Ehrlich II –2nd World Conference on Magic Bullets

Percutaneous absorption of crotamiton in man following single and multiple dosing. DIKA E ¹, TOSTI A¹, FANTI¹, MAIBACH HI². 1. Università degli Studi di Bologna, Dipartimento di Medicina Clinica Specialistica e Sperimentale Sezione di Clinica Dermatologica, Italy. [e-mail] 2. University of California, San Francisco Purpose: Crotamiton is a topical drug used in the treatment of scabies and pruritus. We determined its percutaneous absorption following single and multiple dosing in normal skin. Methods: We used in vivo measurement of percutaneous absorption of [14C] crotamiton in a multidose regimen by measuring urinary excretion and liquid scintillation counting in three groups of four healthy volunteers. The Feldmann urinary excretion method was utilized to ascertain percutaneous absorption. Our results showed that tape stripping does not increase percutaneous absorption of crotamiton; upon repeated application.

Dissociation of Multi-molecular Drug Complexes and Multi-site Binding to 7-TM Receptors:  Protection, Delivery and Enhancement of Adrenergic Activation by Ascorbate.  DILLON PF and ROOT-BERNSTEIN RS. Department of Physiology, Michigan State University. Using capillary electrophoresis (CE), we developed a new method of measuring molecular dissociation constants.  Molecular complexes will dissociate in an electric field.  The relative concentrations of ascorbate (Asc), norepinephrine (NE) and an Asc-NE complex were detected using CE in the 0-200 V/cm range.  By varying the relative concentrations of ascorbate (Asc) and norepinephrine (NE), a dissociation constant at a constant electric field, the Ke, was determined.  Extrapolating multiple log Ke’s to 0 electric field generated the Kd for the complex.  The addition of ascorbate to NE solutions activating smooth muscle shifted the NE dose-response curve to the left by 0.5 log units, indicating enhancement of NE activity by ascorbate.  Since binding of NE by Asc would decrease the free NE concentration, the Asc must dissociate from NE prior to binding.  The electric field generated by the cell membrane will exceed 200 V/cm for 8-9 nm from the membrane, spanning nearly the entire space between adjacent cells, thus causing dissociation of Asc-NE complexes in the interstitial fluid.  Protein receptors create an electronic shadow protecting the agonist binding site from electric field dissociation.  Asc binds to adrenergic receptors in the ìM Asc range, binding to a site between the first and second extracellular loops and trapping the adrenergic molecule in its active site, thus effecting the enhancement. This process enhances both alpha and beta 2 adrenergic receptors, increasing contractions of blood vessels and the relaxation of bronchioles, and has been shown to effectively enhance treatment for asthma conditions in horses and sheep.  Similar results have been found for Asc enhancement of histamine smooth muscle activation. This finding that both molecules in a molecular complex can both bind to a receptor and maximize that receptor’s activity may be a general phenomenon.  Membrane electric fields will dissociate any non-covalent complexes as they approach the membrane.

Role of the cell envelope in the antibacterial activities of cyclic polypeptides against Escherichia coli DIXON RA Department of Forensic and Biomedical Sciences University of Lincoln, Lincoln, United Kingdom Background: The role of membrane permeabilisation and disruption in the mechanism of action of some polymyxin analogues against Gram-negative organisms is contentious. The effects of polymyxin B (PMB) and its analogue polymyxin B nonapeptide (PMBN) on Escherichia coli envelope integrity should correlate but previous work by other workers suggest different modes of action. Methods and Results: This work has reassessed the biochemical techniques used before, and shown that in contrast to previous studies, PMBN (a well-characterized antibacterial synergist) readily releases periplasmic proteins and LPS from treated E.coli at sub-inhibitory concentrations in normal physiological buffer conditions. Conclusions: We conclude that PMBN when tested with appropriate methodology, closely correlates with the early effects of PMB on the cell envelope of E.coli and this study shows that it is now consistent with the accepted interactions of membrane-active agents on Gram-negative cells.

