Ehrlich II –2nd World Conference on Magic Bullets

In Vitro Fungicidal Properties of the Plant Saponin, CAY-1, with (1) Two CAY-1 Structurally Related Saponins and (2) Synergism with Silver. DE LUCCA AJ^1, BOUE SM^1, SIEN T^2, WALSH TJ^2 ^1U.S. Department of Agriculture, New Orleans, LA, USA. ^2National Institutes of Health, Bethesda, MD, USA. Background: CAY-1, 1081 and 919 are structurally similar saponins in Capsicum frutescens fruit. CAY-1 is fungicidal or inhibitory for many fungi including Aspergillus, Fusarium, Microsporum and Trichophyton species. In contrast, 1081 and 919 are weakly antifungal and inactive, respectively. Historically, silver has been used as an antimicrobial. This study determined the (1) fungicidal properties of CAY-1 alone and combined with 1081 and 919 and (2a) fungicidal properties of silver and CAY-1 both alone and mixed and (2b) synergy of silver and CAY-1. Methods: (1) The fungicidal activity of CAY-1, 1081 and 919 mixed in ratios of 8:1:1, 6:2:2 and 4:3:3 (found in Capsicum), were compared to equal weights of CAY-1 alone. Nongerminated (NG) and germinating (G) conidia of A. flavus, A. niger, A. fumigatus, F. solani, F. oxysporum and F. moniliforme were tested to determine activity against such conidial types. Separate bioassays (3) were performed (n=12) for each species and conidial type with results analyzed statistically using SigmaStat. (2) This fungicidal protocol and Minimum Inhibitory Concentration (MIC) bioassays were performed with dissolved silver and CAY-1 both alone and mixed. Results: (1) Overall, mixture 4:3:3 was the most active. For A. flavus and A. niger G conidia, CAY-1 alone was significantly (p < 0.001) lethal at 5.3 and 6.6 μg/ml, respectively, and for mixture ratio 4:3:3 at 5.0 and 6.1 μg/ml, respectively. Significant lethality for F. solani G conidia was achieved with CAY-1 alone and 4:3:3 mixture at 19.8 and 6.6 μg/ml, respectively. CAY-1 was inactive against F. moniliforme but mixture 4:3:3 was lethal at 3.0 μg/ml. (2) CAY-1 significantly reduced the G conidial viability of A. flavus, A. niger and F. solani at 1.3, 0.64 and 12.4 μg/ml, respectively. Silver (0.64-79.4 μg/ml) was significantly lethal for all fungal G conidia and the NG conidia of F. oxysporum and F. solani. Combined, silver and CAY-1 were significantly lethal for all fungi at concentrations inactive when tested separately. MIC data showed combined CAY-1 and silver had an additive synergistic effect. Conclusions: Results suggest that the amount of CAY-1 needed for significant antifungal activity is reduced up to 60% by addition of inactive levels of related saponins or dissolved silver.

Validation of a RP-HPLC Method with Fluorescence Detection for the Bioequivalence Study of Norfloxacin in Plasma Samples MARIA BERNADETE de SOUSA MAIA^A,ISMAEL LEITE MARTINS^B, DEMÉTRIUS FERNANDES do NASCIMENTO^B, ADRIANO NUNES CUNHA^B, FRANCISCO EVANIR GONÇALVES de LIMA^B, FERNANDO ANTÔNIO FROTA BEZERRA^B, MANOEL ODORICO MORAES^B, MARIA ELISABETE AMARAL MORAES^B* ^aDepartamento de Fisiologia e Farmacologia, Universidade Federal de Pernambuco-UFPE, Cidade Universitária, 50670-901, Recife-PE, Brazil; ^bUnidade de Farmacologia Clínica (UNIFAC) – Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceara, Rua Cel. Nunes de Melo, 1127, Rodolfo Teófilo, 60431-970, Fortaleza-CE, Brazil A robust method for the determination of norfloxacin in human plasma using reversed-phase high performance liquid chromatography (RP-HPLC) with fluorescence detection has been developed. The method involved precipitation of plasma protein with acetonitrile using ciprofloxacin as internal standard (IS). Chromatographic separations were performed on a Synergi MAX-RP 150mmx4.6mm, 4 column with an elution system consisting of a mixture of phosphate buffer-acetonitrile (85:15, v/v). The calibration curve was linear in the range of 30 – 3500 ng/mL. The recoveries at concentrations of 90, 1400 and 2800 ng/mL were 103.5%, 100.2% and 100.2%, respectively. The quantification limit for norfloxacin was 30 ng/mL per 10 µL injection employing fluorescence detection with excitation and emission set at 300 and 450 nm, respectively. The method validation included examining the within-run and between-run precision and accuracy and ensuring that these were within accepted limits; in summary, the precision was <8.6% and accuracy ranged from 95.8%-104.1% for concentration from 90-2800 ng/mL. The precision and accuracy for the lowest calibration standard (30 ng/mL) was well within accepted limits for LOQ. The method was then applied in a bioequivalence study in healthy volunteers given 400 mg doses of reference and test formulations of norfloxacin in random order and including a 7-day washout phase.

The Pharmacokinetics and Pharmacodynamics of Miltefosine for Leishmaniasis. DE VRIES PJ^1, DORLO TPC^1,2, BEIJNEN JH. ^2 ^1Div, Infectious Diseases, Tropical Medicine & AIDS, Academic Medical Center, Amsterdam, the Netherlands; ^2Department of Pharmacy & Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Amsterdam the Netherlands Background: Miltefosine is an alkylphosphocholine with activity against leishmaniasis. Pharmacokinetics and -dynamics have little been studied. Methods: A review will be given, combining data from literature and our own data. Results: The pharmacokinetics of miltefosine, administered orally to healthy subjects and patients with cutaneous leishmaniasis, is characterised by a rapid absorption and a very slow, two compartment, elimination proces with elimination half lives of 7 and 31 days. After four weeks of oral treatment, miltefosine remains detectable in plasma for up to 5 or 6 months. The effects of hepatic leishmaniasis on drug disposition have not been studied yet. Toxicity of miltefosine is determined by dose related gastrointestinal adverse effects that are amenable by concurrent food intake and disappear soon after discontinuation of drug administration. The relation between miltefosine plasma concentrations and other adverse effects has not been fully investigated. The pharmacodynamics of antileishmanial activity of miltefosine is poorly documented. In vitro data show different sensitivity of Leishmania species but it is difficult to extrapolate this to clinical efficacy. Efficacy is usually expressed as cure rates in clinical trials which may be higher than 95% for Indian visceral leishmaniasis but lower for other Leishmania infections. The few available data on parasite clearance indicate that this continues until after discontinuation of miltefosine administration. The pharmacokinetics and -dynamics of systemic treatment for cutaneous leishmaniasis and of topical treatment have not been studied. The little available data on eradication time, parasite sensitivity, host immunity, other pharmacodynamic determinants and the sparse data on drug interactions will be will be reviewed. Conclusions: The pharmacokinetics and -dynamics of miltefosine need further study to design rational treatment regimens that address (selection of) resistant parasites, cutaneous leishmaniasis and combination with other antileishmanial agents and to study options for topical therapy.

