Ehrlich II –2nd World Conference on Magic Bullets

Conclusion: This trimodal treatment leads to excellent response rates at irradiated soft tissue sarcomas, it suppresses distant metastases and prolongs survival.

Methicillin Resistance In Staphylococcus Aureus And Coagulase-Negative Staphylococci RIBEIRO de SOUZA da CUNHA ML, MARTINS A, USTULIN DR, PEREIRA VC Instituto de Biociências, Universidade Estadual Paulista - UNESP, Botucatu-SP, Brasil Background: Staphylococcus species are divided into coagulase-positive staphylococci, represented by S. aureus and coagulase-negative staphylococci (CNS). Oxacillin has been one of the main drugs used for the treatment of staphylococcal infections; however, a large number of S. aureus and CNS isolates of nosocomial origin are resistant to this drug. Methicillin resistance is encoded by the mecA gene which is inserted in the SCCmec cassette. Aims: 1) To detect the resistance to meticillin in S. aureus and CNS isolates from the Clinical Hospital of Botucatu Medical School, UNESP, Brazil, 2) To determine the minimum inhibitory concentration (MIC) to erythromycin, netilmycin, sulfamethoxazole-trimethoprim and vancomycin in S. aureus and CNS strains by the E-test technique. Methods: A total of 150 samples were analyzed regarding methicillin resistance by detection of the mecA gene, agar diffusion technique using cefoxitin and oxacillin disks, screening test on Mueller-Hinton agar supplemented withoxacillin 6 µg/mL and 4% sodium chloride and E-test. Results: A total of 102 isolates were identified as S. aureus and 48 as CNS. A total of 46 (45.1%) S. aureus and 37 CNS (81.2%) were mecA-positive. S. epidermidis was the most frequently isolated CNS species corresponding to 87.5% of all CNS strains investigated and 83.3% were mecA-positive. S. aureus and CNS also were tested in relation to erythromycin, with 56 (54.9%) and 31 (64.6%) isolates resistant to this drug, netilmycin, with 42 (41.2%) and 30 (62.5%) isolates resistant, trimetoprim-sulfametoxazol with 46 (45.1%) and 35 (72.9%) isolates resistant, respectively. All the isolates of S.aureus were sensitive to vancomycin and 12(25%) of CNS were vancomycin-intermediate. Conclusions: 1) Most of the strains of S. aureus and CNS was mecA-positive. 2) S. epidermidis was the specie with a higher percentage of resistance to methicillin. 3) Among phenotypic methods, E-test yielded the best results in the detection of methicillin resistance. 4) The distribution of resistance to drugs showed difference between methicillin-resistant Staphylococcus and methicillin-sensible isolates, with higher percentages of resistance to erythromycin, netilmycin and trimetoprim-sulfametoxazol in methicillin-resistant strains. Financial support: FAPESP and FUNDUNESP	Coagulase-Negative Staphylococci Oxacillin Resistant And Toxigenic Isolated In Brazil RIBEIRO de SOUZA da CUNHA ML, CALSOLARI RAO, ARAÚJO-JÚNIOR JP Instituto de Biociências, Universidade Estadual Paulista - UNESP, Botucatu-SP, Brasil Background: Oxacillin resistance in isolates of coagulase-negative staphylococci (CNS) have emerged over the last years in neonatal intensive care units. Aims: 1) To identify CNS species. 2) To detect the resistance to meticillin in CNS strains. 3) To detect gene and mRNA expression of toxins by RT-PCR in clinical samples obtained from newborns hospitalized at the Neonatal Unit, University Hospital, Botucatu Medical School, Brazil. Methods: In the present study, 90 CNS strains isolated from different clinical materials of newborns were investigated for the testing of susceptibility to oxacillin and by PCR for the presence of genes encoding staphylococcal toxins A (sea), B (seb), C (sec-1) and D (sed) and TSST-1 (tst). Strains positive for the presence of one or more genes were tested by RT-PCR for the expression of mRNA encoding the respective toxins. Results: S. epidermidis was the most frequently isolated organism, corresponding to 71.1% of all CNS strains investigated and 63.5% showed oxacillin resistance. The results showed a total of 49 (45.4%) CNS strains that were positive by PCR and 65.3% were oxacillin resistant. Analysis of mRNA expression by RT-PCR detected six (14.0%) CNS strains producing SEA and SEC, with 83.3% these isolates presenting resistance to oxacillin. Conclusions: 1) S. epidermidis was the most frequently isolated CNS species. 