Ehrlich II –2nd World Conference on Magic Bullets

The two main problems in evaluating resistance to antiparasitic drugs in populations of naturally infected hosts: efficacy variability and cut-off value for resistance CABARET J, ANTOINE T INRA, IASP 1232, 37380 Nouzilly, France Background: Variability of parasite/host response to antiparasitic drugs is large in field conditions depending on drug and galenic formulation. Resistance to anthelmintics has been well documented in sheep and goats, partly in cattle and less frequently in man. We dispose of different evaluations of efficacy for anthelmintics, none of them being a golden standard. We do not really know up to what level of efficacy we consider that we are facing a resistance phenomenon. In that respect it has been considered that less than 90% (horses) or 95% (ruminants) efficacy was the limit for stating on resistance. We are in great need of evaluation of efficacy of drugs and cut-off value for resistance. Methods: Since distribution of efficacy values is not known and is clearly not a Gaussian distribution, we propose a bootstrap confidence evaluation, using a freeware we constructed (Bootstreat available on demand or on internet). The bootstrap evaluation is based on different evaluation formulas for efficacy (before /after treatment with and without untreated control, after treatment in treated and controls, either using arithmetic or geometric means). The evaluation of cut-off for resistance is a completely open problem. We propose a two steps’ method: transformation of individual efficacy data that results in a Gaussian distribution and then when efficacy is not real we suppose that Gaussian distribution is acceptable and progressively with increasing doses and /or efficacy, there is a departure from such a distribution. We use data from anthelmintic treatments (tetramisole, macrocyclic lactones, benzimidazoles) in cattle or sheep. Results: Bootstrap results indicate that usual procedure for estimating efficacy are not accurate since confidence intervals reach sometimes more than 20% of the mean. The cut-off values are highly dependent on drug/dose (example in tetramisole) and galenic form used (injectable or per os ivermectin). We propose a strategy for evaluating resistance in the field.

Botulinum Toxin in restrictive strabismus CÁCERES TOLEDO M Clinical-Surgical Hospital. Hermanos Ameijeiras. Department of Ophthalmology, Havana-Cuba SUMMARY The restrictive strabismus is the most frequent sequel in Thyroid Orbitopathy Objective. We want to determinate the effectiveness of botulinumToxin (botox) in correct restrictive strabismus and eliminate the diplopia in primary position of look and reading. Methods. we carried out a descriptive, prospective and longitudinal study to 10 patients (17 eyes) with restrictive strabismus of March to June of the 2008, using botulinum Toxin with direct method, without any electrical control, we have registered the effect and stability in strabismus and influence in the systemic disease. Results. BotulinumToxin´injection in the affected muscle was useful to correct restrictive strabismus and offers excellent results in the elimination of the diplopia and in the patient's immediate comfort; we don't observe any influence in thyroid systemic diseases. The treatment can be to correct strabismus completely or waiting to go to surgery in a better moment. The botulinumToxin´s effectiveness could be for 4 to 6 months, but in some patients is for ever. We concludes that the therapeutic is a very good choice to treat strabismus not only for the efficacy but also for the safe and innocuous of the direct method of application

H pylori: Treatment for the patient only or the whole family? SARI YS, CAN D, TUNALI V, SAHIN O, KOC O, BENDER O. SB Istanbul Training Hospital, Istanbul, Turkey. [e-mail] Aim: To compare the effects of treatment of H pylori-infected individuals with the effects of treatment of individuals as well as all H pylori-infected family members. Methods: H pylori-positive patients with similar demographic specifications were prospectively randomized with respect to treatment, with a triple regimen of either patients and all H pylori-positive family members living togetherI (group I) or patients only (group II). Nine months after treatment, all patients were assessed for H pylori positivity. Results: There were 70 H pylori-positive patients in each group; patients in groups I and II lived with 175 and 190 H pylori-positive relatives, respectively. Age, sex and H pylori positivity rate were similar in both groups of relatives. Nine months after 14 d standard triple therapy, H pylori positivity was 7.1% in group I patients and 38.6% in group II patients [P < 0.01, OR = 8.61 95% confidence interval (CI): 2.91-22.84]. Conclusion: The present results indicate bad environmental hygienic conditions and close intra-familial relationships are important in H pylori contamination. These findings indicate all family members of H pylori-positive individuals should be assessed for H pylori positivity, particularly in developing countries where H pylori prevalence is high; they also suggest patients, their spouses and all H pylori-positive family members of H pylori-positive individuals should be treated for H pylori infection.

