Validation and application of Caco-2 assays for the in vitro evaluation of development candidate drugs as substrates or inhibitors of P-glycoprotein to support regulatory submissions.

1. An understanding of the role that transporters, in particular P-glycoprotein (P-gp), can play in the absorption, distribution, metabolism and excretion (ADME) of candidate drugs, and an assessment of how these processes might impact on toxicity and the potential for drug-drug interactions in the clinic, is required to support drug development and registration. It is therefore necessary to validate preclinical assays for the in vitro evaluation of candidate drugs as substrates or inhibitors of human P-gp. 2. The present study has characterized a Caco-2 cell monolayer model by determining the bi-directional apparent permeabilities and efflux ratios of the known P-gp substrates ([(3)H]-digoxin, [(3)H]-ketoconazole, [(3)H]-verapamil, [(3)H]-quinidine, dipyridamole and loratidine; 1-100 microM) a non-substrate ([(3)H]-propranolol; 10 microM), or by determining the inhibitory potencies (IC(50)) of inhibitors (verapamil, ketoconazole, quinidine, dipyridamole and probenecid; 0.1-100 microM) on the basolateral-to-apical transport of [(3)H]-digoxin (5 microM), in order to validate methodologies for the identification of substrates or inhibitors of P-gp, respectively. 3. The reproducibility of the [(3)H]-digoxin or verapamil data determined from replicate monolayers across different cell passages indicates that the functional expression of P-gp is consistent across the range of passages (25-40) utilized for transport experiments and that the determination of bi-directional apparent permeability, or IC(50) for inhibition of P-gp, respectively, need only be performed on one occasion for a test compound. [(3)H]-digoxin and [(3)H]-propranolol or verapamil and probenecid were considered to be appropriate positive and negative controls of P-gp-mediated transport, or inhibition of P-gp, respectively, to ensure performance of the assays when assessing candidate drugs. Additionally, the low IC(50) values determined for ketoconazole and quinidine indicated that these inhibitors were suitable to use to confirm the role of P-gp in the efflux of a test compound. 4. These validated Caco-2 assays are robust, reproducible and suitable for routine in vitro evaluation of candidate drugs. They have been successfully applied to development projects resulting in the identification of two candidate drugs as substrates and inhibitors of P-gp, whereas a third was neither a substrate nor an inhibitor of this transporter.

Mechanisms of recovery from sublethal damage and potentially lethal damage induced by BrdUrd/313 nm light treatment: alkali-labile lesions.

BrdUrd pulse labelling of synchronous Chinese hamster cell cultures was used to correlate repair of sublethal damage with removal of alkali-labile lesions. Both processes were modified in a quantitatively similar manner by cysteamine. In addition, the age responses for repair of sublethal damage and for cysteamine reduction of repair agreed. Through the use of thymidine as an S-phase-blocking agent it was further demonstrated that progression past the S-phase of the cell cycle was required for the loss of resistance to UVB light in BrdUrd-substituted cells. Similarly, a thymidine block administered before synthesis upon the BrdUrd-substituted template prevented the cell from acquiring the sensitivity to UVB light normally associated with synthesis on a lesioned template. The UVB-light-sensitive mutant V79-UC was shown to have reduced capacities both for the accumulation of sublethal injury and for the removal of alkali-labile lesions. These data support the notion that alkali-labile lesions are responsible for sublethal damage in BrdUrd pulse-labelled Chinese hamster cells.