS), nucleus caudatus (NC) and hippocampus (Hip) of chronicaly ovariectomized three month-old female rats (18 animals/brain structure/experimet). Binding of E2 to isolated synaptosomal plasma membranes (SPM) and synaptosomal mitochondria were calculated by subtracting non-specific bound (in the presence of 100-fould excess unlabeled E2) from total bound of ^3H-labeled E2 (10^-10 - 10^-7 M). For Ca^2+ flux measurement synaptosomes were preincubated in the presence or absence of E2 (10^-9- 10^-7 M) for 15 min. The voltage-dependent ^45Ca^2+ influx were measured in the presence of 50mM KCl and the Na-dependent Ca^2+ efflux in resting conditions (4 mM KCl) for 30 sec. Retained ^45Ca^2+ in the synaptosomes were determined by radioactivity measurement after filtrating synaptosomal suspension through nitrocellulose filters 0.45 m pore size. Synaptosomal mitochondria were preincubated with or without E2 (10^-12- 10^-7 M) for 10 min and ^45Ca^2+ influx through uniporter as well Na-dependent Ca^2+ extrusion in the ^45Ca^2+preloaded mitochondria in presence of 20 mM NaCl and 0.2 mM EDTA were measured. Results: E2 bound to two specific sites on SPM from NC (Bmax1 0.68 pmol/mg, Km1 26 nM and Bmax2 0.16 pmol/mg, Km2 3.6 nM), Hip (Bmax1 0.88 pmol/mg, Km1 30 nM and Bmax2 0.07 pmol/mg, Km2 2.6 nM) and BS (Bmax1 0.3 pmol/mg, Km1 26 nM and Bmax2 0.06 pmol/mg, Km2 4 nM) and one binding site in WB (Bmax 2 pmol/mg, Km 40 nM). In synaptosomal mitochondria we detected one specific binding site in NC (Bmax 48 pmol/mg, Km 32 nM), Hip (Bmax 17 pmol/mg, Km 17 nM) BS (Bmax 3.4 pmol/mg, Km 1.8 nM) and WB (Bmax 0.05 pmol/mg, Km 0.46 nM). E2 at concentrations up to 10^-8 M increased whereas at higher concentrations decreased voltage-dependent Ca^2+ influx in synaptosomes of all brain structures, while at concentrations 5x10^-9 and 10^-8 M increased Na-dependent efflux of Ca^2+ in NC and Hip. Mitochondrial influx of Ca^2+ was not affected by E2 while physiological concentrations of E2 decreased mitochondrial Na-dependent efflux. Conclusions: E2 at physiologial concentrations specificaly bound to SPM and synaptosomal mitochondria from discret brain regions and at same concentrations modulate [Ca^2+]i by affecting voltage-dependent influx and Na-dependent extrusion of calcium in synaptosomes and decreasing mitochondrial Ca release.

Nongenomic Corticosteroid-adrenergic Drug Interactions in the Airway HORVATH G^1,2, MENDES ES^2, SCHMID N^2, SCHMID A^2, CONNER GE^2, SALATHE M^2, WANNER A^2 ^1Semmelweis Univ., Budapest, Hungary; ^2Univ. of Miami, Miami, FL, USA Background: Organic cation transporters (OCTs) have an important role in tissue distribution and elimination of cationic drugs. Carrier-mediated disposal of cationic bronchodilators in the airway tissue, however, is incompletely understood. Aim: To assess the uptake of long-acting 2-agonist bronchodilators by bronchial and vascular smooth muscle cells (SMCs). Methods: Human airway cells and tissues obtained from organ donors were evaluated for cationic drug transporter expression by quantitative RT-PCR and immunofluorescence. For in vitro functional studies, [^3H]-formoterol (FORM) and [^3H]-salmeterol (SALM) uptake by bronchial and vascular SMCs was measured. Results: RT-PCR analysis indicated high mRNA levels for the corticosteroid-sensitive OCT3 in bronchial and vascular SMC. Immunofluorescence staining of airway sections confirmed OCT3 expression in these cells. In bronchial SMC, uptake of the cationic FORM was inhibited with OCT inhibitors. Corticosteroids also inhibited FORM uptake through a rapid (within 15 min) nongenomic action, with the following rank order (relative potency): des-ciclesonide (11.1) > hydrocortisone (5.2) > budesonide (3.8) > beclomethasone 17-monopropionate (1.8) > beclomethasone dipropionate (1.7) > ciclesonide (1.4) > fluticasone (1). The corticosteroid-induced inhibition was significantly higher in vascular than bronchial SMCs. In comparison to FORM, uptake of the noncharged lipophilic SALM was about 10-fold higher (28.4 ± 1.7 vs. 327.5 ± 13.7 pmol/mg/15 min; p < 0.05), and insensitive to OCT inhibitors and corticosteroids. Conclusions: Our findings suggest that corticosteroids, through OCT3 inhibition, rapidly interfere with drug disposal mechanisms in the airway. Increased tissue retention of inhaled cationic bronchodilators due by the corticosteroid-sensitive disposal mechanism could acutely improve bronchodilator responses. This novel immediate interaction supports the use of such combinations in asthma therapy.

