Ehrlich II –2nd World Conference on Magic Bullets

Current knowledge on membrane transporters of vitamin A and its precursors DURING A^1, HARRISON EH^2 ^1Université catholique de Louvain, Louvain-la-Neuve, Belgium; ^2The Ohio State University, Columbus, USA. Background: Humans must obtain vitamin A (or retinol (ROL)) from the diet either as preformed ROL or as provitamin A carotenoid precursors to maintain some vital functions. Both deficiency and excess of ROL are known to cause pathologies. Thus, a better understanding of the mechanisms of intestinal vitamin A absorption is important to optimize amounts in the diet. Aims: 1) To define mechanisms of absorption of ROL/carotenoids. 2) To focus on potential membrane transporters. Methods: In presence of oleate/taurocholate, differentiated Caco-2 cell monolayers on inserts are able to produce chylomicrons (CM). ROL and carotenoids were delivered to cells using Tween 40 (0.1%). Glyburide was used at 0.2 or 1.0 mM. Lipoprotein fractions (CMs and VLDL) in the basolateral medium (BM) were isolated by ultracentrifugation. [^3H]-Glycerol was used to label newly formed triglycerides and follow CM. Lipids (including retinoids/carotenoids) in cells and media were extracted by solvents, triglycerides isolated by TLC and [^3H] counted, and retinoids and carotenoids analysed by HPLC. Inhibitions of SR-B1, NPC1L1, and ABCA1 protein expression were done by siRNAs. Results: When cells were incubated with ROL for varying times, cellular ROL plateaued within 2h, whereas retinyl ester (RE) formation increased continuously. ROL and RE efflux into BM increased linearly with time; ROL in the non lipoprotein fraction and REs in CM. In contrast to carotenoids, ROL uptake was proportional to ROL concentration (0.5-110M). ROL efflux into BM occurred via a saturable process at low concentrations (<10M) and a non-saturable process at higher concentrations. When ROL-loaded cells were maintained on retinoid-free medium, free ROL, but not REs, was secreted into BM. Glyburide significantly reduced ROL efflux, but not ROL uptake. Inhibition of ABCA1 protein expression decreased ROL efflux, but not carotenoid efflux. SR-B1 inhibition did not affect ROL transport, but decreased carotenoid uptake. Conclusions: 1) ROL enters intestinal cells by diffusion. 2) ROL efflux is partly facilitated, probably by the basolateral transporter ABCA1. 3) Newly-synthesized REs, but not preformed esters, are incorporated into CM and secreted. 4) Carotenoid uptake is mediated by the apical transporter SR-B1 and carotenoid efflux occurs exclusively via their secretion in CM.

Sulfated oligosaccharides as main targets in cruzipain, the major cysteine proteinase of Trypanosoma cruzi ACOSTA DM^1, SOPRANO LL^1, ESTEVA MI^1, KOVENSKY J^2, COUTO AS^3 DUSCHAK VG^1 ^1Inst Nac Parasitol “Dr Mario Fatala Chaben”, Ministerio de Salud, Argentina; Laboratoire des glucides, Universite Joules Verne, Amiens, France; ^2CHIDECAR, FCEyN, UBA, Argentina. Background: Trypanosoma cruzi, the agent of Chagas disease contains a major cysteine proteinase, cruzipain (Cz). This lysosomal enzyme bears an unusual C-terminal domain (C-T) that contains post-translational modifications and most antibodies in natural and experimental infections are directed against it. Methods: To address the structure of the N-linked oligosaccharides present in the C-T domain, UV-MALDI-TOF mass spectrometry was used in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography. In order to evaluate the immune responses to sulfated moieties on Cz, and the involvement of anionic charged structures in the immune recognition of sulfated glycoproteins, BALB/c mice were immunized with purified Cz and C-T prior and after desulfation treatment. Results: The MALDI-TOF MS analysis allowed us to identify and characterize a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. The humoral immune response to sulfates on Cz or C-T was mainly IgG2b. IgG2b reactivity was abolished when desulfated antigens were used as immunogens showing that sulfates are absolutely required for eliciting IgG2b response to Cz. A significant reduction of C-T-specific delayed-type hypersensitivity reaction in C-T-immunized mice was observed when desulfated C-T was challenged, suggesting the involvement of sulfate groups in the generation of memory T-cell responses. Moreover, immunization with C-T elicited ultrastructural abnormalities in heart tissue. Surprisingly, hearts from sulfate-depleted C-T-immunized mice did not show pathological alterations. In contrast to anti-desulfated Cz mice serum, anti-Cz serum recognized sulfated poligalacturonic acid with relation So4^2-/COO^- = 1(+++); 0.67 (++); 0.4 (+); 0 (-) and Glucose phosphate in a lower degree (+). Conclusions: We show for the first time 1) the presence of sulfated glycoproteins in Trypanosomatids; 2) that sulfates are able to elicit specific immune responses and appeared to be involved in the generation of heart tissue damage. 