2) All strains identified as CNS by the phenotypic technique were confirmed by the genotypic method. 3) The toxin genes sea, seb, sec-1 and/or tst were detected alone or in combination in all CNS species isolated, except for S. simulans, with the most found to be oxacillin resistant. 4) Analysis of the production of staphylococcal toxins in CNS by RT-PCR confirmed the toxigenic capacity of S. epidermidis and S. lugdunensis. 5) Most of the S. epidermidis that was positive for expression of mRNA that encode staphylococcal toxin were found to be oxacillin resistant. Financial support: FAPESP and FUNDUNESP
Pharmacokinetics (PK) And Pharmacodynamics (PD) Of Fosfomycin (FOF) For Central Nervous System (CNS) Infections RIBES S^1, CABELLOS C^2, GUDIOL F^2, NAU R^1,3 ^1Department of Neurology, University of Göttingen, Göttingen, Germany; ^2Laboratory of Experimental Infection, Infectious Diseases Service, IDIBELL, Hospital Universitari de Bellvitge, Feixa Llarga s/n, L’Hospitalet, Spain; ^3Department of Geriatrics, Evangelisches Krankenhaus Göttingen-Weende, Göttingen, Germany Background: The emergence of infections caused by multiresistant bacteria call for “new” therapies. FOF could be used as part of a combined therapy for CNS infections since it shows favourable PK properties such as low molecular mass, negligible protein binding and good penetration into the uninflamed and inflamed cerebrospinal fluid (CSF). On the other hand, PD data of FOF in the CNS are scarce. Methods: We conducted a selective literature research to compile in vivo PK/PD data of FOF in CNS infections. Results: Few reports have described PD data of FOF in CNS infections in humans and in animal models. FOF peak CSF levels, half life (t ½) and penetration into CSF are shown in the table. In the rabbit meningitis model, a bactericidal effect on a penicillin-susceptible and a cephalosporin-resistant Streptococccus pneumoniae was only reached when FOF levels in the CSF were 8-10 times higher than the MIC value of tested strains (Nau R, et al, J. Antimicrob. Chemother. 1995; Ribes S, et al, J. Antimicrob. Chemother. 2006). Dose Peak CSF levels (g/ml) t ½ CSF (h)/ t ½ serum (h) % Penetration AUCCSF/ AUCserum Reference Human 5 g/8h 45.5 24.03/3.43 45.21* Friedrich H, et al, Immun.Infekt.1987 5g single dose 10. 1± 1.5 ND/2 9.3 Pfeifer G, et al, J. Clin. Pharmacol. Res. 1985 8g single dose 43 ± 20 ND/3.0 23 Pfausler B, et al, J. Antimicrob. Chemother. 2004 8g/8h for 3d 62 ± 38 ND/4.0 27 Rabbit 300 mg/kg/6h 58.40  21.81 5.01/3.09 49.2 Ribes S, et al, J. Antimicrob. Chemother. 2006 * calculated from reported data Conclusions: The MIC of the causative strain is of paramount importance for the PD of FOF in CNS infections since the achievement of appropriate CSF drug concentrations are associated to clinical outcome.	Asparagine Synthetase Inhibitors With Nanomolar Potency: An Unexplored Approach To Treating Drug-Resistant Leukemia RICHARDS NGJ^1, GUTIERREZ-AMOS JA^1, PAN XY^1, MEYER ME^1, HUMKEY RN^1, KILBERG MS^1, HIRATAKE J^2 ^1University of Florida, Gainesville, USA; ^2Institute for Chemical Research, Kyoto, Japan Background: Clinical studies have identified an intriguing inverse correlation between the levels of asparagine in the blood and the susceptibility of leukemia cells to chemotherapy. Leukemia cells seem to become drug-resistant by over-expressing asparagine synthetase (ASNS), an enzyme that catalyzes the intracellular synthesis of asparagine. Compounds that inhibit human ASNS represent potential drugs for treating acute lymphoblastic leukemia (ALL). Aims: 1) To examine the effects of a potent ASNS inhibitor on the growth of a MOLT-4 cell line. 2) To develop an atomistic understanding of interactions formed between the inhibitor and ASNS. Methods: The ASNS inhibitor was incubated at 0.1-1.0 M concentration with asparaginase-resistant MOLT-4 cells, which exhibit unregulated levels of ASNS expression. Cell