Ester prodrugs of ampicillin : cellular pharmacokinetics and intracellular activity
(2003)
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- Authors
- Supervisors
- Tulkens, Paul M.
- Abstract
- Beta-lactam antibiotics are not accumulated by eucaryotic cells and therefore are not good candidate to treat intracellular infections. This lack of accumulation is ascribed to their acidic character. However, the carboxylic function carried by beta-lactams is important for them to exert their antimicrobial activity. Since basic compounds are known to be accumulated in acidic compartments of the cell, new basic derivatives of beta-lactams were synthesised by masking the carboxylic function with a groupment. A first derivative of penicillin G, N-(3-dimethylaminopropyl)-benzylpenicillinamide, was shown to be accumulated by macrophages and to be mainly localised in lysosomes. However, this prodrug was not cleaved by peptidases and remained therefore inactive. For this purpose, less stable prodrugs were thereafter synthesised. Indeed, phthalimidomethylampicillin [PIMA], an original ester, and pivaloylampicillin [PIVA], a commercial prodrug have been described to be accumulated highly in J774 macrophages and to release high amount of ampicillin in buffer. The first part of our study was to characterise the cellular accumulation of PIMA and PIVA. For this purpose, the behaviour of both prodrugs were compared to that of azithromycin and chloroquine, two weak bases known to be accumulated in lysosomes by proton-trapping. Although being weak bases, PIVA and PIMA behave in almost all respect in complete contrast with azithromycin and chloroquine : (i) accumulation at 4°C, (ii) fast release from cells at 37°C and 4°C, (iii) saturation at low concentration, (iv) no effect of the extracellular pH, (v) high binding to liposomes at pH 7.4. This strongly suggests that both PIVA and PIMA are mainly localised in the pericellular membrane. In the second part, we evaluated if this large accumulation of prodrugs in the cells enhances the intracellular activity of ampicillin. To this aim, the activity of PIVA, PIMA and ampicillin was studied against intracellular Listeria monocytogenes. At low extracellular concentration (0.5´ MIC of ampicillin); only PIVA is slightly bactericidal (0.5 log decrease in 5h) but at higher concentration (10´ MIC of ampicillin), the three drugs are slightly bactericidal (0.5 log decrease in 5h). In a prolonged infection model (20h), PIVA could maintain its bactericidal activity at low concentration only if the medium was replaced with a new one every 5h. The intracellular release of ampicillin by prodrugs was also studied. PIVA is able to release high intracellular amount of ampicillin whereas PIMA does not. However, this high amount of ampicillin released by PIVA is transient and decreased rapidly over time if the medium is not replaced every 5h with a new one. Our results obtained on macrophages (localisation of prodrugs in pericellular membrane) cannot explain the in vivo observation that PIVA is able to increase the oral bioavailability of ampicillin. Therefore, we evaluated the transepithelial transport of PIVA in comparison with PIMA and ampicillin, using a model of caco-2 polarized intestinal cells. In apical->basolateral way, PIVA enhances the transepithelial transport of ampicillin whereas PIMA does not. However, in basolateral->apical way, there is no difference in the transport of ampicillin when using PIVA, PIMA or ampicillin. In addition, detailed studies of PIVA in the apical->basolateral way, have shown that (i) PIVA is highly associated to cells and releases high amount of intracellular ampicillin, (ii) neither inhibitors of PEPT1 or OCTN2, nor ATP-depletion affect the accumulation of PIVA and subsequent release of ampicillin (iii) PIVA is rapidly released from cells whatever the temperature (iv) at 37°C, intracellular ampicillin is preferably effluxed in the basolateral media, whereas it remains inside the cells at 4°C (v) the transepithelial transport of ampicillin through the use of PIVA is decreased after ATP depletion. These results suggest that PIVA easily diffuses through apical membrane and once in the cytosol is hydrolysed in ampicillin. This ampicillin is then submitted to a specific efflux to the basolateral side by an ATP-dependent transport system. On the other hand, PIMA does not improve the transepithelial transport of ampicillin. This property is due to the fact that PIMA is not efficiently hydrolised in ampicillin probably because of the absence of recognition of the ester bond by esterase.
- Affiliations
Citations
Chanteux, H. (2003). Ester prodrugs of ampicillin : cellular pharmacokinetics and intracellular activity. https://hdl.handle.net/2078.5/110778