Ehrlich II –2nd World Conference on Magic Bullets

Steroid Sulfatase Inhibitors – Novel Therapeutic Agents for Hormone Dependent Cancers
PUROHIT A¹, WOO LWL², STANWAY SJ¹, POTTER BVL², REED MJ^1
1Imperial College London, London, UK; ²University of Bath, Bath, UK.
Background: Inhibition of steroid sulfatase (STS), the enzyme responsible for the hydrolysis of steroid sulfates, represents a potential novel treatment for postmenopausal women with hormone-dependent breast cancer. Estrone and DHEA are formed by the sulfatase pathway and can be converted to steroids (estradiol and androstenediol, respectively), which have potent estrogenic properties.

Methods: STX64, a tricyclic coumarin-based sulfamate that irreversibly inhibits STS activity, was selected for the first in class Phase I trial of a STS inhibitor in postmenopausal women with breast cancer. STX64 was administered orally (nine patients at 5mg and five patients at 20mg) as an initial dose followed 1 week later by 3 x 2 weekly cycles, with each cycle comprising daily dosing for 5 days followed by 9 days off treatment. Blood and tumor tissue samples were collected for the assessment of STS activity and serum was obtained for steroid hormone measurements before and after treatment.

Results: The median inhibition of STS activity by STX64 was 98% in lymphocytes and 99% in breast tumor tissue at the end of the 5-day dosing period. Serum concentrations of estrone, estradiol, androstenediol and DHEA all decreased significantly from pretreatment levels. Unexpectedly, androstenedione and testosterone concentrations also decreased. Four patients, all of whom had previously progressed on aromatase inhibitors, showed evidence of stable disease for 2.75 to 7 months. The drug was well tolerated.

Conclusions: STX64 is a potent, well tolerated STS inhibitor which causes significant decreases in serum concentrations of estrogenic steroids. Conversion of second generation STS inhibitors to Magic Bullets will be highlighted.

Synthesis Of Fluorescent Heteroaromatic Compounds Using Dehydroamino Acids As Building Blocks, Studies Of DNA And Biomembranes Interactions. Evaluation Of Antiproliferative Effects On Tumor Cell Lines QUEIROZ MJRP^1, FERREIRA PMT^1, ABREU AS^1, CARVALHO MSD^1, CASTANHEIRA E^1, VALE-SILVA L^2, PINTO E^2, NASCIMENTO MSJ^2 ^1Univ. do Minho, Braga, Portugal; ^2Univ. do Porto, Porto, Portugal Background: The synthesis of new anticancer agents is an important goal. Aims: 1) To synthesize new fluorescent compounds, a 3-(dibenzothien-4-yl)indole (1), a 3-(benzothien-3-yl)benzothieno[2,3-b]pyrrole (2) and a 3-(benzothien-2-yl)benzothieno[3,2-b]pyrrole (3). 2) To study their interaction with DNA and liposomes. 3) To evaluate their effects in tumor cell lines. Methods:. Absorption and fluorescence spectroscopies were used to study their photophysical properties in different solvents and their interactions with salmon sperm ds-DNA including fluorescence quenching experiments with iodide ion. Their interaction with liposomes of dipalmitoyl phosphatidylcoline (DPPC) prepared by injection, was studied by fluorescence. The antiproliferative effects on tumor cell lines of breast adenocarcinoma (MCF-7), glioblastoma (SF-268) and non-small cell lung cancer (NCI-H460) were evaluated after a continuous exposure of 48h, using the protein-binding dye sulforhodamine B. Results represents means ± SEM of 3 exp. performed in duplicate. Results: Compounds 1-3 were synthesized in good yields. In the fluorescence spectra a red shift in the λem (nm) is observed from apolar to polar solvents. In the fluorescence spectra using increasing [DNA]/[compound] ratios an increase in the emission intensity is observed. The fraction of molecules accessible to iodide ion was very low. In DPPC liposomes (25 ^oC) the emission spectra are very similar to the ones in cyclohexane. The results of the antiproliferative effects are shown below. Compound GI50 (µM) MCF-7 SF-268 NCI-H460 1 11.00±0.60 17.0±1.20 12.70±1.50 2 7.88±0.08 7.85±1.26 14.13±1.73 3 19.10±11.50 38.70±8.90 3.90±0.30 Conclusions: 1) The preferred mode of binding with DNA is the intercalation. 3) Their location in liposomes of DPPC is the hydrophobic region. 4) A good to high inhibitory effect on the growth of the tested cell lines was observed. Compound 3 shows a high specificity for the NCI-H460 cell line. Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) and FEDER to financial support through the research centres, the research project POCI/59407/2004 and pos-Doc grants attributed to A.S.Abreu (SFRH/BPD/24548/2005) and to L.V.-S. (SFRH/BPD/29112/2006).

