Ehrlich II –2nd World Conference on Magic Bullets

3-O-Methylfunicone, A Fungitoxic And Antitumor Extrolite Produced By Penicillium Pinophilum NICOLETTI R^1, DE STEFANO M^2, BUOMMINO E^2, CIAVATTA ML^3, TUFANO MA^2 ^1Council for Research and Experimentation in Agriculture (CRA), Scafati, Italy; ^2The Second University of Naples, Italy; ^3CNR-Institute of Biomolecular Chemistry, Pozzuoli, Italy. The soil fungus Penicillium pinophilum has been described as an ecological antagonist and a mycoparasite of the widespread plant pathogen Rhizoctonia solani. Its antifungal properties have been reported in relation to the production of 3-O-methylfunicone (OMF), an extrolite characterized by liquid cultures of an isolate of this species (LT4) that is patented by CRA (Italian patent no. 01308189, 2001). OMF is able to inhibit hyphal growth of R. solani and other plant pathogenic and dermatophytic fungi in vitro at a concentration of 0.1 mg/ml. Fungitoxicity is also evident after treatment of actively growing cultures, where the integrity of the hyphal structure appear to be compromised, cells collapse, and their membranes are degraded. In addition, antiproliferative and pro-apoptotic properties have resulted on several human tumour cell-lines (Hep-2, HeLa, MCF-7, A549, A375M). Particularly, the compound is responsible of a cytostatic effect associated to evident morphological changes and modifications in the organization of tubulin fibres. In fact, chemical structure of OMF is based on a substituted -pyrone ring connected to a tri-methoxylated aryl nucleus; this moiety, which also characterizes other known antitumor compounds such as combretastatin, staganacin, the podophyllotoxins, and the chalcones, is thought to interact with tubulins by binding at their sulphydryl groups. OMF also affects expression of genes involved in the cell cycle, which is arrested in the G1 phase, with a significant increase in p21 mRNA expression, and a decrease in cyclin D1 and Cdk4 mRNA. Apoptosis induction has been confirmed by DNA-laddering, annexin assay, and cytofluorimetric analysis of the DNA content of the sub-G1 fraction; the triggered apoptotic pathway is p53 independent. In MCF-7 cells, pro-apoptotic effects also depend on a down-regulation of tumor-associated antigens, such as survivin and hTERT. Finally, anti-metastatic effects may derive by the ability of OMF to affect cell motility, in connexion with down-regulation of v5 integrin and inhibition of matrix metalloproteinases. The reported effects of OMF are indicative of a potential for the development of a new agent for cancer chemotherapy.

Hormone-Immunotherapy Significantly Prolongs Clinical Benefit And Median Overall Survival Of Metastatic Endocrine Dependent Breast Cancer Patients NICOLINI A^1, FERRARI P^1, ANSELMI L^1, CONTE M^1, BERTI P^1, MICCOLI P^1 ^1University of Pisa, Pisa, Italy Background: Metastatic disease from breast cancer is incurable. In endocrine-dependent patients antiestrogens are commonly administered as first and second line therapy. Regrettably, tumour growth becomes resistant to this relatively well tolerated therapy. In the past, beta-interferon was added to tamoxifen to induce estrogen receptor enhancement. However, clinical data did not significantly confirm experimental data. In mice, interleukin-2 added to tamoxifen increased their mutual antitumor activities. Nevertheless, no effective clinical application has been developed. We started an exploratory clinical trial based on the association of these immunostimulating cytokines with antiestrogens for first line salvage therapy of hormone dependent metastatic breast cancer. Methods: Thirty three consecutive breast cancer patients with distant metastases, most of them with involvement of multiple organs, were studied for responsiveness to treatment with first-line salvage anti-estrogen therapy, combined with beta-interferon and interleukin-2 immunotherapy. Clinical response and survival were compared with that of 30 consecutive historical control patients treated with anti-estrogen therapy alone. Results: Controls showed, as expected, a median duration of response, a median survival time after treatment, and after diagnosis of distant metastases, of 16, 31 and 34 months, respectively. After a mean follow-up of 74 + 39 months (range 24-209) from the beginning of first line antiestrogen salvage therapy, the interval times in the studied patients were 33 (p<0.001), 74 (p<0.001) and 79 (p<0.001) months. One long-term survivor appeared to be cured after 155 months from the time of diagnosis with multiple bone metastases. Eleven (33%) of the patients treated with beta-interferon and interleukin-2 have survived. Conclusion: These data suggest that immunotherapy, given in an outpatient setting in addition to conventional antiestrogen salvage therapy, is very well tolerated and provides an important benefit in endocrine-dependent metastatic breast cancer.