Dioxin like compounds for anti-infective agents of know as its degrading enzyme from its resistant Geobacillus midousuji thermophile HOSHINA S, YOSHIDA H Department of Laboratory Medicine, Institute of Clinical Medicine and Research, The Jikei University School of Medicine, Tokyo, Japan. Background: Dioxins are mostly created by human activity and can be classified in the family of halogenated organic compounds, have been shown to bioaccumulate in humans.The highest toxicity is the 2,3,7,8- tetrachloro dibenzo -1,4- dioxin (TCDD). We have noticed Dioxins are kinds of anti-inflamatory and anti-proliferative agents through its resistant bacteria with its respiratory activity. Materials and Methods: The fibroblast was given from Dr. Emiko Sano of Proteios Laboratory (Yokohama, Japan) of human forehead epithelial primary culture. MIC assay is designed for 2,000 cells /100uL a well. After 24 hours pre-culture, serum was depleted as starving culture in 30 hours. In conditioned medium of TCDD and solvent DMSO, cells were cultured in 18 hours, then luciferin and luciferase RLU(relative light unit) was measured by Luminometer. Results: MIC of TCDD by human cell viability assay is estimated as 1pg/mL by the Government. In this study MIC of ATP production showed more sensitive result around 10-100fg/mL. The reductive enzyme has been unique glutathione S transferase of B. midousuji. Conclusions : TCDD has inhibitory activity of cellular ATP production, as new anti-inflammatory, anti-proliferative compound with core skeletal structure. Acknowledgement: This study was granted by Ministry of Environment of Japan.

The prevalence and the resistance mechanism of floroquinolones in bacteria isolated from Bangladesh AKHTER F, OSMAN KT, AMIN R, AKTHER F, ADNAL N, HOSSAIN MA Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh Background: Antimicrobials have been a much misused product in the world. In Bangladesh only 8% of the total used antibiotics are prescribed by a physician; and in poultry, veterinary and aquaculture are using unknown quantities of large amount antibiotics in an uncontrolled manner. This gives rise to the emergence of antibiotic-resistant bacteria. Our group has been working on understanding the origin of antibiotic resistance, resistance mechanism and route of spreading in Bangladesh. Here, we report contribution of medical college hospitals for originating antibiotics-floroquinolones resistant bacteria and its mechanism of actions. Methods: To address the problems, we studied following parameters in hospitals wastewater (HWW)- i. correlation of antibiotics used by the patients and resistant bacteria development; ii. effect of wastewater on sensitive bacteria; and iii. determination of floroquinolones resistance mechanisms using DNA-sequencing technology. Results: The bioassay using sensitive Escherichia coli^s clearly revealed that HWW contained active antibiotics at higher concentration than MIC50 for sensitive bacteria. Enumeration of total resistant bacterial count showed that the count was about 5-log higher in HWW. We randomly selected 52 E. coli isolates with high multi-drug resistance including ciprofloxacin (>MBC100 600 µg/mL). DNA sequencing data revealed that ciprofloxacin resistance in bacteria occurs due to acquisition of mutations in gyrA gene. Computer modeling of the mutant and wild-DNA gyraseA based on available DNA gyraseA crystal structure suggests that acquisition of double mutation that leads to alteration of the ciprofloxacin binding pocket may be the reason of high resistance properties shown by the isolates. Conclusions: In conclusion, our findings clearly demonstrate that – i. HWW have ecotoxicological effect in spreading resistant bacteria as well as active antibiotics in the environments; and ii. floroquinolones resistance in bacteria isolated from Bangladesh are due to acquisition of mutation in gyrA gene rather plasmid born.

