Method for qualifying microbial removal performance of 0.1 micron rated filters. Part II: preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for use as a standard challenge organism to qualify 0.1 micron rated filters.

In this article, we report on the preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for potential use as a standard challenge organism to qualify 0.1 microm rated filters. Filter-cloned H. pseudoflava (ATCC 700892) was easily cultured in a low nutrient broth (R2A broth) under standard laboratory conditions, reaching high titers of 10(8)-10(9) cfu/mL within 48-65 hours of incubation at 25+/-5 degrees C. Under these conditions, H. pseudoflava is a rod-shaped bacterium, averaging 0.25+/-0.03 microm by 1.65+/-0.35 microm, and appears to be smaller than Brevundimonas diminuta in width (0.31+/-0.03 microm), but somewhat longer in length (0.88+/-0.19 microm), which may partly explain the observed penetration. In total, thirty-five 0.2/0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from two different filter manufacturers were challenged with H. pseudoflava. In all cases, H. pseudoflava was shown to consistently penetrate every 0.2/0.22 microm rated filter disc tested. These tests also spanned three different challenge durations, including short-term challenges (30-40 minutes), and two different challenge fluids. The use of serial (double) 0.22 mm rated filters, which is a common industry practice to reduce the prefiltration bioburden to the final "sterilizing" filter, was also shown to be inadequate to fully retain H. pseudoflava under the challenge condition used. In contrast, two different 0.1 microm rated filter types functionally qualified with a specified high titer reduction claim for Acholeplasma laidlawii, were shown to consistently and fully retain H. pseudoflava, and retention by these two filter types was shown to be robust and independent of the challenge duration.     

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part I: characterization of water isolates for potential use as standard challenge organisms to qualify 0.1 micron rated filters.

Although 0.1 microm rated filters intended for pharmaceutical sterilization applications have been commercially available for at least 15 years, there is no industry-wide standard for qualifying the microbial removal performance of these filters. In this article, we report on the bacterial challenge methodology used to screen four bacterial species for potential utility as a standard challenge organism to qualify 0.1 microm rated filters. These isolates were, in their natural state, demonstrated to penetrate 0.2/0.22 microm rated filters in prior studies. In the screening challenges described in this study, three out of these four candidates tested demonstrated consistent penetration of one 0.22 microm rated filter type tested (when cultured in a low nutrient medium under standard laboratory conditions). These included 6204-22 (FAME ID Acidovorax avenae citrulli), 6266-15 (FAME ID Comamonas acidovorans), and 6266-34 (FAME ID Hydrogenophaga pseudoflava). Of these, H. pseudoflava (6266-34) was chosen for additional experiments with other 0.2 microm rated filter membranes. In total, seventeen 0.2 and 0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from three different filter manufacturers were tested. H. pseudoflava penetration was observed for every filter tested. Under the same challenge conditions, H. pseudoflava was consistently retained by a 0.1 microm rated hydrophilic PVDF (polyvinylidenefluoride) filter with a specified high titer reduction claim for Acholeplasma laidlawii. In order to ensure selection of the most stable penetrative phenotype (i.e., select for nonrevertants), H. pseudoflava was subjected to three rounds of "filter cloning," and these results are described herein. The advantages of using H. pseudoflava for qualifying the microbial removal performance of 0.1 microm rated filters are also discussed.     

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part IV: Retention of hydrogenophaga pseudoflava (ATCC 700892) and Ralstonia pickettii (ATCC 700591) by 0.2 and 0.22 micron rated filters