Corpora Non Agunt Nisi Fixata: Tailored Drug Targeting By Intelligent Active Principles Or Intelligent Transporters, State Of Art And Our Experience Nieddu E^1, Pasa S^2, Avalle L^1, Merello L^1, Sanna VA^3, Brigati C^2 ^1Univ. of Genoa, Genoa, Italy; ^2Nat Cancer Inst, Genoa, Italy; ^3Prion DGN, Alghero (SS), Italy. Background: Matter: insufficient therapeutic index and too big gap between research and clinic for certain diseases. Aims: integrate different scientific knowledge (molecular interaction maps, peptide interfering with protein-protein interaction, nanotechnology) to obtain smart active drugs or smart transporters of active drugs saving healthy cells and killing altered ones. Methods: 1) synthesis of inorganic nanoshells incorporating commercially available drugs, functionalization with docking peptide, binding with specific antibody. 2) serum derivative given in vivo to animals and then to patients (pz), 2 phials/die for 10 days (case A orthopaedics 20 pz, case B surgery 339 pz, case C leprosy 20 pz). In vitro every 24h in monoculture of fibroblasts and fibrosarcoma and in co-culture 1:1 of them. Number of each cell line was registered for monoculture and ratio between normal and cancer cells was considered for co-culture (I). Results: 1) final drug-delivery construct in fig 1A (not scale). 2) 100% atoxic in animals and pz evident by unaltered haematic parameters and absence of reactive effects. Anti-inflammatory, anti-exudative, anti-infective, antalgic and healing activity in 100% case A , 89% case B, significant improvement in 100% case C. In vitro original activity: no difference between treated and not treated cells for monocultures, whereas inversion in ratio (I) was clear in co-culture as shown in fig 1B. Conclusions: 1) we intend to rapidly commercialize our intelligent drug delivery system born from interdisciplinarity 2) strict interaction between clinic and research gave a promising intelligent drug, but we need enlightened scientists of laboratories to go ahead. Authors’ disclosure statement: This presentation both gives a general view of the state of the art in tailored therapies, especially in cancer, and in part contains our novel and unpublished data. A clarification: we are working without any specific funding, also with our private money, often adopting unconventional and simple reasoning, against the indifference and unintelligible hostility of many colleagues and superiors, but we are sure we must use all the knowledge, even not standard, against still incurable diseases. We invite enlightened scientists to collaborate for a serious, scientific and exciting research. Thanks.

The Potential Of Oxoglaucine, A New Antiviral Compound, To Inhibit Enterovirus Replication When Applied Alone Or In Combination Nikolaeva-Glomb L^1, FilipovS^2, Galabov AS^1 ^1The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria ^2Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria Background: Despite the fact that enteroviruses are implicated in a variety of diseases, there is no drug for clinical use yet. A clear need exists for development of new antivirals and for new approaches for application of the already known ones. Aims: 1) To characterize the antiviral parameters in vitro of oxoglaucine, a novel inhibitor developed in our laboratory. 2) To combine oxoglaucine and disoxaril in search of a synergistic combination. Methods: The cytopathic effect inhibition test and the plaque-inhibition assay were used for evaluation of the antiviral effect in vitro. The mode of action was revealed by time-of-addition tests. Resistant progeny was derived by serial passages in the presence of the compound. The combined antiviral effect in vitro of oxoglaucine and disoxaril was determined by the 3D model for analyzing drug interactions of Prichard and Shipman. Results: Oxoglaucine inhibited the replication in vitro of 16 enteroviruses exerting a strong antiviral effect on all of them with a selectivity index greater than 100 for the most of the tested viruses. Time-of-addition study showed that the susceptible period was the latent and the lag phase of the virus cycle. Resistant virus strains were derived for poliovirus type 1 and the six coxsackie B viruses. A correlation was established between the sensitivity and the necessary number of passages in the presence of the compound for selection of resistant progeny. Reversion to sensitivity occured when the selective pressure of oxoglaucine was diminished. Evaluation of the combined effect of oxoglaucine and disoxaril revealed that the effect was additive and at some dose combinations it was moderately synergistic. Conclusions: 1) Oxoglaucine is a newly developed antiviral compound with a strong and selective antienterovirus activity. 2) Viruses which reveal the greatest sensitivity to oxoglaucine develop most rapidly resistant progeny. 3) The combination of oxoglaucine and disoxaril is additive with areas of moderate synergy. 4) There is a promise that oxoglaucine, alone or in combination with other antiviral agents, could be developed to a “magic bullet” for the treatment of enterovirus infections.