Targeting CLEC5a/MDL1-1 for he Treatment of Dengue Hemorrhagic Fever CHEN ST^1, LIN YL^2,3, HUANG MT^1, WU MF^1, CHENG SC^1, LEI HY^4, LEE CK^5, CHIOU TY^6, WONG CH^3, HSIEH SL^1,3,7. ^1Dept. and Inst. of Microbiol. and Immunol, Natl. Yang-Ming Univ.; ^2Inst. of Biomed. Sciences, Academia Sinica; ^3Genomics Research Center, Academia Sinica; ^4Dept. and Inst. of Microbiol. and Immunol., National Cheng Kung University; ^5Inst. of Immunol, National Taiwan University; ^6Dept. of Life Science and Inst. of Biotech., National Dong Hwa University; ^7Immunology Research Center, National Yang-Ming University & Taipei Veterans General Hospital, Taiwan Background: Members of non Toll-like receptors (NTLRs) have been demonstrated as pattern recognition receptors to bacteria and fungi, such as mannose receptor and dectin-1 receptor, respectively. However, the ligands and the functions of most of NTLR, including C-type lectin, C-type-like lectin, Ig-like receptors [TREM and TREM-like transcript (TLT)] are still unknown. Method: The extracellular domain of 22 NTLRs expressed on myeloid lineages were cloned by RT-PCR, and was fused with the Fc portion of human IgG1 as fusion gene. After expression in eukaryotic system, the fusion proteins were immobilized on ELISA plates to capture dengue virus Results: Three C-type lectin receptors—DC-SIGN, DC-SIGNR, and CLEC5A/MDL-1—, are able to interact with dengue virion (DV) directly. Further study indicates that DV triggers both CLEC5A and TLR7/8 to produce proinflammatory cytokines synergistically, but CLEC5A is not involved in the production of interferon-alpha (IFN-). Incubation of macrophages (M) with DV induced proinflammatory cytokines release, and blockade of DV-CLEC5A interaction by shRNA or antagonistic mAb attenuates proinflammatory cytokine release without suppressing IFN- secretion in vitro. We established an murine model system to induce dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), and we found that antagonistic anti-antibody inhibits plasma leakage and increases the survival rate of mice infected by DV. Conclusion: In addition to Toll-like receptors, the NTLRs are the promising candidates involved in recognition of pathogen-associated molecular patterns. Our results demonstrates that virions acts as a ligand to activate macrophage via surface receptor, and blockade of NTLR-virion interaction might become a novel strategy to prevent dengue hemorrhagic fever as well as to decrease the mortality of patients suffered from other viral infections in the future.

The Role of Metabolism, Pharmacokinetics and Mass Spectrometry in Selection of a Thrombin Receptor Antagonist for Development HSIEH Y Drug metabolism and pharmacokinetic department, Schering-Plough Research Institute, Kenilworth, USA The rising costs and time associated with bringing new medicines to the market have created a need for a new paradigm for reducing the attrition rates of drug candidates in both preclinical and clinical development stages. Early appraisal of drug metabolism and pharmacokinetic (DMPK) parameters is now possible due to several higher throughput in vitro and in vivo screens. This knowledge of DMPK properties should not only shorten the timelines for the selection of drug candidates but also enhance the probability of their success for development. The role of DMPK researchers in the drug discovery paradigm should not be limited to screening a large array of compounds during the lead optimization process but should strive for an understanding of the absorption, distribution, metabolism, excretion, and potential drug-related toxicities of a chemical series. As an example, in this presentation the role of DMPK and mass spectrometry-based techniques in support of the Thrombin Receptor Antagonist program will be presented.

