Ehrlich II –2nd World Conference on Magic Bullets

Conclusions: This data suggests that polymorphic CYP1A1 and GSTT1 genes appear to affect susceptibility to CML.

Mode of Action of a Naphthoquinone on Tuberculosis TASTAN BISHOP AO^1, MEYER JJM^2 ^1University of Pretoria, Bioinformatics and Computational Biology Unit, Pretoria, South Africa; ^2University of Pretoria, Department of Plant Science, Pretoria, South Africa. Background: Euclea natalensis is a shrub or small to medium size tree, which occurs in southern Africa. The roots of it are used by the indigenous people of southern Africa for various bacterial infections. It was showed that six naphthoquinones from this tree have activity against Mycobacterium tuberculosis. One of them, 7-methyljuglone, has very similar structure to menaquione. In the electron transport process of Mycobacterium species, electrons are transported by menaquinone. Aim: To generate homology model of one of the electron transport system enzymes, cytochrome b from M. tuberculosis, and to investigate the enzyme’s possible interactions with the bioactive anti-tuberculosis compound isolated from plant Euclea natalensis. Methods: Preliminary homology model of M. tuberculosis cytochrome b is calculated by the program MODELLER by using the crystal structure of R. sphaeroides. Results: Preliminary results revealed that part of the ligand stigmatellin located in the cytochrome b of R. sphaeroides crystal structure has a very similar structure to our compound. In laboratory side, it was shown that the minimum inhibitory concentration (MIC) value on the H37Rv M. tuberculosis strain, of the compound is 0.5 g/ml which is comparable to positive drug control rifampicin (0.125g/ml) and better than ethambutol (1.250 g/ml).
And what if we used for therapeutic drug delivery, the "magic bullets" that Dictyostelium discoideum cells expel as a multidrug resistance mechanism? TATISCHEFF^1I, LAVIALLE^1F, ALFSEN ^2° ^1BioMoCeTi, CNRS UMR 7033, Université Pierre et Marie Curie, Génopole campus 1, 91030 Evry cedex ^2CNRS, UMR 8104, INSERM, U567, Institut Cochin, Département de Biologie Cellulaire, Université Paris-Descartes, 75014 Paris, France In 1935, Raper brought the non-pathogenic eukaryotic amoebae Dictyostelium discoideum to the attention of the microbiological scientific community. More than 6 decades later, in 1999, the NIH (National Institutes of Health, USA) decided to add this micro-organism as a new model for biomedical research. Already in 1990, we used Dictyostelium to find out by which mechanism the cells get rid of benzo(a)pyrene, the potent carcinogenic compound of tobacco smoke. We found that, like many human multidrug-resistant cells, Dictyostelium cells do have a multidrug resistance pump, the P-glycoprotein (170 kDa). However, this pump proved to be non-active and therefore not responsible for drug resistance in Dictyostelium cells. Thus, we had to search for a new mechanism mediating drug resistance. We found that Hoechst 33342 (HO342), a DNA-specific marker, was not vitally staining the nuclei, thus giving evidence of Dictyostelium cell nuclear resistance. We observed that when Dictyostelium cells are grown in the presence of HO342, they expel extracellularly vesicles loaded with the dye. Such a vesicular pathway of detoxification was indeed the new resistance mechanism we had been looking for. The next question became: could these biological dye-loaded vesicles be used as a dye-vector to by-pass Dictyostelium nuclear resistance? Experimental data obtained with living naïve Dictyostelium cells (never in contact with the dye before) and also with K562r cells, a human leukaemia multidrug-resistant cell line, allowed us to propose Dictyostelium vesicles as a new promising non-viral drug delivery tool*. As a first approach to therapeutic application, we chose hypericin, a photosensitizer intended for antitumoral photodynamic therapy. The biological loading of vesicles with hypericin was first controlled. Then, the vesicle-mediated drug transfer was studied, using two human cell lines as target cells, skin fibroblast (HS68) and cervix carcinoma cells (HeLa). Altogether, these data probe that the Dictyostelium extracellular vesicles could be used as “magic bullets” for transferring a therapeutic molecule within human cells. ^ Pending European Patent (DRITT SAIC Université Pierre et Marie Curie) no. WO2005004925 20-01-2005, I. Tatischeff, A. Alfsen, F. Lavialle, Extracellular vesicles from non-pathogenic amoeba useful as vehicle for transferring a molecule of interest to an eukaryotic cell.	Recombinant Expression of the Biosynthetic Enzyme for the Biotechnological Production of Tetrahydrocannabinol, a Revisited Magic Bullet from Marijuana TAURA F Kyushu Univ., Fukuoka, Japan. Background: In recent years, tetrahydrocannabinol (THC), the psychoactive principle of marijuana, has attracted a renewed interest as a kind of Magic Bullet. Actually, in some countries, THC has been approved as a medicine for suppressing nausea and vomiting caused by cancer chemotherapy, and in addition, Sativex, a marijuana-based preparation containing THC, was licensed in Canada as a neuropathic pain reliever for adult patients with multiple sclerosis. In marijuana plants, THC generates from the acidic precursor tetrahydrocannabinolic-acid (THCA) via non-enzymatic decarboxylation. The biosynthetic enzyme, THCA synthase, is an attractive target for the biotechnological production of THC because THCA is readily decarboxylized into THC by heating. Aims: 1) To develop an effective expression system for THCA synthase. 2) To develop novel production system for THC using the recombinant enzyme. Methods: The gene encoding THCA synthase was cloned into plasmid pPICZ for yeast transformation. The transgenic yeast Pichia pastoris was developed by homologous recombination with the vector at the alcohol oxidase promoter region. Cannabigerolic-acid (CBGA), the substrate for THCA synthase, was chemically synthesized, and fed to the Pichia culture for the enzymatic synthesis of THCA. The THCA produced was extracted with ethyl acetate, and decarboxylated into THC by heating at 120 degree C. Results: THCA synthase was functionally expressed and secreted from Pichia cells. The optimal expression level (1.32 nkat/liter) was obtained when the cells were cultured in buffered complex medium containing supplements including riboflavin and casamino acids. When the substrate CBGA was directly added to the Pichia culture, THCA production could be detected, but in poor level (<10 % conversion). In contrast, the culture supernatant, from which the cells were removed, could effectively convert CBGA into THCA with a maximum conversion rate of ~98%, and a yield of ~33 mg/liter. THC was prepared by heat treatment of THCA extracted with ethyl acetate from the culture supernatant. The yield of THC was ~24 mg from 1 liter incubation. Conclusions: 1) The culture supernatant containing the recombinant THCA synthase could synthesize THCA, which is decarboxylized into THC. 2) This is the first biotechnological production of THC.