From immunity theory to anti-infectious chemotherapy. Why awarding two Nobel prices in 1908 to Paul Ehrlich (from Germany) and Elie Metchnikoff (from France). What consequences for medical research? DEBUE-BARAZER C EHESS Paris, France. By 1908 Elie Metchnikoff and Paul Ehrlich jointly obtained the Nobel Prize of physiology and medicine for their work on immunity. Metchnikoff explained the phenomenon of the phagocytosis in observing the starfish larvae (1880), whereas Ehrlich developed the theory of fixing antigen/antibody. The end of the XIX^e century is dominated by the Pasteurian theories which were a  great successes in the field of vaccination and serotherapy. First vaccines against variola, then the development of the diphtheria anti serotherapy had indeed nourished immense hope in the fight against the infectious diseases. Work on immunity thus were developed to understand the mechanisms brought into play and to generalize the immunological methods with the unit of the infectious diseases. Unfortunately, this very tempting ideal model is put in failure in the case of many infectious diseases and parasitic (tuberculosis, malaria, trypasonomiasis…). Ehrlich proposes another original model then. He considers that the antigen/antibody reaction is a chemical reaction. So, to fight against a microbe why not  introduce into the host, an external chemical body which, like magic bullet neutralizes the cellular target? He carries out its first experiments with a dye: the methylene blue then with the trypanroth, whereas at the Pasteur institute of Paris Roux, Metchnikoff, Mesnil, Nicolle, and Laveran were also working on various dyes (Trypanroth, blue trypan, afridol…). As the dyes fixe on the micro-organisms, they immobilize or kill them. 1903 is a very important year because H. Wolferstan Thomas (English man) proposes to use an arsenical derivative, Atoxyl, to fight against the African trypanosomiasis. By 1905 he publishes his results in the British Medical Journal. The therapeutic of the infectious disease get in then the era of chemotherapy. Discovered in 1863 by  Antoine Béchamp (French chemist), Atoxyl is the molecule which leads Ehrlich to the development of the salvarsan or 606. If one compares the Nobel conference of the two scientists, that of Metchnikoff is in direct connection with phagocytosis, whereas that of Ehrlich treats already arsenical treatment, that is to say: chemotherapy. Chemotherapy arised from work on immunity. The intellectual logic of the magic bullet and the cellular target prevailed on the anti-infectious discoveries of chemotherapy and to decree two Nobel Prize in 1908 was thus based a new discipline which was leaded the revolution the XX^e century: the chemotherapy.

Neuronal trafficking of proteins involved in synaptic plasticity: a GFP-based approach DECIMO I.*^1, FORMAGGIO E.^1, BERSAN E.^1, RONCARATI R.^3, M CLEMENS^2, CHIAMULERA C.^1, FUMAGALLI G.^1. ^1 Sect. Pharmacology, Dept. Medicine & Public Health, Univ. of Verona (Italy); ^2INAF, National Institute for Astrophysics Astronomical Observatory of Padua, Padova (Italy);^3 Sienabiotech, Discovery research Via Fiorentina 1, 53100 Siena, Italy Background: Number and distribution of ion channels and receptors involved in synaptic plasticity is modulated by several factors including post-translational control of their intracellular traffic. The improvement of molecular biology techniques and the introduction of GFP-based chimeras has boosted our understanding of the molecular determinants of protein sequences that are relevant for selective protein function. We have combined these molecular biology techniques to fluorescence microscopy and videoimaging to analyse the critical steps of assembly, membrane insertion and traffic of small conductance potassium channel type 3 (SK3)and of the p75 neurotrophin receptor. Methods:Hippocampal cell cultures were transfected with fusion proteins between GFP and different SK3 subunit truncations or P75 GFP-tagged constructs. The distribution of the fluorescent recombinant proteins were analyzed by immunofluorescence confocal microscopy or using a living imaging microscope apparatus. Results: We analysed full length, truncated versions or mutated constructs of SK3. Qualitative and quantitative image analysis indicated that the full length ion channel distributed in soma, axon and in dendrites, whereas GFPΔ578-736 (deletion of the entire C-terminal domain), GFPΔCaMBD (deletion of the calmodulin-binding site) and GFPΔN (deletion of the N-terminal domain) forms accumulated in the cell body compartment and colocalized with ER marker. The GFPΔ640-736 form (deletion of the distal C-terminal domain) had a distribution similar to control. The N-terminal deleted construct negatively affected transport and assembly of the full length channel. Movements of intracellular p75GFP were followed by cell imaging at 35°C and found to be sustained by tubulo-vesicular structures acting both anterogradely (0.1-0.5mum/s) and retrogradely (0.1-1.1mum/s), with the retrograde transport characterized by two components. Conclusions: GFP based approach is important for studying molecular and spatial properties of SK3 channel and p75 receptor in CNS neurons. Our data indicate the presence of molecular determinants within the aminoacid sequence of SK3 protein that are relevant for its intracellular processing; the approach can be extended to the analysis of domains involved in function and pharmacological properties of the channel.

Battling pharmacoresistance in epilepsy patients: pharmacodynamic and pharmacokinetic approaches DECKERS C SEIN Zwolle, the Netherlands Pharmacoresistance in epilepsy has been defined as resistance to multiple antiepileptic drugs (AEDs) and to AEDs with different mechanisms of action. The causes of drug resistance can be patient-related (i.e. pharmacogenetic), disease-related (type of etiology, disease progression, disruption of the blood-brain barrier, alteration of drug target) and drug-related (i.e. inefficacious mechanisms of action, development of functional tolerance). Immunological mechanisms can also play a role. However, in individual patients it is still unclear what causes their pharmacoresistance. Research efforts have focused on the “transporter hypothesis”, where altered drug transporters such as P glycoprotein are thought to play a key role, and on the “target hypothesis”, which entails altered sensitivity to AEDs. However, pharmacogenetic studies that have evaluated polymorphisms leading to changed function of P glycoprotein or changed function of drug receptors have thus far mainly yielded negative or contradictory results. A clinical study into the effects of verapamil on carbamazepine resistance is being performed. The classical approach to pharmacoresistance in epilepsy has been resective surgery and the indications for this kind of therapy have broadened in the last decade. Also, several new AEDs have been introduced in the last 15 years that seem to have improved prognosis somewhat. Novel approaches include studies into the role of the blood-brain barrier disruption, AEDs with novel mechanisms of action and neuromodulation techniques.