Addition Of Local Antiseptic Spray To Antibiotic Regime Reduces The Incidence Of Stomal Infection Following Percutaneous Endoscopic Gastrostomy (PEG) – A Randomised Controlled RADHAKRISHNAN NV, SHENOY AH, CARTMILL I, SHARMA RK, GEORGE R, FOSTER DN, QUEST LJ Rochdale Infirmary, Acute Pennine Hospitals Trust, Rochdale, Lancs, UK Background: Stomal infection (SI) following PEG is commonly due to bacteria coming from oro-pharynx and or skin surface. We hypothesise that by combining parenteral antibiotic with local anti-septic spray reduces the incidence of stomal infection by reducing the bioburden at the skin surface. Aim: To study the effectiveness of local antiseptic spray with or without 3 dose parenteral antibiotic in the prevention of SI following PEG. Methods: 96 patients randomised into 3 groups. Group A – intravenous (IV) Cefuroxime 750 mgs just before the procedure followed by 2 further doses 8 hourly. Group B – single application of Povidone – Iodine local antiseptic spray (Betadine). Group C –combination of A & B. Stomal site examined at midweek (3^rd/4^th day) and on day 7 for evidence of SI using a scoring system. Fisher’s Exact Test used for analysis of primary end point [SI at midweek (MW) and end of week (EOW) 1]. Logistic regression (LR) models used to consider effects of age, sex, diabetes, acid suppressants and steroid therapy on outcome. Results: Total 96 patients. Group A (n=34) M:F 18:16, mean age (MA) 74. Group B (n=28) M:F 15:13, MA 72. Group C (n=34) M:F 17:17, MA 74. Indications in A,B,C were broadly comparable. SI at MW in A,B,C were 6 %, 32 %, 9 % and at EOW 1 were 32 %, 32 % & 3 % respectively. SI at MW higher in B (32 %) with 6 % in A & 9 % in C (p=0.0114) and at EOW 1 lower in C (3%) with 32% each in A & B (P=0.0013). Cumulative infections (n) at EOW 1 in A, B, C overall were 11, 12, 3 with significant reduction in S I in C (p=0.003). No significant difference in numbers given antibiotics for other indications between the 3 groups (p=0.363). LR showed only diabetes to have a significant effect on SI (OR at MW 33.34, 95% ci: 4.33 – 256.7) Conclusion: Cefuroxime + Betadine spray significantly reduces both midweek and end of week 1 stomal infection following PEG. Betadine spray on its own does not reduce stomal infection at midweek and end of week 1. Prophylaxis with 3 doses of IV Cefuroxime reduces stomal infection at midweek but not at end of week 1.	Synthesis And Biological Evaluation Of Some New 2-Cinnamamidobenzamides As Potential Antagonists Of The HDM2-P53 Protein-Protein Interactions RAFFA D^1, PLESCIA S^1, CASCIOFERRO S^1, CUSIMANO MG^1, TOLOMEO M^2 ^1Dipartimento di Chimica e Tecnologie Farmaceutiche, Palermo, Italy, ^2Centro Interdipartimentale di Ricerca in Oncologia Clinica (C.I.R.O.C.), Policlinico, Università di Palermo, Palermo, Italy HDM2 is a negative regulator of the tumor suppressor p53. HDM2-p53 interaction maintains p53 in the “off” position and supports its degradation. Because HDM2 overexpressed in many cancers that retain wild-type p53, small molecules that target HDM2 are useful candidates to obtain pharmaceutically acceptable drugs. Among these, substituted 1,4-benzodiazepin-2,5-diones 1 are α-helix mimetic antagonists of the HDM2-p53 protein-protein interaction [1]. We synthesized 2-cinnamammidobenzamides 2 structurally correlated to compounds 1 with the aim to ascertain if they could be inhibitors of HDM2-p53 interaction. Compounds 2 were preliminary tested for their in vitro antiproliferative activity against p53 mutant K562 (human chronic myelogenous leukaemia) and p53 overexpressed HBT-144 (choriocarcinom placenta) cell lines. Some 2-cinnamamidobenzamides 2 posses a significant antiproliferative activity against both K562 and HBT-144 cell lines and, among these the most active was the 5-iodo-2-cinamamidobenzamide (R=I, R1=H) with an IC50 of 0.57 M and 0.28 M against K562 and HBT-144 respectively. Moreover, the presence of substitutions in the cinnamamido moiety lowered the antiproliferative activity only against the K562 cell line. Infact, 5-bromo-2-cinnamamido-4’-chlorobenzamide (R=Br, R1=Cl) showed an antiproliferative activity at 10 M of 18% and 80% against K562 and HTB-144 cell lines respectively. Biological studies to verify if the HMD2-p53 interaction is the real molecular target of compounds 2 are in progress. References: Cummings, M.D.; Schubert, C.; Parks, D.J.; Calvo, V.L.; La France L.V.; Lattanze, J.; Milkiewicz, K.L., Tinbao Lu Chem. Biol. Drug Des. 2006, 67, 201-205.