Magic Bullets Made Easily Using Nano-particulate Membrane Bioreactors TAYLOR AP^12 ^1Australian Membrane Technologies Pty. Ltd. (AMT), Sydney, Australia; ^2Australian Nuclear Science and Technology Organisation (ANSTO), Sydney, Australia. Background: Without genetic modification most secondary metabolites produced by fungi, actinomycets and members of the genus Bacillus are only produced when grown in solid-state culture. Aims: 1) To compare primary and secondary metabolisms in a membrane-surface liquid culture bioreactor called a nano-particulate membrane bioreactor (NMB) to sparged liquid cultures. 2) To assay the influence of membrane thickness on metabolic activities. 3) To scale the NMB up to a size suitable for commercial applications. Methods: A variety of experiments were conducted to measure antibiotic biosynthesis, nutrient consumption and biomass growth in various NMB rigs, and were compared directly to sparges submerged cultures fed identical nutrient broths and incubated at the same temperature. The effect of varying the thickenss of the membranes in the NMB was also assayed with respect to primary and secondary metabolisms. The NMB was modified from a pouch form to a gill form, scaled up and applied to a variety of application. Results: Penicillin yields, nutrient consumption and growth rates were significantly higher/faster in the NMB. Yields and production rates in the NMB were up to 2.4 and 6.0 times the greater than in sparged liquid cultures, respectively. The biomass was reused nine times in an NMB with the yields increasing through the experiment. After the first batch there was no lag before biosynthesis resumed, so primary and secondary metabolisms were concurrent. Scale-up has reached 1,150 m^2, capable of treating 3-40 kL/day depending on the application. The NMB can be repeatedly steam sterilized and can be made of materials compatible with pharmaceuticals manufacture. Conclusions: 1) The concurrent solid and liquid phases in the NMB enables obligate aerobic microbes to grow in direct contact with air, while communicating with a liquid phase for easy control of pH, temperature and nutrient and precurssor concentrations, easy separation of products downstream, efficient biomass retention, and greater yields and production rates, which makes the NMB an ideal technology for biosynthesis of primary and secondary metabolites. 2) Productivity in the NMB increases with thinner membranes. 3) The NMB has been scaled up to be suitable for a wide variety of commercial applications.	Comparison of therapeutic effects of Reboxetine and Methylphenidate in children and adolescents with Attention-Deficit/Hyperactivity Disorder TEHRANI-DOOST M, MOALLEMI SH, SHAHRIVAR Z Roozbeh Hospital, Department of Psychiatry, Tehran University of Medical Sciences, Tehran, Iran Background: Dysregulation of biogenic amines especially of norepinephrine and dopamine has been proposed in pathophysiology of attention-deficit/hyperactivity disorder (ADHD). According to this hypothesis patients with ADHD may benefit from drugs which increase noradrenalin such as reboxetine (a specific noradrenalin reuptake inhibitor).the main aim of this open-label study was to assess the efficacy and tolerability of reboxetine compared to methylphenidate in children and adolescents with ADHD. Method: twenty boys diagnosed with ADHD as the reboxetine group and eighteen as the methylphenidate group, matched on their age, were enrolled in a 6-week open trial treatment. Assessment included the Conners' Parent Rating Scale-Revised, Short Version (CPRS-R-S) which was administered at baseline and weeks 2, 4, and 6. The dose of reboxetine was between 3 and 6 mg/day and of methylphenidate was 1mg/kg/day. Results: it was found a significant reduction in ADHD symptoms as measured by CPRS-R-S in both groups (P<0.01), without any significant difference between two groups after 6 weeks of treatment. Reboxetine was relatively well tolerated. The most common adverse effects of reboxetine were decreased appetite, constipation, sleep problems, and dry mouth. Conclusion: this open-label comparison trial suggests the efficacy of reboxetine in the treatment of ADHD in youth and it is comparable to methylphenidate. Controlled studies with larger samples are needed to confirm this finding.