3) Our findings suggest that this effect could not be specifically due to sulfates but to anionic charged structures. Authors’ disclosure statement: Sera from chronically T. cruzi-infected subjects with mild disease displayed higher levels of total IgG and IgG2 antibodies specific for sulfated epitopes compared with those in more severe forms of the disease.

Rheumatoid arthritis, Proteus and “magic bullets”. EBRINGER A, RASHID T. Analytical Sciences Group, King’s College, 150 Stamford Street, LONDON SE1 9NN, U.K. Rheumatoid arthritis (RA) affects over 5 million people in the European community and over 20 million throughout the world. Stastny’s discovery in 1976, that RA patients carry HLA-D4 led to the identification of the “shared epitope” EQR(K)RAA found in HLA-DR1/4 individuals. Over 80% of RA patients belong to the HLA-DR1/4 groups, whilst the frequency of these genes in the general population is about 35% Immunological and molecular analysis shows that the sequence ESRRAL, which resembles the “shared epitope”, is found in Proteus haemolysin whilst another sequence in Proteus urease crossreacts with type XI collagen found in hyaline cartilage. Antibodies to Proteus bacteria have been found in RA patients from 14 different countries and RA sera have cytopathic properties against sheep red cells coated with EQR(K)RAA peptides. It would appear that RA is caused by an upper urinary tract infection by Proteus bacteria and this would explain why this disease occurs more frequently in women than men. Therefore antibiotics or Ehrlich “magic bullets” against Proteus bacteria should be used as specific therapeutic agents together with non-specific drugs, such as anti-TNF biologicals, DMARDs and NSAIDs in the treatment of RA. The assessment of specific anti-Proteus magic bullets in the treatment of RA is long overdue.

Microemulsions Of Amphotericin B: A Way To Change Its Profile Of Activity And Toxicity DAMASCENO BPGL^1,2, DOMINICI VA^2, URBANO IA^2, ARAÚJO IB^2, OLIVEIRA AG^3, SANTOS-MAGALHÃES NS^4, SILVA AKA^2, MEDEIROS AC^2, EGITO EST^2 ^1State University of Paraíba (UEPB), João Pessoa-PB-Brazil; ^2Federal University of Rio Grande do Norte (UFRN), Natal-RN-Brazil; ^3 Federal University of Pernambuco (UFPE), Recife-PE, Brazil; ^4 UNESP - Araraquara-SP-Brazil. Background: The intrinsical nephrotoxicity of amphotericin B (AmB), still a drug of choice for antifungal treatment, can be reduced by incorporating it in lipidic carriers. Aims: 1) To develop an AmB microemulsion (ME). 2) To analyze its physicochemical properties. 3)To analyse its in vitro pharmacotoxicity. Methods: The obtained ME system was physicochemically characterized by its visual appearance, refraction index, pH, and particle size. The binding and strength of AmB aggregation to the ME was also evaluated. The in vitro pharmacotoxicity of AmB-ME was assessed by using red blood cells (RBC) and Candida albicans. Results: The proposed protocol generated a quite stable ME of AmB (Table 1), which was less toxic and as active as the traditional Fungizon^TM. The observation of a monomeric band located at 405 nm, along all the concentration of study, revealed that the AmB molecules were strongly and individually bound to the ME droplets. Table 1. Mean particle size of ME and AmB-ME by photon correlation spectroscopy (Light Scattering) Conclusions: 1) A novel formulation of AmB-ME was prepared by a straightforward and fast procedure. 2) This new formulation presented the same efficacy of the Fungizon^TM against C. albicans and a lower toxicity against human RBCs. 3) Taken together, these results suggested that ME is an eligible drug carrier for AmB or other water insoluble molecules, and it has potential applications.