Anti neoplastic properties of tea catechins are associated with pro differentiation caspase 14 gene expression: implementation for novel therapies DULEBOHN R^1, DICKINSON D^1, HUFF F^1, KODANI I^2, WU M^3, MESSER R^1, AL-SHABRAWY M^1, TEWFIK A^1, OGBUREKE K^1, LEWIS J^1, AND HSU S^1. ^1Medical College of Georgia, Augusta. USA. ^2Totorri University Hospital, Yonago, Japan. ^3Zhejiang University, Hangzhou, China. Background: Exploration of novel approaches with innovative therapies is needed to combat epithelial cancer. We previously reported that caspase-14, a gene expresses during terminal differentiation of certain epithelial cells, is induced by green tea polyphenols. Our hypothesis is that the expression of caspase 14 induces tumor cell death without damaging normal epithelial cells. Therefore, caspase 14 is a suitable candidate for novel gene therapy to treat a variety of cancers. Objectives: to express exogenous human caspase 14 in human epithelial cancer cells (OSC2) by plasmid transfection and adenovirus delivery, and determine the effects of caspase-14 expression on cell growth, cell death, and tumorigenicity. Methods: The human cancer cell lines A431, HSG, and OSC2 were either transfected with caspase 14 expressing plasmid, or infected by adenovirus expressing caspase 14 cDNA. Expression of caspase 14 was confirmed by Western blotting. Cell morphology was monitored by microscopic photography, cell growth was measured by cell counting and BrdU assay, and cell viability was determined by MTT assay. In addition, the cancer cells were xenografted into athymic mice to determine the tumorigenicity. Results: expression of caspase-14 induced an undefined cell death in these cancer cells compared to the control cells. Cell growth and cell viability were inhibited significantly by caspase-14 expression. Xenograft of caspase-14-expressing cancer cells into athymic mice resulted in significantly reduced tumorigenicity, which could due to an inhibitory effect of caspase 14 on tumor vascularization. Conclusions: human epithelial cancer cells undergo growth inhibition and cell death when exogenous caspase-14 was expressed in these undifferentiated tumor cells. Caspase-14 expression in these cells also reduced tumorigenicity in vivo. Further effort is warranted to explore if caspase 14-expressing adenovirus could be used as a potential therapeutic approach to treat human cancers. This study was supported in part by the Dental Research Foundation of the Medical College of Georgia.

Development of species-specific STAMPs (specific targeted antimicrobial peptide) that target and kill only Streptococcus pneumoniae within a polymicrobial community ^1HU FZ, ^2SHI W, ^1HILLER NL, ^1HOGG JS, ^1JANTO B, ^1AHMED A, ^1BOISSY R, ^1POWELL E, ^1YU S, ^1POST JC, ^1EHRLICH GD ^1Center for Genomic Sciences (CGS), Allegheny Singer Research Institute, 320 E North Ave, Pittsburgh, PA, 15212, USA; ^2Depart of Oral Biology, UCLA School of Dentistry, 10833 Le Conte Ave, Los Angeles, CA 90095, USA Background: STAMP technology uses a tripartite polypeptide with two functional moieties: a killing moiety made of a nonspecific antimicrobial peptide; and a targeting moiety made of a species-specific binding peptide that are joined together by a linker peptide that provides free rotation. The targeting moiety provides specific binding to a selected pathogen and facilitates the targeted delivery of the attached antimicrobial peptide which significantly enhances its killing kinetics compared to the untargeted antimicrobial peptide. STAMPs developed against S. mutans and Pseudomonas aeruginosa have high microbiocidal activity against their targets, but low or no activity against other microbes Methods: Using data from the pneumococcal core and supragenomic analyses performed at the CGS we identified several pneumococcal-specific surface-exposed protein targets including the competence stimulating peptide (CSP) receptors and the Blps. Using bioinformatic approaches a dozen candidate Sp-specific targeting peptides were designed to interact with these targets, and were then synthesized and tested for binding against multiple bacterial species Results: One of these peptides, a truncated form of Sp’s CSP, binds very avidly to Sp biofilms, but does not show any binding to H. influenzae,­ a major co-colonizer of the nasopharynx, nor does it bind to the related S. mutans to any appreciable degree. Another Sp-specific peptide (Sp687) specifically binds to both Sp biofilms as well as planktonic bacteria. Out of 12 Sp-encoded peptide pheromones and related peptides identified using comparative genomics, 7 gave binding results equivalent to or better than the CSP and Sp687 peptides. Conclusions: The ability of these targeting peptides to bind to biofilm envirovars is of key importance as they represent the major target in chronic infections. By combining the antibiofilm affects of the previously characterized antibacterial peptides with the specificity of the pneumococcal targeting peptides we will be able to construct an anti-Sp biofilm magic bullet.

