1 Unité de Pharmacologie Cellulaire et Moléculaire, UCL 7370, avenue E. Mounier 73, B-1200 Brussels; 2 Unité de Chimie Pharmaceutique et de Radiopharmacie, Université Catholique de Louvain, B-1200 Brussels, Belgium
Received 1 May 2003; returned 16 July 2003; revised 23 July 2003; accepted 28 July 2003
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Abstract |
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Methods: Quantitative evaluation of the activity of ampicillin, phthalimidomethylampicillin (PIMA) or pivaloyloxymethylampicillin (PIVA) against intracellular L. monocytogenes, and direct measurement of cellular ampicillin concentration in J774 macrophages.
Results: Ampicillin, PIMA and PIVA caused a 0.5 log decrease in cell-associated cfu within 5 h when used at an extracellular concentration of 3.6 µM [10 x MIC of ampicillin (1.25 mg/L); 1.83 mg/L for PIMA and 1.67 mg/L for PIVA]. Addition of ß-lactamase in the extracellular milieu abolished the activity of ampicillin and of PIMA but not that of PIVA. At low extracellular concentrations [0.5 x MIC ampicillin (62.5 µg/L); equimolar concentrations for PIMA (91.5 µg/L) and PIVA (83.5 µg/L)], ampicillin and PIMA lost all activity (compared with controls), but PIVA remained as active as at the higher concentration. Incubation of cells with PIVA at the low concentration (83.5 µg/L) for 20 h caused a 2 log reduction of cfu if the medium was changed every 5 h (to compensate for the degradation of extracellular PIVA). Incubation of cells with PIVA allowed for a marked (four- to 25-fold) cell accumulation of ampicillin, whereas no ampicillin accumulation was seen for cells incubated with ampicillin or with PIMA.
Conclusions: This is the first demonstration that PIVA (a prodrug of ampicillin) can be used to promote ampicillin cellular accumulation and, thereby to increase ampicillin intracellular activity. PIVA could be useful for control of the intracellular multiplication of L. monocytogenes.
Keywords: prodrugs, ß-lactams, ß-lactamases, infections
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Introduction |
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Materials and methods |
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Figure 1 shows the structural formulae of ampicillin and of its pivaloyloxymethyl (PIVA) and phthalimidomethyl (PIMA) esters. The synthesis of both esters has been described.8,9 Their stability in buffered media (pH 7.4) was found to be comparable (half-lives of 92 and 62 min, respectively, at 37°C), with a release of both free ampicillin and degradation products in a molar ratio of 68:32 and 64:36, respectively8 (as was previously reported for pivampicillin14).
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Because of intrinsic instability, PIMA and PIVA were kept in their dry state at 4°C. For each experiment, an aliquot was weighed, dissolved in 100% ethanol, and diluted in ice-cold 20 mM sodium acetate buffer (pH 5.4) to prevent degradation. This solution was further diluted in culture medium to the desired final concentration, and used immediately. Ampicillin was prepared as a fresh solution in water for each series of experiments and used within a few hours of suitable dilution.
Bacteria and MIC determination
We used a haemolysin-producing strain of L. monocytogenes (serotype 1/2a) obtained, maintained and characterized as described previously.15 The MICs of ampicillin and gentamicin, as determined in tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, MD, USA) by geometric dilution and using standard methods, were 0.125 and 1 mg/L, respectively.
Cell culture, infection and intracellular activities of antibiotics
All experiments were performed with J774 macrophages (a murine continuous reticulosarcoma cell line), which were cultured and maintained as described previously.3 In brief, cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine, seeded in 6-well dishes at a density of 5 x 104 cells/cm2 and used upon confluence (2 days of culture). Infection with L. monocytogenes was performed as described recently,16 based on a method previously developed for THP-1 macrophages.13,15 In brief, infection was obtained with untreated bacteria (at an initial bacteria to macrophage ratio of 5) maintained in contact with cells for 1 h at 37°C. Extracellular bacteria were removed by extensive washing; cells were then returned to fresh medium, and the intracellular growth evaluated after increasing periods of incubation at 37°C. For all incubation periods of more than 5 h, the medium contained gentamicin 2 mg/L (2 x MIC) to prevent the extracellular growth of Listeria.17 For collection, cells were washed with ice-cold PBS, scraped off the culture dish into distilled sterile water with a Teflon spatula, and subjected to vigorous mechanical mixing to achieve homogeneity. Lysates were then used for total cell protein measurement,18 and, in parallel, for cfu counting by plating on tryptic soy agar after appropriate dilution. Colonies were counted using a Gel Doc 2000 apparatus (Bio-Rad, Hercules, CA, USA) operated with Quantity One software (Bio-Rad). We checked that the carried-over antibiotic (i.e. the amount of ampicillin or gentamicin brought into the dishes with the cell samples) was too low to interfere significantly with L. monocytogenes growth in this assay.
