Ehrlich II –2nd World Conference on Magic Bullets

Treatment Outcome Of Otomycosis With 1% Clotrimazole Cream OLOGE FE, NWABUISI C University of Ilorin, Ilorin, Nigeria Among 141 patients suspected of having otomycosis, 76(53.9%) were mycologically confirmed. The fungi isolated were Aspergillus sp (63.4%), Candida (35.5%) and Mucor (1.3%). Ninety-six per cent were symptom free within 2 weeks of topical application of 1% cIotrimazole cream, after thorough cleaning of debri in the ear canal. Treatment failures were minimal, including recurrence (2.6%), 'acute otitis externa (1.3%), foreign body in the ear (1.3%) and blocking of ear by therapeutic agent (2.6%). This treatment regimen is simple, efficacious, cost effective and safe; hence it is recommended for adoption in the management of otomycosis.

B16 And Cloudman S91 Mouse Melanoma Cells Susceptibility To Apoptosis After Dacarbazine And Doxorubicin Treatment Examined In Three Cytotoxicity Tests: Cell Counting, MTT And Flow Cytometry OLSZEWSKA-SŁONINA D, STYCZYŃSKI J, DREWA T Collegium Medicum of Nicolaus Copernicus University, Bydgoszcz, Poland Background: Considering the necessity of an individual choice of cytostatic drugs for patients with cancer disease and tumor cells' resistance to these compounds, their ability to induction of apoptosis should be investigated. The aim of this study was to determine the influence of dacarbazine (DTIC) and doxorubicin on morphology and kinetics of proliferation of B16 and Cloudman S91 cells. Methods: CELL CULTURE: Two rodent melanoma cell lines were used in cytotoxicity tests. Melanoma cells of both lines differ in the degree of pigmentation and in the growth rate in vivo. Cell were cultured in the Dulbecco medium with the addition of glucose, fetal bovine serum, penicilin, streptomycin, gentamycin and diflucan and harvested using 0.04% EDTA in PBS devoid of ions Ca^+2 and Mg^+2. CYTOTOXICITY STUDIES: Dacarbazine was used at concentrations 0.001-3.568M and doxorubicin at concentrations 0.0002 – 0.0431M.Cell counting – melanoma cell were cultured in 12 well plates 24h and then incubated with tested drug in order of decreasing concentration. After 24 h drug solutions was removed and the fraction of viable cells was a pellet. Fixed by washing with 70% ethyl alcohol cells were dried and counted. MTT test was carried out in a 96-well round bottom microtitre plates. Apoptotic and necrotic assay by flow cytometry: B16 and Cloudman S91 cells were stained with annexin V-FITC and propidium iodide and analyzed by the flow cytometer EPICS XL (Coulter). Results: Cell morphology studies: After treating with dacarbazine (1.098M) B16 cells were converted to the oval shape. Characteristic for melanomas in culture spindle-like cells were absent in both of examined cell lines. Cellular atypia of B16 cells was characterized by abnormal structure of nuclei (giant) and hyperchromatosis. Doxorubicin has changed the morphology of both cell lines too. They became dendritic-like cells with long branches. Irregularity of nuclei was also observed. Cells viability after exposure to cytostatic drugs were established by three methods and the assesing value EC50 being compared.methods of direct counting of alive cells and colorimetric methods assesing cell viability are characterized by a relatively low accuracy. The highest percentage of B16 and Cloudman S91 cells resistant to cytostatic drugs was received by flow cytometry and this method is recommended as the most accurate for drug cytotoxicity evaluation. Cloudman S91 cells were more resistant to dacarbazine (EC50 = 2.740M) and doxorubicin (EC50 = 0.0040M) than B16 (respectively EC50 = 1.644M and EC50 = 0.0024M). Maximum percentage of apoptotic B16 cells after DTIC exposure (11%) was lower than Cloudman S91 cells (22.2%). B16 cells underwent apoptosis after exposure to lower doxorubicin concentration than Cloudman S91. Maximum percentage of apoptotic B16 cells after doxorubicin exposure (64.7%) was higher than Cloudman S91 cells (59%). DTIC induces cell arrest in the G2/M and S cell cycle phase, doxorubicin arrested both investigated cell lines at phase S. Conclusions: In our work, it was shown that doxorubicin is radical-generating agent, induced cell death in B16 and Cloudman S91 cells via apoptosis pathway. Our results may suggest that the inefficient therapy of human malignant melanoma can be connected to very low apoptotic cells number after DTIC treatment.

