Conclusions: The strategy is to implement these assays in the early phase of toxicity testing. This means that all these the assays are performed for a ranking ot the lead optimisation compounds. However, due to the high demands on purity of the compound for genotoxicity testing and the overall amount of compound needed for all these assays, i.e. 10 to 20 mg, in principle only after the first positive identification of the pharmaceutical activity in vivo and in vitro.
Non-steroidal Anti-inflammatory Drugs; a Potential New Way to Treat Human Cytomegalovirus (HCMV) Disease
SCHRÖER J1;2, SHENK TE2
1 University Regensburg, Regensburg, Germany; 2Princeton University Princeton, NJ, USA
Background: HCMV is responsible for a great amount of personal suffering as well as for significant economical damage. As of today there are three systemic drugs approved for HCMV treatment: ganciclovir, foscarnet, and cidofovir. These drugs have provided major advances in HCMV disease management, although they are limited by toxicities, oral bioavailability and efficacy. Furthermore there are reports of cross-resistance for the major drugs. Therefore new drugs, preferably in oral formulations, are needed. Of particular need are drugs which could be used in congenital HCMV disease in neonates.
Methods: Experiments have been conducted in primary human foreskin fibroblasts with the HCMV strain AD169. Cells were pre-treated with medium containing the indicated amount of tolfenamic acid for 24 h before inoculation with the indicated amount of virus. The inoculum was replaced by fresh drug-containing medium, which was replaced with fresh drug-containing medium every 24 h. Data were collected via fluorescenct focus assay, immunofluorescence, quantitative PCR & quantitative RT-PCR.
Results: Human cytomegalovirus has previously been shown to induce the accumulation of cyclooxygenase-2 RNA, protein and enzyme activity. High doses of cyclooxygenase enzyme inhibitors substantially block viral replication in cultured fibroblasts. Here we demonstrate that two nonsteroidal anti-inflammatory drugs, tolfenamic acid and indomethacin, reduce direct cell-to-cell spread of HCMV in cultured fibroblasts by about 50 %. The block occurs at concentrations of drug commonly used for human therapy (~ 20 µM), and it is reversed by addition of prostaglandin E2 (PGE2), proving that it results from the action of the drugs on cyclooxygenase activity. It is noteworthy that Tolfenamic acid is well bioavailable and has usually mild side effects even in higher doses over a prolonged period of time and there are trials in pediatric patients.
Conclusions: Our study has been designed to ascertain the therapeutic potential of cyclooxygenase inhibitors as an alternative antiviral treatment. Since direct cell-to-cell spread likely contributes importantly to HCMV pathogenesis, we suggest that non-steroidal anti-inflammatory drugs might help to control HCMV infections, either as a monotherapy or in conjunction with other anti-viral treatments.
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PK/PD Modeling of Time-Kill Curves: Addressing Biphasic Killing Patterns
SCHUCK EL1 & DERENDORF H2
1Eisai Research Institute, Andover, MA, USA; 2University of Florida, Gainesville, FL, USA
Background: Bacterial time-kill curves can be normally modeled with simple modified indirect response Emax models. These models have been successfully applied to describe effects of β-lactams (penicillins, cephalosporins, and penem drugs), against various strains of bacteria. However, quinolones have been shown to exhibit a biphasic killing pattern, which cannot be described by the models applied to β-Lactams. In this work we evaluated different Pharmacokinetic/Pharmacodynamic (PK/PD) models to describe the biphasic killing pattern exhibited by ciprofloxacin (CIP), a fluoroquinolone, in in vitro time-kill experiments.
Methods: E. coli, P. aeruginosa, and S. aureus were exposed to changing concentrations of CIP in an in vitro model of infection, in which the 4 hour in vivo half-life of CIP was simulated. Static concentration kill curves for CIP were also constructed againt E. coli and S. pneumoniae. In all experiments, samples were collected at predetermined time points. Three 10 to 100-fold serial dilutions were plated in duplicates and incubated overnight at 37º C. Time-kill curves were obtained by plotting the number of CFU/mL against time. Different PK/PD models were applied to describe the in vitro time-kill kinetics of CIP in these experiments.