viability was determined after 48 h using a WST-1 proliferation assay. The mean cell titer of treated samples was calculated, as the mean  SD of triplicate experiments, relative to control cells. A model of the ASNS/inhibitor complex was built by homology to the structure of Escherichia coli AS-B, with the inhibitor being placed in the synthetase active site using flexible docking and molecular dynamics methods. Site-specific mutants of Glu-348 in Escherichia coli AS-B were expressed and purified by standard methods, and assayed using both steady-state kinetics and NMR-based ^18O-transfer experiments. Results: In the presence of L-asparaginase, the ASNS inhibitor suppressed the proliferation of an asparaginase-resistant MOLT-4 cell line in a dose-dependent fashion. Molecular docking gave a model for the ASNS/inhibitor complex showing an interaction between a conserved glutamate side chain and a critical methyl group on the inhibitor. Assays of AS-B mutants in which this glutamate is replaced by aspartate and alanine support the assignment of Glu-348 as a general base thereby validating the computational model of the ASNS/inhibitor complex. Conclusions: 1) This study is the first direct demonstration that ASNS inhibitors can suppress proliferation of a drug-resistant MOLT-4 cell line in a dose-dependent manner. 2) The ASNS/inhibitor model provides a firm basis for discovering novel compounds with improved activity against ALL.
Liquid Chromatography-Mass Spectrometry; A Unique Tool In Drug Development And Patient Care Via Quality Assurance, Validation Of Biomarkers And Databases, Tissue Analysis, Assessing Prognosis And Monitoring Drug Interactions RITCHIE M^1,2, WONG V^3, GILMOUR A^4, GATES S^5, BREACH R^2,MORTON M^6 ^1Napier University, Scotland,^2Arbroath High School, Scotland, ^3Aberdeen University, Scotland, ^4Manchester University, England, ^5Cambridge University, England,^6Cardiff University, Wales Background: Liquid Chromatography-Mass Spectrometry (LC-MS) is a unique tool in drug development, biomarker validation, tissue analysis, assessing prognosis and monitoring drug interactions. This is due to the accuracy and reliability of LC-MS as an analytical technique and use of more sophisticated systems capable of analysing large numbers of samples. Methods: Initially our laboratory underwent quality assurance by analysing samples of urine and plasma for phytoestrogens (PE). Results were compared with other international institutions. As a consequence, we were certified for LC-MS analysis of PE internationally. A database of PE content of foods was validated by comparing estimated values from the PE database with LC-MS analysis of duplicate diets^1. Identification of biomarkers PE intake was completed by LC-MS analysis of duplicate diets and corresponding 24 hour urine collections and timed blood samples^2. Biomarkers of PE intake were validated over time using LC-MS analysis of timed spot urine samples over a six month period^3,4. The PE database was used to investigate the effect of PE intake on prostate cancer risk in men^5. Both the database and biomarkers of intake were used to monitor effect of PE intake on prognosis in breast cancer patients across Scotland^6 and to monitor potential effects of dietary PE on patients’ responses to cancer treatments. Uptake of PE in tumour and normal tissue was also measured^7. Results: We were the first group in the world to produce a validated PE database and validated biomarkers of PE intake using LC-MS and employ both in prospective and retrospective studies involving cancer patients. Conclusion: LC-MS is a unique tool for use in health care by identifying novel drugs, validating biomarkers and monitoring their actions and interactions in patients. 1) Ritchie M* Br J Nutr 2006 95:204-13. 