Nicotine Dramatically Increases Impulsive Behavior–Can Nicotinic Acethylcholine Receptor Antagonist Suppress Impulsive Behavior? OHMURA Y^1, TAKAHASHI T^2, TSUTSUI I^1, YAMAGUCHI T^1, IZUMI T, YOSHIOKA M^1 ^1Hokkaido Univ. Dept Neuropharmacol, Sapporo, Japan; ^2Hokkaido Univ. Dept Behav Sci, Sapporo, Japan Background: Impulsivity has been associated with drug addiction, aggression, criminal involvement, and suicide. Impulsive behavior is becoming a social problem in many countries. Recently, the relationship between nicotine and impulsive behavior is attracting attention. Aims: 1) To determine the relationship between the estimated dose of nicotine intake per day and impulsive behavior in human. 2) To examine the acute effect of nicotine on impulsive behavior in rats. 3) To examine whether nicotinic acethylcholine receptor (nAChR) antagonist could suppress impulsive behavior in rats. Methods: Twenty seven habitual smokers and 23 never smokers between 21 and 33 years of age participated in human study. In animal studies, subjects were male Wistar-strain rats (10-13 weeks old). In human study, delay and probability discounting task was employed to assess impulsivity. In animal studies, 3-choice serial reaction time task was used to assess impulsivity and the effects of nicotine (0, 0.05, 0.1, 0.2, 0.4 mg/kg, s.c.) were tested. Moreover, the effects of nAChR antagonist were tested. Results: In human study, the degree of delay discounting of gain was significantly and positively correlated with both the number of cigarettes smoked and the estimated dose of nicotine intake per day (r=0.57, p < 0.05). However, there was no relationship between smoking and the other types of discounting, probability discounting and discounting of loss. In animal studies, nicotine (0.1, 0.2, 0.4 mg/kg) significantly increased premature response, an index of impulsive behavior. Moreover, nAChR antagonist suppressed impulsive behavior. Conclusions: 1) Nicotine increases impulsive behavior regarding gain. 2) However, nicotine does not affect impulsivity regarding loss. 3) nAChR antagonist could be an agent to suppress impulsive behavior.

