J. Van Gelder

Species-dependent and site-specific intestinal metabolism of ester prodrugs

In order to select a species for drug absorption studies of ester prodrugs and to identify a possible absorption window with low esterase activity and hence increased absorption of the ester prodrug, the esterase activity was investigated in homogenates from various intestinal segments of different species. p-Nitrophenyl acetate and tenofovir disoproxil [bis(POC)-PMPA] were used as substrates for esterases. p-Nitrophenyl acetate is a model substrate for esterase activity, while tenofovir disoproxil (fumarate salt) is an ester prodrug of the potent antiviral nucleoside phosphonate analogue tenofovir. As esterase-mediated degradation during transepithelial transport may be a limiting factor for its oral absorption, targeting the prodrug to a region of the intestine with lower esterase activity may lead to an increase in oral absorption of the prodrug. The results obtained with p-nitrophenyl acetate and tenofovir disoproxil showed both a site-specific (duodenum > or = jejunum > ileum > or = colon) and species-dependent (rat > man > pig) degradation in intestinal homogenates. Degradation of tenofovir disoproxil in homogenates from Caco-2 monolayers (0.016+/-0.003 nmol. s(-1). mg protein(-1)) was low compared to its degradation in homogenates from human ileum (0.177+/-0.052 nmol. s(-1). mg protein(-1)). Rat ileum appears to be a suitable model to evaluate the influence of esterase activity on the oral absorption of the ester prodrug, as the degradation rate for tenofovir disoproxil (0.245+/-0.054 nmol. s(-1). mg protein(-1)) in rat ileum was similar to degradation in human ileum. The results also suggest that colon targeting may be a useful strategy to reduce the esterase-mediated degradation of ester prodrugs, hence resulting in a possible increase in their oral absorption.

Evaluation of the potential of ion pair formation to improve the oral absorption of two potent antiviral compounds, AMD3100 and PMPA.

9-(2-phosphonyomethoxypropyl)adenine (PMPA) and AMD3100 are highly potent and selective antiretroviral agents. Since PMPA is negatively charged and AMD3100 positively charged at physiological pH, their transepithelial transport and their potential for oral drug delivery is very low. In this study, ion pair formation was evaluated as a possible strategy to enhance transepithelial transport of PMPA and AMD3100. Positively charged counter ions such as t-hexyl-, t-heptyl-, t-octylammonium bromide and dodecyl-, tetradecyl-, hexadecyltrimethylammonium bromide were used to form ion pairs with PMPA, while sodium taurodeoxycholate (in vitro experiments) and sodium taurocholate (in vivo experiments) were used as counter ions for AMD3100. The effect of counter ions on transepithelial transport of PMPA (1 mM) and AMD3100 (1 mM) was investigated by measuring the flux across Caco-2 monolayers. An enhancement in drug transport could be observed at a concentration of 2 mM of hexadecyltrimethylammonium bromide (counter ion for PMPA) and 10 mM of sodium taurodeoxycholate (counter ion for AMD3100), but at the concentrations used, the absorption enhancing effect could be attributed to a reduction of the integrity of the monolayers. When AMD3100 transport was tested at a concentration of 200 microM, no flux was observed, even in the presence of relatively high concentrations of counter ion (20 times the concentration of AMD3100). Results obtained from partitioning studies of the drugs in the presence or absence of counter ion revealed that competition by other ions was responsible for the absence of an effect: when pure water was used as the aqueous phase, a reduction up to 24.4+/-1.4% and 17.0+/-1.3% of the initial aqueous concentration was observed for PMPA and AMD3100, respectively; however, as soon as other ions were present in the aqueous phase, the effect of the counter ion was diminished (25-50 mOsm) or completely abolished (270-305 mOsm). The absorption enhancing effect of counter ions was also studied in vivo: pharmacokinetic studies in rabbits showed that the oral bioavailability of AMD3100 in the presence of 4 equivalents of taurocholic acid remained very low and was only 3.2-fold better (i.e. 3.6%) in comparison to pure AMD3100. In view of the results obtained in the Caco-2 system, this absorption enhancement can be attributed to an effect on monolayer integrity rather than to the potential to form ion pairs. We can conclude that the formation of ion pairs may not be very efficient as a strategy to enhance transepithelial transport of charged hydrophilic compounds, as competition by other ions may abolish the beneficial effect of counter ions.