Superoxide Dismutase Activity in the Kidney of Mice Exposed to Acute Cadmium Intoxication: Protective Effect of Magnesium Pretreatment DJUKIĆ-ĆOSIĆ D^1, ĆURČIĆ M^1, NINKOVIĆ M^2, MALIČEVIĆ Ž^2, PLAMENAC BULAT Z^1,MATOVIĆ V^1 ^1Institute of Toxicology, Faculty of Pharmacy, Belgrade, Serbia; ^2Institute of Medical Research, Military Medical Academy, Belgrade, Serbia. Background: Cadmium (Cd) is not a Fenton metal, but it can indirectly increase production of reactive oxygen species. Literature data indicate that short-term exposure to cadmium can decrease the activities of antioxidant enzymes, one of them being superoxide dismutase (SOD), which contains copper (Cu) and zinc (Zn) in its active site and catalyses the conversion of superoxide anion radical to molecular oxygen and hydrogen peroxide. On the other hand, investigations indicate that excessive intake of bioelements, particularly magnesium (Mg), may antagonize cadmium effects. The objective of the study was to determine the effect of increased oral magnesium pretreatment on superoxide dismutase activity in kidney of mice exposed to acute cadmium intoxication. Methods: Swiss albino male mice were divided into three groups: I - control group - not treated animals; II - Cd group: animals given single oral dose of 20 mg Cd/kg b.w. as aqueous solution of CdCl2; III – Mg+Cd group: mice given orally 40 Mg/kg b.w. as aqueous solution of Mg(CH3COO)2 1 h before Cd treatment. The animals were sacrificed by decapitation at 4, 6, 12, 24 and 48 h and superoxide dismutase activity was determined by method of Misra and Fridovich. Statistical analysis was performed using a one-way analysis of variance (ANOVA), followed by the LSD multiple range test. Results: The obtained results show that acute Cd intoxication induced significantly decreased SOD activity in kidney after 6 (P<0.001), 12 (P<0.05) and 24 (P<0.001) h. In the kidney of mice pretreated with Mg, SOD activities were not altered if compared with control group. Conclusion: These results imply a positive role of Mg pretreatment on renal SOD activity in the kidney of mice exposed to acute cadmium intoxication. This could be expalined by the fact that under the same experimental conditions Mg had beneficial effect on Cu and Zn kidney content, as we confirmed in our recent investigations: Cd induced decrease of Cu and Zn (which are necessary for SOD function), while Mg pretreatment increased their levels.

WGA Functionalized Chitosan-Ca-Alginate Microparticles for Targeted 5-FU Delivery in Colon Region DODOV GM^1, HINCAL AA^2, CALIS S^2, STEFFANSEN B^3, SIMONOSKA M^1, GORACINOVA K^1 ^1Faculty of Pharmacy, Cyril & Methodius University, Skopje, Macedonia; ^2Faculty of Pharmacy, Hacettepe University, Ankara, Turkey; ^3Faculty of Pharmaceutical Science, University of Copenhagen, Copenhagen, Denmark. Background: Experimental efforts on the field of peroral anticancer polymer drug delivery systems will probably enable this therapy to become a reality in a near future. In that course, the development of complex drug carriers i.e. bioadhesive hydrogel microparticles (MP`s) for local colon delivery is an approach by combining different principles of targeting and controlled release with muco/bioadhesivity of the system. By providing direct binding to mucosal surface and modifying the residence time and drug release rate, active uptake of therapeutics and better therapeutic efficacy might be achieved. Methods: Using novel one step spray-drying process, enteric coated 5-fluorouracil (5-FU) nanoparticles were incorporated within chitosan-Ca-alginate network in order to enhance the delivery and residence time at targeted site of GIT, simultaneously avoiding the early drug leaking till reaching the site of action. Prepared particles were further functionalized with WGA in order to increase the therapeutic benefit and to improve site-specific 5-FU delivery. Physico-chemical characterization and cell culture transport and efficacy studies were performed. Results: Particles with diameter of 8.5m, high encapsulation efficiency (75.5%), positive surface charge and pH dependent swelling were prepared. FTIR and DSC scans suggested that 5-FU was effectively nano-encapsulated and probably entrapped into MP`s at a molecular level. In vitro studies using Caco-2 cells demonstrated the feasibility of MP`s to affects the transport of 5-FU across the cell model, leading to pronounced presence of 5-FU into the cells. By incorporation of 5-FU into MP`s and further functionalization, the uptake of [methyl-^3H] timidine was reduced for about 30% in comparison with 5-FU solution. These observations could be attributed to improved MP/cell interaction due to the cytoadhesion of the carrier and enhanced tissue accumulation of 5-FU when delivered from MP`s. Conclusion: Prepared formulations could be suitable candidates for controlled colon-specific delivery of 5-FU, opening a new therapeutic potential for this carriers for local treatment of colon cancer.