Anticancer Activity and Lack of Toxicity of CZ48 Administered Orally To Nude Mice Carrying Xenografts of 22 Human Tumors CAO, Z, KOZIELSKI, T, VARDEMAN, GIOVANELLA BC CHRISTUS Stehlin Foundation for Cancer Research, 1918 Chenevert Street,Houston, TX 77003 USA Background: Camptothecin, an alkaloid extracted from the tree Camptotheca acuminata and some of its derivatives were found to have potent anticancer activity against human tumors growing as xenografts in nude mice. However, these compounds were much less active against tumors of cancer patients. Further studies established that all the camptothecins were converted to their inactive form in the human blood by the opening of their lactone ring caused by the presence of human serum albumin. Methods and Results: In order to prevent such an opening, our laboratory has developed CZ48 (Camptothecin-20(S)-propionate hydrate). We have tested it for anticancer activity administering it orally to nude mice carrying xenotransplants of 22 human cancers (4 colon, 2 lung, 5 pancreas, 5 breast, 2 melanomas, and 2 sarcomas, 1 prostate and 1 bladder) at doses of 50 mg/kg/day to 2000 mg/kg/day daily, 5 to 7 days/week. The tumors were growing subcutaneously on the back of 3 month old mice. The treatment started when tumors were measurable by caliper (200-300 mm^3). CZ48 was administered suspended in cottonseed oil. Conclusion: Two conclusions clearly emerge from this study: 1) CZ48 is totally devoid of toxicity at the maximum doses used which were administered for more than 300 days; and 2) CZ48 is a potent anticancer drug against human cancer xenografts with a wide spectrum of action which caused complete growth inhibition in 20 out of 22 human cancers treated.

SAPPHIRE: A Structural-energetic Approach to B-cell Epitope Prediction CAOILI SE University of the Philippines Manila, Manila, Philippines. Background: B-cell epitope prediction facilitates the design of antibody-binding constructs for the development of novel vaccines and immunodiagnostics. This work aimed to gain insights into the problem of B-cell epitope prediction using structural energetics. Methods: Structural-energetic analysis was applied to peptide and protein antigens. A possible rate-limiting process of local epitope unfolding was considered for the cross-reaction of antipeptide antibody with protein antigen. Immunodominance was treated as a thermodynamically determined hierarchical steric-exclusion phenomenon. The algorithm thus developed was implemented as the computer program SAPPHIRE (Structural-energetic Analysis Program for Predicting Humoral Immune Response Epitopes), with the estimated affinity for antibody as the main criterion for epitope prediction. Predictions were rendered on the cross-reactivities of polyclonal antibodies to 38 peptides with 15 globular proteins of known structure and evaluated against published experimental data comprising 18 positive and 20 negative binding interactions. Results: Structural-energetic parameters could not be unambiguously assigned to cysteine in view of its capacity for disulfide bond formation. The energetic contribution of histidine could not be determined in view of the uncertainty of its protonation state at physiologic pH. The binding contexts defined by the types of participating antibodies (antipeptide or antiprotein) and antigens (peptide or protein) were all fundamentally different from one another. Predictions on genuine antibody-antigen cross-reactivity could be evaluated against empirical data only with regard to interaction between antipeptide antibody and protein antigen. Maximum areas under the receiver operator characteristic curves were approximately 0.71 either with or without consideration of immunodominance. Conclusions: 1) B-cell epitope prediction is potentially complicated by the presence of cysteine and histidine. 2) The evaluation of B-cell epitope predictions against empirical data is meaningful only if the two pertain to exactly the same types of antibody (antipeptide or antiprotein) and antigen (peptide or protein). 3) Predictions on genuine antibody-antigen cross-reactivity can be evaluated against empirical data in the case of interaction between antipeptide antibody and protein antigen.