Carrier Mechanisms Involved in the Transepithelial Transport of bis(POM)-PMEA and Its Metabolites Across Caco-2 Monolayers

AbstractPurpose. To investigate the role of carrier mechanisms in: [1] the polarized transport of the bis(pivaloyloxymethyl)- [bis(POM)-] ester prodrug of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA] and [2] the directional secretion of its metabolites. Methods. Caco-2 monolayers were used to study the modulating effect of carriers on the transport of bis(POM)-PMEA and the efflux of intracellularly formed metabolites mono(POM)-PMEA and PMEA from the cells. The interaction of bis(POM)-PMEA and its metabolites with the efflux mechanisms present in Caco-2 monolayers was investigated by testing the effect of various concentrations of verapamil (30, 100, 300 μM) or indomethacin (10-500 μM) on transport and efflux. Results. Polarity in transport of bis(POM)-PMEA (50 μM) across Caco-2 monolayers was noted: transport of total PMEA [=bis(POM)-PMEA, mono(POM)-PMEA and PMEA] was significantly higher in basolateral (BL) to apical (AP) direction (14.5 ± 0.4%) than transport in the opposite (AP to BL) direction (1.7 ± 0.2%). This difference was reduced in a concentration dependent way when verapamil (0−100 μM) was included in both AP and BL incubation media. After loading the cells with bis(POM)-PMEA (100 μM) for 1 hr, studies on efflux of PMEA and mono(POM)-PMEA from the Caco-2 monolayers over a 3 hr period, revealed that both metabolites were preferentially secreted towards the AP compartment. Efflux of PMEA towards AP and BL compartments amounted to 14.6 ± 1.1 % and 5.3 ± 0.4%, respectively, of the initial intracellular amount of total PMEA, while efflux of mono(POM)-PMEA towards AP and BL compartments was limited to 2.3 ± 0.1 % and 0.5 ± 0.1 %, respectively. When 10 μM indomethacin was included in the AP incubation medium, efflux of PMEA was decreased to 7.8 ± 0.3% and 3.3 ± 0.3% towards the AP and BL compartments, respectively. The decrease in efflux by indomethacin was concentration-dependent up to 100 μM. Transepithelial transport of total PMEA was also reduced in the presence of 30 μM indomethacin, as reflected in smaller concentrations of PMEA and mono(POM)-PMEA in the acceptor compartment, irrespective of the transport direction. Conclusions. The data obtained in this study suggest that bis(POM)-PMEA is substrate for a P-glycoprotein-like carrier mechanism in Caco-2 monolayers, while its metabolites mono(POM)-PMEA and PMEA are transported by a non-P-glycoprotein efflux protein.

Intestinal absorption characteristics of the low solubility thiocarboxanilide UC-781

The aim of this study was to determine the intestinal absorption characteristics of the antiviral agent UC-781 and to optimize the experimental conditions of the in vitro system for low solubility compounds. The absorption potential of UC-781 was studied with the Caco-2 system and with the rat intestinal perfusion technique. The low solubility of UC-781 required the use of solubility/dissolution rate enhancing agents (e.g. VitE-TPGS, Gelucire 44/14). The creation of sink conditions in the receiver compartment of the Caco-2 system was a prerequisite to reliably study the transport of this poorly soluble compound. After inclusion of VitE-TPGS in the acceptor solution, UC-781 could be characterized as a class II drug of the Biopharmaceutical Classification System (low solubility, high permeation across membranes). A significant concentration-dependent decrease in transport of UC-781 was observed upon increasing the concentration of VitE-TPGS in the apical compartment. This observation contrasts to the absorption enhancing properties of VitE-TPGS, and can probably be attributed to a decrease in the concentration of free UC-781 when using higher concentrations of the solubility/dissolution rate enhancing agents. The use of Gelucire 44/14 as a solubilizing agent resulted in a batch-dependent degradation of UC-781. The inclusion of the solubility/dissolution rate-enhancing agent VitE-TPGS did not result in absorption enhancement in the intestinal perfusion technique.