2) Ritchie M* Brit J Nutr 2004 91: 447-457 3) Ritchie M* J E-B I Med 2004 1:2, 101-112 4) Ritchie M* Eur J Clin Nutr 2004 58:1286-9 5) Heald C, Ritchie M* Br J Nutr 2007 98:388-96 6) Gilmour A, Ritchie M* EMSA 2007 7) Ritchie M Melville Review 2007 *Et al	Receptors For Magic Bullets. Ehrlich, Precursor Of Receptor-Mediated Drug Action. Application To Age-Related Pathologies. A Review ROBERT L, LABAT-ROBERT J Ophtalmol. Res. Lab, Hotel Dieu Hosp, Univ Paris v, Paris, France Background: Medical pharmacology is based on receptor actions, using specific agonist and/or antagonist. The famous concept of Ehrlich:«corpora non agunt nisi fixata» can be considered as a founding principle of receptor theory. No drug, no magic bullet can act without beeing «recognized» by a specific molecule which mediates either its penetration in the cell of the transfer of a specific «message» to the cell interior. Without this concept no specific drug action could have been developed. Ehrlich’s concept developed in the frame of his immunological experimentation, «the side-chain» concept was progresively extended to nearly all drug-cell interaction. One of the consequences of this conceptual advance was the recent increase of life expectancy, below 50years at the turn of the 19th to the 20th century to above 80 years recently. Experimental basis:This progress was based on the application of Ehrlich’s original concept to the pharmacology of age-related diseases, cardiovascular and respiratory pathologies. More recently a number of receptors were shown to decrease with age and some others «uncoupled» in aging cells from their normal intracellular transmisssion pathway with loss of its beneficial effects (Robert L, J. Gerontol, 1998,44:307-317). An example is the elastin receptor recognizing elastin sequences and triggering a Calcium transient followed by modifications of several cell functions, chemotactic movements, NO release and vasodilation but also increase of elastase and free radical release. In «old» cells the Calcium transient is dampened, Cai increase producing apoptotic cell death (Robert L, Labat-Robert J, Biogerontology, 2000,1:123-131). The coupling of the elastin receptor to iNOS is interrupted (Faury et al, Mech. Age. Dev.,1997,95:31-42). We could show that these alterations play a critical role in the age-dependent decline of the cardiovascular and respiratory functions (Robert et al, Biogerontology, DOI:10.1007/s10522-007-9122-6) Conclusions: The original receptor concept of Ehrlich could be applied to aging biology and to its crucial components the mediation and modulation of the interactions of cells with macromolecules of the extracellular matrix. With this enlargement, Ehrlich’s concept could be applied to aging biology and pathology and pharmacology.
Piperacillin Dosing In ICU Patients – New Magic For Old Bullets ROBERTS JA^1, KIRKPATRICK CMJ^1, ROBERTS MS^1, LIPMAN J^1 ^1Univ. of Queensland, Queensland, Australia Background: The poor outcomes for intensive care unit (ICU) patients with sepsis require higher level research to be conducted to ensure antibiotic therapy is optimized. Altered dosing strategies may be the key to further improving patient outcomes. Aims: 1) To identify and analyze relevant pharmacokinetic studies of piperacillin in ICU patients. 2) To discuss pharmacodynamic optimization of piperacillin in ICU patients using ‘non-standard’ dosing strategies. Methods: Relevant articles were identified from searches of Pubmed and the extensive files of the authors including submitted and accepted original research articles not Pubmed listed. Pharmacokinetic parameters between different patient populations were identified and compared. The effect of different dosing strategies on achieving pharmacodynamic endpoints (free piperacillin concentration maintained above the minimum inhibitory concentration of bacteria for at least 50% of dosing schedule; 50% f T>MIC) was evaluated. Results: Piperacillin has been investigated in various different patient populations with differences noted between various pharmacokinetic parameters, particularly clearance and volume of distribution. Piperacillin has non-linear clearance, although this becomes effectively linear at the doses used clinically in ICU patients.. ICU patients have a higher volume of distribution and clearance than other patient populations, which results in bolus dosing producing a reduced capacity to achieve 50% f T>MIC. In contrast administration by continuous infusion enables superior achievement of pharmacodynamic endpoints then bolus dosing (4gm six-hourly bolus, 58%, vs continuous infusion 16gm/day, 94%). Conclusions: 1) Various studies were identified showing altered piperacillin clearance and volume of distribution in various patient populations. 