Trimetazidine Revisited: Current And Future Applications Of Metabolic Modulation Of The Heart ONAY-BESIKCI A, Guner S, Arioglu E, Ozakca I, Ozcelikay AT, Altan VM Ankara University, Faculty of Pharmacy, Ankara, Turkey Background: Trimetazidine (TMZ) is an effective drug mainly used in angina pectoris. The beneficial effects of TMZ are related to the alteration of the substrate preference of the heart in favor of glucose usage and a reduction of the oxygen requirement for energy production. Increased rates of fatty acid oxidation impair contractile function in both healthy and diabetic hearts. Fatty acid oxidation dominates in Type I diabetic hearts for energy production at the expense of an increase in oxygen requirement. The objective of this study was to examine the effect of chronic treatment with TMZ on cardiac mechanical function and fatty acid oxidation in diabetic rats. Methods: A total of 40 rats were randomly divided in 4 groups: diabetic, TMZ-treated diabetic, control, TMZ-treated control. Diabetes was induced by a single intravenous injection. TMZ (7 mg/kg/day) was supplied in drinking water for 13 weeks. The hearts were perfusedin isolated working heartmode at 11.5 mm Hg left atrial preload and 80 mm Hg aortic afterload with a modified KH solution containing 100 μU/ml insulin, 11 mM glucose, and 0.8 mM palmitate (including [9,10-^3H]palmitate) pre-bound to bovine serum albumin. Heart rate and peak systolic pressure were measured by a pressure transducer. Cardiac and aortic output were obtained by monitoring the flows into the left atria and from the afterload line. Palmitate oxidation was determined by measuring ^3H2O produced from [9,10-^3H]palmitate using standard scintillation counting procedures. mRNA expression of long-chain 3-ketoacyl-CoA thiolase (3-KAT) was determined by RT-PCR. Results: Coronary flow was increased with TMZ treatment. Cardiac works of diabetic hearts were significantly lower compared to non-diabetics. Fatty acid oxidation was increased in both diabetic groups. Diabetes induced the mRNA expression of 3-KAT. Conclusions: 1) Diabetes increased the rates of fatty acid oxidation that was accompanied by deteriorated cardiac function 2-3) The inhibitory effect of TMZ on fatty acid oxidation was not detected at 0.8 mM palmitate in the perfusate. This finding along with the increase in 3-KAT expression suggest that higher enzyme expression required a higher concentration of TMZ. Also, a detailed kinetic analysis is of 3-KAT with its substrates needed which is currently performed in our laboratory. Authors’ disclosure statement: This study was supported by a grant (SBAG-2752) for Dr. Arzu Onay-Besikci from The Scientific and Technological Research Council of Turkey (TUBİTAK) and published in CJPP.

Comparison And Evaluation Of The Antiviral Potentials Among Different Soluble Forms Of Nectin-1 To Herpesvirus Infection ONO E^1, TOMIOKA Y^2, OZAKI K^1, MORIMATSU M^2, CHEREL P^3 ^1Kyushu University, Fukuoka, Japan; ^2HokkaidoUniversity, Sapporo, Japan;^3France Hybrids, St Jean De Braye, France Background: Nectin-1 is an alphaherpesvirus receptor that binds to virion glycoprotein D (gD) and mediates entry of alphaherpesviruses including HSV-1 and pseudorabies virus (PRV). We have reported that expression of soluble forms of nectin-1 provided a significant resistance against alphaherpesvirus infection to cell lines and transgenic mice. In order to compare the antiviral potentials of four different soluble forms of nectin-1 in vivo, transgenic mice expressing each of them were challenged with PRV. Methods: Antiviral potentials of two soluble forms of the entire ectodomain of nectin-1 (VCChIg and VCCpIg) consisting of the entire ectodomain and the Fc portion of human or porcine IgG and two soluble forms of the first Ig-like domain of nectin-1 (VhIg and VpIg) consisting of the first Ig-like domain and the human or porcine Fc portion were compared by performing experimental infection. Survival of mice was recorded for 14 days. Results: The average survival rates of transgenic mice expressing each of four different soluble forms of nectin-1 were as follows; 98% in VCChIg, 90% in VCCpIg 100% in VhIg and 79% in VpIg for the intraperitoneal challenge with 20LD50; 83%, 73%, 89% and 40% for 100 LD50; 74%, 68%, 58% and 7% for 1000LD50; 72%, 60%, 97% and 40% for the intranasal challenge with 10LD50. In contrast, more than 90% of all control littermates in each challenge died within 14 days. Conclusions: 1) Transgenic mice expressing each of four different soluble forms of nectin-1 showed a significant resistance to PRV infection via intraperitoneal and intranasal routes, indicating that all of soluble forms of nectin-1 are able to exert a significant antiviral effect against alphaherpesvirus infection in vivo. 2) Mice transgenic for a chimera that carried the human Fc portion (VCChIg and VhIg) were more resistant than those transgenic for a chimera that carried the porcine Fc portion (VCCpIg and VpIg). 3) Mice transgenic for a chimera that carried the entire ectodomain of nectin-1 were more resistant than those transgenic for a chimera that carried the first Ig-like domain, when the extracellular domains were fused to the porcine Fc portion.