Molecular strategies to improve the anti-tumour activity of Zoledronic acid in prostate cancer cells CARAGLIA M^1*, MARRA M^1, ZAPPAVIGNA S^1, LOMBARDI A^1, SANTINI D^2, ABBRUZZESE A^1 Department of Biochemistry and Biophysics, Second University of Naples, Via Costantinopoli 16, 80138, Naples; ^2Campus Biomedico University, Section of Oncology, Rome, Italy. * Corresponding author Background: Zoledronic acid (ZOL) is an aminobisphosphonate able to inhibit the prenylation of intracellular proteins through the inhibition of farnesylpirophosphate synthase. Prenylation is essential for the maintenance of the activation of components of signal transduction pathways regulating apoptosis and proliferation such as ras and ras-related proteins. ZOL has demonstrated a direct anti-tumour effect in vitro and in preclinical models and its ability in preventing skeletal related events is proven in patients with bone metastases from different origins. Clinical evidence on its direct anti-proliferative effects is emerging, but its activity is limited by the excessive accumulation in the bone. Methods: We describe several strategies in order to improve the anti-tumour activity of ZOL based on preclinical and biological rationales. Results: The first strategy is to combine ZOL with either cytotoxic drugs or other biological agents such as the farnesyl-transferase inhibitor tipifarnib focusing also on the importance of the sequence of administration of these drugs. The synergistic interaction with other agents could lower the active concentrations of ZOL allowing the achievement of anti-tumour concentrations also in extra-bone sites. The second strategy is to find new molecular targets of ZOL through the use of technological platforms such as DNA microarrays. We have analysed the gene modulation induced by ZOL in androgen-resistant prostate cancer PC3 cells with cDNA microarray platform to identify new molecular targets of ZOL in prostate cancer. The gene coding for cysteine-rich, angiogenic inducer, 61 (CYR61), often over-expressed in tumour cells, resulted highly down-regulated with a fold-change of 5.58. Therefore, we have studied the effects of different concentrations of ZOL on CYR61 protein product and we have found that CYR61 protein expression was significantly decreased after exposure to ZOL on both PC3 and DU145 cell lines. The effect of ZOL on Cyr61 expression was dose and time-dependent and was due to a reduced transcriptional activity of Cyr61 promoter as demonstrated by transfection with a plasmid encoding for luc-Cyr61 promoter. Interestingly, other signal transduction inhibitors did not induce or induced less effect on Cyr61 modulation if compared to ZOL. Moreover, ZOL reduced CYR61 expression through decreased activation of ras-raf-1-dependent pathway that was dependent from isoprenylation inhibition since they were antagonized by the addition of either farnesol or geranylgeraniol. Finally, we have investigated the role of Cyr61 in the regulation of growth inhibition and invasion/motility of PC3 cells using a shRNA for Cyr61 in order to down-regulate the expression of Cyr61 protein. We have found that shCyr61 enhanced inhibition of proliferation induced by ZOL. Since Cyr61 was reported to be involved in the resistance to taxanes we have evaluated if ZOL could sensitize PC3 cells to Docetaxel (DTX). We have found a sequence-dependent synergism induced by the combination between ZOL and DTX on PC3 cell growth inhibition and similar results were recorded after transfection of PC3 cells with shCyr61. Conclusions: In conclusion, it is possible to design new molecular rationale-based therapeutic strategies in androgen-independent prostate cancer based on the use of ZOL.

The dynamic hypothesis of latent tuberculosis infection offers a new rational to develop future therapeutic strategies. CARDONA P-J Unitat de Tuberculosi Experimental. Institut Germans Trias i Pujol. Universitat Autònoma de Barcelona. Badalona. Catalonia. Spain. www.ute.galenicom.com; e-mail: [e-mail] Background: It has been postulated that once infected by Mycobacterium tuberculosis, its latent form can be retained for the whole life, dormant in old lesions. With the aid of resuscitation factors these bacilli can reactivate towards active tuberculosis. These assumptions raise at least three relevant questions to answer: (1) how can dormant bacilli remain in the lungs for the whole life? Considering the constant cellular turnover and healing of injured tissues; (2) How are the resuscitation factors provided to the dormant bacilli, while immersed in old lesions?; (3) why can a 9-month treatment with isoniazid eliminate the dormant bacilli? As isoniazid is active only against growing bacilli, this treatment should be provided for life, to avoid the reactivation of dormant bacilli. Methods: Using experimental models of latent tuberculosis infection (LTBI) in mice, we have demonstrated that the granulomas are characterized by: Results: (1) the drainage of nonreplicating bacilli by the foamy macrophages towards the alveolar spaces; (2) the constant formation of new foamy macrophages; (3) the presence of local immunodepression, characterized by a high apoptosis, lack of lymphocytic proliferation and anergy; (4) the reduction of the immunological response and foamy macrophages accumulation after a short-term chemotherapy, and the bacilli reactivation once is finished; (5) administration of a vaccine based on fragments of M. tuberculosis (RUTI) allows the control of this reactivation by inducing a polyantigenic response against secreted and structural antigens. Conclusions: The “dynamic hypothesis” suggests that LTBI would be caused by the constant endogenous reinfection of nonreplicating bacilli. This hypothesis is the only one that may explain the efficacy of the 9-month isoniazid treatment, and supports a therapy based in the elimination of the local immunosuppression by a short-term chemotherapy, followed by a therapeutic vaccination able to generate immunity against structural antigens to detect the resting nonreplicating bacilli and to avoid its reactivation.