Results: Biphasic killing pattern was observed against all three bacteria in the in vitro model of infection, as well as for E. coli in the static concentration time-kill experiment. The biphasic killing pattern against these strains was successfully described by both a novel Adaptive Emax model, which included a term to account for the change in the kill rate after approximately 4 hours, as well as a two sub-population Emax model. EC50 values of 0.0035 mg/L for E. coli, 0.0129 mg/L for P. aeruginosa, and 0.078 mg/L for S. aureus were obtained. The model allowed good individual fits for multiple-dose data extracted from the literature. However, kill-curves against S. penumoniae did not present the biphasic pattern, and were successfully modeled by the same modified indirect response Emax models applied to β-lactams.
Conclusions: 1) Biphasic killing kinetics was confirmed for CIP against 4 different strains of bacteria, but not against S. pneomoniae; 2) The biphasic killing pattern was described successfully by 2 very different models; 3) These results do not provide insight into the mechanism of this observation.
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Bringing Light Into the Dark: Influence of Fluorescence Labeling on Protein Nanoparticles for in-vivo Use
BROERMANN P, SCHULTES S, COESTER C
Ludwig Maximilians University, Munich, Germany
Background: Protein nanoparticles (NP) are one of the most promising tools for drug delivery in the field of tumor therapy. While scientific research advances from basic in-vitro tests towards complex in-vivo studies, the visualization of those NP within various animal systems becomes of increased interest. Several groups have used fluorescent dyes or quantum dots with the drawbacks of unintendedly changing the NP properties and very low signal intensities in-vivo. We investigated several labeling techniques and dyes for their influence on size, aggregation and surface charge.
Methods: NP were prepared from gelatin type A by two-step desolvation. Different commercially available fluorescent labels were incubated with the protein solution prior to NP formation or with unmodified NP. All covalent labeling steps were done according to the manufacturer’s protocol, while dextran dyes were incorporated into the gelatin matrix. Cationisation was done with cholamine hydrochloride or with diethylaminoethanol-dextran. The read out of fluorescence intensity of the standards and NP was conducted with a fluorimeter and a fluorescence microscope. Size and zeta were analysed with dynamic light scattering prior and after each modification step.
Results: The signal intensity was comparable to the extinction coefficient from the dye manufacturers. The signal was factor 11.2 (SD 3.4) to 83.2 (SD 10.2) lower depending on the dye. NP in full blood showed even weaker fluorescent signals that could be increased by max. 24.5% with cell lysis. In a range of 0.02 mg/ml to 1 mg/ml dye no significant changes in NP size and surface charge were observed if neutral dyes were used. pH sensitive dyes lead to an increase in NP size and a drop in zeta potential of the cationic NP to the level of unmodified NP. Matrix incorporation resulted in a reduction of the zeta potential but in a growth from 189 nm (SD 2.9) to 201 nm (SD 4.2). Statistical analysis of the data was performed by a one way analysis of variance.
Conclusions: 1) NP were labeled successfully with strong fluorescent dyes without major changes in the inherent properties at low concentrations and if applied prior to NP formation. 2) NP characteristics are influenced by the charge, steric properties and pka values of the used fluorophores. 3) Finally the signal intensity of our NP was maximized towards fast and reliable in-vivo detection.
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Transferable Resistance to Five Different Classes of Protein Biosynthesis Inhibitors Including Oxazolidinones Mediated by the Gene cfr in Staphylococci
KEHRENBERG C1, Long K2, Poehlsgaard J3, Vester B3, Schwarz S1
1Institute of Farm Animal Genetics (FLI), Neustadt-Mariensee, Germany; 2University of Copenhagen, Copenhagen, Denmark; 3University of Southern Denmark, Odense, Denmark
Background: Analysis of staphylococci with elevated MICs of florfenicol (32 - ≥128 mg/L) identified two novel resistance genes, fexA and cfr. While the gene fexA codes for a phenicol specific exporter, the mechanism of cfr-mediated resistance was unknown. Since enzymatic inactivation and active efflux was excluded experimentally, target site modification appeared to be the most likely resistance mechanism.
Methods: Reduced drug binding to ribosomes and modification in 23S rRNA in the presence of Cfr was investigated by CMCT modification assays and primer extension analysis. The type of modification was identified by MALDI-TOF mass spectrometry. MIC testing of cfr-carrying strains followed the CLSI recommendations. Location of cfr on plasmids was shown by transfer experiments.