Developement Of A Novel DNA Vaccine Targeting Macrophage Migration Inhibitory Factpr And Its Efficacy On Murine Models Of Inflammatory Diseases ONODERA S^1, KOYAMA Y^2, NISHIHIRA J^2, ABE R^1, TOHYAMA H^1, YASUDA K^1 ^1Hokkaido Univ. Graduate School of Medicine, Sapporo, Japan; ^2Hokkaido Information Univ., Ebetsu, Japan Background: Previous studies have demonstrated that neutralization of macrophage migration inhibitory factor (MIF) by anti-MIF antibody ameriolated the disease severity or mortality in several animal models of inflammatory diseases. This report describes a simple and effective method for active immunization aimed at eliciting neutralizing anti-MIF autoantibody, and its efficacy on the murine models　of inflammatory diseases including arthritis, sepsis, and atopic dermatitis (AD). Methods: We developed a MIF-deoxyribonucleic acid (DNA) vaccine by introducing oligonucleotides encoding helper T epitope into the cDNA sequence of murine MIF cloned in the mammarian expression plasmid pCAGGS, and administered it by in-vivo electroporation. Vacant plasmid was administered as a control vaccine (CV). Therapeutic effect of this approach against murine arthritis models induced by collagen antibody (CAIA) or in interleukin-1 receptor antagonist knockout mice (IL-1Ra KO), sepsis models induced by lipopolysaccharide (LPS) or cecal ligation and puncture (CLP), and murine AD models in DS-Nh or NC/Nga, were evaluated. Results: Mice administered with MIF-DNA vaccine significantly raised high titers of autoantibody which reacted to native MIF. The symptoms of CAIA and IL-1Ra KO were significantly ameliorated by MIF-DNA vaccination. This approach also significantly improved the survival of mice with LPS- or CLP- induced sepsis (p<0.05, v.s. CV), which was associated with suppressed mRNA levels of inflammatory cytokines and toll-like receptor (TLR) 4 in the lung. Finally, MIF-DNA vaccination significantly prevented the onset of dermatitis in DS-Nh or NC/Nga (p<0.05, v.s. CV). Of note, this approach also significantly improved the symptoms of established dermatitis in these AD models (p<0.05, v.s. CV). Conclusions: 1) MIF-DNA vaccination showed a prophylaxic effect against two models of arthritis, two models of sepsis, and two models of AD in mice. 2) MIF-DNA vaccination showed a therapeutic effect against established symptons of dermatistis in the two AD models. 3) DNA vaccine targeting MIF may provide a new strategy for the prevention and management of inflammatory diseases including arthritis, sepsis, and AD.

