Ehrlich II –2nd World Conference on Magic Bullets

Value of pharmacokinetic/pharmacodynamic in dose management of ceftazidime and imipenem in ICUs AUBERT G^1, CARRICAJO A^1, AUBOYER C^2, ZENI F^3 ^1Antibiology Laboratory, Univ. North Hosp. - Saint-Etienne - France; ^2Intensive Care Unit, Univ. North Hosp. - Saint-Etienne - France; ^3Intensive Care Unit, Univ. Bellevue Hosp. - Saint-Etienne. - France. Background: The purpose of our study was to assess the value of serum assay of ceftazidime (CAZ) and imipenem (IMI) in patients in the intensive care unit (ICU) of the Saint-Etienne University Teaching Hospital and in other ICUs in the region with regard to optimisation of treatment management. Methods: Between 01/11/05 and 29/02/08, in patients hospitalised in ICUs, not on dialysis and undergoing treatment with CAZ given in a continuous infusion or with IMI (non-continuous), serum assay of the respective antibiotics were performed 36 hours after the start treatment using a single serum sample for CAZ and determination of trough and peak concentrations for IMI. Assays were performed using the microbiology technique with results 18 hours after sample collection. CAZ 2 g was given systematically in a bolus at the start of treatment. Results: Assays were performed in 92 and 105 patients respectively for CAZ and IMI. Mean patient age was 66 years (19 to 89 years) and mean weight was 73 kg (33 to 122 kg). The dosage was between 1 g and 6 g/24 h for CAZ and between 1 g and 6 g/24 h for IMI. The mean serum CAZ concentration was 46.9 mg/L (7.4 to 162.3 mg/L). Serum CAZ concentrations were as follows: 35 to 65 mg/L in 37% of patients, < 35 mg/L in 43.2% and > 65 mg/L in 19.8%. Infection was established in 51 patients, with 42 strains of P. aeruginosa detected. The serum concentration / MIC ratio was >= 5 for 84.3% of patients and > 10 for 65.6% of patients. Trough concentrations of IMI were < 0.5 mg/L for 14.7% of patients, between 0.5 and 2 mg/L for 43.2%, and > 2 mg/L for 42.1%. The mean peak concentration of IMI was 19.9 mg/L (3 to 78 mg/L). Infection was recorded in 47 patients, including 34 enterobacteria and 11 P. aeruginosa. Antibiotic dosage was adjusted respectively for CAZ and IMI in 19.8% and 28.4% of patients based on the initial assay results. Conclusion: Our study shows that assays are needed in ICUs to confirm the efficacy of time-dependent antibiotics (ß-lactams), to avoid treatment toxicity, to achieve efficacy as rapidly as possible and to avoid selection of resistant mutants, particularly in strains having limited susceptibility to antibiotics.

The Role of Endothelial Cell Heterogeneity in the Search for Anti-angiogenic Agents AUERBACH R, AUERBACH W Laboratory of Developmental Biology, Department of Zoology and Institute on Aging, University of Wisconsin, Madison, WI 53706 USA Background: In the late 1800s, Paul Ehrlich documented the clear differences among endothelial cells from different organs by describing the uniqueness of the brain-associated vasculature, the blood-brain barrier. Nevertheless, endothelial cell heterogeneity, aptly demonstrated by the blood-brain barrier, was not generally appreciated until the last decade and even now has not become a major consideration in developing angiogenic and anti-angiogenic therapies. Recognizing the importance of this heterogeneity is critical for the development of "magic bullets" against cancer or immune-mediated diseases, inasmuch as there is no single uniform target in the vasculature. Methods: There are multiple methods for studying angiogenic and anti-angiogenic agents, ranging from in vitro models to various in vivo assays. Critical to the choice of targets is the recognition of endothelial cell heterogeneity. We and others have developed numerous endothelial cell lines from various microvascular organ beds, from large arteries, from specific veins, and from tumors. Results: Responses to different agents in vitro varies with the source of endothelial cells used in the assays. In vivo results depend on the site of injection, the physical topography, and the organ environment. The vasculature of developing embryos differs from that of adults, and there are critical distinctions between vessels of the lymphatic system and those of the blood vasculature. Examples of both in vitro and in vivo studies of effects of various pharmacological agents will be presented. Conclusions: Endothelial cell heterogeneity presents an important challenge to the development of pharmacological reagents ("Magic Bullets") when attempting to target the vasculature of specific organs, different tumors, or selected sites of inflammation. Paul Ehrlich pioneered in an area of pathology whose importance is only now beginning to be appreciated. Dedication: This contribution is presented in honor of Dr. Judah Folkman, long-time colleague and friend you died unexpectedly earlier this year.