Antithrombotics that do not induce bleeding: the “holy grail” found by interfering with von Willebrand factor DECKMYN H, VANHOORELBEKE K Laboratory for Thrombosis Research, KU Leuven campus Kortrijk, E. Sabbelaan, 53, B-8500 Kortrijk, Belgium Background: Platelet accumulation at sites of blood vessel wall damage, e.g. as a consequence of the rupture of an atherosclerotic plaque, is the first step in the formation of an arterial thrombus, cause of amongst others myocardial infarction and stroke. Currently used antiplatelet agents are beneficial in the prevention of such events, however without exception suffer from side effects due to an enhanced bleeding risk. In arterial thrombosis, adhering platelets have to withstand high shear forces induced by the fast flowing blood in stenotic areas, for which they rely on von Willebrand Factor (VWF). VWF is a large multimeric protein present a.o. in plasma, that under normal conditions does not interact with its platelet receptor, glycoprotein Ib. The cryptic GPIb binding site within VWF becomes exposed, when VWF, bound to collagen exposed in the damaged artery, is stretched by the high shear forces. Our starting hypothesis was that in such scenario, interfering with VWF functions would have an antithrombotic effect specifically targeted to the (stenotic) arterial side, leaving haemostasis in the slower vessels unaffected, likely resulting in a lower bleeding risk. Results: We produced monoclonal antibodies that inhibit either the VWF-collagen or the VWF-platelet interaction, proved their shear rate dependent activity in in vitro flow chambers where blood is pumped at different speeds over a coated collagen surface and determined their epi- and paratope at the atomic level. Finally we tested the antibodies in a high shear arterial thrombosis model in baboons (collab. U Vrystaat, Bloemfontein, SA) where a strong antithrombotic effect was obtained, without prolongation of the bleeding time or increase of blood loss from a standardised incision, and this in stark contrast to currently used antiplatelet agents. Conclusions: we may have found the “holy grail” of antithrombotic research: a class of compounds with a large therapeutic window between effects on thrombosis and on haemostasis, by interfering with shear-dependent VWF-function. Clearly clinical studies are needed to fully proof that this may open up safe treatment options for especially stroke.

Sculpting the Immunological Response to Dengue Fever by Polytopic Vaccination DEEM MW Rice University, Houston, TX, USA Background: The twin challenges of immunodominance and heterologous immunity have hampered discovery of an effective vaccine against all four dengue viruses. Immunization with one dengue virus is protective against future challenge with the immunizing virus. However, immunity built up after infection by one dengue virus protects only modestly or even negatively against reinfection by the other dengue viruses. In particular, the risk of dengue hemorrhagic fever from one of the dengue viruses during a secondary infection appears to rise significantly if there was a previous primary infection from one of the other dengue viruses. This 'original antigenic sin' implies that an effective vaccine for dengue must induce protective immunity against all four dengue viruses. To date, no such vaccine has been developed. Methods: We here explore the possibility of using polytopic, or multi-site, vaccination to induce an effective T cell immune response against all four dengue viruses. We investigate whether injection of the epitopes from each of the four viruses in different physical locations sculpts a broader TCR response, by inducing TCR selection for each epitope in different lymph nodes. We determine whether polytopic vaccination reduces immunodominance and increases recognition of the four dengue viruses. Results: We show that specific lysis against the four dengue strains is super in the multi-site protocol. By physically separating the TCR selection and reducing the pressure on TCR resource competition within each lymph node, the TCR repertoire can be sculpted toward the subdominant epitopes, and so there is a reduction in immunodominance. Conclusions: By combining polytopic injection with subdominant epitope priming, a vaccination protocol for sculpting the immune response to dengue is suggested. This new protocol reduces immunodominance more fully that does polytopic injection or subdominant epitope priming alone, both of which reduce immunodominance more than does a traditional four-component dengue vaccine. Subdominant epitope priming followed by secondary polytopic injection of epitopes appears to be a promising vaccination strategy for dengue fever and other multi-strain diseases. H. Zhou and M. W. Deem, ``Sculpting the Immunological Response to Dengue Fever by Polytopic Vaccination,'' Vaccine 24 (2006) 2451-2459

Open Chemical Databases and Ontologies in the Genomic Age DEGTYARENKO KN European Bioinformatics Institute, Cambridge, UK Modern research in biochemistry and pharmacology depends on availability of chemical data, which until few years ago were almost exclusively concentrated in commercial databases. Not only the scientific community but the humankind as a whole will benefit from open access to chemical data in standard computer-readable format, just as is the case with bioinformatics and genomics. The open access is necessary but not sufficient: for example, patent documents may host a treasure of chemical data, but this treasure is well buried and is not a trivial task to extract even with the cutting-edge text mining techniques. A number of open-access databases that emerged during the last five years, such as PubChem, ChemSpider and eMolecules, provide free access to millions of structures. However the quality of data (both structure and annotation) depends on community efforts of chemical data curation. The challenges and achievements in the standardisation of chemical language in biological databases will be presented, with emphasis on three aspects of curation: naming: correct, unambiguous and usable nomenclature drawing: unambiguous, computer- and human-readable 2-D diagrams; ontology: linking the entity of interest by defined logical relationships to other entities. I am going to use the open access chemical databases to illustrate these aspects, with focus on ChEBI, a definitive, freely available dictionary of ChemicalEntities of Biological Interest. ChEBI provides standardised descriptions of molecular entities that enable other databases at the EBI and worldwide to annotate their entries in a consistent fashion. The challenge (1) is illustrated by cases of conflicting nomenclature systems, such as Preferred IUPAC Names (PINs) and International Nonproprietary Names (INNs). Challenge (2) can be met by software implementation of the IUPAC recommendations of graphical representation of chemical structures. The formalization of chemical ontology remains to be addressed. ChEBI ontology, like other Open Biomedical Ontologies (OBO), is manually built with only limited validation. However, the fundamental difference between chemical ontology and biological ontologies is that the former can be formalised using the features derived from connectivity tables. In order to be usable by wider scientific community, the chemical ontology should be scalable so the new compounds could be automatically assigned the ontological relationships.

