Report from the EPAA workshop: in vitro ADME in safety testing used by EPAA industry sectors.

There are now numerous in vitro and in silico ADME alternatives to in vivo assays but how do different industries incorporate them into their decision tree approaches for risk assessment, bearing in mind that the chemicals tested are intended for widely varying purposes? The extent of the use of animal tests is mainly driven by regulations or by the lack of a suitable in vitro model. Therefore, what considerations are needed for alternative models and how can they be improved so that they can be used as part of the risk assessment process? To address these issues, the European Partnership for Alternative Approaches to Animal Testing (EPAA) working group on prioritization, promotion and implementation of the 3Rs research held a workshop in November, 2008 in Duesseldorf, Germany. Participants included different industry sectors such as pharmaceuticals, cosmetics, industrial- and agro-chemicals. This report describes the outcome of the discussions and recommendations (a) to reduce the number of animals used for determining the ADME properties of chemicals and (b) for considerations and actions regarding in vitro and in silico assays. These included: standardisation and promotion of in vitro assays so that they may become accepted by regulators; increased availability of industry in vivo kinetic data for a central database to increase the power of in silico predictions; expansion of the applicability domains of in vitro and in silico tools (which are not necessarily more applicable or even exclusive to one particular sector) and continued collaborations between regulators, academia and industry. A recommended immediate course of action was to establish an expert panel of users, developers and regulators to define the testing scope of models for different chemical classes. It was agreed by all participants that improvement and harmonization of alternative approaches is needed for all sectors and this will most effectively be achieved by stakeholders from different sectors sharing data.

In vitro evaluation of BAY Y3118, a new full-spectrum fluoroquinolone.

BAY Y3118, 1-cyclopropyl-7-(2,8-diazabicyclo[4.3.0]non-8-yl)-6-fluoro-8- chloro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride, is a new fluoroquinolone with antibacterial activity against an expanded spectrum of species including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus pyogenes, and also anaerobes such as Bacteriodes fragilis and Clostridium perfringens. MIC90s for S. aureus, S. epidermidis, E. faecalis, and S. pneumoniae clinical isolates were 0.125, 0.25, 0.125 and 0.25 micrograms/ml, respectively. Against methicillin- and/or quinolone-resistant S. aureus, MIC50 levels of BAY Y3118 were 10- to 100-fold lower than those of tosufloxacin, sparfloxacin, or ciprofloxacin. The potency of BAY Y3118 against all members of the Enterobacteriaceae generally was equal to or 2-fold greater than that of ciprofloxacin or tosufloxacin. BAY Y3118 was also highly active against Haemophilus influenzae, Moraxella catarrhalis, Acinetobacter spp. and Pseudomonas aeruginosa. Increasing inoculum concentrations had a minimal effect on MIC determinations. The drug was determined to be bactericidal based upon reference MBCs and time-kill curves. From the results presented here, it was concluded that this new compound surpasses other known 4-quinolones both in spectrum and activity and that its further evaluation by in vitro, in vivo, and clinical studies seems warranted.

Modulation of leukotriene metabolism from human polymorphonuclear granulocytes by bifonazole.

The influence of bifonazole and other azoles on the leukotriene-metabolism of human neutrophil granulocytes (PMN) was investigated. The cells were stimulated with the Ca-ionophore A23187 over 15 minutes in the presence of the azoles. Subsequently the supernatants were analyzed for their leukotriene content by reversed-phase HPLC. In order to confirm that the observed effects are not due to cytotoxic phenomena tests were performed to evaluate the viability of the granulocytes. The chemotactic active leukotriene B4 (LTB4) and its inactive omega-metabolites were detected and quantified by HPLC analysis. The results clearly demonstrated that bifonazole inhibited the omega-oxidation already at concentrations as low as 0.5 microgram ml-1. As a consequence the LTB4-level became elevated. At concentrations up to 64 micrograms ml-1 bifonazole totally blocked the formation of leukotrienes in human granulocytes. Identical experiments were performed with other azoles and with various anti-inflammatory drugs just to have a comparison with bifonazole. It is evident that bifonazole in addition to its well known antimycotic action shows a unique modulation of the leukotriene-metabolism, which is important for its anti-inflammatory potency.