Disinfectant effect of Garcinia kola extract on Staph. Aureus EGWUATU TO;*^1OGUNSOLA FT;^1 AGOMO AN;^1AREWA DG;^1BAMIDELE TJ^2; EGWUATU CA^3 ^1Department of Medical Microbiology and Parasitology College of Medicine, University of Lagos, Lagos, Nigeria. ^2Biotechnology Division, Department of Genetics, Federal Institute of Research and Industrial Oshodi, Lagos Nigeria. ^3Lifegate Specialist Hospital, Ogba, Lagos Nigeria As the search for newer drugs or other innovative measures for treating or controlling Staphylococcus aureus infections intensifies, we investigated the disinfectant effect of Garcinia kola on this organism as a hand pathogen. Garcinia kola is a plant widely consumed among local people in Nigeria as a stimulant and as a therapy for some ailments such as laryngitis, colics, bronchitis, dysentery and diarrhea. The methanolic and aqueous extracts of Garcinia kola seeds were tested in-vitro using the agar dilution method. The minimum inhibition (MIC) was determined as the plate with the lowest concentration on which there was no evident of growth. The methonalic extract was more effective than the aqueous extract with MIC of 12.5mg/ml. For hand washing studies, the G kola activity was compared to hibiscrub (4% chlorhexidine in detergent). Combining G. kola extract with soap and water for the hand washing showed significant antimicrobial activity on S. aureus. (P < 0.05) and this achieved a 2 log 10 reduction compared to Hibiscrub which achieved a 3 log 10 reduction. In conclusion, this study shows that crude methanolic extracts of Garcinia kola possess antibacterial activity and thus may have a potential role as active biological substances for new drugs against S. aureus infections which have developed resistance to most of the antibiotics used for their treatment.

Prediction of species-specific targets for the development of STAMPs based on analyses of bacterial species-level supragenomes EHRLICH GD, HOGG JS, JANTO B, AHMED, A, HILLER NL, BOISSY R, POWELL E, YU S, POST JC, HU FZ Center for Genomic Sciences, Allegheny Singer Research Institute 320 E North Ave, Pittsburgh, PA, 15212, USA Background: Current antimicrobial strategies will fail as they rely on broad-spectrum antibiotics that target common cellular functions resulting in the selection and dissemination of genetically encoded resistances that are passed, via horizontal gene transfer (HGT) mechanisms, among pathogens and commensal flora. Methods: To break this chemical arms race we decided to identify species- and strain-specific targets using 454-based whole genome sequencing and a suite of in-house developed comparative genomic tools to analyze dozens of strains of thehuman pathogens Haemophilus influenzae, Pseudomonas aeruginosa, Escherichia coli, Shigella flexneri, Staphylococcus aureus, and Streptococcus pneumoniae. In each case we chose strains that were associated with a spectrum of clinical presentations to obtain the most complete species-level supragenome possible. Results: For all species the core genome (those genes shared among all strains) contained only a minority (20-30%) of the genes present within the species supragenome. Thus, the distributed genome was 2.5 to 4X that of the core genome, and each strain of a species contains a unique set of distributed genes with respect to all other strains. Importantly, each species only carried a small number of genes that were completely unique to that species. On average, for all species examined, the mean number of genic differences for any pair of strains within a species was between 350 & 600 genes, such that 20-33% of each strain’s genome is unique as compared to all other strains of the species. Each of the core genes for each species gives a unique phylogeny, proving that HGT is the major mode of evolution and that intraspecific phylogenetic studies are meaningless. Conclusions: We are using our comparative genomics pipeline together with a suite of annotation and metabolomic programs to identify species- and disease phenotype-specific targets for antimicrobial strategies. We will then develop STAMPS (selectively-targeted antimicrobial peptides) directed towards these diagnostic molecular moieties to specifically target particular pathogens without the risk of broad-spectrum targeting that promotes the rise and spread of antimicrobial resistance cassettes.