N,N-bis(alkanol)amine aryl esters and N,N-bis(cyclohexanol)amine aryl esters: identification of a new class of Pgp-dependent multidrug resistance (MDR) reverters endowed with potencies in the nanomolar range DEI S^1, MARTELLI C^1, TEODORI E^1, MANETTI D^1, CORONNELLO M^2, SALERNO M^3, SGARAGLI G^4, GUALTIERI F^1 ^1Dipartimento di Scienze Farmaceutiche, Università di Firenze, via U. Schiff 6, 50129 Sesto Fiorentino (FI), Italy; ^2Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze, viale G. Pieraccini 6, 50139 Firenze, Italy; ^3Laboratoire BioMoCeTi, Université Paris 13, 73 rue Marcel Cachin , 93017 Bobigny, France; ^4Dipartimento di Neuroscienze, Università di Siena, via A. Moro 2, 53100 Siena, Italy Background: Multidrug resistance (MDR) is an acquired drug resistance of cancer cells and microorganisms to a variety of chemotherapeutic drugs. Classical MDR derives from the over expression of proteins such as P-glicoprotein (Pgp), that act as extrusion pumps using ATP as energy source. Inhibition of the functions of Pgp is considered a suitable approach to circumvent MDR, even if no drug has been yet approved for therapy. All information collected so far on the structure of Pgp point to the existence of a large, polymorphous drug recognition domain, where a variety of molecules can be accommodated in a plurality of binding modes. Methods: We have obtained a series of molecules where a basic linker tethers, at different distances, two aromatic moieties (A), and a second series of compounds where the flexible moiety of the series A was substituted with a N,N-bis-cyclohexylamine scaffold (B), giving origin to a series of geometrical isomers, that represent restricted conformation analogs of the A compounds. The MDR modulating activity was measured by monitoring the pirarubicin uptake on anthracycline-resistant erythroleukemia K562 cells. Selected compounds were tested for their doxorubucin citotoxicity potentiation (RF: reversal fold) on the same cells. Results: Flexible derivatives A generally show a good efficacy with potencies ranging from 1.0 to 0.10 M. Some of the B compounds are even more interesting, the best one presenting an efficacy close to 1, and a potency of 0.012 M. This compound shows also the best profile in the RF test (36.4). Conclusions: Applying the frozen analog approach to a series of flexible MDR reverters we have identified a new series of drugs that show very low nanomolar potency and high efficacy: one of them is a promising lead for the development of potent and safe MDR reverters.

Effect of Valeriana officinalis in [^3H]Glutamate Binding Lisa M. Del Valle-Mojica^1, Yoshira M. Ayala-Marín^2, Bianca A. Torres-Hernández^1 and José G. Ortíz^1 ^1University of Puerto Rico - School of Medicine, San Juan, PR; ^2University of Puerto Rico – Humacao Campus; Humacao, Puerto Rico. Background: Valeriana officinalis root extracts have been used as a sedative and anxiolytic for more than 2,000 years. The most accepted theory establishes that Valerian root extracts stimulate GABA inhibitory neurotransmission. Alternatively, relaxation and sleepiness can be produced if Valerian reduces the activation of glutamate receptors; ionotropic (iGlu) and metabotropic (mGlu) receptors. The objective of our study is to determine the effects of Valerian extracts preparations on the excitatory neurotransmission through [^3H]Glutamate binding to mGlu and iGlu receptors. We hypothesized that valerian extracts affect the glutamatergic neurotransmission through the selective interaction with mGlu group II receptors. Methods: Valerian roots were obtained from Pacific Botanicals, Oregon. Valerian was extracted in ultra pure water (~23ºC) and stirred during 1 hour. Aliquots were centrifuged before being analyzed. Assays were done using synaptic membranes of cerebral cortex from female rats of approximately two months of age. The reaction was initiated by the addition of tissue (100 µg protein) to tubes containing [^3H]Glutamic Acid (20nM) in a final volume of 500 µL of 50 mM Tris HCl/ 100 mM KCl buffer, pH 7.4. For the dose response curve, valerian extract concentrations of 4ng/ml – 12mg/ml were used. Non-specific binding was determined in the presence of glutamate 1 mM. All samples were incubated on ice for 40 minutes. The assay was stopped by centrifugation for 30 min at 11,000 rpm, then the supernatant is extracted and the pellet washed with 1 mL of ice-cold buffer. After that, the pellets are resuspended and the radioactivity of the samples was quantified in a liquid scintillation counter with 1 mL of scintillation cocktail. Results: Aqueous valerian extract (1x10^-7 – 4x10^-2 mg/ml) increase [^3H]Glutamate binding up to a maximum of 60%. At 0.05mg/ml aqueous Valerian extracts specifically interact with KA (*P < 0.05) but not NMDA, AMPA, L-AP4 and quisqualic acid. In contrast, DCG-IV and EGLU markedly decreased, 37% (***P< 0.001) and 26% (*P< 0.05), respectively, the [^3H]Glutamate binding in presence of valerian extracts (400ng/ml - 10mg/ml) demonstrating that there is a high selectivity for mGluII receptor interactions. Conclusions: 1) The present study demonstrated that Valeriana officinalis extracts selectively interact with mGluII receptors. 2) This selective interaction of Valerian with mGluII receptors may represent an alternative explanation for the anxiolytic properties of this plant.