Breaching The Barriers Of The Brain: From Physics To Cures RAGHAVAN R, BRADY M Therataxis LLC, Baltimore, USA Background: Getting the right dose to the right target is critical to any form of delivery for brain therapies, and the blood-brain barrier is central to regulating this. In individuals, diseases and therapies modify key factors of molecular motion, thus complicating the problem. We model the physics of brain to predict response to new protocols, devices, and therapeutics. Delivery methods range from systemic delivery of small molecules, to direct injection of large ones into tissue. Aims: 1) Model key determinants of molecular transport within and into brain tissue using inputs from in-vivo imaging. 2) From this, obtain time-and space-dependent maps of molecular motion in response to therapeutic interventions. 3) Use this tool to develop and analyze individualized optimal delivery protocols and devices for application by a range of health care providers as well as drug developers. Methods: We developed transport models, solution methods, and estimates of key parameters in the equations, for individuals from in-vivo imaging. Interstitial transport is validated using radiological contrast agents, and transcapillary transport via microdialyis. Direct delivery in tissue was studied in six pigs with concentration of a contrast agent measured with new quantitative imaging, and in seven humans using radioactive iodine. Monkey studies are ongoing. Systemic delivery is studied using microdialysis in humans. Results: 1) Modeling and display suggest alterations of widely accepted therapy protocols and device placement, e.g. in brain cancer, we propose earlier administration of temodar (systemic) and entirely new catheters and placement (peritumoral infusions). 2) The extracellular spaces can expand to more than double their resting value in response to disease-induced edema or therapeutic infusions, having a major impact on flow of fluid and particulates in brain. 3) Determinants of transport can be quantified by specialized, but clinically acceptable, in-vivo imaging. Conclusions: 1) Patient-specific estimation of distribution of therapeutics can be valuable in maximizing the chance of success of clinical trials, and in therapeutic outcome. 2) Inadequate dosing is a serious issue in therapeutics that requires collaboration of diverse disciplines and technologies. 3) Understanding the physics (poroelasticity, microhydrodynamics) of the brain is an important companion to pharmacology in getting drugs to desired locations. Authors’ disclosure statement:The experiments were conducted by collaborators at the Virginia Commonwealth University (pigs), Duke University (humans with radioactive markers), the Johns Hopkins University (humans with microdialysis), and the University of California in San Francisco (monkeys). The collaborators will be named in the talk. The work was supported in part by BrainLAB AG, which has a financial interest in the software developed in the course of the studies.	