2) Dosing by extended infusion or continuous infusion achieves superior pharmacodynamic outcomes, which supports data on improved outcomes observed in previous retrospective clinical studies.	Enhancing Radiotherapy Of Cancer Using Agents That Target Thrombospondin-1 Signaling Via CD47 ROBERTS DD^1, ISENBERG JS^1, MAXHIMER JB^1, HYODO F^2, PENDRAK ML^1, RIDNOUR LA^2, DEGRAFF WG^2, TSOKOS M^1, WINK DA^2 ^1Laboratory of Pathology & ^2Radiation Biology Branch, NCI,NIH, Bethesda, MD, USA Background: Radiation is a primary mode of cancer therapy that acutely damages cellular macromolecules and elicits stress responses leading to cell death. The known cytoprotective activity of nitric oxide is blocked by thrombospondin-1 (TSP1), a potent antagonist of nitric oxide/cGMP signaling, suggesting that TSP1 signaling via its receptor CD47 could correspondingly increase radiosensitivity and that antagonists of this pathway could be effective radioprotectants. Methods: Wild type, TSP1-, TSP2-, and CD47-null mice were subjected to 25 Gy hindlimb irradiation and studied over 8 weeks. Vascular cells isolated from the respective mice were irradiated in vitro at 10 to 40 Gy and analyzed for survival and proliferation. Radioprotective activities of antagonists were studied in vitro in vascular cells and in vivo using wild type mice. Results: Twelve hours after 25 Gy hindlimb irradiation, TSP1 null mice showed significantly less cell death in muscle and bone marrow. Two months following irradiation, skin and muscle units in the null mice showed minimal histological evidence of radiation injury and near full retention of mitochondrial function. Tissue perfusion and acute vascular responses to NO are also preserved in irradiated TSP1 null hindlimbs. The role of TSP1 in radiosensitization is specific in that TSP2 null mice were not protected. However, mice lacking the TSP1 receptor CD47 showed similar radioresistance as TSP1 null mice. TSP1- and CD47-dependent radiosensitization is cell autonomous because vascular cells isolated from the respective null mice showed dramatically increased survival and improved proliferative capacity following irradiation in vitro. Antisense suppression of CD47 expression effectively protected endothelial cells in vitro from radiation-induced death and preserved skin and muscle integrity and function in irradiated hindlimbs of wild type mice. Conclusions: Soft tissues in TSP1 and CD47 null mice are remarkably resistant to radiation injury. This is due to a cell-autonomous radiosensitizing signal arising from TSP1 binding to its receptor CD47. Antagonists of this pathway can protect soft tissues from the deleterious effects of irradiation and thereby enable use of increased radiation doses for cancer therapy.
Present Knowledge On Pharmacogenetics Of Antiretrovirals ROCA B Infectious Disease Division, Departmen of Medicine, Hospital General of Castellón, University of Valencia, Spain Background: HIV infection is a serious but treatable disease, yet current treatment is limited by development of resistance and high rates of adverse drug reactions. Antiretroviral therapy is especially suitable for pharmacogenetic investigation as both drug exposure and treatment response can be reliably measured. Methods: A systematic review of the literature was carried out. PubMed’s Medline was searched at http://www.ncbi.nlm.nih.gov/sites/entrez/ with the following profile: (Pharmacogenetics OR pharmacogenomics) AND (HIV OR antiretrovir*). A total of 147 articles were found. All titles were read, and a total of 19 articles were selected because of their major relevance. Abstracts of all 19 articles were studied, and among them 8 articles, according with scientific importance and recentness of publication, were finally selected. The full text of those 8 most important articles was studied in detail.