Targeted delivery of cisplatin using polymeric nanoparticles AVGOUSTAKIS K^1 ^1Univ. of Patras, Patras, Greece Background: Cisplatin use is associated with serious side effects. The more selective delivery (targeting) of cisplatin to tumor cells would reduce drug toxicity, improving its therapeutic index. Aim: To develop long-circulating cisplatin-loaded nanoparticles and test their anticancer activity in mice cancer models. Methods: A) Nanoparticles preparation: Cisplatin-loaded poly(lactide-co-glycolide)-polyethyleneglycol (PLGA-PEG) nanoparticles were prepared by a double emulsion method. B) Determination of cisplatin profiles in blood circulation: BALB/c mice were administered intravenously with PLGA-PEG/cisplatin nanoparticles and at predetermined time intervals blood samples were obtained and assayed for platinum. C) Evaluation of the tolerance of BALB/c mice to the PLGA-PEG/cisplatin nanoparticles: 5 mice groups (n=3) received 3 intravenous injections at 7-day intervals. Three groups of mice received PLGA-PEG/cisplatin nanoparticles with a cisplatin content of 2, 5 or 10 mg cisplatin/Kg. The fourth group of mice received blank nanoparticles and the fifth group of mice (n=3) received 100 μl saline. D) Evaluation of anticancer activity of PLGA-PEG/cisplatin nanoparticles: HT 29 tumor cells were injected sub-cutaneously into the left flank of SCID mice. Fifteen days later, the mice (n= 6-8) were injected intravenously 5 times at weekly intervals with free cisplatin or cisplatin-loaded nanoparticles at the same dose (5 mg/kg on a cisplatin basis). Results: The entrapment of cisplatin in the nanoparticles resulted in a significant prolongation of cisplatin presence in blood. Balb/c mice tolerated 3 weekly intravenous injections of a relatively high dose of blank PLGA-mPEG nanoparticles (500 mg/Kg) and 3 weekly intravenous injections of a high dose of nanoparticle-entrapped cisplatin (10 mg/Kg). The cisplatin-loaded PLGA-mPEG nanoparticles was effective at delaying tumor growth in HT 29 tumor-bearing SCID mice. The group of mice treated with cisplatin-loaded nanoparticles exhibited higher survival rate compared to the free cisplatin group Conclusions: 1) PLGA-mPEG/cisplatin nanoparticles could prolong cisplatin residence in blood and they were well tolerated by normal Balb/c mice even when relatively high doses were administered to mice. 2) The PLGA-mPEG/cisplatin nanoparticles reduced tumor growth in SCID mice with HT29 xenografts, and these mice exhibited higher survival rate than free cisplatin. Au

The Effect of Melatonin and Zinc on the Immune Response in Experimental Toxoplasma Retinochoroiditis AVUNDUK AM ^1, AVUNDUK MC ^2, BALTACİ AK ^2, MOGULKOC R ^2 ^1Karadeniz Technical University. Trabzon, Turkey ^2Selçuk University. Konya, Turkey Background: Toxoplasmosis is a zoonotic disease and the most common cause of infection of the human retina . In humans and animals, the primary control mechanism of Toxoplasma gondii (T. gondii) infection involves T lymphocytes. One of the potential beneficial approaches to improve the immune defense against T. gondii infestation is zinc supplementation, since due to its catalytic and regulatory functions; zinc can enhance resistance to infections. Other potential approaches that deserve further investigation in this respect are the effect of melatonin (MEL) deficiency and artificial MEL supplementation on the immune response to T. gondii infection. MEL is a hormone secreted by pineal gland that has both direct and indirect effects on the immune system. In the current study, we investigated the impact of MEL and/or zinc deficiencies and artificial MEL and/or zinc supplementations on immune and inflammatory responses in the rat model of toxoplasma retinochoroiditis. Methods: Eighty-four Sprague Dawley male rats were divided into 12 equal groups. All groups, except controls were infected with T. gondii parasite by intraperitoneal injection. Combinations of zinc deficient diet, pinealectomy (Px), artificial zinc, and MEL were supplied during a 1 month period. At the end of the experiment, retinal and choroidal total lymphocytes, CD3+, CD4+, and CD8+ cell numbers were counted in histological sections. Results: The highest amount of cellular infiltration (lymphocytes, CD3+, CD4+, CD8+ cells) in the choroid and retina were detected in infected+MEL+zinc treated rats and the least amount of cellular infiltration was observed in Px+zinc deficient diet treated rats. Although single zinc or MEL supplementation had no significant impact on the cellular infiltration in the retina and choroid in Px rats, combined therapy significantly improved these responses. Conclusion: Artificial supplementation of MEL and zinc should be considered as an adjunctive therapy to classic treatment of Toxoplasma retinochoroiditis especially in immunosuppressed and elderly patients if our data will be confirmed in a clinical setting. Authors’ disclosure statement: Supported in part by a Research Fund of Selcuk University Grant no: TF 98/042. Published in Ophthalmologica. 2007;221(6):421-5 S.Karger AG, Basel. Presented in part at the World Ophthalmology Congress, Feb 19 - 24, 2006,São Paulo, Brazil.

Preventing Immune Evasion as a Strategy for Enhancing the Effectiveness of Herpes Simplex Virus Vaccine. Awasthi S, Lubinski j M, and Friedman H m Infectious Disease Division, Department of Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Background: Herpes simplex virus-1 (HSV-1) and HSV-2 glycoprotein C (gC) are immune evasion proteins that inhibit complement activation by binding complement component C3b. We previously reported that mice passively immunized with a gC1 monoclonal antibody (MAb) were protected from HSV-1 challenge only if the MAb blocked the interaction between gC1 and C3b. We also demonstrated that immunizing mice with gC1 protein induced antibodies that blocked C3b binding and protected mice against HSV-1 challenge by blocking immune evasion. Method: We use the mouse flank (epidermal disease) model to evaluate whether immunizing with gC1 protein increases the efficacy of a glycoprotein D (gD1) subunit vaccine when challenged with HSV-1. We first defined a gD1 immunizing dose that produced partial protection against HSV-1 challenge. Our rationale for partial protection was that the gD2 subunit vaccine used in human trials provided only limited protection, and we wanted to reproduce these results in mice. We then immunized mice with either gD alone or with both gD and gC and challenged with HSV-1. Results: We found that when gC1 was added to gD1 immunizations, mice were significantly protected from epidermal disease compared with gD alone. Importantly, the combined immunizations were more effective than gD alone in preventing infection of dorsal root ganglia. Passive immunization of anti-gC1 IgG obtained from mice immunized with gC1 protein protected complement intact mice, but not C3 knockout mice. Conclusion: We conclude that immunizing with gC1 blocks immune evasion and enhances the efficacy of a gD1 subunit vaccine.