Use of retinoids as environmental contamination biomarkers in aquatic ecosystems EL BOUHALI B^1, NASRI I^2 ^1National Laboratory of Forensic Science, Casablanca, Morocco; ^2Hassan II University, FST, Laboratory of Biochemistry, Mohammedia, Morocco. Background: Xenobiotics discharged into the aquatic environment are important to monitor. There are several advantages in using biomarkers instead of traditional chemical analysis. Biomarkers measure integrated responses in time and space and can reveal the nature of the pollutant. Exposure to adverse xenobiotics in wildlife alters retinoid status that decrease reproductive success, altere immune system, and causes dermatologic anomalies and developmental deformities. Aims: 1) To assess damage caused by industrial and human wastes and chemicals intensive uses by agriculture at aquatic and estuarine ecosystems. 2) To use the rates of retinol (R) and retinyl palmitate (RP) as biochemical markers of this environment health. Methods: The sampling of Gambusia holbrooki fish was made in the Fouarat Lake situated in north-east of Morocco and Sebou estuary. At the laboratory (L), a sample was bred and fed under poor retinol diet. Another sample was bred in external basin in natural conditions. The HPLC assays were performed on a Gilson model 307 with using UV/Vis detector at 325 nm. Separation was made using a Nucleosil C-18 column (250 x 4,6 mm). The isocratic elution was realised with methanol:water (90:10, v/v), flow rate 1,6 ml/min for the (R) separation and 100 % ethanol at a flow rate of 1,5 ml/min for the (RP). Results: (R) determination in the body informs about plasmatic rate whereas the (RP) quantification informs about hepatic retinoid reserve. The comparison of different concentrations in (RP) indicates that fish of the estuary have the highest significant hepatic reserve in comparison with that from other sites. The laboratory’s sample possesses the lowest hepatic reserve. (R) concentrations of Fouarat Lake and Sebou Estuary are nearly identical and significantly higher than those of (L) and external bread. Conclusions: 1) (RP) and the (R) may be useful as sensitive biomarkers for monitoring chemical contaminants in Gambusia in freshwater and estuarine sites. 2) Use of (RP) and (R) indicate if exposure to chemicals was resperctively chronical or acute. 3) A simple, rapid and inexpensive method was developed for extraction and analysis of (R) and (RP) in Gambusia by reversed phase HPLC.