Vascular disruption: an old mechanism rediscovered for targeting tumor blood supply CARLSON RO, PLEIMAN CP, BAICHWAL V, MATHER G, SWABB E, YU M Myriad Pharmaceuticals, Inc., Salt Lake City, UT, USA Background: Vascular disrupting agents (VDAs) are cancer drugs that act through destruction of tumor neovasculature. The majority of known VDAs act through tubulin binding which leads to destabilization of microtubules in neovascular endothelial cells, increased vascular permeability, loss of perfusion and tumor necrosis. Many VDAs are currently in all phases of clinical development. The preclinical and clinical history of MPC-6827, a quinazoline-based tubulin targeting agent under development by Myriad Pharmaceuticals, is presented as a case study for discovery and development of a VDA. Methods: MPC-6827 was identified through a drug discovery process that began with a screen for apoptosis induction in a cancer cell line and continued through biochemical, cellular and animal model characterization of activity. Subsequently, safety and efficacy of MPC-6827 was studied in two Phase I clinical trials. Results: MPC-6827 was found to compete with colchicine for tubulin binding and to destabilize tubulin assembly using biochemical and cellular assays. MPC-6827 was also observed to potently kill tumor and endothelial cells in culture and rapidly damage tumor neovasculature, induce tumor necrosis and inhibit tumor growth in xenograft models. Furthermore, MPC-6827 did not show decreased potency in tumor cells overexpressing the ABC transporters, P-gp (MDR-1), MRP-1 and BCRP-1, which mediate multidrug resistance and maintain the blood-brain barrier. MPC-6827 demonstrated high brain availability in mice, with exposure in excess of 14-times that of plasma. A maximum-tolerated dose (MTD) of 3.3 mg/m^2 was elucidated in a Phase I clinical trial. Conclusions: MPC-6827 is a potent cytotoxic agent that inhibits tumor growth primarily through vascular disruption. MPC-6827 is also not a substrate for ABC transporters, which likely explains the extensive brain exposure in mice. Based upon the preclinical data and upon identification of an MTD in humans, MPC-6827 has been advanced to Phase Ib/II trials in glioblastoma and melanoma.

Comparison of oral pharmacokinetics of nifedipine in different populations CARRASCO-PORTUGAL MC^1, FLORES-MURRIETA FJ^1,2 ^1Unidad de Investigación en Farmacología, INER, Mexico City, Mexico; ^2Escuela Superior de Medicina del IPN, Mexico City, Mexico Background: Nifedipine is a calcium channel blocker that is widely used in the treatment of hypertension and angina pectoris. It has been described that this drug is importantly metabolized by CYP3A4 and differences in its pharmacokinetics have been described. In fact, it has been suggested that plasma levels are higher in Nigerians, South Asians and Mexicans when compared with Caucasians. In order to extend this information, the purpose of this study was to evaluate the oral pharmacokinetics of nifedipine in Mexicans and to compare the pharmacokinetic parameters reported in different populations by means of a meta-analysis. Methods: Twenty male healthy volunteers were enrolled in this study that was approved by the Institutional Research and Ethics Committees. All were fit according to medical history, clinical examination and suitable laboratory tests. After an overnight fast, subjects received an oral dose of 10 mg nifedipine and blood samples were collected during 8 hours. Plasma was analysed by a validated HPLC method. Pharmacokinetic parameters were obtained by non-compartmental approach and compared by meta-analysis with those reported in different populations. Results: Nifedipine was rapidly absorbed reaching the maximum between 30 to 60 minutes, then decayed with a half-life of about 5 hours. When pharmacokinetic parameters obtained in this study were compared with those reported in other populations by meta-analysis, it was observed that AUC reached in non-caucasian (Asian, African and Mexican) populations was almost twice the reported in Caucasians, indicating interethnic differences in the oral pharmacokinetics of this drug. Conclusions: It is confirmed the existence of interethnic differences in the oral pharmacokinetics of nifedipine, non-caucasians reaching higher levels than Caucasians and therefore, blindy extrapolation of dosage regimens between populations is not adequate for this drug.