Assay of ampicillin
Ampicillin in cell culture media was assayed by high-performance liquid chromatography using the method described previously for PIMA and PIVA,11 but with a modified mobile phase (acetonitrile:10 mM acetate buffer pH 5, 1:9). Cell-associated ampicillin was assayed using a specific and sensitive enzymatic method based on the ability of ß-lactams to bind to and irreversibly inhibit the bacterial DD-carboxypeptidase.19 In brief, the activity of the enzyme (purified from Streptomyces R39) was measured by the release of free D-Ala from N,N-diacetyl-L-Lys-D-Ala-D-Ala [lowest limit of detection of ampicillin 20 ng/mL; linearity up to 100 ng/mL (R2 =0.9931)].
Calculation of the apparent cellular concentration of ampicillin
The total cellular ampicillin content was systematically expressed by reference to the protein content of the corresponding samples. The apparent cellular ampicillin concentration was then calculated using a conversion factor of 5 µL of cell volume per milligram of cell protein, as in our previous publications dealing with the cellular accumulation of other antibiotics or drugs.3,20 We refer to this concentration as an apparent one, since we do not know, from the present experiments, where ampicillin is located within the cell. Previous studies using penicillin G, however, have shown that free ß-lactams are distributed in the cytosol.3
Materials
PIMA was obtained as the chloride salt with a purity of >95%.8 PIVA [99.5% purity and complying with the specifications of the Pharmacopée Européenne21 was obtained from Leo Laboratories Ltd (Dublin, Ireland) on behalf of Leo Pharmaceuticals Product Ltd A/S (Ballerup, Denmark). The N,N-diacetyl-L-Lys-D-Ala-D-Ala (the substrate of the DD-carboxypeptidase) and the purified Streptomyces R39 DD-carboxypeptidase were kindly prepared and donated by J.-M. Frère (Centre dIngénierie des Protéines, Université de Liège, Liége, Belgium). ß-Lactamase [from Enterobacter cloacae (0.3 U/mg protein based on benzylpenicillin degradation)] and ampicillin (sodium salt) were purchased from Sigma-Aldrich (St Louis, MO, USA), cell culture media and sera from Life Technologies (Paisley, UK), and all other reagents from E. Merck (Darmstadt, Germany).
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Results |
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In a series of initial experiments, we examined and compared the activities of free ampicillin, PIMA and PIVA over a 5 h period using a suboptimal concentration of ampicillin (0.5 x MIC in broth; 62.5 µg/L) and equimolar concentrations of PIMA and PIVA [0.18 µM; 91.5 and 83.5 µg/L]. The results are shown in Figure 2(a). As anticipated, ampicillin was inactive compared with controls (no antibiotic added). PIMA did not show any activity either, but PIVA caused a reduction in cell-associated cfu (0.5 log) that was essentially similar to what we described earlier for L. monocytogenes-infected THP-1 macrophages incubated with 50 mg/L ampicillin.13 Figure 2(b) shows that increasing the extracellular concentration of ampicillin to 10 x MIC, and that of PIMA and PIVA to equimolar concentrations, allowed control of intracellular L. monocytogenes growth, with no meaningful difference between each of the three compounds. Since both PIMA and PIVA can regenerate free ampicillin extracellularly, we examined whether inactivating the extracellular drug would modify these results. As shown in Figure 2(c), addition of ß-lactamase in the culture medium caused a complete loss of activity of ampicillin and PIMA. In contrast, the activity of PIVA towards intracellular L. monocytogenes remained essentially unchanged.