Ralstonia pickettii has emerged as a bioburden microorganism of considerable importance in pharmaceutical processes utilizing conventional 0.2 or 0.22 micron rated "sterilizing grade" filters. In this article, we re-evaluated and studied the retention efficiencies of 0.2 micron rated nylon 6.6 and 0.22 microns rated modified polyvinylidene fluoride (PVDF) filters for Hydrogenophaga pseudoflava (ATCC 700892) and R. pickettii (ATCC 700591). Out of a total of forty-four 0.2/0.22 micron rated filters discs tested in this study (spanning different challenge fluids, different challenge conditions, and different filter types), H. pseudoflava penetration was observed for every filter disc tested. Log titer reduction (LTR) values ranged from 0.3 to 2.0 logs for 20-48 hour challenges conducted in Water for Injection (WFI), and 3.8-7.1 logs for 6-hour challenges conducted in Minimal Media Davis (MMD). For 0.2 micron nylon 6.6 filter discs, penetration by R. pickettii was observed only in WFI challenges and was dependent on the culture and challenge conditions used. Penetration by R. pickettii was also restricted to only those membrane discs that were very close to the filter manufacturer's production integrity test (the Quantitative Bubble Point, QBP, test) limit. Where R. pickettii penetration was observed, LTR values were significantly higher than those observed for H. pseudoflava with the same filter discs. This study: 1) supports the use of H. pseudoflava as a worst-case challenge model for R. pickettii in process- and product-specific bacterial retention testing; 2) provides experimental evidence, for the first time, for the need to include filter membrane lots that have a physical integrity test value at or near the filter manufacturer's production (lower) limit in these tests; and 3) demonstrates how a standardized membrane integrity test (such as the QBP test) can be used select such "worst-case" membranes and to verify the inclusion of such "worst-case" membranes in these tests, thus serving as the link between the membrane disc used in bacterial retention validation testing and the production process filter.     

Retention of water-borne bacteria by membrane filters. Part III: Bacterial challenge tests on 0.1 micron rated filters

Clear performance differences were observed between different 0.1 micron rated filters in terms of their microbial removal efficiency when challenged with naturally occurring waterborne bacteria from a water source. Penetration occurred with three 0.1 micron rated "sterilizing grade" filter types tested, from three different filter manufacturers, that did not have a specific high titer reduction claim for Acholeplasma laidlawii. Bacteria shown to penetrate these 0.1 micron rated filters were quite similar to those recovered downstream of 0.2.0.22 micron rated filters (described in Part II). All of the isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp. and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems. In contrast, four different 0.1 micron rated "sterilizing grade" filter types from two different manufacturers, which had been qualified with both B. diminuta and A. laidlawii, consistently produced sterile effluents under similar test conditions. This study thus highlights the need for an industry or regulatory standard method of defining the microbial removal performance of 0.1 micron rated filters, and supports the use of functionally qualified 0.1 micron rated filters as sterilizing grade filters in pharmaceutical operations for enhanced sterility assurance.     

Retention of water-borne bacteria by membrane filters. Part I: Bacterial challenge tests on 0.2 and 0.22 micron rated filters

The results of bacterial challenge tests conducted on several 0.2 and 0.22 micron rated "sterilizing grade" filter cartridge types with bacteria from a natural water source are presented. Eight different 0.2/0.22 micron rated "sterilizing grade" filter types from four different filter manufacturers, claimed to be capable of retaining Brevundimonas diminuta at a challenge level of 10(7) CFU/cm2, were tested. The filters tested included nylon 6.6 and polyamide filters from two manufacturers, modified or hydrophilic PVDF filters from two manufacturers, modified or asymmetric PES filters from three manufacturers, and cellulose acetate filters from a single manufacturer. Consistent bacterial penetration was observed, over the 18-24 h challenge period, for all twenty-five integral 0.2 and 0.22 micron rated filter cartridges tested, at challenge levels of about 10(1)-10(4) CFU/cm2, indicating that natural waterborne bacteria were more penetrative than B. diminuta. The observed penetration was thus qualitatively independent of filter media type or manufacturer. These results add to the growing body of evidence that shows 0.2 and 0.22 micron rated filters may not remove all microorganisms under all conditions. These results further establish that bacterial penetration of 0.2/0.22 micron rated filters is not limited just to (1) specific membrane types, or (2) extended duration challenges (> 24 h), or (3) extremely high challenge levels, or (4) bacteria that can only exist in a penetrative state in an artificial laboratory setting.     