Inhibition of experimental Sjögren’s syndrome through immunization with Hsp 60kDa and its peptide aa437-460 - predicting treatment efficacy using multi-plex biomarker profiling DELALEU N ^1, MADUREIRA AC ^1, IMMERVOLL H ^2,3, JONSSON R ^1,4,5 ^1Broegelmann Research Laboratory, The Gade Institute,University of Bergen, Bergen, Norway, ^2 Section of Pathology, The Gade Institute, University of Bergen, Bergen, Norway, ^3Department of Pathology, Haukeland University Hospital, Bergen, Norway, ^4Department of Rheumatology and ^5Department of Otolaryngology, Head and Neck Surgery, Haukeland University Hospital, Bergen, Norway. Background: The aim of this study was to investigate a potential immunomodulatory effect of heat-shock protein 60kDa (Hsp60) on spontaneous experimental Sjögren’s syndrome (SS). Methods: 7-week old non-obese diabetic (NOD) mice were immunized with eukaryotic Hsp60 or a Hsp60-derived peptide (amino-acid residue (aa)437-460). At 21 weeks of age, non-diabetic mice were investigated for salivary gland inflammation, exocrine function and extraglandular disease manifestations. In addition, biomarker profiles comprising 87 analytes in serum and 75 in saliva were analyzed. Results: Immunization with Hsp60 and aa437-460 significantly reduced SS related histopathology compared to NOD controls. In addition, 50% of Hsp60 and 33% of aa437-460 injected mice retained normal exocrine function. Both treatments induced similar changes in biomarker profiles. Notably, circulating IFN--induced protein (IP-10) and eotaxin decreased significantly as a consequence of the treatment. Anti-muscarinic m3 receptor (M3R) IgG1, IL-10 and leptin in contrast discriminated best between the different treatment groups. Successful prevention of hyposalivation was accompanied by quantitative alterations in 36 biomarkers, of which 19 inflammatory mediators declined to levels comparable to Balb/c. Low secreted vascular endothelial growth factor (VEGF)-A predicted most accurately successful prevention of hyposalivation. Low salivary granulocyte chemotactic protein (GCP)-2 was identified as the best predictor of normal secretory function across the strains. Conclusion: Immunization with Hsp60 and its peptide aa437-460 led to inhibition of SS in NOD mice. Comprehensive analyses revealed specific biomarker signatures capable of predicting treatment group and treatment outcome. Molecules involved in inflammatory chemotaxis, neovascularization and regulatory pathways coined the differences displayed by the biomarker profiles.

Synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE vesicles and DOTAP/DOPE lipoplexes LUCIMARA GAZIOLA DE LA TORRE^1, ROGÉRIO SILVA ROSADA^2, ARLETE A.M. COELHO-CASTELO^2, CELIO LOPES SILVA^2, MARIA HELENA ANDRADE SANTANA^1 ^1 Departamento de Processos Biotecnológicos, Faculdade de Engenharia Química Universidade Estadual de Campinas, UNICAMP,CP 6066, 13083-970, Campinas-SP, Brasil; ^2Núcleo de Pesquisas em Tuberculose, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 14049-900, São Paulo, Brasil. Background: The delivery of nucleic acids using cationic lipids as carriers has been a promising area of research since Felgner in 1987 demonstrated its viability in vitro. We present a comparative characterization of physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-bound DNA on EPC/DOTAP/DOPE and DOTAP/DOPE lipoplexes. Methods: The DOTAP/DOPE (50:50% molar) and EPC/DOTAP/DOPE liposomes were prepared according to the procedure described by Bangham. EPC/DOTAP/DOPE liposomes were frozen, freeze-dried and rehydrated. The complexation with pVAXhsp65 was carried out at a final molar charge rate (+/-) 10 and final NaCl concentration of 0.9%. Characterizations: average hydrodynamic diameter, zeta potential; plasmid integrity, determination of the molar charge ratio for complete DNA incorporation into the lipid structure, morphology, plasmid accessibility; phase transition; in vitro cytotoxicity. After 15 or 30 days of mouse vaccination, IgG1 and IgG2a production were evaluated. Results: The EPC inclusion stabilized the DOTAP/DOPE structure, producing higher phase temperature and lower zeta potential despite a close mean hydrodynamic diameter. Similar morphologies were identified in both structures, but a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response induced polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Conclusions: The in vivo antibody production does not correlate with in vitro cytotoxicity, or with structural stability alone. The synergistic effect of structural stability and DNA electrostatic binding on the surface of structures explains the immunological effects, and produced the required condition for DNA delivery.

Synthesis of Some New 1,3,4-Thiadiazol-2-ylmethyl-1,2,4-Triazole Derivatives and Investigation of Their Antimicrobial Activities DEMIRBAS N*, DEMIRBAS A, SAHIN D, BAYRAK H, KARAOGLU S ^aKaradeniz Technical University, Department of Chemistry 61080 Trabzon-Turkey The therapeutic effects of 1,2,4-triazoles have been well studied for diverse patological conditions including inflammation, cancer, pain, tuberculosis and hypertension. In the past decades, the problem of multi-drug resistant microorganisms has reached on alarming level around the world. For the treatment of microbial infections, the synthesis of new anti-infectious compounds has become an urgent need. For this purpose, several compounds that contain a piperazine or morpholine nucleus possessing antimicrobial activity have been synthesized; some which contains an azole ring also. For instance, while Eperezolid, and AZD2563, which are the members of oxazolidinone class antibiotics, consist of morpholine and oxazolidinone rings bearing with each other via a fluorophenilene linkage, another antibiotic, Linezolid, contains a piperazine ring instead of morpholine. On the other hand, Itraconazole, posaconazole and ketoconazole that are using for the treatment of fungal infections, contains a piperazine and one or more azole ring in their structures. In recent years, various antitumor drugs have been developed for the treatment of cancer. Among these, some 1,2,4-triazole derivatives incorporating Shiff Base structure were synthesized as antitumor agents in our laboratory. However, cancer is still a major health problem because of the insufficiency of the conventional methods. Small and linear molecules are suitable for heterocyclic ring syntheses. In this study, some 1,2,4-triazole derivatives were synthesized from the reactions of ester ethoxycarbonylhidrazones (1) and screened for their antimicrobial activities. All the newly synthesized compounds displayed IR, ^1H NMR, ^13C NMR, mass spectral data and elemental analysis consistent with their structures. N. Demirbas, S. A. Karaoglu, A. Demirbas, K. Sancak, Eur. J. Med. Chem., 39 (2004) 793-804. C. G. Bonde, N. J. Gaikwad, Bioorg. Med. Chem. 12 (2004) 2151-2161. N. Demirbas R. Ugurluoglu, A. Demirbas, Bioorg. Med. Chem., 10 (2002) 3717-3723.

Magical interactions between cisplatin, fluorouracil and radiation benefit oesophageal cancer patients DENHAM JW^1 ^1 University of Newcastle, Newcastle, Australia Background: “Chemo-radiation” using bolus cis-platinum, infusional fluorouracil with concurrent radiation has been in use since the early 1980s and has resulted in major improvements in outcome for oesophageal cancer patients. Methods and Results: This presentation reviews the author’s 20 years trials experience in this area in the context of experience elsewhere in the world. Issues discussed include:- differences in pre-clinical and clinical findings dose-response relationships for tumour control and for toxicity variations in response relating to gender and age the possibility of better combinations of the same agents. Conclusions: Many of the beneficial interactions between these agents remain incompletely understood and therefore still might be considered “magical”. Better combinations of the same agents should be sought before these agents are discarded.

