Comparative Stability Studies of Antipseudomonal {beta}-Lactams for Potential Administration through Portable Elastomeric Pumps (Home Therapy for Cystic Fibrosis Patients) and Motor-Operated Syringes (Intensive Care Units)

The stability of antipseudomonal ß-lactams in concentratedsolutions was examined in view of their potential administrationby continuous infusion with external pumps (for intensive carepatients) or with portable pumps carried under clothing (forcystic fibrosis patients). Aztreonam (100 g/liter), piperacillin(128 g/liter, with tazobactam), and azlocillin (128 g/liter)remained 90% stable for up to more than 24 h at 37°C (mezlocillin[128 g/liter] was stable at 25°C but not at 37°C). Ceftazidime(120 g/liter), cefpirome (32 g/liter), and cefepime (50 g/liter)remained 90% stable for up to 24, 23.7, and 20.5 h at 25°Cbut only for 8, 7.25, and 13 h at 37°C, respectively. Thecontrol of temperature therefore appears to be critical forall three cephalosporins that cannot be recommended for usein portable pumps carried under clothes for prolonged periodsfor reasons of stability. Cefpirome and cefepime solutions developedan important color change (from light yellow to dark red) uponexposure when stored at 30°C or higher. Degradation of ceftazidimewas accompanied by the liberation of pyridine which, at 37°C,was in excess of what is allowed by the U.S. Pharmacopeia, i.e.,1.1 mg/liter, after 8 and 12 h for drug concentrations of 12and 8.3%, respectively. Imipenem and meropenem are too unstable(10% degradation at 25°C after 3.5 and 5.15 h, respectively)to be recommended for use by continuous infusion. Faropenem,examined in comparison with imipenem and meropenem, proved asstable as aztreonam or piperacillin.

INTRODUCTION

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Introduction

ß-Lactams are one of the most typical antibiotic classesfor which the pharmacodynamic data, collected in vitro, as wellas in animal studies, clearly indicate a potential advantageof a continuous infusion over the more conventional, discretemode of administration (see references 6, 22, 23, 38, and 41for reviews). Accordingly, several clinical trials have beeninitiated over the last half-decade, most notably in difficult-to-treatsituations such as suspected gram-negative infections in criticallyill patients (3), severe infections in intensive care patients(8, 16, 21, 24, 37), septicemia (1), severe sepsis (20), andcystic fibrosis (29, 40). Yet, as clearly pointed out by oneof the first promoters of the continuous infusion of ß-lactams(6), this mode of administration, through programmable pumpsor other similar devices, requires the drug to remain sufficientlystable in solution over the projected duration of the infusion.In a previous study (30), we examined the stability of ceftazidimein solution and found it to be critically dependent on the temperaturewithin the 20 to 37°C range (68 to 99°F). This setsclear limitations on the potential use of ceftazidime by continuousinfusion if administrations every 24 h are contemplated withoutperiodic changes of the solutions. Chemical considerations suggestthat the stability of the main ß-lactams currentlyregistered for human medicine use may vary to a meaningful extent.Unfortunately, these variations, and their consequences as faras continuous infusion is concerned in the clinics, cannot alwaysbe deduced in a straightforward fashion from the manufacturer'sstability data. The latter, indeed, mainly pertains to shelfstorage conditions. In the present study, we examined and comparedthe stability of nine parenteral ß-lactams (one monobactam,three ureidopenicillins, three broad-spectrum cephalosporins,and two carbapenems) with potential use for continuous infusionin severe infections in intensive care patients and in cysticfibrosis. All of these drugs show activity against gram-negativeorganisms, especially Pseudomonas aeruginosa, and have registeredindications in this context. For the sake of comparison, wealso included faropenem based on pilot studies with meropenemand imipenem and chemical considerations.