Retention of water-borne bacteria by membrane filters. Part II: Scanning electron microscopy (SEM) and fatty acid methyl ester (FAME) characterization of bacterial species recovered downstream of 0.2/0.22 micron rated filters

The results of scanning electron microscopic (SEM) and fatty acid methyl ester (FAME) characterization of the bacterial species shown to penetrate conventional 0.2/0.22 micron rated "sterilizing grade" filters are presented. SEM data suggest that retention of bacteria by these filters appears to be strongly influenced by the morphology, and especially the width of bacteria and less so by length. When the bacterial cell width is small, less than 0.3 micron or so, the cell length does not appear to limit the ability to penetrate 0.2/0.22 micron rated filters. As the bacterial width increases, there is also a strong, almost exponential, decrease in the allowable length for penetration, with most penetrative cells tending to be coccoid beyond a width of 0.5 micron. Significant percentages of the bacteria (40-50%) that were observed downstream of these filters were larger than B. diminuta, the standard organism used to qualify 0.2/0.22 micron rated filters. The average sizes of natural waterborne bacteria that penetrated the filters tested were 20-40% larger in width, and 40-70% larger in length, compared to B. diminuta. These results indicate that size exclusion is not the sole mechanism governing bacterial retention. All isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp. and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems. Most of the bacteria recovered downstream of 0.2/0.22 micron rated filters were gram negative, oxidase positive, motile, nonfermentors.     

Application of membrane filtration for removal of diminutive bioburden organisms in pharmaceutical products and processes

In this report, we present results of a recent investigation in our laboratories demonstrated the effect of process conditions and/or drug product composition on the ability of 0.2 micron and 0.22 micron sterilizing grade filters to fully retain Ralstonia (formerly Burkholderia, formerly Pseudomonas) pickettii. R. pickettii is a opportunistic pathogen widely distributed in nature as well as clinical specimens and there have been several reports of nosocomial infections due to intrinsic manufacture-related R. pickettii contamination in filter-sterilized parenteral fluids. This study documents the penetration of 0.2 micron nylon 66 and 0.22 micron modified PVDF sterilizing grade filters by R. pickettii (grown and challenged) in a drug solution under conditions that simulated a pharmaceutical filling operation. Penetration was not observed for every filter disc tested, and this may be explained, in part, by the stochastic nature (i.e., governed by the rules of probability) of the retention mechanisms involved. Scanning electron microscopy revealed significant changes in the microorganism's size and morphology as a result of exposure to the drug solution; these changes are consistent with those reported for bacteria subjected to nutrient deprivation. The SEM analyses of R. pickettii challenge suspensions in the drug solution showed that the average cell length decreased from 1.25 +/- 0.27 microns to 0.84 +/- 0.17 micron between zero and 24 hours. In addition, significant changes were observed in the size (length) distributions, with approximately 35% of the cells at 24 hours being smaller than any cell observed at the start of the challenge. These data suggest that the significant reduction in bioburden size and morphology that occurred as a result of exposure to the drug solution may play a role in the reduced ability of the 0.2 micron and 0.22 micron filters tested in this study to retain these organisms. Under the same test conditions where penetration of 0.2/0.22 micron filters was observed, 0.1 micron rated membrane filters qualified with both B. diminuta and Acholeplasma laidlawii mycoplasma consistently provided sterile effluent. Bacterial penetration of 0.2 (or 0.22) micron sterilizing grade filters was not observed under identical test conditions with either R. pickettii in a standardized solution (saline lactose broth) routinely used in challenge testing filters, or with the standard test organism, B. diminuta, in the drug solution. This study thus supports the renewed emphasis on both product- and process specific validation as well as routine bioburden monitoring expressed by regulatory agencies, and the use of enhanced bacterial removal efficiency 0.1 micron rated filters to provide enhanced sterility assurance in pharmaceutical processes.     