Antibiotic Resistance of Food Chain Related Bifidobacteria DOMIG KJ, MAYRHOFER S, MAIR C, AMTMANN E, KNEIFEL W BOKU – University of Natural Resources and Applied Life Sciences, Vienna, Department of Food Sciences and Technology, Division of Food Microbiology and Hygiene Background: Representatives of the genus Bifidobacterium play an important role in the human and animal gastrointestinal microflora. The bifidobacterial microbiota is site and host-specific and of beneficial relevance. Therefore several probiotic bifidobacteria strains were studied and are due to their established health benefits of importance in the food and feed industry. The use of antibiotics for prophylactic and therapeutic purposes in humans, animals and agriculture has led to an increase in antibiotic resistance in bacteria and the absence of acquired antimicrobial resistance has become an important criterion to evaluate the biosafety of bifidobacteria used as industrial starter or probiotic cultures. As there are no established standard methods available, a large number of isolated bifidobacteria were tested on LSM media (Klare et al., 2005) by using different defined methods, and the MIC data were analysed in relation to the bifidobacterial species. Methods: A total of about 250 Bifidobacterium strains isolated from the beef and pork production chain were identified by phenotypic and genotypic methods and typed to strain level by molecular methods. The antimicrobial susceptibility against tetracycline, erythromycin, clindamycin, streptomycin, gentamycin, ampilicin and vancomycin was determined by broth microdilution and agar disc diffusion. Resistance genes were detected by PCR-based techniques. Results: The received data support the need for defining microbiological breakpoints to distinguish between the native population and the resistant suppopulation within each bifidobacterial species. Strains atypically resistant to clindamycin, erythromycin and tetracycline were identified. The PCR-based screening results indicate the presence of tet(O), tet(W) and erm(X) genes. The comparision of the two applied susceptibility testing methods (broth microdilution and agar disk diffusion) showed a clear relationship between the two methods. Conclusions: For the first time, the antimicrobial susceptibility of a large number of B. thermophilum and B. pseudolongum isolates from animal origin was studied. The obtained MIC distribution will have some impact on determining the microbiological breakpoint of each tested Bifidobacterium species. Overall, isolates originating from a porcine source showed higher resistances than bacteria from a bovine source. This is maybe due to the higher incidence of antibiotic use in swine farming. The assessed susceptibility methods showed interpretable agreement within this study. The disc diffusion zone diameters are highly reproducible and indicate that this method is a useful alternative to broth microdilution for antimicrobial susceptibility screening of bifidobacterial isolates. Hence, some of the analysed strains may act as a potential reservoir for antibiotic resistance genes, which may be explained that these strains have originated from from an environment of higher antibiotic selective pressure. Domig K.J., et al., (2007) Antibiotic susceptibility testing of Bifidobacterium thermophilum and Bifidobacterium pseudolongum strains: Broth microdilution vs. agar disc diffusion assay. International Journal of Food Microbiology 120, 191-195. Klare I. et al., (2005) Evaluation of new broth mediafür microdilution antibiotic susceptibility testing of lactobacilli, pediococci, lactococci, and bifidobacteria. Applied and Environmental Microbiology 71, 8982-8986. Mättö J. et al., (2007) Susceptibility of human and probiotic Bifidobacterium spp. to selected antibiotics as determined by the Etest method. International Dairy Journal 17, 1123-1131. Mayrhofer S. et al., (2007) Antibiotic susceptibility of Bifidobacterium thermophilum and Bifidobacterium pseudolongum isolates from animal sources. Journal of Food Protection 70, 119-124.