Application of high-performance liquid chromatography of some antibiotics in clinical microbiology.

During recent years high-performance liquid chromatography has become an excellent tool for the determination of antibiotics in biological fluids. Compared with biological assays, the major benefits of this method are specificity and rapidity. In particular, the determination of biologically inactive metabolites emphasizes that this technique plays an outstanding role for the analysis of antibiotics. This paper describes how the method can be used in the analysis of several antibiotics and demonstrates the efficacy of this method for clinical microbiology. Methods for the determination in biological fluids of acylaminopenicillins (azlocillin, mezlocillin, piperacillin and aspoxicillin), quinolones (ciprofloxacine, norfloxacine and ofloxacine), a penem (imipenem) and a cephalosporin (cefixime) are summarized. Furthermore, their application to in vitro studies and their trial in clinical studies are described.

Degradation of acylaminopenicillins with regard to their pH dependency.

Determination of antibiotic concentration is performed in many biological fluids and tissues which all have different pH values. Therefore, we investigated the in vitro stability of three acylaminopenicillins (azlocillin, mezlocillin and piperacillin) in borate buffer by the HLPC technique with regard to pH dependency. HPLC allows the detection of all three substances together with their metabolites, penicilloate and penilloate, within 15 min. Decomposition was monitored at 37 degrees C during a 24 h incubation period (pH values ranged between pH 3.0 and 10.0). The highest degradation rates were observed with a buffer solution of pH = 10.0: 50% of the azlocillin and 83% of the mezlocillin were decomposed after 8 h while under the same conditions, piperacillin was completely decomposed already after 1 h. The highest stability was detected in borate buffer at pH values of 4.0, 5.0, and 6.0. At pH = 3.0, degradation was determined as follows: 31% of the piperacillin, 39% of the mezlocillin, and 45% of the azlocillin were decomposed after 24 h. Penilloic acid was identified as the main metabolite in contrast to buffer solutions with higher pH values which only revealed negligible amounts of this compound.

Concentration of azlocillin in human chondral tissue.

The concentration of azlocillin was determined using high performance liquid chromatography in serum and chondral tissue after intravenous infusion of azlocillin (75 mg/kg body weight). In serum the levels of ten patients (2 to 27 years of age, body weight 12 to 69 kg) decreased from 478 (30 min post infusion) to 120 micrograms/ml (120 min). In contrast, the concentrations in chondral tissue ranged between 24 and 35 mg/g tissue at the corresponding times. Although the mean levels suggest a remarkable penetration of azlocillin into chondral tissue, the high individual differences observed in the tissue levels (2.1 to 138 micrograms/g tissue) require a higher dosage to ensure sufficient antimicrobial therapy in all patients.

Modulation of granulocyte functions by bacterial exotoxin and endotoxins.

The modulation of granulocyte functions by bacterial exotoxins (Streptolysin O, alveolysin, theta toxin) and endotoxins from salmonella and lipid A is described here. Incubation of polymorphonuclear granulocytes with thiol-activated toxins resulted in an increased leukotriene generation. Toxin-pretreated PMNs revealed an increased omega oxidation of LTB4, which may explain why toxin-stimulated cells release more LTC4 than LTB4. Furthermore, toxin-pretreated PMNs showed a decreased leukotriene generation on subsequent stimulation with the Ca-ionophore A 23187 or opsonized zymosan.

Determination of aspoxicillin (TA-058) by high-performance liquid chromatography. Stability at different temperatures.