Modulation Of The Structure Of A Lipid Membrane For Selective Interactions With A Drug NIKOLELIS DP, NIKOLELI GP, RAFTOPOULOU G, PSAROUDAKIS N Laboratory of Environmental Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis-Kouponia, Athens, Greece This presentation will show how the structure of a lipid film can be modified to respond selectively to a specific drug. For this purpose, microporous filters composed of glass fibers were used as supports for the stabilization of lipid films. The lipid films were formed on the filter by polymerization using UV radiation. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2-azobis-(2-methylpropionitrile) was the initiator. Proteins [i.e., enzymes (i.e.,acetylcholinesterase, urease, etc) and natural receptors (ABP1, etc)] were incorporated within this mixture prior to polymerization and the results showed that these proteins retain their activity for repetitive uses. This method for preparation of stabilized lipid membranes was investigated using Raman spectroscopy. The results have indicated that the kinetics of polymerization are completed within 4 hours. These lipid films were used to study the interactions of several drugs (i.e., atenolol) and other compounds.(i.e., sweeteners) with bilayer lipid membranes.( BLMs). The interactions of atenolol and artificial sweeteners with BLMs produced transient electrochemical ion current signals that reproducible appeared within a few seconds after the exposure of the membranes to the drug.or the sweetener. The current signals were related to the concentration of atenolol (20 to 200 μM) in electrolyte solution ssDNA incorporated into BLMs can interact with atenolol, and decreased the detection limit of this drug by one order of magnitude. The oligomers used were single stranded deoxyribonucleic acids: thymidylic acid icosanucleotide terminated with a C-16 alkyl chain to assist incorporation into s-BLMs (5′-hexadecyl-deoxythymidylic acid icosanucleotide, dT20-C16). The selectivity of the lipid films for the direct interaction with a drug can only partly be modulated by modification of the composition of the lipid film. Further results will also present the interactions of lipid films with incorporated artificial receptors (i.e., resorcin[4]arene or methoxy-calixerene receptor) with doping materials (i.e., adrenaline, dopamine and ephedrine). These BLMs modified with the resorcin[4]arene receptor were used as sensors for the direct electrochemical sensing of these stimulating substances. The interactions of these compounds with the lipid membranes were found to be electrochemically transduced in the form of a transient current signal with a duration of seconds, which reproducibly appeared within a few s after exposure of the membranes to these doping materials.. The selectivity of response could be modulated with an alteration of the structure of the artificial receptor The mechanism of signal generation was investigated by differential scanning calorimetric studies. These studies revealed that the adsorption of the receptor is through the hydrophobic tails of the receptor, whereas hydrophilic groups of the receptor were directed towards the electrolyte solution enhancing the ion transport through the lipid membranes. Raman spectroscopy was further used to investigate the location of the proteins into the structure of the lipid films and how are attached with the bilayer lipid membranes. This will allow the practical use of the techniques for the preparation of stable lipisomes with an incorporated drug that can selectively b e directed to a specific target.

A Novel And Effective Antibody Therapy For Brain Infarction Targeting Alarmin/HMGB1 NISHIBORI M^1, LIU K^1, TAKAHASHI HK, WAKE H^1, ZHANG J^1, TOMONO Y^4, ADACHI N^5, YOSHINO T, OHTSUKA A, MORI S^1,6 Department of Pharmacology^1, Pathology^2 and Human Morphology^3, Okayama Univ Grad Schl Med, Okayama, Japan; ^4Shigei Medical Inst, Japan; ^5Ehime Grad Schl Med, Matsuyama, Japan; ^6Shujitsu Univ Schl Pharmacy, Okayama, Japan Background: The high mobility group box-1 (HMGB1), originally identified as an architectural nuclear protein, exhibits an inflammatory cytokine-like activity in the extracellular space. HMGB1 is released from necrotic cells and activated macrophages in different manners. The cytokine profile of HMGB1 has shed light on the role of nuclear proteins and promoted the studies on roles of this unique factor in different disease conditions such as sepsis, acute lung injury, ischemic liver injury and arthritis. In the present study, we examined the possible involvement of HMGB1 in the development of brain infarction in middle cerebral artery occlusion (MCAO) rats using neutralizing anti-HMGB1 monoclonal antibody (mAb). Methods: MCAO was performed by the insertion of silicone-coated 4.0 nylon thread from the bifurcation of the internal and external carotid arteries under anesthesia. After 2 hour occlusion, reperfusion was started. Intravenous injection of mAb was done twice immediately and 6 h after reperfusion. Results: Treatment with neutralizing anti-HMGB1 mAb remarkably ameliorated brain infarction induced by 2-hour MCAO in rats, even when the mAb was administered after the start of reperfusion. Consistent with the 90 % reduction in infarct size detected by TTC staining, the accompanying neurological deficits in locomotor function were significantly improved. Anti-HMGB1 mAb inhibited the increased permeability of the blood-brain barrier, the activation of microglia, the expression of TNF-alpha and iNOS and the morphological changes in BBB at electron microscopic levels, whereas it had little effect on blood flow. Conclusions: These results as a whole indicate that HMGB1 plays a critical role in the development of brain infarction through the amplification of plural inflammatory responses in the ischemic region and could be an outstandingly suitable target for the treatment. Intravenous injection of neutralizing anti-HMGB1 mAb provides a novel therapeutic strategy for ischemic stroke and may make an innovation in the treatment.