Malaria-induced up and down-regulation of Cytochrome P450: Implications for Pharmacotherapy de-Oliveira ACAX, Paumgartten FJR, Da-Matta AC, Poça KS, Santos MJS. Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil. Background: During the last three decades, experimental and clinical studies have shown that infections and inflammatory conditions down-modulate expression and activity of cytochrome P450 enzymes (CYP). A few studies suggested that drug metabolism is depressed in malaria as well. This study was undertaken to extend these observations by investigating the effects of malaria on different CYP isoforms. Additionally, we evaluated whether malaria modulated the effects of alkylating agents including that of an antineoplastic drug. Methods: Female adult DBA-2 (D2, N=16) and C57BL/6 (BL6, N=19) mice were infected (I) with Plasmodium berghei (ANKA) and their parasitemia rates (P) were determined. An equal number of non-infected mice matched for age and sex was the control group (C). Monooxygenase activities (CYP1A: ethoxyresorufin-O-deethylase, EROD; CYP2B: benzyloxyresorufin-O-debenzylase, BROD and CYP2A5: coumarin 7-hydroxylase, COH) were determined in liver microsomes in mice with P higher than 30% (D2) or 20% (BL6). CYP1A apoprotein levels were evaluated by immunoblotting with an anti-CYP1A antibody. Genotoxic effects (micronuclei in bone marrow cells - BMC) of cyclophosphamide (CPA, 25 mg/kg body wt, CYP2B and 3A- activated), dimethylbenzanthracene (DMBA, 50 mg/kg bw, CYP1A-activated) and ethylmethanesulphonate (EMS, 150 mg/kg bw, direct-acting clastogen) were also investigated in BL6 mice. BMC were harvested 24 h (EMS and CPA) or 48 h (DMBA) after treatment. Results: Results (mean±SE, ANOVA Dunnett *: p< 0.05, I x C) were in D2: EROD: 60±4.6* x 92±12; BROD: 52±5* x 80.5±7; COH: 170±20* x 93±10 and in BL6: EROD: 53±4* x 118±7.8; BROD: 42.5±5* x 85±11; COH: 11.8±1.6* x 7.9±0.7. Immnublotting showed that levels of CYP1A protein in liver microsomes of infected mice were lower than levels in controls. Data also indicated that malaria attenuated effects of CPA and DMBA and enhanced that of EMS. Conclusions: 1) Malaria up-modulates CYP2A5 and down-modulates CYP1A and 2B in the liver. 2) Effects of alkylating agents activated by CYP1A, 2B and 3A were depressed while that of a direct-acting agent was enhanced. Taken together these findings suggested that malaria up and down regulates CYP and that it may either increase or decrease effects of drugs depending on the CYP isoforms involved in the activation and or clearance of the compound.

Synthesis, Anti-rhinovirus Activity and Mechanism of Action of New Chromene and Chroman Derivatives DESIDERI N and CONTI C Università ?Sapienza?, P.le A. Moro, 00185 Rome, Italy. Background: The human rhinoviruses (HRV) are important pathogens causing most of the upper respiratory tract infections in humans. Although these infections are often mild and self-limiting, the impact on human productivity and on medical costs is enormous. Since more than one hundred serotypes of HRVs make the development of a vaccine impractical, extensive efforts have been focused on the development of effective antiviral agents for the treatment of HRV infections. However, despite the in vitro activity of several compounds, to date only few drugs have shown efficacy in humans and none have been approved for clinical use. Several flavanoids and flavonoids studied by us exhibited a broad antipicornavirus spectrum. In continuation of the search for more potent and highly selective analogues, we designed, synthesized and tested new (Z)-3-benzylidenechromans, 3-benzyl-2H-chromenes and 3-benzylchromans related to the most active synthetic 3(2H)-isoflavenes and homoisoflavones previously studied by us. Methods: In preliminary studies, the cytotoxicity of all the compounds was evaluated by measuring the effect on morphology, viability and growth of HeLa (Ohio) cells.The inhibitory activity on HRV 1B and 14 replication was evaluated in a plaque reduction assay, starting from the maximum non-cytotoxic concentration (MNTC). HRV 1B and 14 were selected as representative serotypes for group B and A, respectively. Group B contains twice as many serotypes as group A, and accounts for five times as many colds as serotypes group A. Results: All the compounds tested showed a potent and selective anti-HRV 1B activity within micro or submicromolar range (IC50s ranging from 0.11 to 6.62 mM). The low cytotoxicities resulted in high therapeutic indexes for all these compounds. In contrast, only a modest inhibition of HRV 14 replication was observed up to MNTC. On the basis of the high activity and therapeutic index (IC50 = 0.12 mM and TI = 625.00, respectively), (Z)-3-(4-chlorobenzylidene) chroman (2b) was chosen to clarify the mechanism of action. The effects of the presence of 2b on different stages of HRV1B growth show that this chroman interferes with an early event of virus life cycle, similarly to previously studied flavanoids. Moreover, the decreased susceptibility of the virus to inactivation by mild acid pH or heat, suggest a stabilizing action of 2b on virion capside conformation.

Platinum and Non-Pt Anticancer Drugs: Insights from High-Level Computations DEUBEL DV^1, CHIORESCU I^1, ARION VB^1, KEPPLER BK^1, CHIFOTIDES HT^2 ^1University of Vienna, Austria; ^2Texas A&M University, College Station, TX, USA. [e-mail] Background: The breakthrough of metals in medicine was the discovery and clinical use of cisplatin as an anticancer drug, which increased the cure rate of testicular cancer from nearly 0 to over 90 percent. The therapeutic spectrum of cisplatin is limited, however, as frequent types of cancer are resistant to the drug or become resistant during therapy. Many patients undergo cycles of partial disease remission, resistance, relapse, and treatment with another drug, until further therapy is considered ineffective. Therefore, the search for new anticancer drugs has continued and extended to compounds of non-platinum metals, most notably ruthenium and rhodium. The importance of high-level computation in these research efforts is rapidly increasing. Methods: Platinum, ruthenium, and rhodium anticancer agents and their reactions with small models of biomolecules were investigated using computational methods, including quantum chemistry and statistical mechanics for calculating free energies of isolated molecules as well as continuum dielectric methods for calculating energy changes arising from the biological environment. Results: 1. The computations predict the reactivity of platinum anticancer drugs such as cisplatin towards various functional groups of biomolecules and help identify active drug metabolites. 2. The standard reduction potential (SRP) of ruthenium(III) anticancer compounds such as KP1019, which are likely activated upon reduction to their ruthenium(II) analogs in the hypoxic environment of tumors, can be predicted with a remarkable accuracy of 0.16 V (or 3.7 kcal/mol) using our best computational approach. 3. The complicated molecular mechanism of the binding of the anticancer compound dirhodium tetraacetate to the nucleobases guanine and adenine has been clarified by computation. The mechanism was elusive despite many experimental studies. Conclusions: High-level computations are complementary to experimental work; they provide promising tools for future guidance of experiments in the development of novel metallo-drugs.