Isatin (2.3-dioxo-indole) and its Analogues as New Antipyretic Agents TELEGDY G, ADAMIK A, GLOVER V¹ Department of Pathophysiology, University of Szeged, Szeged, Hungary , ¹Institute of Reproductive and Developmental Biology, Imperial College, London, UK. Background: Most of the antipyretic drugs available are acting mainly on blocking the arachidonic cascade forming PGE2. From our previous experience isatin (2,3 dioxo-indole) was able to prevent hyperthermia by blocking the action of PGE2 also. Methods: In the present work the action of isatin and isatin analogues (5-methylisatin, 6-hydroxyisatin, 7-ethylisatin, N-acetylisatin) have been tested on prostaglandin E2 (PGE2)-induced hyperthermia in mice and rats. Two forms of induced hyperthermia have been tested. When the PGE2 was given simultaneously with the isatin or analogues, the development of hyperthermia was tested, when the test compounds were given 3o min following PGE2 administration, the action on already existing hyperthermia was measured in mice and in rats. The temperature of the animals was measured in the colon. Results: The results demonstrate that isatin in a dose of 12.5 mg/kg ip is able to block the initiation and and in a dose of 25.0 mg/kg ip attenuate the PGE2-induced existing hyperthermia in rats. In mice 3.12 mg/kg ip isatin can block the development of PGE2-induced hyperthermia, while in a dose of 12.5 mg/kg ip can attenuate the existing hyperthermia. 5-Methylisatin in a dose of 3.36 mg/kg ip can block the development of hyperthermia and in the dose of 13.44 mg/kg ip can attenuate the existing PGE2-induced hyperthermia in rats. In a dose of 0.21 mg/kg ip can block the initiation of hyperthermia and in a dose of 6.72 mg/kg ip attenuates the existing hyperthermia in mice.6-Hydroxy-isatin in a dose of lo.40 mg/kg ip blocks the development and also the existing hyperthermia in rats. In mice 5.2 mg/kg is able to block the development of hyperthermia and in a dose of 10.4 mg/kg attenuates the existing hyperthermia. 7-Ethylisatin in the dose of 0,112 mg/kg blocks the initiation and also the existing hyperthermia in rats. In mice both the initiation and also the existing hyperthermia can be blocked by the dose of 0,0288 mg/kg. N-Acethylisatin dose of O,O96 mg/kg blocks the initiation and the dose of 0,384 blocks the existing hyperthermia in rats. O,005 mg/kg blocks the initiation of hyperthermia, while the dose of 1.024 mg/kg blocks the existing hyperthermia in mice. Conclusion. The results demonstrate that not only isatin but the isatin analogues can also block the PGE2-induced hyperthermia and that 7-ethyl- and N-acetylisatin are the most effective compounds in both blocking the development of hyperthermia and also in attenuating the PGE2-induced hyperthermia.	Activated iron(IV)-oxo structures in intermediates of heme enzymes and their models TERNER J^1 Dept. of Chemistry, Virginia Commonwealth University, Richmond, VA 23284-2006, USA Background: Postulated high valent heme active sites of the cytochrome P450 enzymes during catalysis have not been readily observable. High reactivity and instability appear to make their physical characterization difficult. In contrast, high valent intermediates of the related heme enzymes known as peroxidase and catalase, have been under study for many years, and have been used to infer the nature P450 intermediates. Methods: The activated ferryl forms of peroxidases from sources such as horseradish, turnip, baker’s yeast, and other plant and mammalian sources were probed by resonance Raman spectroscopy. The two primary intermediates are known as compounds I and II, formed by two and one-electron oxidation of the ferric resting enzyme. Oxygen-18 and deuterium allow identification of Fe(IV)=O vibrations. In addition to the classical peroxidases, myoglobin has a low level of peroxidase activity. Myoglobin was studied as well since there is much interest in the characterization of ferryl myoglobin. Results: Fe(IV)=O frequencies found for native peroxidases are lower than those found for synthetic model ferryl hemes. For peroxidases which retain activity over a wide pH range, such as horseradish peroxidase, the observed Fe(IV)=O frequencies are highly pH sensitive, indicative of ionization of an amino acid in the proximity of the heme active site. The pH dependent frequencies are also markedly sensitive to deuterium substitution. Conclusions: It was not expected that Fe(IV)=O frequencies in proteins would be generally lower than those observed for the synthetic ferryl porphyrin models of compounds I and II. It has been recently speculated that the Fe(IV)=O groups in the protein active sites might be better described as Fe(IV)-OH. In actual fact, many of the observed Fe(IV)=O frequencies in the proteins are remarkably sensitive to deuterium substitution. However, it appears that the deuteration sensitivity is a result of hydrogen-bonding within the protein active site, that is not mimicked in nonaqueous synthetic systems. Hydrogen-bonding imparts partial single bond character to the protein Fe(IV)=O groups. But with only one known exception, the iron-oxo groups in proteins appear to still be best described as double bonds. ^1Financial support is acknowledged from the U.S. National Institutes of Health.