MATERIALS AND METHODS

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Materials and Methods

Origin of products. All drugs that were commercially available in Belgium were obtainedthrough a hospital pharmacy as brand name products distributedfor clinical use by their original marketing authorization holderas follows: aztreonam (Azactam; Bristol-Myers/Squibb), piperacillin(Pipcil; Wyeth-Lederlee/American Home Products), piperacillin-tazobactam(Tazocin; Wyeth-Lederlee/American Home Products), ceftazidime(Glazidim; Glaxo-SmithKline), cefepime (Maxipime; Bristol-Myers/Squibb),cefpirome (Cefrom; Aventis-Pharma), imipenem-cilastatin (Tienam;Merck, Sharp & Dohme), and meropenem (Meronem; AstraZeneca).Samples of azlocillin and mezlocillin (which are not commerciallyavailable in Belgium) were kindly provided by Bayer AG, Wuppertal,Germany; samples of faropenem were obtained from Suntory, Ltd.,Gunma, Japan. Water for dissolving the drugs was obtained froma a MilliQ Academic ultrapure water system (Millipore Corp.,Bedford, Mass.). All other reagents were of analytical grade(or for high-pressure liquid chromatography [HPLC] analysis)and were provided by E. Merck AG (Darmstadt, Germany) or Sigma-Aldrich(Steinheim, Germany).

Choice of concentrations and conditions of stability. The highest, clinically acceptable daily doses of each drugwere determined based on officially approved recommendationsfor severe infections in intensive care patients or cystic fibrosisor on widely accepted clinical practice. Faropenem (which hasno useful activity against P. aeruginosa) was only used forcomparison purposes with imipenem and meropenem and was usedat the same concentrations as were used for meropenem. All finalfigures are shown in Table 1. For intensive care patients, weconsidered that the daily amount of drug would be placed ina volume of 48 ml (i.e., the standard volume of motor-operatedsyringe pumps) and infused at 25°C (room temperature). Forcystic fibrosis patients, for whom home therapy was contemplated,we considered that the daily drug amount would be placed ina 125-ml motorless elastomeric pump carried under clothing (and,therefore, at temperatures close to 37°C). These considerationsgave us a maximal drug concentration, as well as a range oftemperatures, for practical stability testing. For most drugs,however, this maximal concentration proved to be impractical,either because of the development of an important color changeat 37°C (cefpirome and cefepime at >32 g/liter and >50g/liter, respectively, assessed against a white background bya trained inspector with solutions left at 37°C for 2 hafter the solutions were prepared), unacceptable viscosity forunhindered flow from elastomeric pumps (e.g., meropenem at >64g/liter, aztreonam at >100 g/liter, and ureidopenicillinsat >128 g/liter, with a flow rate falling to <75% of thenominal flow), or limited solubility (imipenem plus cilastatinat >8 g/liter or the presence of nondissolved material whensolutions were examined with the naked eye on a white and ablack background 1 h after preparation). These drugs were thereforetested only at the highest practical concentration. Table 1shows the maximal values actually used in our study.

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TABLE 1. Maximum daily drug dosages projected, maximum concentrations tested, and pH values of the the corresponding solutions

Conditions of experiments. Antibiotics were dissolved in sterile water; diluted to thedesired concentrations; immediately brought to 4, 25, or 37°C;and maintained at that temperature for up to 24 h under normalartificial laboratory light (aimed at mimicking the light environmentof an intensive care unit). No attempt was made to use any diluentother than water since all solutions were highly hypertonicand infusion to patients would always take place at very lowrates (typically between 33 and 86 µl/min). No controlof pH was attempted after we observed that the value reachedfor each drug upon dissolution in water from its clinicallyavailable form or from the sample received from the correspondingmanufacturer (see Table 1) was actually offering the best stability.Samples were taken at appropriate times, and the drug residualconcentration was determined immediately by HPLC after suitabledilution and then compared to freshly prepared standards. Ifthe assays had to be delayed, samples were frozen at -80°Cafter we verified that no degradation would occur for severaldays under these conditions.