Plasmodial Plasma Membrane: isolation and its implication in drug transport ELANDALOUSSI LM^1,2, LINDT M^1, SMITH PJ^1 ^1Division of clinical pharmacology of the Univ. of Cape Town and Groote Schuur Hospital, Cape Town, South Africa; ^2IRTA, Sant Carles de la Rapita, Spain. Background: A model to study drug transport across the plasmodial plasma membrane is important to investigate the mechanism involved in drug accumulation and resistance in the malarial parasite. Aims: 1) to obtain parasite plasma membranes purified from Plasmodium falciparum, 2) to investigate the role of Pgh1 and 3) to study chloroquine accumulation and ATPase activity in the purified plasmodial plasma membrane. Methods: To obtain the parasite plasma membranes in the form of vesicles, trophozoites released by saponin treatment from infected-erythrocytes were purified using anti-erythrocyte antibodies fixed to polystyrene beads and biotynylated to facilitate their recovery with a magnetic system prior to disruption by nitrogen cavitation. The effect of chemosensitisers on ATPase activity and chloroquine accumulation capabilities of the plasma membranes were determined. The effect of anti-Pgh1 antibodies on chloroquine accumulation was also investigated. Pgh1 was identified in plasma membranes isolated from various strains of P. falciparum and the subcellular localisation of Pgh1 in infected-erythrocytes was examined. Results: Subcellular localisation of Pgh1 indicated that this protein is present in P. falciparum plasma membrane but no link between the overexpression of Pgh1 and chloroquine sensitivity of the P. falciparum strains tested could be found. In addition, polyclonal antibodies directed at Pgh1 were unable to inhibit chloroquine accumulation in purified plasma membranes, suggesting that Pgh1 is not involved as a chloroquine transporter in the plasma membrane of P. falciparum. Verapamil and other agents known to reverse chloroquine resistance by increasing chloroquine accumulation in parasitised erythrocytes did not affect either chloroquine accumulation or the ATPase activity of isolated membranes indicating that resistance reversal do not occur at the plasma membrane level. Conclusions: a method for the isolation and purification of the P. falciparum plasma membrane was developed. This work provides evidence for a verapamil-insensitive site of chloroquine accumulation in the plasma membrane and demonstrated that Pgh1 is not involved in the mechanism of chloroquine resistance at the plasma membrane level.

Chemopreventive and Renal Protective Effects for Epigallocatechin-Gallate (EGCG) and Resveratrol (RSVL) in Human and Animal Cancer Models El-Mowafy AM^1, ElmeseryM^1, Alkhalaf M^3, Al-GayyarM^1, Salem HA^2 Departments of ^1Biochemistry and ^2Pharmacology, Faculty of Pharmacy, Mansoura Univ., Mansoura, Egypt and ^3Dept. of Biochemistry, FOM, HSC, Kuwait Univ., Kuwait Background: Catechins (like epigallocatechin-gallate, EGCG) and stilbenes like (Resveratrol, RSVL) are plant-derived polyphenols with numerous health benefits. Aims: we evaluated their chemopreventive effects alone or in combination with cisplatin (CP) in the Ehrlich-ascitis-carcinoma (EAC) solid tumor mouse model, and monitored concomitant changes in serum levels of C-reactive protein (CRP), and lipid-peroxides (malondialdehyde; MDA). Further, in the MDA-MB-231 human breast-cancer cells, the apoptotic potential of RSVL was evaluated by measuring the activation of caspase-3/cleavage of proapoptotic polymerase protein (PARP). Lastly, in rats, we verified the capacity RSVL and EGCG to ameliorate the lethal, CP-induced nephrotoxicity and assessed underlying mechanism/s. Results: EAC-based induction of solid tumor in mice exhibited markedly elevated CRP (11-fold) and MDA levels (2.7-fold). EGCG and RSVL elicited significant, dose-dependent reductions in tumor size (76-98%) and markedly enhanced the chemopreventive effects of CP. These effects were accompanied by reductions in MDA, and CRP levels that correlated well with the antitumor effects (r= 0.9). Further, in human MDA-MB-231 breast-cancer cells RSVL markedly (5-fold) increased caspase-3 activity to induce PARP cleavage and apoptosis. These responses were specifically blocked by inhibiting caspase-3. Moreover, in rats, CP (10mg/kg) induced nephrotoxicity (2-5-fold rise in serum creatinine/urea levels) after 4days, and globally-induced animal fatalities after 7days. Kidney-homogenates from CP-treated rats displayed significantly-elevated MDA, and -reduced GSH levels. Rats treated with EGCG, but not RSVL, survived the lethal effects of CP, and showed a significant recovery of renal function; while their homogenates had markedly-reduced MDA, and -increased GSH levels. Significant association was detected between creatinine level and each of MDA (r=0.83) and GSH (r=-0.86); thus indicating causal relationships. Conclusions: 1) In the EAC-mouse model: EGCG and RSVL elicited prominent chemopreventive effects on their own, and appreciably augmented those of CP as well. The extent of tumor progression was highly reflected by CRP levels. 2) In human MDA-MB-231 breast-cancer cells, RSVL markedly activated caspase-3 to cleave PARP and induce apoptosis. 3) In rats, EGCG, but not RSVL, obliterated the lethal CP-induced nephrotoxicity and renal tissue injury by reducing oxidative stress, and upregulating the endogenous antioxidant machinery, like GSH.