Change in Knowledge of Women about Cervix Cancer, Human Papilloma Virus (HPV) and HPV Vaccination due to Introduction of HPV Vaccines DONDERS GGG^1-4, BELLEN G^4, DECLERQ A^1, BERGER J^1, VAN DEN BOSCH T^1,2, RIPHAGEN I^1, VERJANS M^1 Department of Obstetrics and Gynecology, of the 1) H Hart Hospital Tienen, 2) University Hospital Gasthuisberg Leuven, 3) University of Liège, and 4) Femicare vzw, Clinical Research for Women, Tienen, Belgium Objectives. Test knowledge of HPV, cervix cancer awareness and acceptance of HPV vaccination of women now and a year ago. Methods. Questionnaire were filled out by 305 women visiting 4 gynaecologists of the Regional Hospital Heilig Hart, Tienen, Belgium during two subsequent weeks. Fisher T or Chi² were used as statistical methods to compare the data with the survey of 381 women exactly one year before. Results. Knowledge about HPV as a cause of cervix cancer and the presence of a vaccine rose from roughly 50% in 2007 to over 80% in 2008 (p<0.0001). Level of education and having daughters, boys or no children were no longer of influence in the level of knowledge or willingness to accept the vaccine. Most parents favour the age group 12-16 years as an ideal time for vaccination. In contrast with the 2007 survey, women below 26 had now acquired almost equivalent knowledge to older women about the virus, cervix cancer and the vaccine, but they were far less likely to accept the vaccine due to its cost price, unless it would be reimbursed (OR 4.2 (1.6-11) p=0.0055). Conclusion. One year after introduction of the first two HPV vaccines, over 75% of women attending a ambulatory gynaecology clinic know HPV causes cervix cancer and that you can get vaccinated against it. Compared a year earlier, young and lower educated women had dramatically improved their knowledge. However, women below 26 are less prepared to pay the cost price for vaccination if it is not reimbursed.

Do parasitic nematodes regulate cell apoptosis in the host DONSKOW-SCHMELTER K, DOLIGALSKA M. University of Warsaw, Warsaw, Poland; Background: Parasitic nematodes turn the immune response towards activation of Th2 and Treg lymphocytes, which create tolerogenic or suppressive immune state in the host. Many infections are chronic and therefore in prolonged time may influence or facilitate the outcome of serious disorders especially when innate immunity or activation of Th1-related response is downregulated. Possibly, regulation of cell apoptosis under ongoing infection is a key phenomenon in duration and intensity of the host immune response. The understanding of mechanisms operated during nematode infection is important to predict the immune reactivity not only to other pathogenic factors but also for treatment procedure. Aims: 1) To estimate dynamic of cell apoptosis in different phases of nematode infection in mice infected with Heligmosomoides polygyrus 2) To evaluate differences in the intensity of apoptosis in slow and fast responder strains of mice 3) To identify stage of nematode which inhibits or induces apoptosis 4) To estimate if pro-apoptotic activity of synthetic glucocorticoid – dexamethasone (DEX) is neutralized by H. polygyrus antigens. Methods: This study included male mice of three strains: middle fast responder mice BALB/c, fast responder mice FVB and slow responder mice C57Bl6 infected with intestinal nematode, H. polygyrus. The intensity of cell proliferation, and cytokine production induced by nematode antigens was determined 3, 6, 12, 24 and 30 days post infection Apoptosis of mesenteric lymph node cells (MLNc) ex vivo and in vitro culture was evaluated by FACS. Results: During ongoing infection the percentage of apoptotic cells including CD4^+, was changed and different in evaluated strains of mice (P<0.0001). In C57Bl/6 apoptosis of CD4^+ cells significantly increased in the histotropic phase of infection at 3 day (P<0.0001). In the enteric phase of infection especially on day 12, apoptosis of MLNc was inhibited in each strain of mice. The antigen of infective larvae stage L3 induced apoptosis of MLNc both infected (P<0.05) and uninfected (P<0.05) mice but antigen of the next stage- L4 and adult stage inhibited apoptosis in in vitro culture. Adult stages excretory-secretory antigen partially reduced total and CD4^+ MLNc apoptosis of uninfected (P<0.0001) and infected (P<0.0001) mice provoked by DEX. Conclusions: 1) H. polygyrus antigens evoked different level of inflammatory reaction in slow and fast responder mice; 2) in C57Bl6 mice a weak inflammation appeared with accordance of accelerated CD4^+ cell apoptosis.3) L3 antigens induced apoptosis but L4 and adult worms antigens inhibited apoptosis. 4) The glucocorticoid pro-apoptotic activity was neutralized by H. polygyrus metabolic extracts.