The importance of accurate microorganism identification in microbial challenge tests of membrane filters--part I

Microbial challenge testing is a common procedure to determine the retention efficiency, performance, and validity of a sterilizing-grade filter. The ASTM 838-05 standard describes a bacteria challenge test procedure based on Brevundimonas diminuta (ATCC 19146), routinely used to verify a 0.2 μm rated sterilizing-grade filter. Process validation procedures most often also utilize B. diminuta (ATCC 19146), but instead of the standard procedures and fluids, process, and product parameters are employed to determine whether these parameters influence the retentivity of the filter or changes to the challenge organism, which might result in the penetration of the filter. In certain instances, the native bioburden within the drug manufacturing process is used to perform such process validation challenge tests. Filter penetrations can happen and cause concern; therefore, it is essential to identify the organism species with accuracy to avoid unnecessary confusion. This paper and its follow-up will describe such imprecision and the resulting misconceptions. It will clarify past determinations and put perspective on the findings. LAY ABSTRACT: Sterilizing-grade filters are used to remove microorganisms from biopharmaceutical solutions. To determine the retention performance of such filters, bacteria challenge tests are utilized, often with a standard challenge organism (Brevundimonas diminuta), in instances with native bioburden. The accuracy of the microorganism identification is of importance to avoid flawed results and misinterpretation of the filter's performance.     

Changes in the cell size of Brevundimonas diminuta using different growth agitation rates

Brevundimonas diminuta (ATCC 19146) is a standard organism for validation of sterilizing-grade membrane filters. Cell size is critical for the determination of retention characteristics of 0.2 micron rated membrane filters. In this study, cell size changes of B. diminuta cultured under different physiologic states and variable agitations at 50, 100 and 200 rpm were measured by a particle size analyzer and scanning electron microscope (SEM). The smallest cells were obtained at initial stationary phase in saline lactose broth (SLB) as a shaking culture at 50 rpm. Cells grown under agitation at 50, 100 and 200 rpm showed an increase of specific growth rate (mu), about 2.9, 3.6 and 3.6 fold, respectively, compared to the non-agitated cells in SLB media. These results suggested that the cell size decreased proportionally with increase of the specific growth rate (mu) in SLB. These size changes were associated with penetration through a 0.2 micron rated cellulose acetate filter. A scale-down filtration system was developed and performed bacterial challenge test and bubble point test with cells cultured in SLB. Cells grown under agitation conditions in SLB were not retained by 0.2 micron rated membrane filter.     

Microbial retention characteristics of sterilizing-grade membrane filters with alginate substituted for oil-based products

For oil-based products, FDA recommends substitution of the oil with a compound which has similar viscosity and physical characteristics. In this study, a substitute for oil-based products was screened by measuring the viscosity and filterability, and examined for the presence of cell clumps in the various test fluids using an optical microscopy. The viscosity of the test fluids measured in the range of about 60-75 cP. Brevundimonas diminuta (formerly Pseudomonas diminuta), a standard challenge test organism for validation of 0.2 micron rated membrane filters, formed clumps in oils (corn, olive, sesame, and soybean) and polyethylene glycol (PEG, Molecular Weight (MW) = 400 and 1,000). During the viability test, cells suspended in 80% glycerol showed a ten-fold mortality rate after an exposure for 6 hours, but there was no significant change in viability in alginate (low, medium, and high viscosity) for 24 hours. These results suggested that alginate is better suited as a substitute for oil-based products than 80% glycerol. Since high viscosity fluids take longer to filter, the glycerol mortality rate would influence the challenge test negatively. A scaled-down filtration system has been developed for the described trials, and the bacterial challenge and bubble point tests have been performed in 1.6% alginate (66.7 cP), which was the choice of carrier fluid.     

Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics.

Liposomes were prepared by stepwise extrusion through 5, 1, 0.4, 0.2, 0.1 and 0.05 microm pore sizes using two different filter-extruders, the continuous high pressure device Dispex Maximator (CE) or alternatively the discontinuous Avestin LiposoFast (DE). The liposome dispersions obtained were compared in terms of particle size, lamellarity and encapsulation efficiency of calcein. The liposomes were smaller with CE than DE at all stages due to higher flow rates and pressure drops, except for final filter pore size (0.05 microm) where both preparations had similar sizes. The particle size analysis technique itself had a strong influence on the liposome sizes measured. For bigger liposomes (extruded through 0.4 microm filters) the Nicomp 370 revealed bigger volume-based mean particle sizes along with more stringent differences between volume-based and number-based diameters than the Malvern Zetasizer. In contrast, for small liposomes extruded through 0.05 microm filters, similar liposome sizes were found no matter which of the two PCS techniques or cryo-transmission electron microscopy was used. In congruence to the liposome sizes measured, encapsulation efficiencies were smaller for CE than DE at all filter stages except the final (0.05 microm). No lipid loss occurred and lyso-phosphatidylcholine formation was negligible irrespective of which extrusion technique was used.     