Application of Population Pharmacokinetic Analysis and Monte Carlo Simulations Method in Drug Phenotyping. Assessment of Cytochrome P450 1A2 Activity in a Population of Adult Non-Related Caucasians from Sparse Data TERZIIVANOV DN, BOZHINOVA KV Clinic of Clinical Pharmacology and Pharmacokinetics, Univ Hosp"St.Ivan Rilski", Med Univ, Sofia, Bulgaria Background: Already published data of the authors have unambiguously demonstrated the ability of the Nonparametric Expectation Maximisation (NPEM) method of population pharmacokinetic modelling to deal with sparse data in estimating systemic caffeine clearance (CLS1) for monitoring and evaluation of cytochrome P450 (CYP) 1A2 activity in a population of adult non-related Caucasians. The results of this investigation have shown that NPEM analysis is a reliable and sufficiently sensitive method for clinical testing of liver function even in presence of reduced renal function, using CLS1 as a biomarker. On the other hand, these results have revealled the abilities of the NPEM method as suitable and relevant for large-scale epidemiological studies with regard to phenotyping cancer susceptibility of high risk populations by monitoring their CYP1A2 activity based on random sparse data. The method can deal even with mixed populations (smokers and non-smokers) in discerning multicomponent distributions with departure from normality, which the parametric methods have failed to do. Methods: Large-scale epidemiological study was simulated using the method of Monte Carlo simulations (MCS) in order to explore the applicability of nonparametric population pharmacokinetic modeling in phenotyping of CYP1A2 activity by using of CLS1 as a biomarker. Simulated populations from 500 to 1000 subjects were studied. 250 simulated subjects were randomly sampled according to a 10 points sampling scheme and their simulated “measured” plasma caffeine concentrations were further submitted to nonparametric population pharmacokinetic analysis. Results: The resulted distribution of CLS1 was clearly trimodal. Each of the clusters (“High”, “Intermediate”, and “Low”) had normal distribution of CYP1A2 activity. Obviously, in no clinical setting it would be possible to sample randomly 250 subjects. Conclusions: These results reveal the exceptional usefulness of MCS in drug phenotyping when dealing with clinical setting data which are, by their very nature, limited and sparsely distributed.	Efficacy of 1% topical cyclosporine in the treatment of severe vernal keratoconjunctivitis in childhood SPADAVECCHIA L, TESSE R, FANELLI P, ARMENIO L Department of Biomedicina dell’Età Evolutiva- Pediatric Clinic “Maggiore”-University of Bari, Bari, Italy Introduction: Corticosteroids and high-concentrated cyclosporine eyedrops have been used for treatment of severe vernal keratoconjunctivitis (VKC) cases. The purpose of our study was to verify the efficacy of 1% topical cyclosporine in improving severe form of VKC in childhood and investigate for factors affecting the response to therapy. Material and Methods: We conducted an open trial involving 197 children with severe VKC, who received topical cyclosporine 1% for 4 months. Ocular subjective symptoms and objective signs were scored in all children at entry, 2 weeks and 4 months. Skin prick tests and microscope endothelial cells evaluation were also performed; serum IgE and cyclosporine levels were assessed. Results: The mean score values for severity of subjective symptoms and objective signs were significantly decreased after 2 weeks, and 4 months, compared with those at entry (p<0.001) in all children. Cyclosporine serum levels were not detectable at the end of therapy, nor were endothelial corneal cells damaged. Patients who started the therapy at the beginning of the disease and/or received long-term regimen of treatment with cyclosporine had a faster improvement of ocular signs and symptoms, compared to all other patients. Conclusion: Our findings suggest that 1% cyclosporine concentration administrated topically at the beginning of the disease and for a long-term period might be the most effective treatment to control symptoms and local inflammation in severe forms of VKC in childhood.
Evolution of treatment of kala-azar during last 4 decades in Indai THAKUR CP MD, FRCP (London & Edin), Emeritus Professor of Medicine, Patna Medical College, Member of Parliament, Chairman, Balaji Utthan Sansthan, Uma Complex, Fraser Road- Patna-800 001, Bihar (India), Tel.: [phone], Fax: [phone], Email: [e-mail], [e-mail], [e-mail], Website: www.bus.org.in Abstract: In 1977 there was a massive epidemic of kala-azar with 100,000 patients and 30% of patients were unresponsive to the traditional regimen of sodium antimony gluconate i.e. 6 ml intramasuclar daily for 6 -10 days, starting with ½ ml per day and increasing ½ ml daily to reach 6 ml . By increasing the dose to 6 ml/im daily to 20 days without incremental dose led to cure rate of 92.6 %. This regimen was also approved by World Health Organization. But after some time even with this regimen cure rate decreased and we further rationalized the dose on body wt. basis and 20 mg/kg body wt. for 40 days of treatment gave the best results but toxicity of the drug also increased. 28 days regimen with this dose was agreed upon by World Health Organization and was also adopted by World Health Organization. Later on we found the drug very toxic and ineffications and advised for withdrawl of this drug. We worked also on pentamidine which was found less effective and very toxic causing permament dibetes and we recommended for its withdrawl from the market. We further worked on amphotericin B deoxycholate and found it very useful and recommended its use for general patients. In our experience 1 mg/kg body wt. of this drug given for 20 days gave the best results. We also worked how the toxicity of this drug could be minimized. We also worked on milltefosin and paromomycin Miltefosin has come in the market and paromomycin is underging phase IV trial. Our centre was involved in the trial of amphotericin B lipid complex (Ambisome) and found that even sigle dose of ambisome 15 mg/kg body wt. was well tolerated and cured all patients. We also worked on other oral drugs which were found effective by others but not found effective in our expericne and those drugs were discarded. We are also now working on a drug derived from the wild plant which has been found effective. An oral drug which could be found more effective and less toxic than the current drug in use needs to be developed and is the need of the hour. The principle of treatment and criteria for cure of PKDL, a major source of parasites were changed both with SAG and amphotericine B; 3 to 5 courses of SAG and amphotericine B eleared all lesions. No recurrence was encountered and no patient relapsed contrary to the previous belief. With 20- day regimen of amphotericin B for kala-azar treatment incidence of PKDL has also decreased. Thus there has been a complete change in the understanding of PKDL. Keeping in mind its prevalence in four states of India for the last hundred years. Kala-azar is a big challenge and we are attemting to eliminate it with the help of international co-operation.	