Cross-kingdom Vaccines : dogma and heresy CASSONE A* & TOROSANTUCCI A Department of Infectious, Parasitic and Immuno-mediated Diseases Istituto Superiore di Sanità ROME (Italy) *Presenting Author A vaccine made up by an algal β-glucan (laminarin; β-1-3 glucan with occasional β-1-6 single glucose side chains), conjugated with diphtheria toxoid as a carrier protein component , protects against infections by different fungi and induces antibodies capable of inhibiting fungal growth .This is a sort of “cross-kingdom” vaccine because the immunizing antigen and the vaccination target belong to two different kingdoms, and this is certainly the first case in the field of human vaccines, which are generally based on the dogma “ one or more specific antigens against one disease”. Thus, it is “heretically”possible to convey in a single immunological tool the potential to protect against multiple infections, in our case all those caused by β- glucan -expressing fungi or bacteria. The generation of antibodies with the potential of directly inhibiting the growth of, or killing the fungal cells also opens an exciting perspective for both active and passive vaccination in immunocompromized subjects. The above approach could be theoretically extended to non-fungal infections by selecting the appropriate molecular pattern shared by a given microbial group ( e.g. peptidoglycan for Gram positive bacteria). Noteworthy , the molecular patterns are those microbial molecules which foster natural immunity through their binding to the pattern-recognition structures on host cells. Thus, single-component, molecular pattern-based vaccines would merge the broad target range typical of innate immunity with the highly focussed specificity of the adaptive immunity.

Heparin as anti-inflammatory and anti-metastatic drug- new potentials of an old player CELIE JW^1, BEELEN RH^1 ^1Dept. Molecular Cell Biology & Immunology, VU University Medical Center, Amsterdam, the Netherlands. Background: Heparin is a well-known compound based on its anticoagulant activities. This molecule is a highly sulfated carbohydrate glycosaminoglycan-chain, belonging to the family of heparan sulfates. Heparan sulfates are ubiquitously expressed throughout the body, and are especially known for their roles in cell adhesion and migration, angiogenesis and wound healing. Based on the roles of heparan sulfates in physiological processes, the use of heparin as anti-inflammatory and anti-metastatic drug has gained increasing research interest. Methods: Several studies have examined the effectivity of heparin, low molecular weight heparin and synthetic heparin mimetics in various experimental inflammatory models and tumor growth and metastasis. Results: Heparin and its derivates have been shown to have broad anti-inflammatory properties in models of delayed type hypersensitivity, peritonitis, meningitis, allergic encephalomyelitis, airway (allergic) and cutaneous inflammation, myocarditis, as well as ischemia/reperfusion-induced inflammation in the heart, liver and kidney. The mechanism involved is likely based on the ability of heparins to block leukocyte adhesion and migration through binding of the selectin family of adhesion molecules (especially L-selectin and P-selectin), as well as chemokines and cytokines. In addition, there is increasing evidence that heparins can inhibit tumor growth and metastasis by affecting angiogenesis, cell proliferation, as well as cell-matrix interactions. We will shortly discuss the several lines of evidence. Conclusions: Although heparin has been known and used for decades, it is becoming increasingly clear that this highly potent compound may be effective in several other clinically important settings such as (chronic) inflammation and cancer. Efforts are being made to generate heparin-mimetics with targeted specificities to enhance their potential use in clinic.