A rapid and sensitive HPLC-method has been developed for the determination of serum concentrations of aspoxicillin (TA-058), a new semisynthetic beta-lactam antibiotic. Aspoxicillin was chromatographed with a phosphate buffer/methanol (92:8 v/v) mobile phase and a C-18 reversed phase column and was detected at a wavelength of 220 nm. The stability of aspoxicillin in serum and buffer at different temperatures was studied over a time period of 3 months. Furthermore, the degradation of aspoxicillin versus piperacillin was determined in serum and buffer at 37 degrees C. Aspoxicillin remains stable only at -70 degrees C whereas degradation has been observed at -20 degrees C and 4 degrees C. At 37 degrees C, 20% of aspoxicillin is degraded in serum within 24 h whereas piperacillin is completely degraded under the same conditions.

The role of leukotriene-inducing and -metabolizing enzymes in inflammation.

Leukotrienes are potent mediators of allergy and inflammation. Polymorphonuclear granulocytes which participate in acute and chronic disease processes can be activated by immunological and nonimmunological stimuli to generate leukotrienes. It appears that on activation of the cells 5-lipoxygenase is released into the supernatant and thus may exert a potent role with regard to interdependent cellular interactions. The release of leukotriene-metabolizing enzymes (e.g., gamma-glutamyl-transpeptidase, dipeptidase) is stimulus-dependent. Polymorphonuclear granulocytes alter their release profile once the cells have been prestimulated. Thus, a precise knowledge on the leukotriene-inducing and -metabolizing enzymes is of utmost importance for future immunopharmacological interventions. It appears that the enzyme activity reflects the state of cellular activation.

Determination of ciprofloxacine, norfloxacine, and ofloxacine by high performance liquid chromatography.

A fast, versatile and precise method is described for the determination of quinolone derivatives in biological fluids and tissues by high performance liquid chromatography (HPLC) and fluorescence detection. Using a solvent consisting of acetonitrile/methanol/water/tetrabutylammoniumhydroxide (pH 3.0) and reversed phase material (5C18), ciprofloxacine, norfloxacine and ofloxacine were identified within 10 min, with a detection limit of 10 pg (ofloxacine 40 pg). No interference with other substances was observed. The sample preparation needs only one dilution and centrifugation step (recovery in blood, serum and urine about 100%). Ciprofloxacine, norfloxacine and ofloxacine are not degraded when incubated at 4 degrees C or 37 degrees C for 24 h in serum and urine. In heparinized blood ofloxacine remains stable, whereas a decrease of 25% (ciprofloxacine) and 30% (norfloxacine), respectively, is observed after 24 h at 37 degrees C without the formation of fluorescent metabolites. In serum and urine of treated patients, however, metabolites are detected. These findings suggest a metabolization process in organs, e.g. the liver.

Determination of mezlocillin and its penicilloate and penilloate by high-performance liquid chromatography and stability of mezlocillin at different temperatures.

A method is described for the determination of mezlocillin and its metabolites penicilloic acid and penilloic acid in biological fluids by high-performance liquid chromatography. The stability of mezlocillin in serum and buffer was studied at different temperatures (4, -20, -70, and -196 degrees C) over a period of 6 months. Mezlocillin remained stable at -70 and -196 degrees C, whereas degradation was observed at -20 and 4 degrees C in serum and buffer.

Effect of thiol-activated toxins (streptolysin O, alveolysin, and theta toxin) on the generation of leukotrienes and leukotriene-inducing and -metabolizing enzymes from human polymorphonuclear granulocytes.

The generation of leukotrienes (LTC4, LTD4, LTE4, and LTB4; 12-epi-LTB4 isomer) from human granulocytes by thiol-activated toxins (streptolysin O, alveolysin from Bacillus alvei, and theta toxin from Clostridium perfringens) is described. The release occurs under noncytolytic conditions. Although LTB4 is the major component after calcium ionophore stimulation, more LTC4 as compared with LTB4 is released with the toxins. The 5-lipoxygenase pathway of toxin-mediated activation can effectively be inhibited by caffeic acid, a lipoxygenase inhibitor. The toxins also induce the release of leukotriene-metabolizing enzymes such as gamma-glutamyltranspeptidase, which transfers LTC4 into LTD4, and dipeptidase, which metabolizes LTD4, into LTE4. Dipeptidase activity is more pronounced than the gamma-glutamyltranspeptidase activity but still does not reach the levels obtained when cells were triggered with opsonized zymosan.

