Ester prodrugs of ampicillin : cellular pharmacokinetics and intracellular activity

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.Thèse de doctorat en sciences pharmaceutiques (FARM 3) -- UCL, 200

Stability and compatibility study of cefepime in comparison with ceftazidime for potential administration by continuous infusion under conditions pertinent to ambulatory treatment of cystic fibrosis patients and to administration in intensive care units.

Cefepime has been examined for stability, potential liberation of degradation products and compatibility with other drugs under conditions mimicking its potential use by continuous infusion in cystic fibrosis and intensive care patients (5-12% w/v solutions; temperatures from 20 to 37 degrees C; 1 h contact at 25 degrees C with other drugs frequently co-administered by intravenous route to these types of patients). Ceftazidime was used as a comparator based on a previous normative study with this antibiotic for the same indications. Based on a limit of max. 10% degradation, cefepime can be considered stable for a maximum of 24 h at 25 degrees C, but for only approximately 14 h at 30 degrees C, and for 30 degrees C for >12 h as shown from a marked increase in pH and from the development of a strong red-purple colour. Incompatibilities were observed with erythromycin, propofol, midazolam, phenytoin, piritramide, theophylline, nicardipine, N-acetylcysteine and a concentrated solution of dobutamine. We conclude that: (i) cefepime cannot be used safely by continuous infusion if containers are kept for more than a few hours at 37 degrees C (as will be the case for cystic fibrosis patients if using portable pumps carried under clothes); (ii) caution must be exercised in intensive care patients if the temperature and co-administration of other drugs is not kept under tight control. The nature and safety of the cefepime degradation products need to be studied further

Accumulation and Oriented Transport of Ampicillin in Caco-2 Cells from Its Pivaloyloxymethylester Prodrug, Pivampicillin

Pivampicillin (PIVA), an acyloxymethylester of ampicillin, is thought to enhance the oral bioavailability of ampicillin because of its greater lipophilicity compared to that of ampicillin. The fate of PIVA in intestinal cells and the exact location of its conversion into ampicillin have, however, never been unambiguously established. Polarized Caco-2 cells have been used to examine the handling of PIVA and the release of ampicillin from PIVA by the intestinal epithelium. Experiments were limited to 3 h. Cells incubated with PIVA (apical pole) showed a fast accumulation of ampicillin and transport toward the basolateral medium, whereas PIVA itself was only poorly accumulated and transported. Cells incubated with free ampicillin accumulated and transported only minimal amounts of this drug. Release of ampicillin from cells incubated with PIVA was unaffected by PEPT1 and OCTN2 inhibitors but was sharply decreased after ATP depletion or addition of bis(4-nitrophenyl)-phosphate (BNPP; an esterase inhibitor). PIVA incubated with Caco-2 lysates released free ampicillin, and this release was inhibited by BNPP. Efflux studies showed that the ampicillin that accumulated in cells after incubation with PIVA was preferentially transported out of the cells through the basolateral pole. This efflux was decreased by multidrug resistance-associated protein (MRP) inhibitors (probenecid, MK-571) and by ATP depletion. A phthalimidomethylester of ampicillin that resists cellular esterases failed to cause any significant release (cell lysate) or transport (polarized Caco-2 cells) of ampicillin. These results show that when PIVA is given to Caco-2 cells from their apical pole, ampicillin is released intracellularly and that ampicillin is thereafter preferentially effluxed into the basolateral medium through an MRP-like transporter

Comparative Stability Studies of Antipseudomonal β-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 β-lactams in concentrated solutions was examined in view of their potential administration by continuous infusion with external pumps (for intensive care patients) or with portable pumps carried under clothing (for cystic 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°C but only for 8, 7.25, and 13 h at 37°C, respectively. The control of temperature therefore appears to be critical for all three cephalosporins that cannot be recommended for use in portable pumps carried under clothes for prolonged periods for reasons of stability. Cefpirome and cefepime solutions developed an important color change (from light yellow to dark red) upon exposure when stored at 30°C or higher. Degradation of ceftazidime was 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 12 and 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 as stable as aztreonam or piperacillin

LPS induces IL-10 production by human alveolar macrophages via MAPKinases- and Sp1-dependent mechanisms-0

<p><b>Copyright information:</b></p><p>Taken from "LPS induces IL-10 production by human alveolar macrophages via MAPKinases- and Sp1-dependent mechanisms"</p><p>http://respiratory-research.com/content/8/1/71</p><p>Respiratory Research 2007;8(1):71-71.</p><p>Published online 4 Oct 2007</p><p>PMCID:PMC2080632.</p><p></p>nd assayed for IL-10 content by ELISA for Panel A and by real time PCR for Panel C. Panel B: HAM were stimulated for 24 hours with increasing concentration of LPS and supernatant were assayed for IL-10 by ELISA

LPS induces IL-10 production by human alveolar macrophages via MAPKinases- and Sp1-dependent mechanisms-4

<p><b>Copyright information:</b></p><p>Taken from "LPS induces IL-10 production by human alveolar macrophages via MAPKinases- and Sp1-dependent mechanisms"</p><p>http://respiratory-research.com/content/8/1/71</p><p>Respiratory Research 2007;8(1):71-71.</p><p>Published online 4 Oct 2007</p><p>PMCID:PMC2080632.</p><p></p> μg/ml) during 24 h. Supernantants were assayed by ELISA to determine IL-10 production