Identification Of Novel Hepatitis C Virus Polymerase NS5B Inhibitors Through Structure-Based Virtual Screening And 3-D QSAR Studies RAGNO R^1, MUSMUCA I^1, CAROLI A^1, SIMEONI S^1, MAI A^1, KRISHNAN R^2 , KAUSHIK-BASU N^2 ^1 “La Sapienza” University, Rome, IT, ^2 UMDNJ-New Jersey Medical School, Newark, New Jersey, US Background: Although a great deal of research has beeen focused on the developement of anti-HCV agents, to date no vaccine is available and there is no effective therapy for all genotypes. Thus, there is an urgent need to identify and develop HCV-specific antiviral agents to improve the effectiveness of actual HCV therapy. Nonstructural protein 5B (NS5B), a 66 Kda RNA-dependent RNA polymerase (RdRp) has attracted the attention of medicinal chemists as a target for drug development since it plays a pivotal role in HCV replication. Methods: Here we present the development of structure-based 3-D QSAR models for inhibitors binding either at the thumb or the palm NS5B allosteric sites. The final models proved to be statistically robust showing q^2 and r^2 values in the range of 0.5-0.9. The use of external test sets showed good predictive abilities of the 3-D QSAR models. Through either ligand based (Surflex) or structure based (Autodock) molecular alignment the NCI Diversity Set was then virtually screened and the result were externally scored with the 3-D QSAR models. For each allosteric site, the first 20 molecules predicted more active were selected for biological assay against NS5B. Results: Preliminary biological data proved that among the selected compounds three derivatives showed to be effectively active against NS5B at a fixed dose of 100 µM with percentage of enzyme inhibition in the rang of 60-70%. Details of computational and virtual screening procedures will be reported along with further biological investigations Thum Binding Site Palm Binding Site
The Immunosupressive Drugs- Cyclosporin A, FK506 And Rapamycin Modulate The Functional Expression Of The Na^+-Ca^2+ Exchangers In An Isoform Specific Manner ELBAZ B^1,2, DAVIDOV G^1, GOTTESMAN MM^2, KIMCHI-SARFATY C^3, RAHAMIMOFF H^1,2 ^1Department of Biochemistry Hebrew University – Hadassah Medical School, Jerusalem, Israel, ^2Laboratory of Cell Biology, NCI, NIH, Bethesda MD, USA,^3Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA Background: Treatment of organ transplant patients with immunosupressive drugs leads to complications such as hypertension, nephrotoxicity, neurological symptoms and bone loss that can be linked to impaired cell Ca^2+. The Na^+-Ca^2+exchanger is a major cell Ca^2+regulating protein encoded by three genes: NCX1, NCX2 and NCX3. NCX1 protein is expressed ubiquitously and NCX2 and NCX3 are expressed almost exclusively in the brain. We have investigated the modulation of NCX expression by the immunosupressive drugs Cyclosporin A, FK506 and Rapamycin and the non-immunosupressive PSC833. The drugs were added to the cultured cells. Methods: Functional expression of NCX protein was measured in transfected HEK 293 cells and non-transfected-NCX1-expressing H9c2 cells. Na^+ dependent Ca^2+ fluxes, surface and total NCX protein expression were determined in parallel by FACS analysis, surface biotinylation, Western analysis and quantitative PCR. siRNA targeting Cyclophilin A was used for its knock down. All measurements were done in triplicates and repeated at least 5-7 times. Results: Treatment of NCX1, NCX2 and NCX3-transfected HEK 293 cells with Cyclosporin A and PSC833 results in down regulation of surface expression and transport activity of the protein without a decrease in expression of cell NCX protein. But whereas CsA had no effect on total cell NCX1 protein expression, PSC833 reduced mRNA and cell protein expression of NCX2 and NCX3. FK506 had no effect on NCX1 expression yet it down-regulated NCX2 and NCX3 surface expression and transport activity without any significant effect on cell NCX expression. Rapamycin had no effect on NCX2 and NCX3 protein expression yet it reduced NCX2 and NCX3 transport activity. Knock-down of Cyclophilin A modulated NCX1 expression and the effect of Cyclosporin A. Conclusions: Since all the experimental conditions in our studies were identical, presumably the different drug response is related to structural differences between NCX isoforms. Expression of NCX genes is tissue specific.	