Levetiracetam in the Treatment of Neuropathic Pain: Evidence from Cellular and Behavioral Pain Models AYAR A^1, OZCAN M^2 Firat University, Faculty of Medicine, Departments of ^1Physiology, ^2Biophysics, Elazig, Turkey. Background: Neuropathic pain is a common, complex, costly pain condition, "initiated or caused by a primary lesion or dysfunction in the nervous system”, presenting a major healthcare and social problem. Despite an increasing number of trials there is currently no satisfactorily effective therapy for neuropathic pain. Similarities betweenthe pathophysiological phenomena observed in some epilepsy modelsand in neuropathic pain models justify the use of anticonvulsantsin the symptomatic management of neuropathic pain. We attempted to elucidate the effectiveness of levetiracetam in neuropathic pain state in mice model and subsequently cellular mechanisms involved in a cellular nociceptive model. Methods: The in vivo nociceptive behavioural “hot-plate test” was performed in normal and diabetes (streptozocin 200 mg/kg i.p.)-induced adult male Balb/C mice. Subsequent to behavioral testing, electrophysiological measurements and calcium imaging experiments were performed on cultured neurones of the rat dorsal root ganglia (DRG) using the whole cell patch-clamp technique and the fura-2 ratiometric fluorescence microscopy. Data were analyzed using Kruskal-Wallis One-way Analysis of Variance (ANOVA), Dunnett’s and Students’ t tests, where appropriate. Results: While levetiracetam had (60-900 mg/kg) no significant effect on the nociceptive threshold in normal mouse much lower doses (≤200 mg/kg) significantly restored the pain threshold latency in diabetic mice, in a dose-dependent manner. Current clamp recordings from DRG cells indicated that levetiracetam caused membrane hyperpolarisation and reduction of multiple action potential firing. Estimation of reversal potentials of levetiracetam-induced hyperpolarizing currents indicated involvement of K^+ channels. Furthermore, levetiracetam dose-dependently suppressed the depolarisation (high KCl) -induced intracellular calcium signals in DRG neurons. Conclusions: Results obtained from in vivo behavioral tests and cellular electrophysiological and ratiometric fluorescence measurements in rat sensory neurons with agreement lend support to the validation of the promising therapeutic potential of the new anticonvulsant levetiracetam for the management of neuropathic pain.

Insulin, IGF-1 and Rosiglitazone: How Do They Effect The Glucose Metabolism and Insulin Resistance in Human SH-SY5Y Cells with Alzheimer Key Proteins? AYCAN (YERER) MB^1, ECKERT A^2 ^1 University of Erciyes, Faculty of Pharmacy, Department of Pharmacology, 38039, Kayseri, Turkey. ^2 Psychiatric Univ. Clinics, Neurobiol lab. for Brain Aging, Basel, Switzerland. Background: IGF are potent neurotrophic and neuroprotective factors that reverse the effects of Ab25–35-, 1–40- and amylin induced neurotoxicity in hippocampal cells. IGF-I protects hippocampal neurons against Ab-induced toxicity. Furthermore, insulin resistance leads to a functional decrease in insulin receptor (IR)-mediated signal transduction in the brain, again consistent with the hypothesis that hyperinsulinemia or insulin resistance may potentiate the risk of AD. In this study, the effects of insulin, IGF-1 and rosiglitazone on normal and transfected SH-SY5Y cells (P301L and hTau40) with key proteins involved in Alzheimer’s disease (AD) have been investigated. Methods: The cell lines were cultured in different glucose mediums (0-100mM) to identify the effects of insulin (5µg/ml), IGF-1 (1-10-100ng/ml) and rosiglitazone (20µM) in altered glucose concentrations. ATP synthesis and the mitochondrial membrane potential (MMP,ΔΨm) were measured. Results: Insulin was found to increase the ATP and the MMP significantly in SH-SY5Y cells in all glucose levels. However, insulin didn’t cause any changes in low glucose medium in P301L cells whereas it increased these values in high glucose environment (p<0.05). In hTau40 cells insulin increased the ATP synthesis in low glucose medium whereas the ATP synthesis reduced in these cells in high glucose medium (p<0.05). Rosiglitazone slightly lowered the ATP sythesis and the MMP in normal cells in low glucose medium, and slightly increased in high glucose medium. In hTau40 (p<0.05) and P301L (p<0.001) cells rosiglitazone alone decreased the ATP synthesis whereas the cells were prevented significantly from this reduction by the combination of insulin and rosiglitazone (p<0.05) which reveals that there is a probable insulin resistance in transfected cells where the ATP synthesis may be promoted by rosiglitazone in the presence of insulin. 100ng/ml IGF-1, increased the ATP levels in low glucose medium and this increase was greater then the insulin had in normal SH-SY5Y Neuroblastoma Cells. 1 and 100ng/ml of IGF-1 had slightly lowered the ATP levels in high glucose level medium whereas 10ng/ml of IGF-1 had no significant effect on the ATP synthesis in SH-SY5Y cells. In P301L cells, in the 0 and 25mM glucose medium 100ng/ml IGF-1 lowered the ATP synthesis significantly. In the hTau40 cells there were not any significant effect of IGF-1 in ATP synthesis. When we compare the MMP, all concentrations of IGF-1 had lowered the MMP in 0 and 100 mM glucose mediums but increased the MMP especially in 25mM glucose (p<0.05) in normal SH-SY5Y Neuroblastoma Cells. However, it didn’t have any effects on P301L cells. IGF-1 only lowered the MMP in hTau40 cells in high glucose (100mM) mediums. Conclusion: Since brain possesses high energetic requirements, any decline in brain mitochondria electron chain could have a severe impact on brain function and particularly on the etiology of neurodegenerative diseases. Therefore, to identify the relationships between insulin, IGF-1 and rosiglitazone combination could be beneficial to find out novel treatment alternatives for the insulin resistance in late on-set of AD.