Opening of Blood-Brain Tumor Barrier by Phosphodiesterase Type 5 (PDE5) Inhibitors in a Mice Metastatic Brain Tumor Model HU J, SHU Y, KO MH K, PHILLIPS DJ, KONDA BM, ESPINOZA A, YUAN X, LJUBIMOVA J, BLACK KL Department of Neurosurgery, Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. Background: While adequate delivery of drugs may occur in systemic tumors, the blood-brain tumor barrier (BTB) limits delivery of anti-tumor agents into brain tumors including metastases. In this study, we examined the role of phosphodiesterase type 5 (PDE5) inhibitors in BTB opening in a mice metastatic brain tumor model. Methods: Firstly, we established the metastatic brain tumor model by implanting CRL-5904 cells (human brain metastasis of non-small cell lung cancer) intracranially and flankly. The mice were treated with different tracers with or without vardenafil (Levitra), an inhibitor of cGMP-specific PDE5. Then, we detected the in vivo BTB permeability by using fluorescence microscopy, quantitative autoradiographic method and Xenogene imaging system. We also investigated the in vitro drug uptake by using the transwell system to mimic blood brain barrier. Results: We showed that oral administration of vardenafil significantly increased doxorubicin uptake in brain tumors. Further, vardenafil could increase the tumor permeability of therapeutic antibody Herceptin and big molecule tracer ^14C-Dextran intracranially and flankly, but not in contralateral normal brain. In vitro drug uptake assay demonstrated that vardenafil enhanced the uptake of doxorubicin in both CRL-5904 and human brain microvessel endothelial cells (HBMEC). Sildenafil (Viagra) increased not only the uptake of the tracer ^14C-sucrose but also that of the chemotherapeutic drug ^14C-carboplatin in brain microvessel endothelial cells and tumor cells. Furthermore, filipin, an inhibitor of caveolae endocytosis pathway, could block PDE5 inhibitor-induced drug uptake, suggesting a mechanism via caveolae transcytosis pathway. Conclusions: These findings suggest that PDE5 in metastatic brain tumors may serve as an effective target for pharmacological modulation of BTB permeability to enhance selective delivery of chemotherapeutic drugs to metastatic brain tumors.

Novel Targeted Anticancer Prodrugs of Phosphoramide Mustard HU L Department of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854; The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA Background: Many anticancer agents in current clinical use lack the desired tumor-selectivity and are associated with systemic side effects. Prodrug design is one approach that could potentially improve the target-selectivity of anticancer agents. Efforts in our laboratory have been focused on designing analogs of cyclophosphamide and phosphoramide mustard (PM) to move the site of activation from the liver to tumor tissues through the incorporation of reductive and proteolytic triggers. Methods: A series of nitroaromatic analogs and peptide aminoarylmethyl conjugates was designed to efficiently release the active phosphoramide mustard in a site-specific manner in tumor tissues, thus increasing the selectivity in killing cancer cells and decreasing systemic toxicity. Traceless linkers have been designed and optimized to efficiently release the active phosphoramide mustard through incorporation of fluorine substitutions. We also successfully developed a new amidation reaction for the preparation of peptide-aminoaryl PM conjugates using selenocarboxylate and azides, thus avoiding the use of unstable basic nucleophilic amine intermediates. Results: The prodrugs were shown to be stable under physiological conditions including whole blood. The nitroaryl phosphoramides were shown to be excellent substrates of E. coli nitroreductase and highly cytotoxic towards nitroreductase-expressing V79 and SKOV3 cells. When the peptide used is a substrate of prostate-specific antigen (PSA), the peptide-aminoaryl PM conjugates were activated by PSA and have been shown to be selectively more cytotoxic to PSA-producing LNCaP prostate cancer cells than to DU145 cells that do not express PSA. Conclusions: These prodrugs could potentially be developed into clinically useful chemotherapeutic agents for the targeted treatment of cancer. Authors’ disclosure statement: A world-wide patent application (WO 2008/067495) has been filed to cover the targeted prodrugs and the novel synthetic process.