Analyses. Separations by HPLC for assessment of the stability of the ß-lactamswere made on LiChrospher 100RP-18 column (25 by 0.4 cm; 5 µm;Merck KgaA, Darmstadt, Germany), except for cefepime for whicha XTerrra RP-18 column (0.46 by 15 cm; 5-µm-size beads;Waters, Milford, Mass.) was used. Analyses were made on a SpectraPhysics P2000 system (Thermoquest, San Jose, Calif.) or on aWaters 2690 System (Waters Corp.), both of which were equippedwith a diode array UV detector, and by using a injection volumeof 25 µl. Elution was isocratic for all drugs except forimipenem. (See Table 2 for details of the methodology and referencesto the original publications and the ranges of linearity andintraday coefficients of variation for all drugs [except inspecial circumstances, all assays for each experiment were madeover a single 24-h period].) All conditions were selected toprovide a retention time (R_T) of ca. 5 min and satisfactoryseparation from known degradation products (obtained by keepingsamples for several days at 37°C; for example, the conditionsused for ceftazidime allowed us to unambiguously detect theappearance of its ${Delta}$ ₂ isomer). To ensure that no degradation productwould comigrate with the nondegraded drug, the full spectrumof the major peak (associated with the putatively nondegradeddrug) in all samples showing signs of degradation was systematicallyrecorded and compared to that of the intact drug. In preliminaryexperiments with aztreonam, we also verified that the intactnessof the drug, as assessed by HPLC, corresponded to the maintenanceof an unaltered microbiological activity (based on a disk-plateassay with Escherichia coli as the test organism). Pyridinereleased by ceftazidime was assayed as recently described (11)(XTerrra RP-18 column; mobile phase, 10 mM ammonium acetatebuffer [pH 5]-acetonitrile [94:6, vol/vol]; detection, 257 nm;retention time, 3.38 min [versus 4.44 min for ceftazidime];lowest limit of detection, 5 ng; limit of quantification, 20ng; linearity, up to 1,000 ng [R² = 0.999]).

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TABLE 2. Conditions of assay of ß-lactams by HPLC

RESULTS

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Results

Stability studies. Figure 1 and Table 3 show the rate of degradation of all ß-lactamsinvestigated upon incubation for up to 24 h at 37°C andat the highest concentration tested. Table 3 gives the timeduring which each drug remained >90% stable, which is thelimit set up by the U.S. Pharmacopeia (39) for most ß-lactams,including those studied here. If we consider aztreonam and theureidopenicillins first, it is evident that aztreonam and thepiperacillin-tazobactam mixture had a satisfactory stabilityfor at least 24 h at 37°C. Piperacillin without tazobactamwas less stable, since 10% of the drug was already degradedat 22 h. Mezlocillin was the least stable molecule in this group.Moving to the cephalosporins, it is clear that three drugs testedshowed essentially similar behaviors, being less stable thanaztreonam or piperacillin since they had already undergone 10%degradation within 7 to 13 h. The greatest instability, however,was observed for the two parenteral penems, imipenem and meropenem(imipenem was tested only in the presence of cilastatin sincepharmaceutical preparations of imipenem alone for clinical usewere unavailable). These drugs, indeed, remained 90% stablefor less than 3 h at 37°C and were degraded by up to 60and 70% within 24 h at that temperature. In sharp contrast,however, faropenem displayed a remarkable stability with nomore degradation than that shown by piperacillin-tazobactamwithin 24 h.

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FIG. 1. Stability of the ß-lactams in water at 37°C over time at the maximum concentration tested. (A) Symbols: ${triangleup}$ , 10% aztreonam; ${square}$ , 12.8% piperacillin; ${blacksquare}$ , 12.8% piperacillin plus tazobactam (since the slope for 12.8% azocillin was almost identical to that for piperacillin-tazobactam, it was omitted for the sake of clarity); ${blacktriangledown}$ 12.8% mezlocillin. (B) Symbols: ${blacksquare}$ , 12% ceftazidime; ${square}$ , 5% cefepime; ${blacktriangleup}$ , 3.2% cefpirome. (C) Symbols: ${square}$ , 0.8% imipenem plus cilastatin; ${triangleup}$ , 6.4% meropenem; ${triangledown}$ , 6.4% faropenem. All values are the means of three independent determinations ± the standard deviation (SD; symbols without bars indicate values for which the SD is smaller than the symbol size).

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TABLE 3. Time during which ß-lactams remains >90% stable at the highest concentration tested (see )

These experiments were repeated at 4, 25, and 30°C to morefully delineate the limits of stability of each molecule. Theresults are presented summarized in Fig. 2 (where only one representativedrug in each group is shown for the sake of clarity) and inTable 3 (where only data obtained at 25°C are presentedsince this temperature is of direct interest for practical clinicalusage). It is clear that the degradation of all drugs was temperaturedependent. Degradation could be almost completely abolishedif the drugs were maintained at 4°C, with the notable exceptionof imipenem, which suffered almost as much degradation at 4°Cas at 25°C (ca. 30% in 24 h). At 25°C, aztreonam andthe two ureidopenicillins suffered only minor degradation after24 h, whereas the three cephalosporins showed ca. 10% degradationwithin 20 to 24 h. The two parenteral penems showed a fast degradation(10% in 3 h 30 min and 5 h 15 min, respectively). Yet, faropenemdemonstrated an almost complete stability. Finally, in viewof the slight differences in stability between piperacillinand piperacillin plus tazobactam, all data concerning thesetwo preparations were subjected to statistical evaluation bypaired t test on all matched points (Table 4), but this comparisonshowed only a trend toward a greater instability of the piperacillinalone versus the piperacillin plus tazobactam (lowest P value= 0.0736).