Optimizing New Therapeutic Discoveries from Ethnomedical and Ethnobotanical Data ELVIN-LEWIS M Washington University, St Louis Mo 63130, USA Increasingly, ethnobotanical and ethnomedical data are being utilized to identify new therapeutic agents. This is because more and more pharmacopeias are being appreciated for their value as acceptable alternatives to conventional medicine, and that methods are now available to validate their worth. It is recognized that many useful botanical remedies are moderate in potency and toxicity, and thus expectations of these yielding new pharmaceutical agents are remote. However in the context of use, the combination of compounds they contain often serves to amplify the totality of their healing effects. In order to optimize the discovery of suitable candidates, several methods can be applied both in the field and laboratory to achieve this goal. These activities, often require collaboration of a variety of scientists, and can result in the development of new botanicals, phytopharmaceuticals or pharmaceuticals. This presentation will review current techniques used in this process and discuss the value of disease-targeting, ethnomedical focusing, ethnobotanical and chemical dereplication, and use of both functional and mechanistic assays.

Process Research on a Key Synthetic Intermediate of Clopidogrel EMA T Okayama Univ., Okayama, Japan Background: Clopidogrel is a platelet aggregation inhibitor widely administered to atherosclerotic patients with the risk of a heart attack or stroke that are caused by the formation of a clot in the blood. Worldwide sales of Plavix (clopidogrel bisulfate) amounted to $6.4 billion per year, which ranks second. Here we report the environmentally benign and practical chemoenzymatic synthesis of a key intermediate for clopidogrel, (R)-1, in detail. Methods: -Keto ester 2 was prepared according to the literature, and the whole-cell reduction of 2 was done with the recombinant E. coli. The results are summarized in Table 1. Results: The desired alcohol (R)-1 was obtained at 30 ºC in 76% yield with >99% ee (entry 1). Table 1 outlines how we optimized the productivity by changing the substrate concentration and the reaction temperature. When the reaction temperature was decreased by 5 ºC, the conversion and isolated yield increased (entry 2), which prompted us to double the substrate concentration. Even at the substrate concentration of 0.6 M, the conversion reached 94% (entry 3). Therefore, we further increased the substrate concentration up to 1.0 M, which resulted in 90% conversion (entry 4). Finally, we further lowered the reaction temperature (entries 5 and 6) to find the best temperature giving the highest conversion at the same substrate concentration. Thus, the whole-cell reduction of 1.0 M of 2 at 20 ºC gave 99% conversion and 1.78 g of isolated product (R)-1 (entry 5), which corresponds to the productivity of 178 g/L (weight of isolated product per liter of initial reaction volume). Table 1. Asymmetric reduction of 2 with recombinant E. coli Conclusions: In summary, the present biotransformation provides an efficient and green method for the synthesis of methyl (R)-o-chloromandelate ((R)-1). The hydride source is glucose, which is the cheap biomass, and the catalyst is E. coli, which can be multiplied easily and inexpensively. The reaction is performed in an aqueous solution under air. This is the first example of the direct asymmetric synthesis of (R)-1 with >99% ee. Excellent productivity as high as 178 g/L has been achieved. Because of the pharmaceutical value of the downstream product, clopidogrel, this bioprocess has good potential for an industrial application.