Whooping cough vaccines: a public health and producer's perspective THALEN M^1, DE GRAAF T^2 ^1SynCo Bio Partners, Amsterdam, the Netherlands, Netherlands Vaccine Institute, The Hague, the Netherlands. Background: Despite the high vaccination coverage in most of the Western world, the incidence of whooping cough has been increasing in all age groups for the last 2 decades. The global rise in whooping cough cases did not lessen after the introduction of acellular vaccines a decade ago, or the introduction of booster vaccinations in toddlers. Currently registered vaccines are not well equipped to overcome the rising incidence of whooping cough for a number of reasons. Therefore, there is a definite need for an improved whooping cough vaccine. Methods: This paper selected the following characteristics of an improved vaccine. An improved whooping cough vaccine should (1) enable all age groups to be vaccinated with B. pertussis circulating strain antigens, (2) protect against whooping cough induced by B. parapertussis, (3) enable infants to be vaccinated earlier, (4) cause minimal adverse events after repeated vaccination, and (5) protect longer than currently registered vaccines. A number of scenario's and corresponding vaccine compositions that comply with (part of) these characteristics were examined, using time to market, costs and risks as constraints. Results & conclusions: The most likely candidates to fulfill all characteristics are oral or intranasal vaccines consisting of inactivated whole B. pertussis cells, given the fact that an oral vaccine has already shown proof of protection in a phase III study without adverse events, and that an intra-nasal vaccine has shown proof of concept in a phase I study. Live attenuated vaccines fulfill many of the 5 characteristics as well, but will most likely take longer to reach the market. At this point it is not clear if a B. parapertussis component should also be included in an improved whooping cough vaccine, and what the cost-benefit ratio would be.
Nephrotoxicity of immunosuppressive drugs, new insight THERVET E Hopital Necker, Paris, France Background: Organ transplantation is the best treatment, if not the only, of end stage organ failure. However, even though the incidence of acute rejection episode has dramatically lowered after renal transplantation with a short term graft survival above 90 %, long-term results have not improved consequently. Conversly, non renal transplant, chronic renal failure (CRF) is a common complication. One of the major causes of CRF, namely nephrotoxicity, is a major side effect of the immunosuppressive drugs used and may precluded long-term outcome. This side effect is well-recognized for calcineurine inhibitors but sirolimus (SRL), a mTOR inhibitor, also exhibit nephrotoxicity especially after delayed graft function. New diagnostic tools can be used. They include pharmacokinetic approach such as abbreviated aera under the curve, automatic quantification of interstitial fibrosis or pharmacometabonomic. New insights of nephrotoxicity mechanisms also have been demonstrated. For calcineurine inhibitors, in vivo and in vitro data demonstrate that P-glycoproteine may play a critical role in protecting renal epithelial cells from cyclosporine A (CsA) toxicity. Regarding the molecular mechanisms, transcriptional profiles of human proximal tubular cells exposed to CsA found that it preferentially alters biological processes located at the cell membrane, such as ion transport or signal transduction. Wide expression analysis suggested that CsA may induce an endoplasmic reticulum (ER) stress in tubular cells in vitro and in vivo, because of cyclophilin A inhibition. Furthermore, CsA induces epithelial phenotypic changes reminiscent of an incomplete epithelial-to-mesenchymal transition in a TGF- independent manner, as did various ER stress inducers. Finally, in primary cultured human tubular cells CsA induces autophagy dependant of ER stress. SRL modifies biological processes within the nucleus and related to transcriptional activity. It inhibits the proliferative response to mitogenic stimuli, and causes cell cycle arrest in the early G(1) phase, by a nonspecific process due to inhibition of the p70(S6k) pathway and by a direct effect on cyclin D3 mRNA stability. Finally, new therapeutic approach such as calcineurine inhibitors mimization conversion or avoidance, have demonstrated contradictory results because of the increased risk of acute rejection.	Evaluating our Performance: Tissue Penetration Paradigm Shift THEURETZBACHER U Center for Anti-Infective Agents, Vienna, Austria The term paradigm shift, as introduced by Thomas Kuhn, describes a change in basic assumptions within a ruling theory of science. Such a change in a certain thought-pattern is usually the result of a long process. Applying the paradigm shift model to the measurement of concentrations of anti-infective drugs at the site of infection, we indeed observe a remarkably slow shift in thinking and acting even in the face of compelling reasons for change. For several decades anti-infective drug concentrations at the site of infection have been primarliy measured by taking a whole tissue biopsy, grinding it, determining the total concentration in the homogenate, comparing it with the corresponding blood sample, and then judging a drug’s clinical value and performance from such a measurement. As has been shown, homogenizing biopsy samples results in a mixture of distinct pharmacological compartments, of bound and unbound drug, and, thus, fails to give meaningful information about concentrations at the infection site. Surprisingly, despite our knowledge of the many well-known problems with interpreting tissue concentrations from biopsy samples, this method remains in fairly common use over many years after the flaws were described. According to Kuhn’s definition of a paradigm shift, the anti-infective research community is - like other research communities - reluctant to change and slow to adopt new thinking. This can be attributed to some extent to the lack of reliable alternatives or to challenging issues with new methods. However, if the new paradigm of repeatedly measuring free concentrations in distinct pharmacological compartments - such as extracellular fluid of tissues or various body fluids - is generally accepted, thoroughly explored and executed, the old paradigm will be replaced. We seem to be close to this paradigm shift and thus to more accurate measurement and interpretation of tissue concentrations.