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FIG. 2. Influence of temperature on the degradation of selected ß-lactams (24 h of incubation) at the maximum concentration tested as follows: 12.8% piperacillin ( ${circ}$ ), 5% cefepime ( ${triangleup}$ ), 6.4% meropenem ( ${blacksquare}$ ), and 0.8% imipenem plus cilastatin ( ${square}$ ). All values are the means of three independent determinations ± the SD (symbols without bars indicate values for which the SD is smaller than the symbol size).

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TABLE 4. Influence of tazobactam on the degradation of piperacillin at 37°C

Release of pyridine from ceftazidime. Ceftazidime contains a pyridinium moiety in its C3 side chainand is known to release pyridine upon chemical degradation (42).The U.S. Pharmacopeia (39) states that the pyridine contentof ceftazidime solutions for intravenous administration cannotexceed 1.1 mg/ml. We therefore tested for release of pyridineby quantitative methods. As shown in Fig. 3, pyridine was foundto be released in a time-, temperature-, and drug concentration-dependentfashion. While only minimal amounts of pyridine (<0.1 mg/ml)could be detected in freshly prepared samples, the storage ofceftazidime (12% [wt/vol]) at 37°C for 24 h caused a pyridinerelease of >4 mg/ml. The limit set forth by the U.S. Pharmacopeia(1.1 mg/ml) was actually already attained after less than 8h at 37°C and between 16 and 24 h at 25°C at the highestconcentration tested (i.e., 12%). This limit was not reachedat 25°C within the 24-h time span if a lower drug concentration(8.3%) was used.

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FIG. 3. Release of pyridine from ceftazidime upon incubation at 4°C ( ${square}$ ), 25°C (), and 37°C ( ${blacksquare}$ ) at two different initial concentrations (left panel, 8.3% [wt/vol]; right panel, 12% [wt/vol]). Freshly prepared samples contained <0.1 mg of pyridine/ml. The dotted line indicates the upper limit allowed for pyridine content in ceftazidime solutions according to the U.S. Pharmacopeia (39).

DISCUSSION

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Discussion

In vitro and animal pharmacodynamic studies have clearly demonstratedthat ß-lactams are time-dependent antibiotics thatare more effective when administered by continuous infusion(7). However, these drugs tend to be intrinsically unstablein aqueous media because of the high reactivity of their four-memberß-lactam ring (which is critical for their antibacterialaction [17, 33]). This ring is indeed very susceptible to hydrolysisthrough base catalysis combined with nucleophilic attack onthe nitrogen atom (25). It is therefore no surprise to observesignificant degradation of ß-lactams after dissolutionin water-based media, especially if maintained at temperaturesthat are too high. Three key findings, with regard to the practicaluse of ß-lactams, made here are that (i) at 37°Ca significant degradation may indeed occur for some ß-lactams,a finding that has immediate implications in daily practice;(ii) degradation rates vary widely among ß-lactamsin present clinical use, making determinations of the stabilityof each molecule essential in the context of its potential useby continuous infusion; and (iii) clear warnings must issuedconcerning the unsuitability of some ß-lactams andcertain clinical conditions for this type of administration.We know of no easily accessible and validated information inthis context.