Electrochemical Behavior of Flavonoids in the Presence of Metal Ions CERDÁ MF^1, KREMER C^2, TORRES J^2, HEINZEN H^2, BERTUCCI A^2, DOMINGUEZ S^3 ^1Facultad de Ciencias, UdelaR, Montevideo, Uruguay; ^2Facultad de Química, UdelaR, Montevideo, Uruguay; ^3Universidad de La Laguna, España Background: Flavonoids are a large group of phytochemicals ubiquitously found in many food products of vegetable origin. They have been reported to have a broad spectrum of pharmacological activity. In addition, due to the presence of hydroxyl groups in their molecular structure, flavonoids exhibit strong antioxidant properties. Previous works in this field show that their antioxidant activity is modified in the presence of metal ions by the formation of soluble complex species which change the ability to scavenge free radicals. The goal of this work was to (i) make a comparative study on the electrochemical behavior (cyclic voltammetry) of the flavonoids quercetin (qrc), rutin (rut), rhamnetin (rha) and isorhamnetin (irh) on gold electrodes, and (ii) to study the influence of the relevant metal ions Cu(II) and Fe(III) on the electrochemical oxidation of the four flavonoids. Methods: Qrc and rut were reagent grade, purchased from commercial sources and used without further purification. Rha and irh were isolated from B. trimera. The isolated compounds were further characterized by HPLC-DAD analysis and HPLC co-chromatography with pure standards purchased from Indofine and Fluka. For cyclic voltammetry experiments, a polycrystalline Au-pc disc (99.999 %, 3 mm diameter) shrouded into a Teflon body and a Pt sheet, 3.0 cm^2 geometric area, were used as working and counter electrodes, respectively. The reference was the Ag/AgCl electrode (3 M KCl, E = 0.230 V vs. SHE). The solutions were prepared by mixing the flavonoid and/or the metal ion in basic media (pH around 9) to reach 1 mM concentration in water. The ionic strength was adjusted up to 0.15 M with NaClO4 assupporting electrolyte, and adjusted to the desired pH with dilute NaOH or HClO4. Potential scan rates within the range 0.005 Vs^-1 ≤ v ≤ 0.05 Vs^-1 were employed. Results: Evaluated free flavonoids present similar features, including many anodic contributions located in the positive potential range. Some of them can be certainly attributed to the redox behavior of the system Au-pc in the supporting electrolyte and the others arise from the electroactive moieties of the flavonoids. Table 1. Anodic contributions ascribed to redox processes involving functional groups of the flavonoids. E(V) vs. SHE Anodic peaks (Ean1) at ca. 0.3 V and 0.5 V could rise from the oxidation of the OH moiety from chatecol to quinone following a two one-electron steps path. Contributions (Ean2) at 0.68 V in rha and 0.87 V in qrc could be assigned to oxidation of the OH located in the same ring than the quinone. This process was not detected in rut, caused by to the blocking of this position with the sugar residue. In the case of irh this signal is also absent, may be due to difficulties to reach the electrode surface or the formation of an intramolecular hydrogen bond with the O of the quinone. Finally, contributions at ca.1 V (Ean3) can be attributed to the oxidation of some of the remaining OH groups. When copper is added, a new contribution (not present for the free flavonoids) is detected at ca. -0.2 V, assigned to the copper redox behavior itself. The strong interaction Cu(II)-flavonoid makes copper reduction less favorable. As a consequence, a poorer participation of the copper ion in the formation and propagation of the free radicals is expected. After addition of Cu, only the Ean1 is mainly affected and appeared at less positive potentials than in the case of the free flavonoids, indicating an increased reduction power of the copper complexes in aqueous solution. When Fe(III) is added to the flavonoid containing solutions, features are quite similar to those observed for the copper complexes. qrc rut rha irh Ean1 0.43 0.38 0.35 0.32 0.56 0.45 0.53 Ean2 0.87 0.68 Ean3 0.97 1.13 1.07 Conclusions: 1) The study of the electrochemical behavior of the flavonoids in aqueous solution is a powerful tool to understand the redox processes related to the Antioxidant Activity of these natural antioxidants. The oxidation of the catechol moiety producing a quinone can be split into two independent signals and individually analyzed. 2) Further oxidation of the remaining OH groups of the quinone can also be studied in this medium, as well as the reduction of the flavonoids. 3) Upon complexation with Cu(II) or Fe(III), the species with 1:1 stoichiometry present a similar voltammetric pattern compared to the free flavonoids. However, the oxidation of the flavonoids is thermodynamically favored, especially in the case of the Cu(II) complexes. 4) The complex species also present changes in the ability of the metal ion to be reduced, i.e., the metal reduction is less favorable in the presence of the flavonoids. The flavonoids could sequester Cu(II) and Fe(III) preventing their reduction and subsequently the formation of free radicals. 5) The influence of Cu(II) on the AA of the flavonoids, at least from a thermodynamic point of view, is more notorious than in the case of Fe(III). On the other hand, the interaction with flavonoids regarding the possibility to reduce the metal ion is also more favorable for Cu(II). This work was accepted for publication in Inorganic Biochemistry: Research Progress, NOVA Publishers, Ed. by J.G. Hughes and A.J. Robinson.