Antimicrobial Resistance by Mycoplasma species in Farm Animals. AYLING RD. Veterinary Laboratories Agency (Weybridge), Addlestone, Surrey, UK. KT15 3NB Background: More than 125 Mycoplasma species have been identified, many being pathogens in farmed animals. Mycoplasmas lack a cell wall, hence they are not susceptible to penicillins and some other antimicrobials. Mycoplasma infections are usually treated with tetracyclines, macrolides, chloramphenicols, aminocyclitols and the fluoroquinolones, however antimicrobial resistance to all of these has been reported. The treatment of mycoplasma infections is rarely successful and often requires repeated treatments. Although Mycoplasma species are not usually zoonotic, this repeated use of antimicrobials and development of resistance may have implications for the critically important antimicrobials used in human health. MIC data from UK bovine and ovine mycoplasma isolates and resistance mechanisms in M. bovis is presented. Methods: In vitro microbroth dilution minimum inhibition concentrations (MIC’s) were determined for 14 antimicrobials against M. bovis and M. ovipneumoniae isolates. Sensitive M. bovis isolates were subcultured at sub-MIC levels in 14 different antimicrobials to induce antimicrobial resistance in vitro. Where antimicrobial resistance had been induced this variant DNA was used to PCR the domains II and V of the 23S rRNA gene and the gyrA and parC genes, which were then sequenced. Results: Isolates of M. bovis and M. ovipneumoniae gave high in vitro MIC values against most classes of antimicrobials, except the fluoroquinolones, although high fluoroquinolone MIC’s have since been recorded in field isolates. The rate of development of antimicrobial resistance varied between isolates and between antimicrobials, but resistance was induced by all isolates to all antimicrobials. Fluoroquinolone resistant isolates showed a single base change (G to A) at position 259 and (C to T) at position 248 in the gyrA gene, observations that have been shown to confer fluoroquinolone resistance in E. coli. Conclusions: It is unlikely that the genetic exchange of resistance genes occurs between Mycoplasma species and zoonotic organisms, however the treatment of Mycoplasma infections in farmed animals requires careful selection and monitoring of antimicrobial use to maximise the effectiveness of treatment and to evade the further development of antimicrobial resistance.

Cell Hydration as a Universal and Extra-Sensitive Biomarker for Determination of the Functional State of the Organism Ayrapetyan S UNESCO Chair-Life Sciences International Postgraduate Educational Center, Yerevan, Armenia Background: Although the functional significance of intracellular water is widely accepted, its messenger role in signal transduction and generation of different diseases is not adequately studied by the researchers. My presentation contains a review of our data on the metabolic regulation of cell hydration and its physiological significance in norm and pathology. Methods: Neuronal, muscle and reproductive cells, isolated tissue and organs of animals and woman breast cancer tissues serves as a subject for investigations. The light microscopic, whole cell and patch clamp, isotope, standard biochemical and genetic engineering methods were used. Results: The data showed a close correlation between cell hydration and number of functionally active membrane proteins, having enzymatic, chemoreceptive and ionoforetic properties. The data on the Table show that the number of ouabain receptors (Na/K pump units) depends on membrane surface, which changes upon the effect of factors causing the increase of membrane permeability. The correlation between the Na/K pump regulating the cell hydration and intracellular signaling system was also shown. It makes the cell hydration as a universal and extrasensitive cell marker determining the cell functional state and a sensor for different extraweak environmental signals. By specific mRNA-induced expression in oocites membrane was shown that the cyclic nucleotide–dependent Na/Ca exchanger plays a crucial role in cell volume regulation, when the Na/K pump is inactive (cell pathology). Conclusions: 1) The number of functionally active protein molecules in cell membrane depends on cell active surface. 2) There are a negative feedback between Na/K pump and cell hydration and a positive feedback between membrane permeability and cell hydration. 3) The cell overhydration is a marker for cell pathology. 5) The Na/K pump regulating the cell hydration is a universal and extrasensitive sensor for various environmental factors. [^3H] Ouabain concentration Incubation medium Hypotonic Isotonic Hypertonic Ach 10^-4M GABA 10^-4M 1x10^-9 32.0±2.2 21.1±1.4 12.2±0.9 30.54±1.55 27.63±3.17 1x10^-8 793±45.6 508±30.1 283±19.4 270.93±28.53 174.48±13.54 Table: Binding of [^3H] Ouabain (x10^8 molecules/mg dry weight) to Helix Pomatia Cell Membrane in different mediums

EM703 a New Derivative of Erythromycin, Inhibits Lung Fibrosis Induced by TGF- Signaling in Murine and Human Lung Fibroblasts AZUMA A, LI YJ, YU CH, ABE S AND USUKI J Department of Internal Medicine - Pulmonary Medicine, Infection, and Oncology, Nippon Medical School, Tokyo, JAPAN Background: 14-membered ring macrolides have been effective in chronic airway inflammation and also prevented lung injury and fibrosis in bleomycin -challenged mice via anti-inflammatory effects. EM703 is a new compound of Erythromycin (EM) without bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in 1) experimental murine fibrosis model induced by bleomycin and 2) murine and human lung fibroblast cell lines. Methods: 1) Seven weeks old male ICR mice (eight mice/group) were used. Bleomycin was administered intravenously to mice at day 0. EM703 was orally administered daily to mice. All groups were examined for cellpopulations in bronchoalveolar lavage fluid, and for induction of mRNA of Smad3 and Smad4 in lung tissues by RT-PCR at day 7. Fibroblasticfoci were assessed histologically and hydroxyproline contentwas chemically determined in lung tissues at day 28. 2) We also assessed proliferation and soluble collagen production, and examined induction of mRNA of smad3 and smad4 by RT-PCR in lung fibroblast cell line MLg2908. We examined smad3, smad4, smad7 and phosphorylated smad2/3 (P-smad2/3) protein assay by western blotting in lung fibroblast cell lines. Results: Bleomycin-induced lung fibrosis, infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. Expression ofsmad3 and smad4 mRNA was clearly attenuated by bleomycin, but recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by TGF. Expression of smad3 and smad4 mRNA in murine lung fibroblasts disappeared by TGF , but recovered by EM703. EM703 inhibited expression of phosphorylated-smad2/3 and smad4 protein in murine lung fibroblasts induced by TGF. In human lung fibroblast, EM703 inhibited the augmentation of Smad3 mRNA induced by TGF. Inhibited Smad3 mRNA by TGF was augmented by co-incubation with EM703. Conclusions: These findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti inflammation and regulation of TGF signaling, which is associated with inhibition of phosphorylation of Smad2,3 through recovery of Smad7 level.