Results: In the presence of cfr, a reduced drug binding to ribosomes could be demonstrated for the phenicols chloramphenicol and florfenicol, the lincosamide clindamycin, the pleuromutilins tiamulin and valnemulin as well as for the streptogramin A antibiotic virginiamycin M1. MIC testing of cfr-carrying strains revealed in part dramatic increases in the MICs not only for the antimicrobial agents mentioned above, but also for the oxazolidinone linezolid. Analysis of the 23S rRNA revealed an additional methylation at position A2305 which is located in the overlapping binding area of phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (PhLOPSA) antibiotics. The gene cfr was mainly located on plasmids. Occasionally, it was integrated via insertion sequences IS21-558 into plasmidic copies of the fexA-carrying transposon Tn558.
Conclusions: 1) The gene cfr codes for a rRNA methyltransferase which targets the adenine residue at position 2305 in 23S rRNA. 2) Methylation at this position is believed to cause resistance by interfering with the correct positioning of the PhLOPSA antibiotics. 3) The gene cfr ist the first gene for transferable resistance to oxazolidinones and pleuromutilins. 4) Although this gene is currently detected very rarely among human and animal staphylococci, its location on plasmids points towards a potential for dissemination.
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Determination of Modafinil (Provigil) in Plasma and Urine by High-Performance Liquid-Chromatography
SCHWERTNER HA
Clinical Research, Wilford Hall Medical Center, San Antonio, Texas USA 78236
Modafinil (Provigil) is a new wake-promoting drug that is being used for the management of excessive sleepiness in patients with narcolepsy. It has pharmacological properties similar to that of amphetamine, but without some of the side effects associated with amphetamine-like stimulants. Since modafinil has the potential to be abused, accurate drug-screening methods are needed. Analytical methods are also needed for therapeutic drug monitoring and for pharmacokinetic studies. In this study, we developed a high-performance liquid-chromatographic procedure (HPLC) for the quantitative analysis of modafinil in plasma and urine. Provigil tablets (Cephalon, Inc.) containing modafinil were pulverized and extracted with methanol. After centrifugation, aliquots of the extract were used to prepare the urine and plasma calibrators. 3-Acetamidophenol and phenylthioacetic acid were used as internal standards for the analysis of plasma and urine, respectively. Modafinil was extracted from plasma and urine with ethyl acetate and analyzed on a C18 reverse phase column with methanol-water-acetic acid (500:500:1, v/v) as the mobile phase. Modafinil and the internal standards were analyzed at 220nm. The lower limit of sensitivity was 1.0 mg/mL, the within-day CV’s were 4.8, 2.5, and 2.4-2.5 percent at 1.0, 5.0, and 10.0 mg/mL for plasma and urine, and recoveries from plasma and urine were 80.0 and 79.6%, respectively. Forty-nine two-hour post dose urine samples from sham controls or from individuals taking 200 or 400 mg of modafinil were analyzed without knowledge of drug administration. All 16 placebo urine samples were correctly classified as negative at a modafinil concentration <0.4 mg/mL. Except for one sample, all 32 two-hour post-dose urine samples tested positive. No interfering substances (> 0.4 mg/mL) were found in plasma samples from 28 randomly selected individuals or in urine samples from 16 individuals not taking modafinil. Likewise, aspirin and acetaminophen did not interfere with the HPLC method. Phenylthioacetic acid and modafinil acid, a major metabolite of modafinil, could not be extracted with ethyl acetate, but could be extracted from plasma and urine with ethyl acetate:acetic acid, 100:1, v/v.
Conclusions: Modafinil could be accurately and reproducibly analyzed in both urine and plasma. The HPLC method can be used for therapeutic drug monitoring, pharmacokinetic studies, and for drug abuse screening.
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Bacterial Tetracycline Resistance: Prevalence, Evolution and Dissemination of Genes
SCOTT KP, PATTERSON AJ, KAZIMIERCZAK K
Microbial Ecology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Greenburn Road, Bucksburn, Aberdeen, UK. AB21 9SB
Background: Bacterial resistance to antibiotics is a global phenomenon. More than 40 distinct tetracycline resistance (TcR) genes confer resistance by four different mechanisms. Continually improving molecular and bacterial cultivation techniques have facilitated the identification of new genes in anaerobic bacteria. Recently discovered mosaic TcR genes appear to have arisen by homologous recombination between parental genes, simulataneously present in the same host bacterium. Mosaic genes are particularly prevalent in environments that have been exposed to tetracycline selective pressure. The first tet(W) gene was located on a large conjugative transposon, and intra-species gene transfer between bacteria is crucial in the spread of antibiotic resistance.