The Role of ABC Transporters in the Pharmacokinetics of Miltefosine for Leishmaniasis DORLO TPC^1,2, VAN TELLINGEN O^3, DE VRIES PJ^2,BEIJNEN JH^1 ^1Department of Pharmacy & Pharmacology, Slotervaart Hospital/Netherlands Cancer Institute, Amsterdam, The Netherlands; ^2Division of Infectious Diseases, Tropical Medicine & AIDS, Academic Medical Center, Amsterdam, The Netherlands; ^3Department of Clinical Chemistry, Netherlands Cancer Institute , Amsterdam, The Netherlands. Background: The ATP-binding cassette (ABC) drug efflux transporters breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) are involved in multidrug resistant cancer. Recently, it was shown that human ABC transporters are possibly also involved in drug resistance of the Leishmania parasite by modulating the human macrophage host cell. We investigated whether miltefosine, originally an anticancer drug and now an antileishmanial, is a substrate for BCRP or P-gp and review the literature on this topic. Methods: The influence of BCRP and P-gp on drug transport, accumulation, and efflux of miltefosine were studied directly in cells overexpressing BCRP1 (MDCKII-Bcrp1), and cells overexpressing MDR1a (LLCPK-MDR1a), grown as a monolayer on a permeable surface. Transport of radioactively labeled was evaluated and compared in transwell-experiments, in triplicate. A review will be given, combining data from literature with our own data. Results: Miltefosine was not a substrate for BCRP in our cell model. On the other hand, miltefosine was a modest substrate for Pgp (MDR1a). The LLCP-MDR1a cells showed twice as much transport of miltefosine from the basolateral to apical compartment, than vice versa (B-A: 7.5% transport, A-B: 3.4%; means after 4h). A large amount of miltefosine was internalized by the cells. We elaborate on the role of Pgp in the pharmacokinetics and drug resistance concerning miltefosine. Conclusions: Miltefosine was shown to be a substrate for Pgp, but not for BCRP. Transport was only influenced by overexpression of Pgp (MDR1a) in our transwell experiments. Further studies are warranted to investigate the role of miltefosine in e.g. antimony resistant leishmaniasis.

Adult stem cells are a source of paracrine factors for tissue regeneration. DORONIN SV^1, POTAPOVA IA^1, GAUDETTE GR^2, BRINK PR^1, ROBINSON RB^3, ROSEN MR^3, COHEN IS^1 ^1Department of Physiology and Biophysics, Institute of Molecular Cardiology, State  University of New York at Stony Brook, Stony Brook , NY 11794 , USA ; ^2Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester , MA 01609 , USA ; ^3Center for Molecular Therapeutics& Departments of Pharmacology and Pediatrics, Columbia University, New York , NY 10032 USA Background: Studies in animal models and clinical trails have shown that mesenchymal stem cells (MSCs) participate in the wound healing process in mammalians. Systemically infused MSCs accumulate at the sites of injury and inflammation. Delivery of MSCs to infarcted heart results in improvement of cardiac functions. Only a small fraction of MSCs differentiates into cardiac myocytes suggesting that the major therapeutic effects of MSC delivery are related to release of anti-apoptotic and pro-angiogenic factors Methods: We investigated interactions of human MSCs with endothelial cells and cardiac myocytes in vitro. Results: We established that MSCs specifically recognize apoptotic endothelial cells. This suggests that endothelial cells undergoing apoptosis may regulate homing of MSCs to the sites of injury. We developed a method to concentrate paracrine factors secreted by MSCs 5-20 times. Concentrated paracrine factors stimulated migration, extracellular matrix invasion, proliferation, and survival of endothelial cells. Co-culture of MSCs with cardiac myocytes improved myocyte survival and triggered mitotic propagation of cardiac myocytes. Conclusions: We suggest that further investigation of therapeutic properties of paracrine factors produced by MSCs will help to understand the role of MSCs in the wound healing and develop novel therapeutic agents for tissue regeneration.