Magnetic Resonance Imaging ‘Pathfinder’ Molecules For Use in Optimizing Antibody and Gene-Directed Enzyme-Prodrug Therapy (ADEPT/GDEPT) Treatments THOMAS NR^1, GALLIARD L^1, SPENCE D^1, KÖCKENBERGER W^2,MAYERA^3 ^1Centre for Biomolecular sciences, Univ. Nottingham, Nottingham, NG7 2RD UK; ^2Sir Peter Mansfield MR Centre, Univ. Nottingham, Nottingham, NG7 2RD UK; ^3Dept of Oncology, Royal Free and University College Medical School, Hampstead Campus, Rowland Hill Street, London NW3 2PF. Background: A number of Antibody and Gene Directed Enzyme Prodrug Therapy (ADEPT/GDEPT) systems have been developed over the past two decades in order to target anti-cancer drugs to tumour cells and several of these have entered clinical trials. Here we report new magnetic resonance imaging (MRI) contrast agents that have been designed to function as ‘pathfinder’ molecules and allow the non-invasive visualization of the localization and catalytic activity of the enzyme carboxypeptidase G2 (CPG2) used in these prodrug activating systems. Methods: Contrast agents are used in ~50% of all human MRI scans in order to improve the contrast between different tissue types in the images generated. Positive contrast agents cause a reduction in the T1 relaxation time of the hydrogens in water molecules that are coordinated to the metal in the contrast agent. We have modified the clinically used gadolinium(III) contrast agents Gd(III)-DOTA (Gadoterate meglumine:Dotarem^®) and Gd(III)-HP-DO3A (Gadoteridol: ProHance^®) to incorporate enzyme cleavable appendages that protect the remaining coordination sites of the gadolinium(III) ion from binding to water molecules until they are cleaved by the enzyme. We have measured the difference in relaxivity for the ‘pro-contrast agent’ and the hydrolysed product that is able to bind one or two water molecules and so ‘lights-up’ in the MR image Results: We have identified gadolinium(III)-based contrast agents that exhibit a 3-5 fold signal enhancement after being cleaved by MFECP1, a recombinant antibody-enzyme fusion protein of ananti–carcinoembryonic antigen single-chain Fv antibodyand the bacterial enzyme carboxypeptidase G2. The structure of these is being optimized to improve the compound’s stability and rate of CPG2 hydrolysis Conclusions: New enzyme-activated MRI contrast agents have been produced that once evaluated for use in humans could be utilised to optimize ADEPT or GDEPT treatments on a patient by patient basis.	Angiotensin Converting Enzyme Inhibitors Determination in Plasma by Enzyme Kinetic and High-Performance Liquid Chromatography THONGNOPNUA P^1, BOTTCHER C^2 ^1Chulalongkorn University, Bangkok, Thailand; ^2Charite (Campus Mitte), Berlin, Germany. Background: The analysis of Angiotensin Converting Enzyme (ACE) inhibitors in plasma is an essential part for ACE inhibitors pharmacokinetics and bioequivalence determination. A reliable and convenient analytical method is needed. Aims: 1) To develop the simple analytical method for analyzing ACE inhibitors in plasma having enalapril and captopril as the representative compounds, by enzyme kinetic principle and high-performance liquid chromatography (HPLC). 2) To apply the method for determining the concentration of enalaprilat in volunteers’ plasma after oral administration of enalapril. Methods: The concentration of ACE inhibitors in plasma was determined by analyzing hippuric acid by HPLC, using 2-methyl hippuric acid as the internal standard. Hippuric acid is the product from the reaction between exogenous ACE and hippuryl-histidyl-leucine. The HPLC analysis of hippuric acid was carried on a C-18 column, and quantified by UV detector at 228 nm. This analytical method was verified by perfoming a bio-analytical method validation. The method was then used for analyzing enalaprilat concentrations in twelve healthy male volunteers after oral administration of enalpril to determine pharmacokinetic parameters. Results: The proposed method is simple, neither sample extraction nor derivatization is required. The HPLC analysis consumes less than 6.5 minutes per sample. By using human plasma as exogenous ACE for ACE inhibitors and analyzing hippuric acid, the concentration of plasma ACE inhibitors could be determined with the lowest concentrations of enalaprilat and captopril of 3.0 and 10.0 ng/ml, respectively. The relative standard intra-day and inter-day deviations of each compound in plasma were less than 9%. The percentage of bias was less than ± 1% which confirmed the accuracy of the method. The method has been proven for its reliability in analyzing plasma enalaprilat concentration after enalapril administration Conclusions: 1) The concentration of ACE inhibitors in plasma could be simply determined by analyzing hippuric acid, the product of ACE and substrate reaction. 2) The use of human plasma as exogenous ACE for enzyme kinetic analysis of ACE inhibitors is convenient and accurate. 3) The method could be applied for all ACE inhibitors. 4) The method was successfully utilized in pharmacokinetic and bioequivalent studies of enalapril.
