Exploring the role of different drug transport routes in permeability screening
ABSTRACT The influence of different drug transport routes in intestinal drug permeability screening assays was studied. Three experimental models were compared: the small-intestine-like 2/4/A1 cell model, which has a leaky paracellular pathway, the Caco-2 cell model, which has a tighter paracellular pathway, and artificial hexadecane membranes (HDMs), which exclusively model the passive transcellular pathway. The models were investigated regarding their ability to divide passively and actively transported compounds into two permeability classes and to rank compounds according to human intestinal absorption. In silico permeability models based on two-dimensional (2D) and three-dimensional (3D) molecular descriptors were also developed and validated using external test sets. The cell-based models classified 80% of the acceptably absorbed compounds (FA >/= 30%) correctly, compared to 60% correct classifications using the HDM model. The best compound ranking was obtained with 2/4/A1 (r(s) = 0.74; r(s) = 0.95 after removing actively transported outliers). The in silico model based on 2/4/A1 permeability gave results of similar quality to those obtained when using experimental permeability, and it was also better than the experimental HDM model at compound ranking (r(s) = 0.85 and 0.47, respectively). We conclude that the paracellular transport pathway present in the cell models plays a significant role in models used for intestinal permeability screening and that 2/4/A1 in vitro and in silico models are promising alternatives for drug discovery permeability screening.
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ABSTRACT: The aim of this study was to investigate the presence of pharmaceutically relevant drug transporters in frog intestine which has been proposed as model for intestinal permeability screening assays of passively absorbed drugs in humans [Trapani, G., Franco, M., Trapani, A., Lopedota, A., Latrofa, A., Gallucci, E., Micelli, S., Liso, G., 2004. Frog intestinal sac: a new in vitro method for the assessment of intestinal permeability. J. Pharm. Sci. 93, 2909-2919]. The expression of transporters in frog intestine was supported by the following observations: (i) the involvement of purine nucleobase transport system was deduced by inhibition of acyclovir transport in the presence of adenine; (ii) baclofen or l-dopa transport was inhibited by the digitalis steroid ouabain and it may be related to the Na(+) electrochemical potential difference, presumably involving amino acid transporters; (iii) the presence of proton-dependent peptide transporters was argued evaluating the effect of the pH change (from pH 5.9 to pH 7.4) on the transport of glutathione; (iv) the possible expression in the frog intestine of an efflux system distinct from P-glycoprotein (Pgp) in the benzylpenicillin transport was deduced using a glucose enriched frog Ringer with or without the known Pgp inhibitor verapamil; (v) the contribution of Pgp-mediated efflux system in determining the frog intestinal absorption of drugs was supported by the specific inhibition of cimetidine or nadolol transport in the presence of verapamil. These results indicate that pharmaceutically relevant drug transporters should be also expressed in frog intestine. In this work, an attempt was also made to compare the measured P(app) values in the frog intestinal model for the aforementioned series of actively/effluxed transported drugs in humans to the corresponding literature values for the fraction absorbed. The P(app) values used in these comparisons were obtained at high concentrations of drugs at which probably saturation of the carrier occurs. Interestingly, it was found that drugs that are completely absorbed had P(app) values >3 x 10(-6)cm/s, while drugs absorbed <90% had P(app) values lower than 1 x 10(-6)cm/s. In these cases, indeed, a borderline region characterized by the apparent permeability coefficient P(app) value between 1 x 10(-6) and 3 x 10(-6)cm/s should be considered for which the prediction of the absorbed fraction after oral administration in humans become more uncertain by the frog intestinal sac system.International Journal of Pharmaceutics 03/2008; 352(1-2):182-8. DOI:10.1016/j.ijpharm.2007.10.027 · 3.79 Impact Factor
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ABSTRACT: Permeability (P(e)) and solubility/dissolution are two major determinants of gastrointestinal (GI) drug absorption. Good prediction of these is crucial for predicting doses, exposures and potential interactions, and for selecting appropriate candidate drugs. The main objective was to evaluate screening methods for prediction of GI P(e), solubility/dissolution and fraction absorbed (f(a)) in humans. The most accurate P(e) models for prediction of f(a) of passively transported and highly soluble compounds appear to be the 2/4/A1 rat small intestinal cell model (in-vitro and in-silico), a newly developed artificial-membrane method, and a semi-empirical approach based on in-vitro membrane affinity to immobilized lipid bilayers, effective molecular weight and physiological GI variables. The predictability of in-vitro Caco-2, in-situ perfusion and other artificial membrane methods seems comparably low. The P(e) and f(a) in humans for compounds that undergo mainly active transport were predicted poorly by all models investigated. However, the rat in-situ perfusion model appears useful for prediction of active uptake potential (complete active uptake is generally well predicted), and Caco-2 cells are useful for studying bidirectional active transport, respectively. Human intestinal in-vitro P(e), which correlates well with f(a) for passively transported compounds, could possibly also have potential to improve/enable predictions of f(a) for actively transported substances. Molecular descriptor data could give an indication of the passive absorption potential. The 'maximum absorbable dose' and 'dose number' approaches, and solubility/dissolution data obtained in aqueous media, appear to underestimate in-vivo dissolution to a considerable extent. Predictions of in-vivo dissolution should preferably be done from in-vitro dissolution data obtained using either real or validated simulated GI fluids.Journal of Pharmacy and Pharmacology 08/2007; 59(7):905-16. DOI:10.1211/jpp.59.7.0001 · 2.16 Impact Factor
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ABSTRACT: Protein solubility ranges from low micrograms per ml to several hundreds of milligrams per ml, and is very compound-specific. Most antibodies are known to reach solubilities of hundreds of mg per ml whereas beta amyloid protein has very low solubility. Small structural changes could lead to significant changes in solubility, for example, cryo-immunoglobulins may be almost insoluble. The dose and route of administration may demand a higher concentration than possible in simple formulations, posing a challenge to the development of a clinically or commercially viable product. One important challenge is that proteins are typically administered via injections due to poor bioavailability by other delivery modes (See review articles in book edited by Audus and Raub, 1993; Pharmaceutical Business Review website, 2005), which restricts the types and levels of excipients (FDA website; Powell et al., 1998; Strickley, 1999, 2000). Further constraints are imparted by the small volume of administration appropriate for subcutaneous and intramuscular delivery routes which need to be consistent with patient compliance and ease of delivery. This can be very different from the volume/ concentration constraints of intravenous administration. For therapeutic doses in the mg/kg levels, the less than approximately 1.2 mL acceptable volume for subcutaneous delivery may necessitate formulations containing hundreds of mg/mL protein. Moreover, toxicological studies may assess approximately 10- fold higher doses than those planned for clinical studies in order to establish a safety window. This necessitates even higher concentrations for non-clinical formulations than for clinical formulations.08/2007: pages 341-357;