L-tryptophan as a Research Compound and Therapeutic Agent DOUGHERTY DM^1, RICHARD DM^1, MATHIAS CW^1, HILL-KAPTURCZAK N^1, DAWES MA^1, BADAWY A.A-B ^2 ^1University of Texas Health Science Center San Antonio, Psychiatry, Division of Alcohol and Drug Addiction, NRLC ^2The Cardiff School of Health Sciences, University of Wales Institute Cardiff (UWIC) L-tryptophan is a dietary amino acid that has been used as a treatment for mood disorders, either alone or in combination with other pharmacological agents, to augment serotonin function. In the research arena, procedures have been used to reduce L-tryptophan availability and consequently deplete serotonin in the brain, to study the relationship between serotonin and accompanying mood states in participants with various psychiatric disorders. Important findings in this arena over the last decade have produced dramatically increased interest in L-tryptophan research. The clinical benefits of L-tryptophan indicate that it could play a significant role in augmentation strategies in the treatment of psychiatric disorders, and findings from studies that alter L-tryptophan availability further underscore its importance for altering mood states.

Circumventing the blood-brain-barrier. Soviet attempts during World War 2. DREIFUSS J-J, TIKHONOV N University Medical Centre, CH-1211 Geneva 4, Switzerland. [e-mail] Background. Following the outbreak of the WW2, Soviet scientists were asked to contribute to the war effort. Lina Stern (LS), the  first and only female member of the Academy of Sciences (AoS), saw an opportunity to serve her country, while at the same time assessing, if what she had learned in animal experiments with by-passing of the blood-brain-barrier, applied also to humankind. She had noted that direct injection of drugs into the spaces containing cerebro-spinal fluid led to different, sometimes even opposing effects, compared to those seen after injections into blood, muscle or peritoneum. Thus, intracisternal (ic) injections could lead to marked stimulation of autonomic functions. Methods. Our research is based on the few articles on human patients LS published during WW2 in British and American medical journals, on Soviet publications aimed at informing the international community about scientific achievements in the USSR, as well as on experimental protocols kept in LS' archives at the AoS. Results. During the war against Finland (1940) and Germany (1941 and thereafter), soldiers suffering from traumatic shock received ic injections of potassium phosphate solutions and a majority benefited from such a treatment. LS was rewarded with a Stalin Prize (1943), although some members of the medical corps of the army remained sceptical of her approach. Significant results were obtained with ic antitoxin injection to tetanos-affected patients, although the data is difficult to interpret, as these patients also received the antitoxin intramuscularly. In 1945-46, LS obtained small amounts of streptomycin, the antibiotic discovered in 1944 in the United States which was the first ever to be active against tuberculosis. She gave it to paediatricians to allow them to treat, with "her method", children affected by meningeal tuberculosis_ which had a very short life expectancy. The "magic bullet" worked, a majority of children survived, however many with hearing deficits, a side effect attributable to the streptomycin. Coda. Early in 1947, LS was accused of treason and terrorist activity, arrested and detained for nearly 4 years in the abominable Lubjanka prison in Moscow. Whether this was in part due to the unauthorized obtaining (through personal international contacts) and importing of streptomyicn into the USSR remains obscure. More important, however, Staline was turning antisemitic in his last years and the Cold War was looming large. Notice that LS was born into a family of German-speaking, well educated, Latvian Jews. In July 1952, 14 prominent members of the Jewish Antifascist Committee were sentenced : 13 to death, LS to 5 years of internal exile. In 1953, only weeks after Stalin's demise, she could return to Moscow, where she lived until her death in 1968, at the age of 90.