Antifungal therapy in cardiothoracic transplant recipients in a new prescribing era DHAR D, NAZARETH D, LYSTER H, HALL A, BANNER NR, CARBY M Background: Both heart and lung transplantation are established treatments for advanced cardiopulmonary disease. Transplant recipients require life long immunosuppression to prevent allograft rejection placing them at risk of opportunistic infections. The incidence of invasive aspergillosis in transplant recipients has been reported at 5-20%. Treatment with amphotericin has resulted in patient survival rates of less than 40%. We report a single centre experience of treating fungal infections with the newer antifungal agents. Methods: We studied 108 patients treated at our hospital from September 2005 to December 2007. Patients requiring inpatient treatment of fungal infections were identified from pharmacy records. Diagnosis required a clinically compatible illness with either the fungus identified in bronchoalveolar lavage specimens, blood or biopsy by microscopy or culture, or radiological evidence of compatible pulmonary lesions after excluding other aetiologies. Outcomes are reported as either successful (Complete Response: Resolution in clinical signs and symptoms with regression of radiological lesions. Partial Response: Clinical improvement with marked improvement in radiological lesions. Stable: Clinical improvement without marked improvement in radiological lesions.) or unsuccessful where patients did not survive to hospital discharge. Results: 108 patients were treated, 86 lung and 22 heart transplants (annual incidence 29.7% and 3.2% respectively). 61 were male. Age at time of treatment was 46 years (range 19 to 65).Time post transplant was 4.5 yrs (range 2 days to 13 years). 54 patients were taking tacrolimus, 21 ciclosporin and 11 sirolimus. 43 were treated with voriconazole alone and 33 caspofungin alone. 31 were treated with both voriconazole and caspofungin. One was also treated with a third agent (liposomal.amphotericin). Duration of treatment varied from 1.5 weeks to 11.5 weeks. The interaction between voriconazole and calcineurin inhibitors led to peak CNI levels on day 3 of treatment. 87 were successfully treated, 70 lung (81%) and 17 heart (77%). 21 were unsuccessful. In lung transplant recipients, most infections occurred either early(during the first year) (30) or late after 5 years(36). Common symptoms in lung transplant recipients were fever (22) dyspnoea 35, cough 25, purulent sputum 12. 14 patients had a fall in FEV1. Conclusions: We found a lower mortality from fungal infections treated with voriconazole and caspofungin than that reported with after treatment with amphoterecin. Our data supports a strategy of early and aggressive treatment based on standard criteria.

Aspirin Non-responders in Thai Ischemic Stroke/TIA Patients DHARMASAROJA P Faculty of Medicine, Thammasat University, Pathumthani, Thailand Background: Aspirin resistance has been defined as inability of aspirin to protect individuals from thrombotic complications or to produce an anticipated effect from laboratory tests of platelet function. Most reported information comes from Western patients with coronary artery disease and aspirin resistance is defined by laboratory criteria. The purpose of the study was to look for aspirin non-responders in Thai patients who presented with acute/subacute ischemic stroke and transient ischemic attack (TIA). Methods: We prospectively included acute ischemic stroke/ TIA patients who were treated at Thammasat hospital during August, 2006- July, 2007 and had already been on aspirin. Information about compliance of medication, reasons for taking aspirin, doses of aspirin, baseline characteristics, stroke subtypes of the patients were collected. Results: There were 194 acute/subacute ischemic stroke/TIA patients during the study period. Forty-six patients (23.7%), who had already been on aspirin (aspirin non-responder) while having new stroke/TIA, were studied. Eighteen patients were on aspirin 300-325 mg and 28 patients were on 81 mg per day. Most patients have taken aspirin 300-325mg/day as secondary prevention, while half of patients taking aspirin 81 mg/d had diabetes mellitus and took aspirin as primary prevention. Conclusions: Aspirin non-responders are more common than we previously thought. Future study is required to clarify mechanisms of aspirin non-responders in Thai patients.

Targeting Mutated Janus Kinases In Myeloproliferative Neoplasms Drug Discovery DIACONU CC^1, DUSA A^2, PEQUET C^2, CHIVU M^1, DRAGOMIR L^1, CONSTANTINESCU SN^2 ^1Stefan S. Nicolau, Institute of Virology, Bucharest, Romania; ^2Ludwig Institute for Cancer Research Brussels Branch, Christian de Duve Institute, Universite Catholique de Louvain, Brussels, Belgium. Background: Janus kinases (JAK) are protein tyrosine kinases which play a crucial role in controlling many cellular processes; dysregulation of JAK expression and activity leads to different disorders as leukemia and lymphoma, auto-immune diseases, and myeloproliferative neoplasms (MPN). Since 2005, different mutated JAK were characterized. A unique mutation in JAK2, leading to a V617F substitution, is a major molecular event in >95% of the Polycythemia Vera (PV), 50% of the essential thrombocythemia (ET), and 50% of the primary myelofibrosis (PMF) patients. No specific therapy exists against any of these diseases. Especially for PMF, the development of a specific therapy would be useful since the evolution of this disease is usually unfavorable. <30% of PV and ET cases evolve towards PMF. All three can evolve towards acute myeloid leukemia. Thus, the JAK2 V617F offers a molecular target for drug discovery. It remains a significant challenge to develop selective inhibitors for JAK given their homology and potential structural plasticity. Since the wild type (wt) JAK2 is important for red blood cell formation and for the action of several cytokines and hormones, ideally, an inhibitor should target selectively JAK2 V617F and not wt JAK2. Methods: Our strategy involved profiling a collection of 1,980 small-molecule compounds from Developmental Therapeutics Program NCI/NIH in a dose–response format against a panel of three JAK dependent cellular assays using growth inhibition screening and overall inhibition of ATP production. Our hits were subsequently analyzed in apoptosis assays, and genetic reporter assay in order to identify better candidates for leads development. Results: Among 2000 selected small molecules, 7% inhibited proliferation of cells driven by JAK2 V617F, but about 5% also inhibited proliferation of the cells driven by JAK2wt and by JAK1 V658F, a constitutively active JAK1 that harbors the homologous V617F substitution of JAK2. 2% of the compounds showed <5 fold selectivity for the cells expressing JAK2 V617F. Conclusions: All JAK2 V617F potential inhibitors respect Lipinsky's “Rule of five” and the ”New Lead-likeness Rule” and may have potential for further development.