New Strategic treatment for cutaneous leishmaniasis by local injection (Glucantime + triamcinolone 1/20ratio) in 250 patient in Bam-Iran EMADI SN 1- Red Crescent Society Research Center, Tehran, Iran 2- Baqiatallah University of Medical Sciences, Tehran, Iran Introduction: Leishmaniasis is a protozoal disease whose diverse clinical manifestations are dependent both on the infecting species of leis mania and immune response of the host. 88 countries and more than 350 million people are at risk and the prevalence of this disease is 12 million. ¾ of leishmaniasis presented with cutaneous leishmaniasis. On 26^thof December 2003 a disastrous earthquake occurred at Bam, a city in Iran. After this disaster an epidemic of cutaneous leishmaniasis occurred in this region. Our objective was to study the following: 1. The clinical feature and demography 2. The result of combination therapy by the new way of local injection glucantime and triamcinolone. Patients and Methods: From December 2004 to March 2006 in a case series study, more than 5000 patients suffering from only cutaneous leishmaniasis (CL) were seen by dermatologists in dermatology clinic of Bam’s hospital (Pastor). The clinical diagnosis of CL was confirmed by a direct smear stained by geimsa .Demographic data, including age, gender, residence (Bam or villages), and domestic were recorded in questionnaires. 250 of the patients with less than 3 lesion and had problem for systemic injection of glucantime treated by complete and carefully planed local glucantime injection (Glucantim + triamcinolone 1/20) and pre and post pictures were taken of all the patients. Results: The mean age of patients was 19y/o. This study of the population included 57% male and 43% female. The locations of lesions in order of frequency were as follows; face hands, upper limbs and feet. The most frequent sites of involvement on the face were the cheeks and then lips, ears and eyelids. The clinical pictures of CL in order of Eczematous and Crusted, Lupoid and recurrent, hyperkeratotic, tumoral, spototrichoid, anthrax like lesions, paronichial lesions, warty and pustular lesions. More than 72% of patients with eczematous, crusted, lupoid, hyperkeratosis, tumoral and warty form were treated by very careful local injection (G.T 1/20). The plan of injection was as follows: 1. Around the lesion on the outer side of the wound (targeted site) where the skin seems fine up to 0.5cm injected in intradermal as long as we had three signs and symptoms namely: bleaching - hardening - pain and we tried to prevent the leakage of G.T outside the targeted site. 2. Insertion site must be 1 (one) if there was need for more insertions the needle was changed to prevent incubation. 3. We did this twice a week for 6 (six) weeks. Discussion and Conclusion: Almost of the lesions have three sections namely 1. Necrosis or wound space 2. Granulation tissue (under wound space) 3. Inflammation space around lesion that looks like healthy - this space is important because Leishmania parasites expand in this space, so the most important target by local injection is around this space. Since CL is an infectoinflammatory disease, low dose of triamcenolone is used to reduce the inflammation and create a more faster and effective working condition for glucontime. Finally: The correct use of drugs especially local injection of glucontime by the physician or technician can prevent side effects of systemic therapy of glucontime and reduce the cost which is beneficial for the patient.