Thus, our study shows that aztreonam and the ureidopenicillins(with the exception of mezlocillin) entirely fulfill the requirementsof stability set forth by the U.S. Pharmacopeia (39), even whenkept for 24 h at temperatures up to 37°C. These moleculescan therefore be used in almost all clinical conditions, includingadministration from portable pumps carried under clothes, asoccurs in the at-home therapy of cystic fibrosis patients. Thesituation is less favorable for the cephalosporins since thethree drugs we tested here (and which probably represent themost useful ones in cases of severe infections) showed significantlyless stability than penicillins. Chemical considerations suggestthat cephalosporins are more reactive than penicillins, andthus less stable in water based-media, because of their C3 substituent,which acts as a leaving group (4, 5) (this hypothesis has, however,been challenged [26]). Since the C3 substituents of ceftazidime,cefepime, and cefpirome (a pyridinium, a pyrrolidinium, anda 5H-cyclopentapyridinium, respectively) are all cationic, acommon behavior of these molecules with respect to stabilityis not surprising. Globally, and of direct interest for theclinician, is the fact that neither of these three cephalosporinscan be used for 24-h infusion if they are maintained at a temperatureof >25°C. This conclusion is somewhat at variance withother studies, wherein it was concluded that ceftazidime andcefepime solutions could be kept at 24°C, or even at 30°C,and at pH 4.6 to 6.5 for ca. 2 days and yet maintain 90% oftheir initial concentration (14, 28, 43). These studies, however,(i) used more diluted solutions, (ii) had the drug preparedin buffered media (which are not available to clinicians ina pharmaceutically validated form and may also not be sufficientin view of the high drug concentrations needed for the projectedclinical use), and (iii) were not specifically designed to testthe drug stability under pertinent conditions of clinical use.

Apart from the loss of active product, the release of potentiallyharmful degradation products upon storage of ß-lactamsmust also be considered. As shown in our previous study (30),the degradation of ceftazidime is accompanied by the releaseof at least three products: its ${Delta}$ ₂ isomer, an ethanal derivative,and pyridine. The latter two compounds arise from degradationof the ß-lactam ring and the ensuing release of theC3 substituent of ceftazidime. While little is known about thesafety of the ethanal derivative, pyridine is a well-known toxic,and the level of its release needs therefore to be accuratelydetermined. This has already been studied by several authors(10, 11, 32, 42), but we considered it useful to reexamine thisissue in order to unambiguously establish the link between druginstability and pyridine release in the projected conditionsof clinical use. Our results not only confirm clearly this relationshipbut also demonstrate that pyridine release exceeds the limitconsidered acceptable by the U.S. Pharmacopeia (39) if the drugis kept at 37°C for more than a few hours. This reinforcesour previous conclusion (30) that ceftazidime should not berecommended for use by continuous infusion at temperatures greaterthan 25°C (see also the comment by Plasse et al. [J. C.Plasse, J. C., C. Chabloz, A. Terrier, and G. Bellon, Letter,Pediatr. Pulmonol. 33:232-233, 2002]). We are aware of a recentstudy (29) in which ceftazidime was infused to cystic fibrosispatients with the aid of portable pumps changed only every 24h. Presumably, these pumps were carried under clothes, and thedrug was therefore exposed to temperatures exceeding 25°C.While no adverse effect was reported, these authors did notaddress specifically the issue of pyridine toxicity nor didthey check for the intactness of ceftazidime and release ofpyridine under these conditions of use. We did not check inthe present study for the release of degradation products fromcefepime and cefpirome. However, the degradation of both cephalosporinshas been shown to be accompanied by the release of compoundsoriginating from their C3 substituents (namely, N-methylpyrrolidinefor cefepime [12] and 6,7-dihydro-5H-1-pyrindine for cefpirome[34]). We could not find public data concerning the toxicityof these compounds. It is also of interest that degradationstudies in water have demonstrated the destruction of the cephemring for cefepime (28) and the release of isomers for cefpirome(epi-cefpirome, ${Delta}$ ₂-cefpirome, and anti-cefpirome [34]). Again,no public information could be found concerning the toxicitypotential of these compounds. Based on all of these considerations,it can be concluded that it is not safe to use ceftazidime,cefepime, or cefpirome for any clinical use that involves thestorage of these drugs in aqueous solution at temperatures higherthan 25°C for more than a few hours. More specifically,portable pumps carried under clothes should not be used in thisway unless their content is renewed at least every 8 h. Conversely,ceftazidime, cefepime, and cefpirome appear to be suitable forinfusion from external pumps in countries and, during periodsof the year, in which the air temperature remains or can bemade to remain lower than 25°C. The degradation of cefepimehas been claimed to be slowed down by avoiding its exposureto light (28), but we consider this approach to be unpracticaland unreliable under clinical conditions.

