Correlation between oral drug absorption in humans, and apparent drug permeability in TC-7 cells, a human epithelial intestinal cell line: comparison with the parental Caco-2 cell line.

PURPOSE: To determine and compare the relationship between in vivo oral absorption in humans and the apparent permeability coefficients (Papp) obtained in vitro on two human intestinal epithelial cell lines, the parental Caco-2 and the TC-7 clone. METHODS: Both cell lines were grown for 5-35 days on tissue culture-treated inserts. Cell monolayers were analysed for their morphology by transmission electron micrography, and for their integrity with respect to transepithelial electrical resistance, mannitol and PEG-4000 transport, and cyclosporin efflux. Papp were determined for 20 compounds exhibiting large differences in chemical structure, molecular weight, transport mechanisms, and percentage of absorption in humans. RESULTS: The TC-7 clone exhibits morphological characteristics similar to those of the parental Caco-2 cell line, concerning apical brush border, microvilli, tight junctions and polarisation of the cell line. The TC-7 clone however appeared more homogenous in terms of cell size. Both cell lines achieved a similar monolayer integrity towards mannitol and PEG-4000. Monolayer integrity was achieved earlier for the TC-7 clone, mainly due to its shorter doubling time, i.e. 26 versus 30 hours for parental Caco-2 cells. When using cyclosporin A as a P-glycoprotein substrate, active efflux was lower in the TC-7 clone than in the parental Caco-2 cells. The Papp and mechanisms of transport (paracellular or transcellular routes, passive diffusion and active transport) were determined for 20 drugs. A relationship was established between the in vivo oral absorption in humans and Papp values, allowing to determine a threshold value for Papp of 2 10(-6) cm/sec, above for which a 100% oral absorption could be expected in humans. Both correlation curves obtained with the two cell types, were almost completely superimposable. These studies also confirmed that the dipeptide transporter is underexpressed in both cell lines. CONCLUSIONS: On the basis of morphological parameters, biochemical activity and drug transport characteristics, the TC-7 clone appeared to be a valuable alternative to the use of parental Caco-2 cells for drug absorption studies.

Development of a two-site immunoassay of recombinant urate oxidase (SR 29142) and its use for determination of pharmacokinetic parameters in rats and baboons.

Two monoclonal antibodies (Mabs), 12C7 and 11G11, both directed against recombinant urate oxidase (SR 29142), were selected for their epitope specificity to develop a two-site immunoassay of urate oxidase in plasma. A quantitative recovery of urate oxidase in plasma was obtained at all the concentrations tested, and the limit of quantification was found to be 0.5 ng/mL. Intra-and interassay coefficients of variation ranged from 1.2 to 6.7% and from 3.5 to 10.8%, respectively. The specificity of the two antibodies was studied in Western-blot experiments. This assay was used successfully to determine urate oxidase pharmacokinetic parameters after intravenous injection in rats and baboons. In these two species, urate oxidase pharmacokinetics was characterized by a low clearance and a low volume of distribution without gender difference.

Importance of the paracellular pathway for the transport of a new bisphosphonate using the human CACO-2 monolayers model.

The transport of a new bisphosphonate, Tiludronate, was investigated on the human adenocarcinoma cell line, CACO-2. Experiments were performed 7-16 days after cells achieved confluence, conditions under which they form well-differentiated monolayers joined by tight junctions. Tiludronate transport rate across CACO-2 monolayers was independent of the temperature (4 degrees versus 37 degrees), of the polarity of the cell membrane (apical-to-basolateral versus basolateral-to-apical), and of the presence of metabolic poisons (sodium azide). Its transport was enhanced by either the presence of EGTA in the incubation buffer, i.e. when extracellular Ca2+ concentration was reduced, or by the pretreatment of monolayers with EGTA, i.e. when the intercellular spaces and the tight junctions were widened. Based on these different observations, we could suggest that Tiludronate mainly used the paracellular pathway to cross the intestinal epithelium. An increase in the Tiludronate permeability coefficient was also observed following treatment of cells with high Tiludronate concentrations, as a consequence of the direct effect of this compound on the extracellular Ca2+ ions. Hence, for high drug concentrations, i.e. 20 mM, we observed a decrease in free extracellular Ca2+ concentration, an increase in the transepithelial electrical resistance and an increase in the transport of [14C]polyethyleneglycol ([14C]PEG400), a probe for the paracellular pathway. The results indicate that Tiludronate is transported across CACO-2 monolayers by the paracellular route. Moreover, it can affect its own transport by its concentration-dependent effect on tight junction widening.

Regulation of cytochrome P450IA1 gene expression in a human intestinal cell line, Caco-2.

The expression and inducibility of cytochrome P450IA1 isozyme was investigated in the human carcinoma cell line Caco-2 cultured between days 7 and 35 in the absence or the presence of various enzyme inducers such as 3-methylcholanthrene, beta-naphthoflavone (beta NF), dioxin, isosafrole, rifampycin, dexamethasone or phenobarbital. 7-Ethoxyresorufin O-deethylase activity (EROD) was maximal at day 25 when the differentiation of Caco-2 cells, characterized by the level of the brush border associated enzymes such as sucrase isomaltase and alkaline phosphatase, was higher. The inducibility of this enzyme activity was found to be maximal when cells were treated between days 7 and 10. After a 3-day treatment of Caco-2 cells with 50 microM beta NF, EROD achieved 36.6 +/- 14.6 pmol/min/mg compared to 2.5 +/- 1.1 pmol/min/mg in untreated cells. This enzyme activity appeared to be supported only by P450IA1 isozyme because: 1) EROD was quantitatively inhibited by alpha-naphthoflavone, a P450IA1-specific inhibitor; otherwise, phenacetin O-deethylation was completely abolished in the presence of alpha-naphthoflavone and not by furafylline, a P450IA2-specific inhibitor; 2) EROD was induced after treatment with 3-methylcholanthrene, beta NF and dioxin, which are P450IA1 inducers, but not by isosafrole, a P450IA2-specific inducer; 3) cytochrome P450IA1 apoprotein could be immunodetected by antibodies directed against rabbit cytochrome P450-LM6, orthologous to P450IA1, in polycyclic hydrocarbon-treated cells; 4) under the latter conditions, P450IA1 mRNA accumulation was specifically detected, but not P450IA2 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papio papio) liver microsomes. Cross reactivity with human forms.

Two cytochrome P450 isozymes, FA and FI, were isolated and characterized from liver microsomes of phenobarbital-induced baboons (Papio papio). The cytochrome FA possesses the same N-terminal amino acid sequence as P450 MK2 from crab-eating monkeys (Macaca irus) and closely resembles the human P450 3A isozymes. This cytochrome was able to oxidize nifedipine and hydroxylate testosterone at the 6 beta position. The second baboon cytochrome (FI) is closely related to the P450 2A subfamily and has the same N-terminal sequence as human P450 2A7. Like human P450 2A forms, it is highly active as a coumarin 7-hydroxylase. Antibodies against P450 FA and FI cross-react with two human liver proteins of 51 kDa and 49 kDa, respectively. The concentration of the first protein in the human samples, was five-times greater than the second. However, the latter showed marked interindividual variation. In primary cultures of human hepatocytes, rifampicin is a strong inducer of the 51-kDa protein and a moderate inducer of the 49-kDa protein, while phenobarbital has the opposite effect on the two proteins.