Discovery of Malaria Vaccine Candidates – Application of the HT Cell-Free Protein Production System Born of the Study on Ricin Toxin ENDO Y Ehime University, Matsuyama, Japan. Background: Selection of expression systems plays a crucial role in the post-genomic studies on the structure and function of proteins. Although a variety of cell-based expression systems have been widely used for a long time, they have inherent limitations in terms of their adaptability to high-throughput screening and production, and the quality of the proteins produced. Many of these limitations can be circumvented by the use of cell-free translation systems. Among them, the wheat germ based system is of special interest for its eukaryotic nature; it has the significant advantage of producing eukaryotic multi-domain proteins in a folded state. All of those conventional cell-free systems, however, were plagued by their short lives and as a result, inefficient protein production. Methods and Results: This shortcoming was overcome by the advent of a new method for the preparation of wheat germ extract, that was based on an idea obtained through my study on molecular mechanism of action of ricin toxin which catalitically inactivates ribosomes. Combining other elementary technologies developed, we established the protocol for the practical use of the wheat cell-free system. The technology consists of (1) in-silico selection of suitable genes from the database, (2) construction of templates for transcription by the split-PCR, and method for (3) transcription and translation reaction. There are two variations of the protocol, one is for genome-wide production and the other is for massive production of protein, have been successfully incorporated into task-specific robots that permit fully automated transcription, translation, and purification overnight. Conclusions: I introduce how the practical cell-free system is built and it’s robotic automation. In addition, I will focus on successful applications to drug discovery such as genome-wide screening of malaria vaccine candidates, the methodology can be extended for selecting effective drugs against mutant enzymes of HIV.

Natural products from plants and its derivatives as CYP19 (aromatase) inhibitors: flavonoids and dihydroisocoumarin ENDRINGER, DC^1, MAITI, A^2, GUIMARÃES, KG^1, CUENDET, M^3, CROY, VL^2, CUSHMAN, M^2, KONDRATYUK, TP^4, PEZZUTO, JM^4, BRAGA, FC^1 ^1 Faculdade de Farmácia, Universidade Federal de Minas Gerais, MG, 31270-90, Brazil ^2 Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and ^3 The Purdue Cancer Center, Purdue University, IN, 47907, USA ^4 College of Pharmacy, University of Hawaii at Hilo, HI, 96720, USA Background: Aromatase is a well-established target for the chemoprevention of breast cancer. A part from the development of synthetic aromatase inhibitors (AIs), there is a continuing search for new classes of natural products that inhibit aromatase to discover novel breast cancer chemopreventives. Methods: CYP19, CYP1A1, CYP2C8, and CYP3A4 inhibition were quantified by measuring the fluorescent intensity of fluorescein, the hydrolysis product of dibenzylfluorescein (DBF), by aromatase. Cell proliferation assay was based on the selective binding of sulforhodamine B with cellular protein (cell lines LU-1, LNCaP, HepG2, and MCF-7). (±)-Abyssinone II and its derivatives were synthetised and the dihydroisocoumarin (3R,4R)-(-)-6-methoxy-1-oxo-3-pentyl-3,4-dihydro-1H-isochromen-4-yl acetate (1) was isolated from an EtOH extract of Xyris pterygoblephara. Results: The synthetic (±)-abyssinone II moderately inhibited CYP19 (IC50 = 40.95 ± 11.3 μM), whereas (1) showed a potent inhibitory activity (IC50 = 1.6 ± 0.1 μM). (1) did not show inhibitory activity with CYP2C8 and CYP3A4 (IC50 > 65 μM) but moderately inhibited CYP1A1 (IC50 = 38.0 ± 2.0 μM). Proliferation suppression of LU-1 and HepG2 cells was insignificant, whereas a weak antiproliferative effect against MCF-7 (IC50 66.9 ± 2.3 μM) and LNCaP cell lines was observed (IC50 57.5 ± 2.0 μM). Amongst the (±)-abyssinone II derivatives, 4’-methoxy (IC50 = 4.08 ± 2.10 μM), 7-methoxy (IC50 = 4.75 ± 0.61 μM), and 4’,7-dimethoxy (IC50 = 3.67 ± 1.61 μM) derivatives were about 10 times more potent than (±)-abyssinone II itself. Conclusions: The results reported demonstrate that dihydroisocoumarin 1 and the methoxy-(±)-abyssinone II derivatives mediate potent in vitro aromatase inhibitory activity. An efficient total synthesis of (±)-abyssinone II and a series of abyssinone II-based flavonoid AIs has been successfully carried out.