Treatment of Hepatic Abscess: Magic Bullets and Beyond CERWENKA H Department of Surgery, Medical University of Graz, Austria. Background: Constant improvements of antibiotics have had crucial influence on clinical management of pyogenic liver abscess (PLA). Advances in microbiological diagnosis as well as in interventional radiology have also contributed to better outcome, but successful treatment of PLA still remains a considerable clinical challenge. Methods: Clinical data of a series of 76 patients with PLA were analyzed. Initially, broad-spectrum antibiotics were given, and treatment was modified according to sensitivity testing as soon as possible. When indicated, additional therapy with percutaneous puncture/drainage, endoscopic papillotomy/stenting or surgical interventions was used. Results: The disease was confined to the right hepatic lobe in 76% of the patients and to the left lobe in 7%; in 17%, both lobes were affected. Fifty-eight patients (76%) had a single PLA and 18 patients had multiple abscesses. Etiology was biliary in 38%, hematogenous in 11%, posttraumatic in 9% and cryptogenic or attributable to rare reasons in the remaining patients. Microbiological culture was sterile in 24%, which was at least partly due to antibiotic pre-treatment. Staphylococci, Streptococci and E.coli were most often identified. Anaerobes were found in 15%. Factors associated with the need for surgery included empyema of the gallbladder, underlying malignancy, perforation, vascular complications (hepatic artery thrombosis) and foreign bodies (e.g., toothpick, infected ventriculo-peritoneal shunt). In patients with biliary fistulae it was important to ensure prompt bile flow (for instance, by sphincterotomy/stenting) Conclusions: In surgical departments, we usually treat a selected group of patients with very severe forms of PLA. “Magic bullets”, adapted due to the results of microbiological testing, constitute the mainstay for treatment. However, additional therapy with interventional radiology and/or surgery was usually required in our patients and successful management of underlying diseases played a decisive role for positive outcome.

Anti-Angiogenic and Anti-Neoplastic Potential of Prostate-Specific Antigen (PSA) CHADHA KC Roswell Park Cancer Institute, Buffalo, NY, USA Background: In prostate cancer, disease progression and prognosis are related to angiogenesis and the degree of tumor vascularization correlates with progression and the development of metastatic disease. PSA protein present within the prostate tissue microenvironment represents the pool most critical to the pathogenesis of prostate cancer. Tissue-PSA (T-PSA) levels correlate with prognosis in prostate cancer, as well as in breast cancer. Hypothesis: PSA down regulates pro-angiogenic growth factors and up-regulates anti-angiogenic growth factors. Methods: In vitro and in vivo studies were carried out using prostate tumor (PC-3M) cells in culture and in nude mice. We analyzed modulation of protein expression in PC-3M cells by PSA using 2D-DIGE analysis coupled with HPLC-MS/MS and SEQUEST data mining. Biological network analysis was carried out using MetaCore integrated software designed for functional analysis of experimental data. Gene expression data for several regulated proteins were confirmed by real-time, quantitative PCR. Anti-angiogenic potential of PSA was also observed using human umbilical vein endothelial cells (HUVEC) in an in vitro anti-angiogenic assay. Results: a. Incubation of PC-3M cells with purified PSA resulted in a significant down-regulation of expression of 147 genes including genes like VEGF, IL-8, EphA2, CYR61, Bcl2, Pim-1 oncogene, and uPA, that are associated with angiogenesis/tumor progression in different cancers and up-regulation of expression of 137 genes including IFN and IFN-related genes and peptide inhibitors of angiogenesis. b. Forty one proteins were significantly (p<0.05) changed in abundance in PC-3M cells treated with PSA. Many down regulated proteins including N8 gene product long isoform, laminin receptor, Vimentin, DJ-1 and Hsp60 are known to be involved in tumor progression. c. PSA inhibited significantly, in a dose dependent manner, the migration/chemotaxis and attachment functions required for tube formation by HUVEC. d. PSA delivered to PC3M xenografts engrafted to nude mice resulted in inhibition of tumor growth. Conclusions: 1. In PC-3M cells, PSA down regulates pro-angiogenic growth factors and up regulates anti-angiogenic growth factors. 2. Enzymatically active and inactive forms of PSA have anti-angiogenic activity in vitro. 3. PSA has antitumor activity against PC-3M xenografts in nude mice.