Degradation of azlocillin in human serum.

We developed a rapid and precise high performance liquid chromatographic method (HPLC) for the determination of azlocillin and its metabolites penicilloate and penilloate in serum and tissue as well as in vivo as in vitro. The linear relationship (r greater than 0.99) of determination ranged between 0.05 and 10 micrograms. No interference with other serum components was observed. The metabolism of azlocillin in vitro was analysed in serum. Within 24 h the amount of penicilloate increased from 1.5% up to 18% and from penilloate up to 1% respectively. Buffer controls revealed a significant lower metabolism (4.7% penicilloate, 0.9% penilloate). These data suggest that the degradation of azlocillin does not only depend on bacterial beta-lactamase activity but is influenced by enzymatic activities within serum and human tissue.

Functional characteristics of leukotriene C4- and D4-metabolizing enzymes (gamma-glutamyl transpeptidase, dipeptidase) within human plasma.

Several properties of the leukotriene C4- and leukotriene D4-metabolizing enzymes within human plasma were studied after fractionation of the plasma proteins using ammonium sulfate precipitation. Leukotriene D4-metabolizing enzymes were widely distributed among the fractions obtained. They showed different pH optima (pH 6.5, pH 7.0 and pH greater than or equal to 8.5) and revealed a different degree of thermal stability. The results indicate the presence of more than one enzyme in plasma which interacts with leukotriene D4. EDTA and L-cysteine inhibited the metabolism of leukotriene D4. Two leukotriene C4-metabolizing activities (gamma-glutamyl transpeptidases) differing in their molecular weights were detected after gel filtration. Their molecular weights were estimated to be Mr greater than or equal to 150 000 and Mr between 55 000 and 100 000.

The metabolism of leukotrienes in blood plasma studied by high-performance liquid chromatography.

The metabolism of leukotrienes (B4, C4, D4, and E4) within human plasma was studied and a simple sample preparation is presented. It was demonstrated that leukotriene E4 and leukotriene B4 were stable during incubation at 37 degrees C using the in vitro system. In contrast, leukotriene C4 was metabolized by gamma-glutamyl transpeptidase activities into leukotriene D4 which was further metabolized by dipeptidase activities of plasma into leukotriene E4. The transition state inhibitor of gamma-glutamyl transpeptidase L-serine-borate decreased the metabolism of leukotriene C4 in plasma. Dilution of plasma demonstrated that the dipeptidase was more active compared to the gamma-glutamyl transpeptidase. The metabolizing activities of plasma were functionally characterized by fractionating the plasma proteins.

Generation of slow-reacting substance (leukotrienes) by endotoxin and lipid A from human polymorphonuclear granulocytes.

Leukotrienes were released from human polymorphonuclear granulocytes on incubation with endotoxins and lipid A. The analysis was performed by their smooth muscle contracting properties, reversed phase high-pressure liquid chromatography and radioimmunoassay for leukotrienes C4 and D4. The active component of the lipopolysaccharides seems to be the lipid A portion.

Generation of leukotrienes from human granulocytes by alveolysin from Bacillus alvei.

We investigated the effect of alveolysin on human granulocytes. Alveolysin is an exoprotein produced by Bacillus alvei and belongs to the group of sulfhydryl-activated cytolysins. Other members of this group are streptolysin O and theta-toxin from Clostridium perfringens. It is demonstrated that alveolysin leads to leukotriene generation from human granulocytes, which exert chemotactic (leukotriene B4) and slow-reacting substance (leukotriene C4, D4, and E4) activity under sublytic concentrations.