Finally, a clear warning must be raised against the use of parenteralcarbapenems (i.e., imipenem and meropenem) for continuous infusionin view of their very unsatisfactory stability. Carbapenemsand penems are usually considered to be intrinsically less stablethan the corresponding carbapenams and penams because of thedouble bond in the five-member thiazoline or dihydropyrrolerings (through a decrease in the basicity of the leaving aminebrought by the introduction of a conjugation in the tetrahedralintermediate [25]). The chemical instability of imipenem isactually well known, and degradation of this drug has been shownto occur rapidly (half-life, <2 h), even in diluted solutions(35). This instability results from complex oligomerizationcombining an intermolecular carboxyl group attack on the ß-lactamgroup (favored by a weakly acidic environment) and an intermolecularreaction between the ß-lactam and formimidoyl groups(favored by a weakly alkaline milieu [31]). Meropenem degradationalso occurs readily in water through hydrolysis and dimerizationfrom intermolecular aminolysis, although it was claimed thatimproved stability, compared to other carbapenems, could resultfrom the protective effect exerted by the 1-ß-methylgroup against attack of the ß-lactam ring (36). Ourdata show that this protection is ineffective under the practicalconditions of clinical use described here. It has been suggestedthat meropenem could be safely used for continuous infusionif kept at 4°C (18). Our data confirm that meropenem isstable for 24 h if kept at 4°C, but the safety and applicabilityof this approach in the clinics remain dubious. Maintainingthis low temperature is an absolute requirement, since >30%of drug degradation will occur at 25°C (we did not investigateintermediate temperatures since these are not easily controlledin a clinical environment). The clinician will therefore needto carefully weigh the potential advantages of the administrationof meropenem by continuous infusion against the difficultiesrelated to this need for a strict control of the temperatureand the risks associated with a significant degradation (andrelease of compounds of unknown toxicity) should these stringentconditions of administration not be met. The situation is evenworse for imipenem, which should not be considered for continuousinfusion at any rate.

It is intriguing that faropenem, which shares with imipenemand meropenem the same unsaturation in its five-member ring,shows a remarkable stability. This could result from the factthat faropenem is not a carbapenem but a penem (i.e., it possessesa S atom in its five-member ring, which may reduce the intra-ringstress). Another possibility could be the absence of protonablegroup in the C3 substituent of faropenem (tetrahydrocyclofuranyl),making this drug a monoanionic compound, whereas the C3 substituentsof imipenem (iminomethylaminoethyl; pK_a = 10.4) and of meropenem(pyrrolidinyldimethylaminocarbonyl; pK_a = 8) make both drugszwitterionic at the pH at which they spontaneously equilibrated(i.e., pH 6.8, 7.8, and 8.2) and were therefore tested (datafrom registry numbers 64221-86-2 [imipenem], 96036-03-02 [meropenem],and 106560-14-9 [faropenem], SciFinder Scholar version 2001[http://www.cas.org/SCIFINDER/SCHOLAR/index.html; Chemical AbstractServices, American Chemical Society, Washington, D.C.]).

In conclusion, we show here that the poor stability of ß-lactamantibiotics in solution should not be underestimated if theiruse by continuous infusion is contemplated. We suggest herethat each clinical situation and each drug be specifically examinednot only in relation to a loss of activity but also for therelease of potentially toxic compounds. It is only if investigationsare conducted in an orderly and practice-oriented fashion thatthe clinician will be provided with specific and useful guidelinesthat will help him or her to safely optimize patient therapybased on sound pharmacodynamic principles.

ACKNOWLEDGMENTS

We thank E. Sonveaux (Unité de Chimie Pharmaceutiqueet Radiopharmacie) and J. Marchand-Brynaert (Unité deChimie Organique et Médicinale) for helpful discussionson ß-lactam chemical stability.

This work was supported by the Fondation Roi Baudouin/KoningBoudewijn Stichting, Brussels, Belgium. H.S. is Boursier ofthe Belgian Fonds pour la Recherche dans l'Industrie et l'Agriculture.H.C. is Aspirant and M.-P.M.-L. is Maître de Recherchesof the Belgian Fonds National de la Recherche Scientifique.We thank the drug suppliers for the kind gift of the correspondingclinical and/or investigational samples.

FOOTNOTES

* Corresponding author. Mailing address: Unité de Pharmacologie Cellulaire et Moléculaire, Université Catholique de Louvain UCL 73.70, Avenue E. Mounier 73, B-1200 Brussels, Belgium. Phone: 32-2-764-73-75. Fax: 32-2-764-73-73. E-mail: eric.viaene{at}facm.ucl.ac.be.

REFERENCES

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