Scaling from discs to pleated devices

Membrane discs offer a convenient format for evaluating membrane performance in normal flow filtration. However, while pleated devices of different sizes tend to scale in close proportion to their contained areas, they do not necessarily scale in direct proportion from flat discs. The objectives of this study are to quantify differences in performance among sterilizing-grade membrane devices as a function of device type and size, to develop an understanding of the factors that affect device scalability, and to develop a mathematical model to predict a cartridge-to-disc scalability factor based on membrane properties and porous support properties and dimensions. Measured and predicted normalized water permeability scalability factors for seven types of pleated cartridges, including 0.1-micro and 0.2-micro rated PES, and 0.2-micro rated polyvinylidene fluoride (PVDF) sterilizing-grade filters in nominal 1-inch to 5-inch lengths, were determined. The results of this study indicate that pleated cartridge performance can be closely predicted based on 47-mm disc performance provided that a number of measured device parameters are properly accounted for, most importantly parasitic pressure losses in the filter device and plumbing connections, intrinsic membrane variability, true effective device filtration area, and the hydraulic properties of all porous support materials. Throughput scalability factors (discs to devices) tend to converge towards unity, especially for highly plugging streams. As the membrane fouls, the resistance through the membrane dominates other resistances, so the flux scales more linearly with membrane area and the overall scaling factor becomes close to one. The results of throughput tests on seven different cartridge types and five different challenge streams (with widely varying fouling characteristics) show that most of the throughput scaling factors were within +/-10% of 1.0. As part of this study, the effects of pressure and temperature were also evaluated. Neither of these factors was found to have a significant effect on scalability.     

Extractables from filter components in model solvent streams for pharmaceutical filtration processes

Drug manufacturers use filters widely in their manufacturing processes for bioburden reduction and sterility assurance. In implementing a pharmaceutical filtration, it is essential to determine and profile the filter extractables that could be introduced into the product. There is an ongoing industry effort to explore the analysis of extractables. Toward this end, the use of complementary analytical techniques was evaluated to aid in extractables identification by determining whether the analysis of filters in the form of components, rather than entire assemblies, facilitated the profiling of extractables. The study examines filter components of 10-inch, 0.22-microm Durapore cartridge filters using Fourier transform infra-red spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR), as well as determines the non-volatile residues (NVR) and total organic carbon contents (TOC) of the extractables. The complementary techniques of high performance liquid chromatography (HPLC/UV) and liquid chromatography nuclear magnetic resonance spectroscopy (LC/NMR) were also used. The results of the tests indicated very low levels of extractables.     

Effects of sterilizing-grade filters on the physico-chemical properties of onion-like vesicles

Spherulites are new promising multilamellar vesicles that we study in a drug delivery context. The sterilization of spherulites suspensions is a necessary step before biological tests and later, before pharmaceutical applications (for example, parenteral or local injections). Among all sterilizing operations, the filtration through 0.22 microm sterilizing-grade filters (of the type Millex (? 4 mm) by Millipore) is easy and rapid, and we decided to study it as a mean to obtain sterile suspensions. The spherulites diameter is usually comprised between 0.2 and 0.5 microm but bigger vesicles occur and reach ? 1 microm. The effects of such filters on vesicles' size and lipids' concentration were then compromised. After examination of this challenging operation, results proved that the sterilizing filtration had no effect on these two parameters whatever the formulation chosen. Then, the possible release of amaranth, an encapsulated hydrophilic dye was followed. With the formulations and in spite of a filter diameter inferior to that of the vesicles, the encapsulation yields were not significantly different before and after the filtration and no leakage could be detected. Finally, the spherulites' functionality after sterilizing filtration was studied under the chemical angle: vesicles containing an amphiphilic reactive anchor (CholE3ONH2) were still able to bind covalently a peptidic molecular recognition pattern. The ligation was quantified by fluorimetry as high as for non-filtrated suspensions. Thus, though spherulites can present a diameter superior to that of the sterilizing filters, their passage through them do not alter the physico-chemical properties of these vesicles.     

