Strategies for absorption screening in drug discovery and development.

This review gives an overview of the current approaches to evaluate drug absorption potential in the different phases of drug discovery and development. Methods discussed include in silico models, artificial membranes as absorption models, in vitro models such as the Ussing chamber and Caco-2 monolayers, in situ rat intestinal perfusion and in vivo absorption studies. In silico models such as iDEA can help optimizing chemical synthesis since the fraction absorbed (Fa) can be predicted based on structural characteristics only. A more accurate prediction of Fa can be obtained by feeding the iDEA model with Caco-2 permeability data and solubility data at various pH's. Permeability experiments with artificial membranes such as the filter-IAM technology are high-throughput and offer the possibility to group compounds according to a low and a high permeability. Highly permeable compounds, however, need to be further evaluated in Caco-2 cells, since artificial membranes lack active transport systems and efflux mechanisms such as P-glycoprotein (PgP). Caco-2 and other "intestinal-like" cell lines (MDCK, TC-7, HT29-MTX, 2/4/A1) permit to perform mechanistic studies and identify drug-drug interactions at the level of PgP. The everted sac and Ussing chamber techniques are more advanced models in the sense that they can provide additional information with respect to intestinal metabolism. In situ rat intestinal perfusion is a reliable technique to investigate drug absorption potential in combination with intestinal metabolism, however, it is time consuming, and therefore not suited for screening purposes. Finally, in vivo absorption in animals can be estimated from bioavailability studies (ratio of the plasma AUC after oral and i.v. administration). The role of the liver in affecting bioavailability can be evaluated by portal vein sampling experiments in dogs.

An ecto-nucleotide pyrophosphatase is one of the main enzymes involved in the extracellular metabolism of ATP in rat C6 glioma.

The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the alpha- and gamma-phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for approximately 50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 microM abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a Km for ATP of 17 +/- 5 microM. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.

Cyclic AMP-mediated induction of the glial fibrillary acidic protein is independent of protein kinase A activation in rat C6 glioma.

N6-O'2-dibutyryl cAMP (dbcAMP), N6-monobutyryl cAMP (N6-mbcAMP), 8-Chloro cAMP (ClcAMP), and O'2-monobutyryl cAMP (O'2-mbcAMP) were used to study glial fibrillary acidic protein (GFAP) induction in rat C6 glioma. With the exception of O'2-mbcAMP, these cAMP analogs induced GFAP after stimulation of cells with a concentration of 0.5-1 mM. Only dbcAMP and N6-mbcAMP increased the intracellular concentration of cAMP. Protein kinase A (PKA) activation is often proposed to be involved in GFAP expression in astrocytes. Ion-exchange chromatography indicated that protein kinase activity is associated with PKA type II in C6. dbcAMP, N6-mbcAMP, and ClcAMP upregulated the amount of cAMP-binding proteins approximately twofold. RI was upregulated in the cytosol and particulate fraction, whereas RII was not affected after stimulation with dbcAMP. Concomitant, the PKA activity decreased approximately 60% and 40% in the cytosol and particulate fraction, respectively. CREB is constitutively expressed in C6 and is downregulated after stimulation with dbcAMP. The membrane-permeable PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89) did not suppress the induction of GFAP-mRNA and its translation into GFAP. On the contrary, depending on the time difference between H89 and dbcAMP addition to C6, GFAP synthesis could even be potentiated more than twofold. Experiments in the presence of cycloheximide showed that protein synthesis is necessary for GFAP transcription. Although all components of the PKA signal transduction pathway are present in C6, GFAP synthesis is not dependent on PKA activation but required the synthesis of an unidentified factor.

Inhibition of nucleoside diphosphate kinase (NDPK/nm23) by cAMP analogues.

Nucleoside diphosphate kinase (NDPK/nm23) ATP/GDP phosphotransferase activity and serine autophosphorylation is inhibited by N6-mbcAMP, 8-ClcAMP and 8-BrcAMP. Inhibition of the enzymatic activity largely depends on the concentration of ATP and becomes significant at ATP concentrations up to 0.5 mM and at effector concentrations measured in C6 cells stimulated with 1 mM cAMP analogue. N6-mbcAMP is a substrate of the enzyme. DbcAMP and 0'2-mbcAMP, cAMP analogues with a modified 0'2-ribose, did not affect the NDPK activity. Cyclic AMP is only a moderate inhibitor of NDPK even at low ATP concentrations. Possible inhibitory effects of cAMP and cAMP analogues on reported extra- and intracellular functions of NDPK/nm23 are discussed.