Antifungals in the Treatment of Candidiasis Eruption in Oral Autoimmune Diseases THONGPRASOM K^1, KANJANABUD P^1, PIBOONRATANAKIT P ^1, SERERAT T^1, LAOTHUMTHUT T ^2 ^1 Chulalongkorn University, Bangkok, Thailand; ^2Mahidol University, Bangkok, Thailand. Background: Candida infection has been reported as the common lesion eruption during treatment of oral autoimmune diseases such as oral lichen planus (OLP), lupus erythematosus (LE), pemphigus vulgaris (PV) and mucous membrane pemphigoid (MMP) with topical and systemic steroids. Aims: 1) To study the incidence of candidiasis eruption after treatment with steroids. 2) To investigate the effectiveness of topical antifungals. 3) To know the side-effects of topical antifungals. Methods: A retrospective study included 297 patients (female=236; male=61), age ranged between 18-80 years (Mean+SD=48.69+13.46). Data of the patients with OLP (268), LE (6) and MMP (6) cases randomly treated with topical steroids such as fluocinolone acetonide, clobetasol propionate, triamcinolone acetonide and dexamethasone were analyzed. In addition, 17 cases of PV including drugs-induced PV treated with topical and systemic steroids were included in this study. All cases were diagnosed clinically and confirmed by histopathological or immunofluorescent studies. The first candidiasis detection after treatment with steroids was recorded and investigated by KOH 10%, Periodic Acid Schiff stain (PAS) or Candida culture. Topical miconazole and nystatin were used in 40 and 25 cases with candidiasis respectively. Oral findings during treatment in all cases and side-effects were recorded. The data were processed by SPSS 13.0 for Windows. Results: Pseudomembranous candidiasis was the most common lesion found on the areas of treatment in 73 out of 297(24.58%); comprising 66/268 (24.63%) in OLP, 5/17 (29.41%) in PV, 2/6 (33.33%) in LE patients but no candidiasis was found in all MMP patients. The duration of candidiasis eruption in those lesions after treatment with steroids varied from 0.23-87 months (Mean+SD=8.40+15.69). However, candidiasis could be eliminated by topical miconazole and nystatin in all cases without any side-effects in long-term follow-up. Conclusions: Our study confirms that steroids can induce candidiasis after treatment of various oral autoimmune diseases and topical antifungals can be effectively treated candidiasis without any side-effects.	Innovative Method For Quality Control of High Molecular Weight Semi-synthetic Vaccines TIETZ D^1 ^1DJT Consultants, 8167 Shoal Creek Drive, Laurel MD 20724, USA Background: Robbins, Schneerson et al. developed conjugated meningitis vaccines (Hib) for infants [1]. Earlier preparations varied in immunogenicity. For accelerated testing of vaccines, analytical methods based on physical parameters were developed from 1983 - 95. These techniques [2] remain a promising tool for vaccine quality control and for predicting vaccine effectiveness (recently reviewed in [3]). Methods: Horizontal 2-D submarine-type agarose electrophoresis was employed, a method developed by Serwer [4] for the separation of intact viruses. Computer programs ElphoFit and GelFit were used for data analysis and image processing [2]. Results: Original images (left) show characteristic patterns for vaccine preparations (I-III) and two carboxylated polystyrene samples (S) used for standardization. These images were transformed (right) to a coordinate system of particle radius vs. free mobility (surface net charge). No distinct zone patterns can be seen (I-III), since sizes of vaccine particles vary continuously over a wide range (polydisperse) due to randomizing processes in the vaccine preparation (crosslinking and sonication). Conclusion: 1) The vaccine patterns are a fingerprint of the preceding vaccine preparation process and, therefore, can be used for purposes of quality control. 2) Results are available within 1-2 days, whereas immunological testing may take several weeks. 3) Samples II and III with particle sizes larger than 30 nm radius were effective vaccines. Sample II contains a mixture of vaccine batches. 4) The technique cannot only be used for Hib conjugated meningitis vaccines, but also for other high molecular weight vaccines. References: [1] Robbins, JB, Schneerson, R, Anderson, P, Smith, DH, JAMA 1996, 276, 1181–1185. [2] Tietz, D, Aldroubi, A, Schneerson, R, Unser, M, Chrambach, A, Electrophoresis 1991, 12, 46-54. [3] Tietz, D, Electrophoresis 2007, 28, 512–524. [4] Serwer, P, Anal. Biochem. 1985, 144, 172-178.