Methods: A macroarray screen containing 23 prevalent tetracycline resistance (TcR) genes was hybridized to DNA extracted from animal, human and soil samples. Sequences flanking specific TcR genes were compared. PCR amplification utilizing full-length and nested primer sets for different TcR genes assessed the prevalence and distribution of mosaic genes.
Results: The macroarray screen indicated that tet(W) is the most prevalent gene in gut ecosystems that are dominated by anaerobic bacteria. In contrast tet(W) was virtually undetectable in oral samples, which were instead dominated by tet(M). Analysis of sequences upstream and downstream of the tet(W) gene in diverse bacteria showed that short regions of 650bp and 100bp respectively are strongly conserved. This 2.7kb region may be a mobile mini-element, and may be, at least in part, responsible for the wide distribution of tet(W).
Mosaic genes (78%) outnumbered wild-type genes in samples obtained from commercial pigs, and constituted 50% of clones obtained from a human facal sample. Close analysis of mosaic genes combining tet(O) and tet(W) motifs illustrated that recombination hotspots exist. The most complex mosaic gene identified was tet(O/W/32/O/W/O).
Conclusions: 1) Different TcR genes dominate oral and faecal samples from the same individuals, reflecting the different microbial communities present 2) Identical mosaic genes have been identified by different research groups from different samples, illustrating their prevalence
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Treatment of chronic myeloid leukaemia (CML) by imatinib mesilate in Togo
KUEVIAKOE MI *; SEGBENA AY*; PADARO E*; DORKENOO M*; BORIES D**
* Laboratories of the Campus Teaching Hospital (Lomé, Togo)
** Cellular and Molecular Haematology Laboratory of Henri Mondor Teaching Hospital (Créteil, France)
Goal: Evaluate the efficacy of imatinib mesilate in chronic myeloïd leukemia (CML) patients followed in the Campus Teaching Hospital of Lomé.
Patients and Methods: Eighty patients followed since 55 months for CML in Lomé and using imatinib mesilate, supported by GIPAP Program, were investigated. BCR-ABL fusion was found in all them by RT-PCR technique in Créteil. Molecular follow up was based on BCR-ABL/GDR ratio.
Results: Eighty patients (6 females and 12 males) were included in the study. The mean age at diagnosis was 40.7 ±15.1 years old with limits between 18-70 years. All patients had splenomegaly at diagnosis. The average number of leucocytes/mm3 was 187905 ± 75602.3 [55700-350000], the mean haemoglobin level was 9.4 g/dl ± 1.4 [5.7 and 11.5 g/dl]. Dose of imatinib administrated varied between 200 and 400 mg. On imatinib, the clinical remission was obtained on average 3 months [7 days-9 months], that of the haematological remission on average 3 months [20 days-14 months]. The long time obtaining haematological remission was due to the therapeutic breaks for major iatrogenic neutropenia. At the diagnosis, 33% of the patients were higher than 10-1 and 67% between 10-1 and 10-2. To date, 5 patients (31.3%) have an undetectable transcrit (10-6) since 11 months, 4 (25%) were between 10-3-10-4, 3 (18.7%) between 10-2 and 10-3 and 4 (25%) between 10-1 and 10-2. Two deaths were noted after blastic transformation (due to very irregular administration of the treatment according to the induced cytopenia). In spite of a daily dose of 300 mg, the sensitivity to the treatment is higher than that described in the literature. Patients using imatinib in first intention was the best responders and presented less iatrogenic signs.
Conclusion: The imatinib mesilate in continuous treatment in the CML allows often early clinical, hematology and cytogenetic remission even in sub-Saharan Africa context.