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The extracellular concentration of ampicillin had to be increased to 17.4 mg/L (50 µM) in order to reliably detect ampicillin in cells, and PIMA and PIVA were therefore studied in comparison with ampicillin at an equimolar concentration (PIMA 25.4 mg/L; PIVA 23.2 mg/L). Figure 3 shows that cells incubated with free ampicillin or PIMA accumulated very little ampicillin (the cellular:extracellular ratio of ampicillin remaining close to 1 for up to 24 h in cells incubated with free ampicillin, and with similarly low amounts of ampicillin in cells incubated with PIMA). In sharp contrast, cells incubated with PIVA showed a rapid and extensive accumulation of ampicillin (reaching an apparent cellular concentration of 500 mg/L (this would correspond to a cellular to extracellular concentration ratio of at least 20:1 if it is assumed that all extracellular PIVA has been quantitatively transformed into free ampicillin). This accumulation was, however, transient, since the cellular ampicillin concentration quickly decreased with a half-life of
2.5 h. Since the half-life of ampicillin in aqueous media at pH 7.4 (presumably the pH of the cell cytosol) is long (>48 h)8 and is not reduced by cell extracts (unpublished results), we reasoned that this sharp decrease in the cellular ampicillin concentration could only be due to the fast degradation of extracellular PIVA. This would indeed cause a rapid decrease in the amount of membrane-bound PIVA,11 thereby decreasing the rate of delivery of ampicillin from PIVA to cells. As a result, cell-associated ampicillin will quickly re-equilibrate with the extracellular ampicillin, thus drastically reducing the intracellular concentrations of the drug (see Figure 3) [detailed studies have shown that J774 macrophages incubated for 5 h with free ampicillin at extracellular concentrations ranging from 10 to 100 mg/L (27270 µM) achieve an apparent cellular to extracellular drug concentration ratio of only 0.4 ± 0.09 (n = 18)].11 We therefore undertook to regularly re-expose the cells to the same concentration of PIVA by changing the medium every 5 h. Figure 3 (dotted line) shows that this protocol allowed maintenance of the cellular concentration of ampicillin at an almost constant level of
200 mg/L (a concentration at least 10-fold larger than the maximum ampicillin concentration that could be present in the extracellular milieu assuming that all extracellular PIVA is converted into free ampicillin).
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The activity of ampicillin against intracellular L. monocytogenes is time dependent, and a 24 h incubation is required to obtain a 2 log decrease in cell-associated cfu in comparison with the original inoculum.17 In view of the results obtained in the 5 h model with low concentrations of PIVA (Figure 2), and the results of the ampicillin accumulation studies over 20 h (Figure 3), we extended our studies with infected cells to 20 h. As shown in Figure 4, PIVA added at an initial concentration of 83.5 µg/L with no further medium change afforded only transient control of bacterial growth. Thus, the number of cell-associated cfu declined over the first 5 h, but then rose sharply between 5 h and 20 h. When the medium was changed every 5 h, however, the number of cell-associated cfu declined steadily reaching a 2 log decrease in 20 h. This decline is similar to that observed with ampicillin 50 mg/L in a model of Listeria-infected THP-1 macrophages over the same period of time.17
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Discussion |
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Moving now to the treatment of listeriosis, our data also offer a somewhat provocative new insight concerning the optimal choice of agents in this context. A combination of ampicillin and gentamicin is widely considered as a first-line therapy,26 based primarily on in vitro susceptibility data but backed by a limited number of clinical comparative studies. However, we have shown previously that gentamicin is essentially inactive against intracellular L. monocytogenes in THP-1 macrophages.13 This observation is extended here to J774 macrophages, since gentamicin present in the 20 h model studies did not prevent the intracellular multiplication of bacteria. We also showed in the THP-1 model that ampicillin is bacteriostatic against intracellular L. monocytogenes in short-term (5 h) incubations, but may become bactericidal if prolonged incubations (24 h) are used.17 In both cases, however, large concentrations of ampicillin (several times above the MIC) are necessary. Large and sustained concentrations of ampicillin cannot easily be obtained in patients with its conventional mode of administration. We show here that low concentrations of PIVA achieve essentially the same effect against intracellular L. monocytogenes as large concentrations of ampicillin, and that a continuous reduction of bacterial counts can be obtained simply by regular restoration of these low concentrations. The data therefore indicate that pivampicillin may provide similar chemotherapeutic effects to ampicillin while not requiring such high serum levels. It must, however, be emphasized that: (i) PIVA will offer such an advantage in vivo only if administered intravenously (pivampicillin administered orally will be hydrolysed in the intestinal wall and in the liver25,27) and at regular intervals (or even by continuous infusion); and (ii) that the administration of another antibiotic acting upon extracellular organisms will still be necessary. Bearing these caveats in mind, PIVA could be further tested in animal models and carefully designed human studies as a means of better controlling the intracellular survival and spread of L. monocytogenes in difficult situations.
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Acknowledgements |
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Footnotes |
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References |
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