The in vitro effects of Thymoquinone on human endometrial adenocarcinoma cells TIMURKAN H^1, BULUT H^1, RISVANLI A^1, AYDIN S^1, GURATES B^1, YEKELER H^2, TIMURKAN O^3 ^1University of Firat, Elazig, Turkey; ^2Hospital of Research and Education Elazig, Turkey; ^3 University of Ankara, Ankara, Turkey Background: In this study, it was investigated whether Thymoquinone (TQ) has the effect on women endometrial adenocarcinoma cells as in vitro. Methods: It was used frozen vial human endometrial adenocarcinoma (KLE) cells that obtained from American Type Culture Collection (ATCC) in this study. KLE cells were treated with TQ for various dosages. Results: According to experiments, it was founded that TQ has toxic effect in all dilutions until that 300 micro molar (µM). Especially, TQ had blocking effect on growing in number of KLE cells. Conclusions: It was determined that TQ that composition of Nigella sativa has blocking effect on growing in number of endometrial adenocarcinoma cells as dose depending in cytotoxicity experiments that in vitro.	Dissecting the DNA Base Excision Repair Pathway: Implications for Cancer Therapy MOHAN RD, TINI M University of Western Ontario, London, Ontario, Canada Background: The base excision repair (BER) pathway removes DNA bases damaged by oxidizing and alkylating agents, as well as mispaired bases. The first step of BER is initiated by DNA glycosylases that recognize and excise aberrant bases. Subsequently, the resulting abasic site is processed by other specialized BER enzymes that generate a single nucleotide gap and replace the missing nucleotide. The BER pathway is clinically important as it prevents deleterious mutations and also processes DNA lesions produced by anticancer drugs. Thymine DNA glycosylase (TDG) is a BER enzyme dedicated to the repair of damaged cytosine-guanine (CpG) dinucleotides. TDG and other BER enzymes have been implicated in epigenetic control of gene expression by gene-specific CpG demethylation. TDG also regulates gene expression by direct interaction with the transcription machinery. Interestingly, this versatile enzyme also mediates removal of 5-fluorouracil from DNA and may determine the sensitivity of cells to this widely used anticancer drug. TDG is posttranslationally modified by sumoylation, phosphorylation and acetylation. In exploring the potential of TDG as a drug target, we have sought to decipher how these modifications regulate various TDG functions. Methods: We have employed cell imaging techniques, cell-based reporter gene assays and biochemical analysis to investigate the regulatory roles of TDG posttranslational modifications. Results: We demonstrate that posttranslational modification of TDG dramatically affects TDG subnuclear localization as well as interactions with DNA and accessory proteins, thereby altering the transcriptional and DNA repair functions of this enzyme. Importantly, we have identified specific kinase signalling pathways responsible for phosphorylation of TDG in living cells and show that phosphorylation acts antagonistically to acetylation to regulate DNA damage processing. These findings have allowed us to design mutant versions of TDG that display more potent DNA repair functions. Conclusions: These findings suggest that multiple post-translational modifications and therefore different signaling pathways regulate the biochemical properties and subcellular localization of TDG. The interplay of these covalent modifications allow for exquisite regulation of a DNA repair pathway integral for genome stability
The Acute Effects of Statins in an Experimental Model of Renal Ischemia-Reperfusion Injury TODOROVIC Z, NESIC Z, PROSTRAN M, STOJANOVIC R, VUCKOVIC S, DIVAC N Department of Pharmacology, Clinical Pharmacology and Toxicology, School of Medicine, University of Belgrade, Belgrade, Serbia Background: Renal ischemia-reperfusion (I/R) injury with a high mortality rate and unresolved questions in pharmacotherapy remains one of the leading causes of acute renal failure. We aimed to compare the acute effects of different statins in an established model of renal I/R injury. Methods: Adult male Wistar rats, anesthetized with sodium thiopentone and subjected to renal I/R injury (45 min of I + 4 h of R), were pretreated with simvastatin or pravastatin (1 mg/kg, i.v.), 30 min before I, 30 min before R or 5 min before R. Control rats (subjected to I/R injury) and sham-operated rats were pretreated with appropriate solvent only (10% DMSO or saline). Blood and kidney tissue samples were taken at the end of experiment and selected parameters of glomerular and tubular function were assessed. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test. Results: Simvastatin-treated rats had significantly reduced serum creatinine concentrations, fractional excretion of sodium and total histological score in comparison with the controls (up to 60%, 80% and 40%, respectively). The acute protective effects of simvastatin (1 mg/kg) did not depend on the time of injection and the dose used. On the other hand, pravastatin (1 mg/kg) was significantly more effective than simvastatin in reducing I/R-induced changes of glomerular and tubular function, especially regarding total histological score. Conclusion: The acute pretreatment with a single dose of statin may ameliorate renal impairment and allow earlier recovery from I/R injury. However, it seems that statins are not equally effective in reducing such an injury.	Clinical Role of Fluorouracil Metabolizing Enzymes and Optimal Duration in Chemotherapy with Tegafur TOKUNAGA Y, MD PhD, SASAKI H, MD PhD, SAITO T, MD PhD Department of Surgery Osaka North Japan Post Hospital