Transporter Mediated Drug-Drug Interactions: Modulation Of Drug Absorption By Environmental Toxicants OOSTERHUIS B^1, VUKMAN K^1, VÁGI E^1, GLAVINAS H^1, JABLONKAI I^2, KRAJCSI P^1 ^1Solvo Biotechnology, Budaörs, Hungary; ^2 Institute of Biomolecular Chemistry, Chemical Research Centre, Hungarian Academy of Sciences, Budapest, Hungary Background: ABC transporter proteins are efflux pumps expressed in cell membranes, which pump their substrates out of the cytosol into the extracellular, through hydrolization of ATP. Due to their expression in barrier organs like the intestine, liver, kidney and blood-brain-barrier, ABC transporters form an important wall of defense against xenobiotics from different sources. Among others, many drugs but also environmental toxicants like pesticides have been shown to interact with ABC transporter proteins. In this study a large number of pesticides was studied for interaction with several different ABC transporters. In follow-up experiments the effect of a subset of compounds on drug absorption and cytotoxicity was studied in more detail. Methods: A large number of pesticides was screened for interaction with human ABC transporter proteins P-gp, MRP1, MRP2 and BCRP in membrane based transporter assays (ATPase, Vesicular Transport). A subset of seven chloroacetanilide herbicides was selected and further investigated for inhibition of Pg-p in a number of methods. The effect of chloroacetanilides on the cellular efflux of calceinAM and cytotoxicity of paclitaxel, both P-gp substrates, was studied in K562-MDR cells. The transport of digoxin on Caco-2 monolayers was used as a model for drug absorption. The apical to basolateral flux of digoxin was measured in the presence and absence of chloroacetanilides. Results: Over 50% of the tested compounds showed interaction with one or more transporters, mainly Pg-p and BCRP. Four out of seven selected chloroacetanilides showed significant stimulation of P-gp specific ATPase activity. These compounds also inhibited calceinAM efflux from K562-MDR cells and sensitized the same cells to the cytostatic agent paclitaxel. Furthermore, those compounds showing interaction with P-gp also increased the permeability of digoxin on Caco-2 monolayers up to 2.5 times. Conclusions: This study showed interaction of structurally diverse pesticides with several human efflux transporters. The results from the experiments with chloroacetanilides demonstrate that environmental toxicants, like herbicides, can inhibit ABC transporter activity, and thereby modulate drug absorption or sensitize cells to cytostatic drugs. Abbreviations: MDR: Multidrug Resistance, MRP: Multidrug Resistance Protein, BCRP: Breast Cancer Resistance Protein, P-gp: P-glycoprotein.

25-Years Of Sight Saving By Preventing Postoperative Scarring With Local Use Of Antimetabolites OPHIR A Hillel-Yaffe Medical Center, Hadera and the Ruth and Bruth Rappaport Faculty of Medicine, the Technion, Haifa, Israel Background: Chronic glaucoma is a progressive blinding disease. A major risk factor of disease progression is an increased intraocular pressure (IOP). When topical and laser treatments are not adequate in halting disease progression, a surgical approach is indicated. Trabeculectomy, the preferred surgical choice, is aimed to open a new, long-lasting route for outflow of intraocular fluids in order to reduce IOP. The most common cause of surgical failure is scarring of the new opening. Postoperative scarring before the local antimetabolite era occurred in 30-70% of cases, depending on the preoperative existence of factors that promote scarring. Many eyes were thus doomed to blindness. Methods: Literature review. Results: Since the introduction of local application of antimetabolites in 1982, initially 5-fluorouracil (5-FU) by us and later mitomycin C (MMC) by others, surgical success rate in all groups commonly exceeds 90%. As an inhibitor of fibroblasts proliferation 5-FU was injected under the conjunctiva twice daily for 14 days after surgery. Following our observations that it is also toxic to existing fibroblasts, few injections are administered and only when imminent scarring is evident. 5-FU side-effects relate to inhibition of neighboring cell proliferation, and are mild and temporary. MMC, the more potent agent, is toxic also to quiescent neighboring cells. It use could be risky, termed “time-bomb”: Gradual thinning of the postoperative conjunctival roof of the new outflow path, called the filtering bleb, led in many eyes to spontaneous ocular perforation and intraocular infection (endophthalmitis). Nowadays, following our understanding of both that MMC toxic mechanism and the modification of the trabeculectomy-MMC approach, MMC may be used safely. Conclusions: 1. The local administration of 5-FU or MMC intraoperatively and/or postoperatively became the turning point that improved glaucoma prognosis substantially during the last decade. This led to an extensive increase in the number of glaucoma operations, that saved sight of many thousands world-wide. 2. The local use of chemotherapeutic agents has spread to avoid failure of other surgical approaches, such as ocular pterygium and lacrimal-path operations, as well as in fields other than ophthalmology, such as the use of a coated cardiac stent.