Cryoimmunology Induced After Reimplantation Of Malignant Bone Tumor Treated With Liquid Nitrogen NISHIDA H^1,2, TSUCHIYA H^1, KAWANO M^1, TOMITA K^1 ^1Kanazawa Univ., Kanazawa, Japan; ^2Fujii Hosp., Kanazawa,Japan Background:1) In the laboratory, systemic antitumor responses following reimplantationof tumor treated with liquid nitrogen +/- dendritic cells were evaluated by measuring immune reactions and counting distant metastases in mouse osteosarcoma models. 2) In the clinical setting, immunological cytokines were measured after reconstruction with liquid nitrogen-treated autografts. Methods: 1) LM8 mouse osteosarcoma cells were implanted subcutaneously into C3H mice. After two weeks, tumors were treated with a) excision (E) (n=15); b) liquid nitrogen (LN) (n=15); or c) liquid nitrogen plus dendritic cells (DCs) (n=15). Four weeks later, serum IFN-γ levels, lung metastases area, and CD8(+) T-lymphocyte counts in lung metastases were measured. 2) IFN-γ and IL-12 levels were measured in blood samples collected before surgery and one and three months after surgery from 23 patients with malignant bone tumors treated with tumor-bearing autograft frozen by liquid nitrogen. Results: 1) Mean serum IFN-γ level was significantly higher in DCs (118.29 ± 5.71; p<0.01) than in both LN (37.22 pg/ml ± 2.74) and E (8.25 ± 2.76); LN was significantly higher than E (p < 0.05). Mean metastasis area was significantly smaller in DCs (5.39 ± 1.49; p < 0.01) than in both LN (13.21m^2 ± 2.59) and E (24.12 ± 3.60); LN was significantly smaller than E (p < 0.01). Mean number of CD8(+) T-lymphocytes was significantly higher in DCs (8.23 ± 2.56; P < 0.05) than in both LN (2.46 cells/mm^2; ± 0.57) and E (0.64 ± 0.56); LN was significantly higher than E (p < 0.01). 2) Mean INF-γ relative concentration of one month after and three months after against before surgery were 149.7 and 268.3%, and mean IL-12 relative concentration were 170 and 432.2%, respectively. Composite values for all 23 patients showed progressive increases in INF-γ and IL-12 levels one and three months after surgery. Conclusions: 1) Treatment of tumor tissue with liquid nitrogen can activate the immune system and inhibit metastatic tumor growth. Dendritic cells enhanced immunological activity synergistically. 2) Patients with malignant bone tumors who received autografts treated with liquid nitrogen showed evidence of immune system activation. 3) These responses suggest that liquid nitrogen treatment of bone tumors may offer certain unique benefits.

Effect Of Insulin Resistance On In-Stent Restenosis After Coronary Stenting In Type 2 Diabetic Patients NISHIO K^1^,2, KOBAYASHI Y^1 ^1Univ. SHOWA, Tokyo, Japan; ^2Yamanashi red-cross Hosp, Yamanashi, Japan Background: Metabolic syndrome is related to cardiovascular diseases. Insulin resistance is one of causes for metabolic syndrome. The aim of this study was to elucidate the mechanism of the effect of insulin resistance on in-stent restenosis after coronary stenting in patients with type 2 diabetes mellitus. Methods: A prospective, randomized trial, involving 54 type 2 diabetic patients referred for coronary stenting who were randomly assigned to either the control or the pioglitazone group, was performed. Quantitative coronary angiography was performed at study entry and at six months follow-up. Endothelial nitric oxide Synthase (eNOS), tumor necrosis factor α, interleukin-6, leptin, and adiponectin were measured at study entry and at six months follow-up. Results: 28 patients were randomly assigned in the control group and 26 patients were assigned in the pioglitazone group. There were no significant differences in glycemic control levels or in lipid levels in the two groups at baseline or at follow up. Insulin, homeostasis model assessment insulin resistance, eNOS, and leptin at follow up were significantly reduced in the pioglitazone group compared with in the control group. The late luminal loss and in-stent restenosis were significantly less in the pioglitazone group than in the control group. Multiple regression analysis showed that leptin independently correlated with late luminal loss. Conclusions: The treatment with pioglitazone in type 2 diabetic patients significantly reduced leptin. This decreased leptin improved insulin resistance and endothelial function with the reduction of insulin. The improved endothelial function affected the reduction of in-stent restenosis.