The Impact of Inflammatory Responses on Taste Bud Cell Turnover and Function. WANG H^1, ZHOU M^1, BRAND J^1, HUANG L^1, ^1Monell Chemical Senses Center, Philadelphia, USA. Background: Taste disorders impact negatively on general health and quality of life. Although many conditions can contribute to or exacerbate taste deficits, viral and bacterial infections are among the most common causes. The underlying molecular and cellular mechanisms, however, are largely unknown. We hypothesize that inflammatory agents, particularly interferons (IFNs), may be involved in the infection-induced taste disorders. Methods: Quantitative real-time polymerase chain reactions (PCR), in situ hybridization and immunohistochemistry were used to determine the presence of IFN signaling pathways in taste buds. Primary tissue cultures of taste bud-containing lingual epithelia were stimulated with recombinant IFNs to assay the activation of these IFN pathways. Animal models mimicking the viral and bacterial infections were established to assess the impact of infection on gene expression in taste buds. Finally, the effect of systemic administration of IFNs on taste bud cell turnover was also evaluated. Results: IFN signaling pathways, including IFN receptors and their downstream components: protein kinases JAK1 and TYK2, and transcription factors STAT1, STAT2 and IRF9 were present in subsets of taste bud cells. Incubation of recombinant IFNs with cultured taste tissues activated the IFN signaling pathways, leading to the phosphorylation of the transcription factors. Intraperitoneal injection of lipopolysaccharide or polyinosinic:polycytidylic acid up-regulated the expression of IFN-inducible genes in taste papillae whereas the systemic administration of recombinant IFNs resulted in the increased apoptosis in the taste buds. Conclusions: These findings suggest that bacterial and viral infection-induced IFNs can act directly on taste bud cells, affecting their cellular function in taste transduciton, and that IFN-induced apoptosis in taste buds may cause abnormal cell turnover and skew the representation of different taste bud cell types, which eventually lead to the development of taste disorders.

Combinational therapeutics Targeting Laboratory and Clinical Isolates of HIV-1 HUANG RC, ABD-ELAZEIM IS, GNABRE J Johns Hopkins University, Baltimore, MD, U.S.A Background: Most if not all viruses, including replication-competent mutants, are host dependent. They require the participation of certain cellular factors to sustain viral growth. Cellular factors, unlike viral proteins, are not under mutational pressure and are generally structurally invariable. Some of the viral motifs interacting with these cellular factors also remain evolutionarily stable. Thus, inhibitors that block the usage of these conserved factors at different stages of vial life cycle are likely to be good candidates for mutation-insensitive antiviral drugs. Methods: A new cocktail of small organic molecules, targeted specifically at the highly conserved motifs of virus/host interactions at different steps of the viral life cycle (viral entry, integration, and pro-viral transcriptions) was developed. Using specific bioassay-guided purification methods, including counter current chromatography, Waters Co’s alliance HPLC 2695 separations module equipped with photodiode array detector, and empower chromatogradify manager, several classes of therapeutically important molecules have been isolated from plants and chemically identified. Results: Structures of three anti-HIV inhibitors, GEN-1, M522, and G4N targeting viral entry, integration, and pro-viral transcription respectively, are shown in Figure 1. When tested alone, GEN-1 has an IC50 of 40 μM, N522 an IC50 of 4.6 μM, and G4N an IC50 of 18 μM. When tested in combination, they can block HIV-1 production in culture CD4+ cells synergistically against a variety of HIV strains at an IC50 of 1.3 μM. They are equally active in targeting mutant strains that are highly resistant to currently available drugs against HIV protease and reverse transcriptase as compared to the wild type viruses. In comparison, cytotoxicity for the three drugs combined in H9 cells was also analyzed using MTT assay. As further shown in Figure 2, cellular toxicity to H9 cells with combined was not detected in the presence of the three drugs, when 12 μM of each was used.