Culture, Susceptibility Testing And Genotyping Of Mycobacterium Tuberculosis Isolated From Tuberculosis Patient In Bangladesh RAHIM Z^1, ZAMAN K^1, van SOOLINGEN D^2, LUBY SP^1, ENDTZ HP^1, SUZUKI Y^3, van der Zanden AGM^4 ^1International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh; ^2National Mycobacteria Reference Laboratory, National Institute of Public Health and the Environment, Bilthoven, The Netherlands; ^3Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan; ^4Medisch Microbiologisch, Onderzoeker, Laboratorium Microbiologie, Enschede, The Netherlands Background: It is essential to study the magnitude of the burden of tuberculosis (TB) in order to control the disease efficiently. Aims: 1) To isolate M. tuberculosis from sputum samples of TB patients, 2) Susceptibility testing of isolates and 3) To record circulating phylogenetic clades of M. tuberculosis. Methods: M. tuberculosis was isolated from the pulmonary TB patients of selected rural and urban areas of Bangladesh. Conventional method was followed to isolate the pathogen from sputum samples and to tests susceptibility with respect to first line anti-TB drugs. Strains were genotyped by spoligo typing technique. Phylogenetic clade designation was performed matching the spligo patterns with that available in the International Database: SpolDB4. Results: This study-included susceptibility testing of 657 isolates. Resistance to isoniazid, rifampicin, streptomycin and ethambutol was 14 %, 6%, 45 % and 8 % respectively. Simultaneous resistance to isoniazid and rifampicin was detected in 6% of the isolates. Randomly selected 224 strains were genotyped by spoligo typing. One hundred and ninety three (86%) of 224 isolates were grouped into 31 clusters containing from 2 to 34 isolates and 31 pattern isolates (14%) were unique. The comparison of spoligopatterns with SpolDB4 indicares that 75% of M. tuberculosis population of this study composed of Principal Genetic Group 1 (PGG1) having clades like; East African Indian (EAI, 44%), Beijing (15%), and the Central-Asian (CAS, 15%). The remaining 25% of the isolates belonged to PGG 2 and 3 having Latin-American-Mediterranean (LAM) clade as predominant. A new pttern signature was detected in 49 out of 224 isolates within the clade EAI (EAI 6 BGD1) and was named Matlab type after the name of the field site where it was isolated for the first itme. Conclusion: Drug resistance was significantly higher among patients previously received anti-TB treatment. Besides, this study provides a first description of the genetic population structure of M. tuberculosis in Bangladesh, where TB patients are infected with a diverse and heterogeneous population of M. tuberculosis without predominance of a single genotype. The newly described Matlab types are suggestive of new South or South-East Asian-linked emerging genotypes.
The Herpes Simplex Virus (HSV) Vaccines: Old Problems, New Challenges RAJCANI J Alpha medical a.s., Institute of Laboratory Diagnostic, Martin; Institute of Virology, Bratislava, Slovak Republic Background: The HSV candidate vaccines tested until know were either purified subunit vaccines or recombinant envelope glycoproteins. In mice, guinea pigs and rabbits immunized with a classical subunit vaccine (Rajčáni, J et al.: Acta virol. 39, 1995, p.37-49), clear-cut protection against virus challenge and reduction of the extent of latency were demonstrated. Methods: To compare our experimental HSV vaccines (based on the purified cell extract and on the recombinant gD polypeptide), Balb/c mice were immunized with the recombinant fusion protein gD1/313 (FpgD1/313, the ectodomain of gD1), with the non-pathogenic ANGpath gE-del virus, with the purified cell extract and with a plasmid (pcDNA3.1-gD) expressing gD1.