Effects of Alcohol and Sucrose Intake on Rat Liver Cyp2e1 AZZALIS LA^1, FONSECA FLA^2, SCHINDLER F^2, GIAVAROTTI L^1, MONTEIRO HP^3, VIDELA LA^4, JUNQUEIRA VBC^5 ^1Universidade Anhembi Morumbi, São Paulo, Brasil, ^2Faculdade de Medicina do ABC, Departamento de Hematologia e Oncologia, Santo André, Brasil, ^3Departamento de Bioquímica, UNIFESP, São Paulo, Brasil,^4Programa de Farmacologia Molecular y Clínica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile. ^5Disciplina de Geriatria, Departamento de Medicina, UNIFESP, São Paulo, Brasil. Background: Ethanol metabolism by CYP2E1 leads to a significant reactive oxygen species (ROS) release, accompanied by the defense systems decrease against oxidative stress. Since expression of CYP2E1 is very much influenced by nutritional factors, specially carbohydrate consumption, and various results concerning the expression of CYP2E1 were obtained with low-carbohydrate alcohol liquid diet or the intragastric tube feeding model that also utilizes a low carbohydrate diet, this study describes the effects of ethanol and sucrose treatment on CYP2E1 levels using an ad lib model. Methods: Male Sprague-Dawley rats were fed ad. lib. for 1, 2, 3 or 4 weeks a commercial diet (Purina Ind., Brazil) plus a 25% ethanol-20% sucrose solution. Control groups were isocalorically pair-feed to the leading ethanol-consuming animals, or received isocaloric amounts of sucrose for pairing only ethanol calories. Eighteen hours before sacrifice ethanol was withdrawal and animals had only free access to tap water or they were offered food and water ad. lib. Results: Our results have shown that ethanol administration was associated with CYP2E1 induction, otherwise CYP2E1 stabilization was more associated to sucrose consumption. Conclusions: Our findings indicate that dietary deficiencies, especially low carbohydrate intake could be crucial in the CYP2E1 stabilization.

Significant Interactions between some Antibiotics and Antimalarial Drugs BABALOLA C.^1, OLANIYI A.^1, IWHEYE G., FASHEDEMI T.^1, AJOKU C.^1, ADENIYI B ^2 ^1Department of Pharmaceutical Chemistry, ^2Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Ibadan, Nigeria Background: Coadministration of antibacterial and antimalarials is common in the tropics as a result of frequent association of malaria with other infections. The combination of ampicillin and cloxacillin (AM-CL) prescribed in many infections produces a broad-spectrum antibiotic activity against both Gram-positive and Gram-negative bacteria. The current studies investigated effects of chloroquine(CQ), proguanil(PG), quinine(QN) and artesunate(ART) on the bioavailability and activity of cloxacillin(Clox) and ampicillin(Amp) in vivo and in vitro. Methods: 7 healthy volunteers received single oral doses of Clox alone followed by Clox + PG, another 8 volunteers received AM-CL alone and AM-CL+ CQ, while another 14 volunteers received AM-CL alone and AM-CL + ART in a cross-over manner. Total urine voided was collected at various time intervals. Clox in urine was determined by HPLC. Effect of CQ and PG on dissolution of Amp and Clox and on their antibacterial activity against E. coli and S. aureus was investigated. Results: CQ showed a significant decrease in total amount (Du∞) and % dose of Clox excreted in urine by 64%, while PG led to 48% decrease. Ongoing study reveals 90% reduction of Clox by QN. In vitro dissolution revealed >40% reduction in % Amp and Clox dissolved in the presence of CQ. Similar results were obtained with PG. The MIC of Amp was increased two- to four-fold from 5.42 µg/ml to 10.83 & 21.66 µg/ml by CQ, an indication of reduction in bactericidal activity of Amp. Similar results were obtained between Clox and CQ. 9 out of 14 subjects (64%) showed 39% decrease in urinary excretion of Clox while only 5 (36%) showed an increase of 27% when AM-CL were coadministered with ART. Conclusion: These results indicate significant drug-drug interactions between AM-CL and antimalarials in a way that correlates in vitro with in vivo findings. The urinary pharmacokinetic studies indicate marked reduction in bioavailability of Clox when coadministered with CQ, PG and QN and ART. The in vivo interactions may be due to interference with the dissolution of Amp and Clox in the body by the antimalarials. Though clinical implications of the findings are inconclusive, there should be caution in prescribing these classes of drugs together to avoid sub-therapeutic levels, which can lead to treatment failure and drug resistance.

Toxins and Adhesion Factors Associated with Staphylococcus Aureus Isolated from Urinary Tracts Infections BABA MOUSSA L^1, ANANI L^2, HAÏKOU N^1, HOUNSOU F^2, COUTURIER M^3, SANNI A^1, MONTEIL H^3, PREVOST G^3 ^1FAST-University of Abomey-Calavi, Bénin; ^2LNSP, Cotonou, Bénin ; ^3UPRES EA-3432, Institut de Bactériologie, Strasbourg France Background: Staphylococcus aureus infections are widely prevalent in West Africa and are often associated with urinary tract infections (UTIs). Virulence factors from S. aureus have rarely been described for such infections. The purpose of the current study was to determine the prevalence of toxins and adhesion factors obtained from S. aureus isolated from presumed primary UTIs at the Cotonou University Hospital (CUH) in Benin as compared with the Strasbourg University Hospital (SUH) in France. Methods: Both ambulatory and hospitalised patients were included in the study. Sixty-five independent strains of S. aureus from CUH and 35 strains from SUH were obtained over a four-month period. Virulence factors were characterised by immunodetection or multiplex polymerase chain reaction, and meticillin susceptibility was recorded. Approximately 50% of all isolates produced at least one enterotoxin. Results: No isolate from SUH produced PantoneValentine leucocidin (PVL), whereas 21.5% of the S. aureus isolates from CUH produced PVL (P < 0.01). Six of 14 (43%) PVL positive isolates were meticillin-resistant. At SUH, the incidence of MRSA (57%) was significantly higher (P < 0.01) than at CUH (14%). Genes encoding clumping factor B, and elastin and laminin binding proteins were detected in almost all isolates (80%), irrespective of the geographical origin. Conclusions: The results for elastin binding protein differed significantly from published data regarding isolates from other clinical origins. Staphylococcal toxins and adhesion factors may be important in the physiopathology of UTI.