Magic Bullets Versus Shotgun Drugs in Cancer Therapy and Prevention CHAKRABARTY AM ^1, FIALHO AM ^2 ^1 University of Illinois, Chicago, USA, ^2 Instituto Superior Tecnico, Lisbon, Portugal Background: The pioneering ideas developed over hundred years ago by Paul Ehrlich led not only to his Nobel Prize but also to the modern day way of rational drug design. The concept of promiscuous drugs, where a single drug can target not only multiple steps in cancer progression and prevention but also infections caused by viruses and parasites, is more recent and contrasts with the shotgun approach of one target – one drug concept. Methods: Bacterial proteins such as azurin or Laz are produced as weapons by pathogenic bacteria to prevent other intruders to invade their habitats during colonization in human tissues. Assays involving induction of apoptosis and cancer cell death as well as protein-protein interactions have shown the ability of azurin or Laz to inhibit the growth of various cancers, HIV/AIDS and malaria/toxoplasmosis-causing parasites, thus acting as a magic bullet. Results: The ability of azurin or Laz to induce apoptosis through complex formation with tumor suppressor protein p53 or inhibit growth of cancer cells by interfering in receptor tyrosine kinase-mediated cell signaling or preventing angiogenesis has been shown. In vivo cancer regression by azurin has also been demonstrated. A chemically-synthesized 28-amino acid peptide derived from azurin, termed p28, is non-toxic to animals including monkeys and is under consideration by the U.S.FDA for phase I human clinical trials as an anticancer agent. Azurin and p28 can also interfere in oncogenic transformation to prevent precancerous lesion formation in mouse alveolar and ductal mammary glands exposed to a carcinogen DMBA. Many patents have been issued to cover these inventions. Conclusions: While Paul Ehrlich emphasized human ingenuity to design new chemicals for the treatment of infections and cancer, such efforts are limited to our ability to develop validated targets. Our understanding about cancer is an evolving process and will take years for a fuller grasp. A single or few targets also allow the disease agents to change such targets to become drug resistant. Bacteria have 3 billion years of evolutionary wisdom and can design proteins as weapons that target multiple disease agents and multiple steps in the disease progression pathway to prevent rapid drug resistance. We believe such multi-targeting bacterial proteins will be our next generation drugs as envisioned by Paul Ehrlich.

Capitalizing on CYP450 polymorphism in HIV treatment optimization TO KW^1, LEE SS^2,3, Chan DPC^3, LIU ST^4, LEE KCK^1, CHEUNG SW^2, CHAN CY^2. ^1Department of Medicine and Therapeutics, ^2Department of Microbiology, ^3Stanley Ho Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, Hong Kong; ^4East Lake Hospital, Shenzhen, PRC Background: The use of highly active antiretroviral therapy (HAART) is now the standard in the clinical management of HIV infection. HIV patients are normally prescribed with 2 nucleoside reverse transcriptase inhibitors (NRTI) plus one of the two other major classes of compounds – protease inhibitors (PI) or non-nucleoside reverse transcriptase inhibitors (NNRTI). As Cytochrome P450 is the major enzyme system for the metabolism of the latter two antiretrovirals, treatment response may vary from one ethnic group to another. Treatment optimization could be effected by exploiting the pharmacokinetic and pharmacoegnomic patterns. Methods: NNRTI is commonly prescribed as a component of first-line regimen in treatment-naïve patients. The pharmacokinetics of efavirenze, an NNRTI is correlated with the CYP450 2B6 polymorphism in a Chinese population. Results: We have studied the prevalence of G516T of CYP450 2B6 gene in healthy Hong Kong Chinese blood donors and found it to be high (TT genotype 23%; allelic frequency of T at 516 0.43) compared to Caucasian populations. Interestingly there’s also variation among different ethnic groups in Chinese population. In a followup study we recruited 68 Chinese HIV patients from a hospital in Southern China. All patients have been receiving a HAART regimen comprising of efavirenz, an NNRTI metabolized largely through CYP450 2B6. The allelic frequency of 516T in this cohort was 0.23. The TT genotype was associated with a higher level of efavirenz at 8-12 hours post-dose, as compared to GT or GG. The pharmacokinetic pattern of TT was likewise different. Conclusions: 1) The determination of CYP genotype and allelic pattern ca be a useful adjunct in the clinical moniyoring of HIV I infection. 2) Adjustment of efavirenz dosage in accordance with CYP genotypic or allelic pattern could be a potentially useful strategy for optimizing treatment outcome and minimizing adverse effects. 3) Clinical guidelines on the use of antiretroviral ompounds may need to be refined according to the pharamcogenomic pattern of the local population.