Chloroquine is Therapeutic in Murine Experimental Model of Paracoccidioidomycosis DIAS-MELICIO LA, CALVI SA, BORDON AP, GOLIM MA, PERAÇOLI MTS, SOARES AMVC. Department of Microbiology and Immunology, Biosciences Institute, São Paulo State University, Botucatu, São Paulo, Brazil. Background: Chloroquine, due to its basic properties, has been shown to prevent release of iron from holotransferrin, thereby interfering with normal iron metabolism in a variety of cell types. Methods: Thus, we have studied the effects of chloroquine on the evolution of experimental paracoccidioidomycosis by evaluating the viable fungal recovery from lung, liver and spleen from infected mice and H2O2, NO production, TNF-alpha, IL-6, IL-10 levels by ELISA and transferrin receptor (TfR) expression by flow cytometry from peritoneal macrophages from uninfected and infected mice. Resuts: Chloroquine caused significant decrease in the viable fungal recovery from all organs tested, during all periods of evaluation. Peritoneal macrophages from chloroquine-treated infected mice showed higher H2O2 production and TfR expression, and decreased levels of NO, endogenous and stimulated-TNF-, IL-6 and IL-10 during the three evaluated periods. Chloroquine-treated infected mice macrophages showed a decrease in NO levels that promoted an increase in TfR expression probably through NO modulatory effect on the iron-regulatory proteins (IRP-1 and IRP-2), which are cytoplasmic proteins responsible for controlling cellular iron storage and uptake by interaction with specific nucleotide sequences, called iron-responsive elements (IREs). Conclusions: Our findings together supported the idea that therapeutic effects of chloroquine on murine experimental paracoccidioidomycosis is due its capacity to interfere with intracellular iron availability that disabled its uptake by the fungus with consequent fungal death, as well as by its suppressor effects on the macrophage functions. Up to now, no data are available on the possible antifungicidal effect of chloroquine in the clinical situation. Thus, chloroquine could be utilized as a potential drug, which could be administrated in association with conventional paracoccidioidomycosis treatment. Financial Support: FAPESP (grant n° 02/03680-8). e-mail: [e-mail]

Cross-Reactivity and Identification of T and B Epitopes in Plant Food Allergens: Peach Pru p 3 as a Model DÍAZ-PERALES A^1*, TORDESILLAS L^1, SANCHEZ-MONGE R^1, SALCEDO G^1 ^1Departamento de Biotecnología, ETS Ingenieros Agrónomos, Universidad Politecnica, Madrid, Spain. *[e-mail] FAX: [phone] Background: Plant non-specific lipid transfer proteins (LTPs) are plant defence proteins that constitute a relevant panallergen family present in both plant foods and pollens. Their high resistance to proteolytic digestion, thus being probably primary sensitizers by ingestion, and association with systemic and severe clinical symptoms, has led to proposed LTPs as a model of true food allergens. Peach Pru p 3, the prototypic member of the family, has been extensively studied at the biochemical, immunological and clinical level. Methods: Crossreactivity among LTPs was analyzed by specific IgE-quantification and ELISA-inhibition assays. Both sequential and conformational B-epitopes of Pru p 3 were defined by analyzing the IgE-binding capacity of synthetic peptides spanning the full Pru p 3 sequence covalently bound to cellulose membranes, and of a random peptide phage display library (mimotopes), complemented by 3D modelling of the allergen. T-cell epitopes of Pru p 3 were identified by testing the proliferation responses of peripheral blood mononuclear cells and Pru p 3-specific T-cell lines to synthetic peptides covering the entire Pru p 3 amino acid sequence. Results: Different degrees of cross-reactivity were found between Pru p 3 and other allergens belonging to the LTP family. Comparison of amino acid sequences and 3D structures of these allergenic LTPs suggested the presence of both common IgE epitopes and relevant specific IgE-binding regions different for each allergen. Two principal B-epitopes, comprising residues 31 to 46 and 70 to 80, were located in the Pru p 3-surface. On the other hand, a major T-cell epitope, Pru p 365-80, was identified. Conclusions: In vitro and structural studies have helped to understand cross reactivities among LTP allergens that induce the so call ‘LTP syndrome’. The identification of B- and T-cell epitopes of Pru p 3 can accelerate the process to design new vaccines and new immunotherapy strategies for patients suffering such ‘LTP syndrome’.

Success story of the first regulatory approval of safety biomarkers, Part II: Consortia, regulatory submissions and translation to human DIETERLE F, MARRER E Novartis Institutes for BioMedical Research Background: A number of promising new renal injury biomarkers have been identified and are now becoming more and more popular not only in the literature but also in pharmaceutical and academic laboratories. Yet these biomarkers are not accepted by health authorities for regulatory decision making such as changing the treatment regimen or selecting patients for treatment. Methods: To demonstrate the superiority of new urinary biomarkers in comparison to the current standards serum Creatinine and BUN, a consortium was founded (Predictive Safety Testing Consortium, PSTC) and data were exchanged between 16 pharma companies and academia involving 22 pre-clinical studies and 7 urinary renal safety biomarkers in a first round. For a maximum level of compariblity, different consortium standards needed to be created, such as a histopathology lexicon, data processing and statistics. Furthermore, published and contributed clinical data were systematically reviewed to be able to translate the biomarker to human. Results: The 7 new biomarkers outperformed the current standards for identifying and monitoring tubular and glomerular injury. Together with the FDA/EMEA, a process was defined, how these biomarkers can be submitted for regulatory assessement and approval in a so-called VXDS (Voluntary eXploratory Data Submission). Conclusions: This story highlights the first successful approval of safety biomarkers by FDA/EMEA and triggered the establishment of standards and of a submission process and qualification process for safety biomarkers. The renal biomarkers submitted were approved by the FDA/EMEA for a regulatory use in a pre-clinical rat GLP study context. In addition, the use of renal biomarkers in clinical trials is to be considered on a case-by-case basis in order to gather further data to qualify their usefulness in monitoring drug-induced renal toxicity in man.