Panton-Valentine leucocidin as a major virulence factor associated to furuncles BABA MOUSSA L^1, PREVOST G^2, COUTURIER M^2, MONTEIL H^2, MOREAU B^3, PRADINAUD R^4, COUPPIE P^4 ^1FAST-University d’Abomey-Calavi, Bénin ; ^2UPRES EA-3432, Institut de Bactériologie, Strasbourg ; ^3Bactériologie ; ^4Dermatologie, C.H.G. Cayenne, France Secretion of the Panton-Valentine leucocidin by S. aureus is associated to furuncles in more than 90% of isolates and in cases of furuncles. Besides, it also is associated with community pneumonia. However, most investigations considered only limited series of virulence factors being produced by these isolates. To determine whether Panton-Valentine Leucocidin is an essential factor associated, we determined production of toxins or the presence of genes encoding enterotoxins, leucotoxins, epidermolysins, Epidermal Differentiation Inhibitor (EDIN) factors, and 8 adhesion factors for 16 isolates from VIH^- patients with furuncles, 9 isolates from VIH^+ patients, and 30 isolates from secondary skin infections, all independent isolates from the Cayenne’s hospital (French Guiana). Antibiotic resistant was also performed. Only one of the isolates from furuncles do not produce Panton-Valentine leucocidin, while only three isolates from the secondary skin infections produce this toxin. The difference between these incidences is statistically significant (Student T test, p<0.001). Concurrently, 14/30 isolates from secondary skin infections and 12/25 isolates from furuncles produced at least one enterotoxin. One isolate amongst all produces Toxic Shock Syndrome Toxin-1, 5/30 isolates from secondary skin infections produce epidermolysin A, and 1/25 from furuncles produces epidermolysin B. No isolate carries any gene encoding Epidermal differentiation Inhibitor factors A, B, or C. Concerning adhesion factors, neither the presence of genes encoding clumping factor B, collagen binding protein, bone-sialoprotein, laminin binding protein, elastin-binding protein, fibronectin and fibrinogen binding proteins significantly differs in each group of isolates. In addition, there was no significant statistical difference for the occurrence of virulence for isolates from VIH- or VIH+ patients. Therefore, Panton-Valentine leucocidin can be presumed as the most essential causative factor for furuncles, and immune disorders as acquired immunodeficiency does not seem to contribute to any susceptibility in developing furuncles.

Transdermal Drug Delivery into and Beneath the Skin - Application to Anti Inflammatory Drugs BABU RJ^1, CHADHA GS^1, PARSONS DL^1, PATLOLLA RR^2, SINGH M^2 ^1Harrison School of Pharmacy, Auburn University, Auburn, USA; ^2College of Pharmacy, Florida A&M University, Tallahassee, USA. Background: α-Melanocyte-stimulating hormone (α-MSH) is an endogenous neuro-immunomodulatory peptide that has potent anti-inflammatory effects. We hypothesize that this compound can be developed as a topical formulation for the therapy of psoriasis and contact dermatitis. The stability of α-MSH, and in vitro and in vivo percutaneous absorption from various dermatological vehicles was investigated in rats. The anti-inflammatory effect of topically delivered -MSH was tested on an allergic contact dermatitis (ACD) mouse model. Methods: The stability of -MSH in ethanol-water (1:9) at various temperatures (40 to 70^oC) and pH conditions (pH 1.0 to 10.0) was examined. The in vitro permeation and skin retention kinetics of -MSH as a saturated solution in various dermatological vehicles were studied using hairless rat skin and Franz diffusion cells. The in vivo skin penetration of α-MSH was studied in rats by a dermal microdialysis technique. The anti-inflammatory effect of selected topical formulations (0.25 and 0.5% of -MSH in 10% n-methyl-2-pyrrolidone and 50% ethanol) was evaluated in the ACD mouse model. Results: Stability studies indicated that α-MSH possess an ~110 day shelf-life (time of 10% degradation) as determined by HPLC. Further, a-MSH demonstrated good stability in the pH region between 3.0 and 8.0. Permeation studies indicated that ethanol, transcutol and propylene glycol (PG) and ethanol vehicles had maximum permeation of α-MSH through the skin (between 5.0 and 7.5 mg / 24 h). Ethanol demonstrated the maximum skin retention (2.0 mg /mg) as compared to all other vehicles for which the skin retention was < 1.0 mg /mg. Dermal microdialysis results show the ethanol formulation produced a maximum concentration (Cmax) in dermis of 6.467±2.11 ng/ml as compared with the PG formulation with a Cmax of 2.565±1.284 ng/ml, demonstrating 2.5 fold higher dermal levels by the ethanol formulation. a-MSH formulations demonstrated significant anti-inflammatory activity in an ACD mouse model. The data indicate that like dexamethasone, α-MSH was effective in reducing the ACD response. Conclusions: Stable -MSH can be formulated for effective topical delivery into skin layers to demonstrate significant anti-inflammatory activity in an ACD mouse model.