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Effect of Pectin-Papain Interactions on Thermo-Mechanical Properties of Pectin Films Applied for the Treatment of Skin Wounds
ILYINA A1, SEGURA CEP.1, BOONE VVD1, ZARAGOZA CA2, FLORES GS 2, VARGAS DCI3
1Universidad Autónoma de Coahuila, Saltillo, Coah., Mexico. Fax: 52-844-415 9534, E-mail: psegura@mail.uadec.mx; anna_ilina@hotmail.com.
2Centro de Investigación en Materiales Avanzados (CIMAV), Chihuahua, Chih., Mexico
3Clinica Magisterio Secc. 38, Saltillo, Coah., Mexico
Background: Pectin is a biodegradable and water-soluble polymer. In this work, pectin obtained from peels of the maracuya (Passion fruit) was applied. It was used as support for papain (EC 3.4.22.2) immobilization to make the films suitable for treatment of skin wounds with this enzymatic debridement agent. However, the mechanical properties of the films must be improved to obtain the material with higher resistance to break. In the present study the effects of pectin-papain interactions on enzyme stability and thermo-mechanical properties of films with and without glycerin (G) and polyvinyl alcohol (PVA) were evaluated.
Methods: Films were prepared using solution of 1 % pectin, 1 mg/ml of papain, G at 0.75% (v/v) or PVA at 0.25% (w/v). The films were studied by Differential Scanning Calorimetry (DSC) and Dynamic Mechanical Analysis (DMA), as well as in enzyme stability study performed spectrophotomerically using casein as substrate. Pectin-papain films with and without glycerin were tested on wounds of voluntary patients.
Results: Different glass transition temperatures (Tg) were detected: 18ºC for pectin system, 15.43º for pectin-papain, 18.48º for pectin-G-papain and 25ºC for pectin-PVA-papainsystem. The values of the tensile breaking for pectin and pectin-papain films were 9.16 and 8.88 MPa, respectively, with an elongation of 1.64% and 2.53%, while for pectin-G-papain films were 14.2 MPa and 9.99%, 11.2 MPa and 2.68% in the presence of PVA. The PVA (but not G) decreased papain activity and stability. Moreover, in assays performed on the voluntary patients treated with pectin-papain and pectin-G-papain films, healing of the wounds were accelerated without any negative secondary effects.
Conclusions: 1. Addition of plasticizers improves the mechanical properties of the pectin films. The comparison of thermo-mechanical parameters demonstrates the presence of interactions leading to a change of mobility of biopolymer chains. 2. Glycerin addition does not influence on papain-pectin film capacity to accelerate healing of the skin wound.
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Variability of the Systemic Availability of Budesonide in Man when Administered Locally at Different Levels in the Gut with Different Doses of Ketoconazole
SEIDEGÅRD J1, BORGÅ O2, NYBERG L2
1AstraZeneca R&D Lund; 2Bioperm AB, Lund, Sweden
Background: A number of studies have demonstrated that the gut mucosa contributes substantially to the overall first-pass metabolism of many drugs. The major active enzyme in these cases is cytochrome P450 3A4 (CYP3A4), which is expressed in the small intestine and the liver. The relative contribution of gut wall and hepatic metabolism is seldom known and there is also a lack of knowledge regarding to what extent different parts of the gastrointestinal tract differ with respect to metabolic activity. Aim: To investigate gut wall first-pass metabolism in various regions of the gut metabolism of budesonide, a CYP3A4 substrate.
Methods: Budesonide (3 mg) as a solution, was given locally in the gut on different occasions to eight healthy men, with and without pretreatment (5 min before) with a low dose of ketoconazole (10 mg) in solution to investigate the gut wall first-pass metabolism in jejunum, ileum, and colon. The solutions were infused through a thin, soft tube that was brought to the desired position by a peristaltically driven capsule. Simultaneously, deuterium-labelled budesonide, 0.2 mg, was given intravenously to make sure that body clearance of the drug was not affected by the low dose of ketoconazole (no hepatic inhibition). Budesonide in plasma was assayed up to 12 h by use of an LC-MS/MS method.
Results: Ketoconazole pretreatment increased systemic availability of budesonide 1.8 times after jejunal and 2.0 times after ileal infusion but it was virtually unaffected by ketoconazole after infusion into the colon. Terminal half-life (T½) and body clearance were similar after the treatments. The systemic availability of budesonide without ketoconazole pretreatment was 12, 16, and 13 % from jejunum, ileum, and colon, respectively.
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