Removal of influenza A virus,phage T1, and PP7 from fluids with a nylon 0.04-μM membrane filter

We tested the ability of a 0.04-μm nylon membrane filter to remove viral agents (influenza A virus, 80–120 nm; phage T1, 50–150 nm; and phage PP7, 25 nm) from the following media: ultrapure water (UPW), Dulbecco's modified Eagle minimum essential medium (DMEM), gelatin phosphate (GP), DMEM with 10% (DMEM-10) fetal bovine serum (FBS), and 100% FBS. When challenged with at least 3.0 × 10⁷ plaque-forming units/mL, no influenza A virus was detected downstream of the filter with any of the fluids tested. The titer reduction (Tr) was determined using the equation: . Higher concentrations of phage T1 were removed from UPW (Tr = 1.6 × 10⁶) and DMEM (Tr = 1.1 × 10⁶) than from GP (Tr = 9.3 × 10³), DMEM-10 (Tr = 1.5 × 10²), and 100% FBS (Tr = 2.4 × 10²). Phage PP7 was removed in significant numbers only in ultrapure water (Tr = 8.5 × 10⁴). The results indicate that adsorption enhanced the titer reduction in fluids containing low levels of protein. The titer reduction in DMEM-10 and 100% FBS may reflect the sieving properties of the 0.04-μm filter. As expected, a much smaller Tr was observed in the filtrate of the 0.2-μm filters, compared to the 0.04 μm filters. In contrast to the 0.04-μm filter, no increase in Tr was detected when the 0.2-μm filters were challenged with virus diluted in UPW compared with virus diluted in GP. These results suggest that the 0.04-μm filter has greater adsorptive properties than the 0.2-μm filter. © 1994 by John Wiley & Sons, Inc..     

LC-MS Determination of remifentanil in maternal and neonatal plasma

An HPLC-MS with electrospray ionization method for the determination of remifentanil in human plasma samples, pre-treated with SPE cartridge, has been developed and validated. Ionisation was performed by positive-ion electrospray and quadrupole filter mass spectrometer operated in the single ion-recording mode. Pre-treatment was performed using Waters Oasis((R)) SPE cartridges. Chromatographic separation was achieved in isocratic elution using a X-Terra C8 5 microm, 150 mm x 2.1 mm i.d. column. The mobile phase consisted of a mixture of water, methanol and acetonitrile (86:10:4, v/v/v) containing 0.1% of formic acid. The method showed to be linear in the range between 0.5 and 48.0 ng/ml, the estimated LOD is 0.18 ng/ml and the LOQ is 0.5 ng/ml. The method can be used to quantify remifentanil in plasma samples taken from adult and newborn patients in a range suitable for clinical studies.     

In line final filters for removing particles from amphotericin B infusions.

The feasibility of using membrane filters to remove particles from intravenous infusions of amphotericin B in dextrose 5% (a colloidal solution) was studied. Six types of commercial membrane filters, ranging in pore size from 0.45-1.0 mum, were used. Because of the effect of pH on the particle size of colloidal solutions, each filter was tested at solution pH 4.7, 5.6 and 6.5. Samples of filtrate were analyzed spectrophotometrically for amphotericin content. All filters of pore size 0.22 mum removed amphotericin B from solution and were inappropriate for use with this product. Solutions at pH 4.7 were turbid, filtered slowly and were generally unacceptable for clinical use. At pH 5.6, only filters with pore sizes of 1.0 mum or greater showed acceptable results. At pH 6.5, filters with pore sizes of 0.45 mum or greater gave acceptable results; the use of a filter with a pore size of not less than 1.0 mum would provide a margin for error to compensate for variations in the colloidal particle size of amphotericin B.     