Is GRP78/BiP, a Master-Regulator of Defensive Unfolded Protein Response, a New “Magic Target”? GRP78/BiP-targeting Cytotoxin and ER Stress-Inducing Drugs Synergize to Kill Cancer Cells Backer MV^1, Paton AW^2, Paton JC^2, Backer JM^1 ^1Sibtech, Inc., Brookfield, CT 06804 USA; ^2School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, 5005, Australia Background: Diverse insults increase the amount of unfolded/misfolded proteins in the endoplasmic reticulum (ER). This leads to activation of a defensive unfolded protein response, which is controlled by ER-resident protein, GRP78/BiP. Whether the cell dies or survives the ER-stress is determined by the amount of available GRP78/BiP. Recent discovery that GRP78/BiP is the only known substrate for the proteolytic A subunit (SubA) of a novel bacterial AB5 toxin provides the first opportunity for targeted destruction of GRP78/BiP. Aims: 1) To establish if targeted delivery of SubA into tumor cells is selectively cytotoxic. 2) To establish if destruction of GRP78/BiP leads to enhanced toxicity of ER-stress inducing drugs. Methods: SubA was genetically fused to human epidermal growth factor (EGF) and the resulting EGF-SubA was expressed in E.coli. Tumor cells (MCF7, PC3, F98, F98-EGFR, MDA231luc) were treated with EGF-SubA alone or in combination with various drugs. Biochemical and cell biology methods were used to characterize cellular EGFR levels, GRP78/BiP cleavage, activation of unfolded protein response, apoptosis, and cell viability. Results: Exposure of cells expressing high levels of EGFR to EGF-SubA results in rapid (~2 hours) EGFR-mediated destruction of GRP78/BiP. Despite the ongoing cleavage, in most cells it results in significant upregulation of GRP78/BiP level by 24h of treatment. EGF-SubA is highly cytotoxic to growing and confluent cells with high level of EGFR (> 10^5 EGFR per cell), with IC50 in the range of 3 to 40 pM, while EGFR-negative cells are at least 500-fold less sensitive. EGF-SubA strongly synergizes with thapsigargin, an ER-stress inducing drug, with ~10-fold enhanced efficacy of the drug combination, relative to each compound alone. Less prominent synergism is observed with drugs that are less stressful for ER. Conclusions: GRP78/BiP might be a new “Magic Target”. Its targeted destruction with subnanomolar concentrations of EGF-SubA is extremely cytotoxic, while non-toxic concentrations of EGF-SubA disarm cellular defense and allow to use virtually non-toxic amounts of ER-stress inducing drugs.

The Strong Growth Advantage in Stationary Phase Phenomenon in Mixed Cultures of Antimicrobial Resistant Escherichia and Salmonella BACUN-DRUZINA V^1, BUTORAC A^1, HALEC I^1, GJURACIC K^2 Faculty of Food Technology and Biotechnology, Zagreb, Croatia; ^2GSK Research Centre Zagreb Ltd., Zagreb, Croatia. Background: During prolonged stationary phase mutants with increased fitness express growth advantage in stationary phase” (GASP) enabling them to grow and displace the parent as the majority population. Aims: 1) To study the GASP phenotype in mixed cultures of Escherichia coli and Salmonella enterica serovar typhimurium wild type and strains harboring either the resistance to nalidixic acid (Nal^R) or to streptomycin (Str^R). 2). To evaluate the influence of multidrug resistance on the appearance of GASP phenotype. 3) To detect if there are genomic differences between both aged wild-type and used resistant mutants (Str^R and Nal^R) of E. coli, the pulsed-field gel electrophoresis (PFGE) of total genomic DNA was conducted. Methods: In a typical GASP competition experiment, cells from a 10-day-old culture are inoculated as a numerical minority (1:100 vol/vol) into a young (1-day-old) culture. The genomic DNA from randomly chosen colonies was digested with SfiI and resolved by pulsed-field gel electrophoresis (PFGE). Results: In the mixture consisting of the aged S. enterica Str^R and young E. coli Nal^R, strong phenotype GASP mutants of S. enterica dominated after the third day of mutual growth. Likewise, but with inversed bacterial resistance, in the mixture of 10-day-old S. enterica Nal^R culture with young culture of E. coli Str^R, the S. enterica Nal^R GASP mutants of strong phenotype dominated the mixture after the fifth day and were maintained at about 1x10^8 CFU/ml. Electrophoretic karyotype of the 10-days-old GASP mutants of E. coli strains carrying the resistance revealed additional bands when compared to the wild-type. Conclusions: 1) The strong GASP phenotype was obtained in mixed cultures with the aged mutant strains, but not when the isogenic antibiotic-sensitive strains were used. 2) The cells in mixed cultures of double mutant E. coli strain Nal^R Val^R bear multiple useful GASP mutations that increase its fitness in comparison with mutants E. coli strain Str^R and S. enterica strain Str^R and showed strong GASP phenotype. 3) The PFGE analysis demonstrated the significant chromosomal rearrangements in 10-day-old bacterial antibiotic-resistant mutated cells that correspond with the appearance of strong GASP phenomenon.

AntiNeoplaston: Synthesis, Biological, and Clinical Evaluation in Egypt BADRIA FA^1*, ABOU-ZEID L^2, MOWAFY A^3, HAWAS S^4, KHAFAGY Y^5. MABED M^6 Pharmacognosy^1, Medicinal Chemistry^2, and Biochemistry^3 Departments, Faculty of Pharmacy Immunology^4, Surgery^5, and Oncology^6 Departments, Faculty of Medicine Mansoura University, Mansoura, Egypt 35516 Background: Antineoplastons, first described by Burzynski, are naturally occurring peptides and amino acid derivatives, which control neoplastic growth. Antineoplaston A-10 (3-phenylacetyl amino-2,6-piperidinedione) is the first chem. identified antineoplaston. We previously reported the utility of antineoplaston-A 10 (3-phenylacetylamino-2,6-piperidinedione) as an endogenous cancer protector and immune modulator in breast cancer patients. Methods: Antineoplaston A-10 level was measured in the urine of 31 breast cancer patients and 17 normal women using high performance thin layer chromatography (HPTLC). Four new piperidinedione A 10 analogs were synthesized and tested for their antimitotic activity on a human breast cancer cell line against the prototype A 10 and the antibreast cancer drug tamoxifen. Moreover, the DNA binding capacity of such compounds. was evaluated against A 10. Apoptosis was measured in patients with breast cancer at time of diagnosis and to correlate urinary antineoplaston A-10 levels with neutrophil apoptosis and to describe the direct effect of A-10 in vitro on neutrophil apoptosis in breast cancer patients. The participants were patients with a histologically confirmed diagnosis of breast cancer. Only those cases without previous treatment for breast cancer were included. Neutrophil apoptosis was assessed in breast cancer patients both morphology and by DNA fragmentation and studied relative to healthy controls. Antineoplaston A-10 was measured using high performance liqiuid chromatography in urine samples collected from the patients. Urine samples from normal women served as controls. Direct effect of antineoplaston A-10 on neutrophil apoptosis was tested in vitro after adding A-10 at a concentration. of 10 ng/mL to the cellular suspensions of breast cancer patients. Non-treated samples served as controls. Results: Significantly lower antineoplaston A-10 levels were detected among patients with breast cancer. (E)-3-(4-Nitrocinnamoylamino)-2,6-piperidinedione and (E)-3-(4-hydroxycinnamoylamino)-2,6-piperidinedione were several-fold more potent antiproliferative agents than A 10 and tamoxifen. They also had significantly higher capacity to bind DNA than A 10. Conversely, (E)-3-(cinnamoylamino)-2,6-piperidinedione and (E)-3-(4-methoxycinnamoylamino)-2,6-piperidinedione had weaker biological profiles than the lead compound A 10. Detailed synthetic, spectroscopic, and biological data are reported. Significantly higher neutrophil apoptosis levels were detected among patients with breast cancer with a P value <0.001. Urinary antineoplaston A-10 level is significantly neg. correlated with high apoptosis levels (P<0.0001). In vitro, antineoplaston A-10 was found to inhibit significantly the neutrophil apoptosis with a P value <0.0001. Conclusions: Antineoplaston A-10 may provide rational basis for designing trials to employ its immune modulatory potentials as adjuvant therapy in breast cancer patients. These findings confirm the presence of immune defects among patients with breast cancer and such results should stimulate the development of new strategies to induce and augment immunity for the treatment of breast cancer.

Antibacterial, Antisecretory and Antihemorrhagic Activity of Azadirachta indica Used to Treat Cholera and Diarrhea in India Bag PK^1, Thakurta P^1, Bhowmik P^1, Mukherjee S^1, Hajra TK^1, Patra A^2 ^1Department of Biochemistry, ^2Department of Chemistry, University of Calcutta, Kolkata, India Background: Indigenous uses of Azadirachta indica A. juss (Maliaceae) (locally known as neem) leaves in different parts of India for curing gastrointestinal disorder such as diarrhea and cholera is wide spread. The objective of the present study was to evaluate the antibacterial and antisecretory activity of neem extract against Vibrio cholerae, a causative agent of watery diarrhea such as cholera. Methods: Methanol extract of neem leaves were tested using the strains of multi-drug resistant V. cholerae belonged to O1, O139 and non-O1, non-O139 serotypes. Antibacterial activity of the extract [10, 25, 50, 100, and 200 mg/ml (200 μl/well)] was determined by agar-diffusion assay. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were assessed using the broth microdilution method. The effect of the extract on fluid secretion and hemorrhage in intestine induced by V. cholerae was studied using mouse model (Male BalB/C mice, 23.8 ± 1.1 g body weight). Mice of each of groups 1 and 2 (n=2 per group in duplicated) were administered 500 μl of bacterial inoculums (2×10^9 CFU/ml) orally. After 1 h mice were fed either with 200 μl of methanol (group 1) or crude extract (group 2; at oral doses between 100 and 1800 mg/Kg in 200 μl of methanol) orally. Mice were sacrificed after 24 h incubation and fluid accumulation (FA) ratio was measured. Results: Crude extract showed inhibitory activity against multi serogroup strains of V. cholerae by agar-diffusion assay with significance (p<0.05). MICs reached by 50% (MIC50) and 90% (MIC90), and MBC for the extract were 2.5 mg/ml, >5mg/ml, and 10 mg/ml respectively. Administration of the extract (1800 mg/kg) did not produce any sign of toxicity in mice. Group 1 mice showed fluid accumulation (FA ratio; between 0.11±0.01 and 0.16±0.02) and hemorrhage in the intestines. Neem extract showed activity with inhibition (of fluid secretion) values of 27.7 ± 7.8, 41.1 ± 3.4, 43.3 ± 1.3, 57.0 ± 5.9, and 77.9 ± 7.2 % at doses of 100, 200, 300, 450, and 1800 mg/kg respectively. Oral administration of the extract inhibited hemorrhage induced by V. cholerae in mouse intestine at a dose ≥300mg/kg (visually observed). Conclusions: 1) Methanolic extract of A. indica leaves was an effective antibacterial agent against V. cholerae, and significantly reduced the fluid secretion and hemorrhage induced by V. cholerae in mouse intestine. 2) The active extract may be employed for the treatment of cholera and as potential source to develop novel antimicrobial compound and antisecretory drug useful to treat cholera and diarrheal patients. Authors’ disclosure statement: This work has been published in J. Ethnopharmacology: Thakurta, P., P. Bhowmik, S. Mukherjee, T. K. Hajra, A. Patra and P. K. Bag. 2007. Antibacterial, antisecretory and antihemorrhagic activity of Azadirachta indica used to treat cholera and diarrhea in India. J. Ethnopharmacology (Elsevier) 111 (3): 607-612.