Determination of clopidogrel metabolite (SR26334) in human plasma by LC-MS

A new, sensitive, specific and reproducible method for determination of clopidogrel metabolite (SR26334) in human plasma has been developed. After liquid-liquid extraction on Chem Elut cartridges with dichloromethane, samples were quantified using reversed-phase high performance liquid chromatography with mass detection. The determination was performed on a Luna C18, 3 microm (75 mmx4.6 mm i.d.) column with an acetonitrile-water-formic acid mixture (60:40:0.1, v/v/v) as a mobile phase. The flow rate was set at 0.2 mL/min. Repaglinide was chosen as an internal standard and the time of analysis was 12 min. For SR26334 the limits of detection and quantification were 7.5 ng/mL and 20 ng/mL, respectively, and the calibration curve was linear up to 3000 ng/mL. The extraction recovery of SR26334 from plasma was within the range of 85-90%. The method has been successfully used to study clopidogrel metabolite pharmacokinetics in healthy volunteers.     

Cytotoxicity assessment of chlorinated bacteria in water using the RNA synthesis inhibition method

Chlorination of drinking water containing organic materials is known to generate toxic by-products. We suggested that such compounds may also be produced by interactions between chlorine and bacteria present in water. To confirm this hypothesis, a method based on RNA synthesis inhibition of HeLa S3 human cells in the presence of toxic compounds was applied. This method is rapid and highly sensitive since the concentration of the samples is not required. Furthermore, it was shown to be a suitable method for measurement of the cytotoxicity of water. Aeromonas hydrophila suspensions, prepared with pyrodistilled water, devoid of any organic material, were chlorinated for a definite contact time. HeLa S3 cells were incubated (20 h, 37 degrees C) in a culture medium prepared with the chlorinated bacteria suspensions. The rate of incorporation of 3H uridine into RNA was used as a measure of RNA synthesis and was evaluated in the presence and absence of chlorinated bacteria suspension. This study showed that chlorinated bacteria suspensions are cytotoxic. We observed that 0.22 microm filters retain cytotoxic compounds but 0.45 microm filters did not. Chlorine concentration and bacteria level influence the cytotoxicity. First, the toxicity level increases with chlorine concentration, then it decreases when chlorine concentration is too high. On another hand, a dose effect relationship between bacteria concentration and cytotoxicity was established.     

Histamine induces cytoskeletal changes in human eosinophils via the H(4) receptor

1. Histamine (0.004-2 microm) induced a concentration-dependent shape change of human eosinophils, but not of neutrophils or basophils, detected as an increase in forward scatter (FSC) in the gated autofluorescence/forward scatter (GAFS) assay. 2. The histamine-induced eosinophil shape change was completely abolished by thioperamide (10 microm), an H3/H4 receptor antagonist, but was not inhibited by pyrilamine or cimetidine (10 microm), H1 and H2 receptor antagonists, respectively. The H4 receptor agonists, clobenpropit and clozapine (0.004-2 microm), which are also H3 receptor antagonists, both induced eosinophil shape change, which was inhibited by thioperamide (10 microm). The H3/H4 receptor agonists, imetit, R-alpha-methyl histamine and N-alpha-methyl histamine (0.004-2 microm) also induced eosinophil shape change. 3. Histamine induced actin polymerisation (0.015-10 microm), intracellular calcium mobilisation (10-100 microm) and a significant upregulation of expression of the cell adhesion molecule CD11b (0.004-10 microm) in eosinophils, all of which were inhibited by thioperamide (10-100 microm). In addition, the H4 receptor agonist/H3 receptor antagonist clozapine (20 microm) stimulated a rise in intracellular calcium in eosinophils. 4. Activation of H4 receptors by histamine (1 microm) primed eosinophils for increased chemotactic responses to eotaxin, but histamine (0.1-10 microm) did not directly induce chemotaxis of eosinophils. 5. Pertussis toxin (1 microg ml-1) inhibited shape change and actin polymerisation responses induced by histamine showing that these effects are mediated by coupling to a Galphai/o G-protein. 6. This study demonstrates that human eosinophils express functional H4 receptors and may provide a novel target for allergic disease therapy.