No abstract

The amyloid-␤ (A␤) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-␤ precursor protein (APP) through consecutive proteolytic cleavages by ␤-site APP-cleaving enzyme and ␥-secretase. Unexpectedly ␥-secretase inhibitors can increase the secretion of A␤ peptides under some circumstances. This "A␤ rise" phenomenon, the same inhibitor causing an increase in A␤ at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the A␤ rise depends on the ␤-secretase-derived C-terminal fragment of APP (␤CTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of A␤, known as "p3," formed by ␣-secretase cleavage, did not exhibit a rise. In addition to the A␤ rise, low ␤CTF or C99 expression decreased ␥-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, ␥-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of A␤ under conditions of low substrate expression. The A␤ rise was also observed in rat brain after dosing with the ␥-secretase inhibitor BMS-299897. The A␤ rise and potency shift are therefore relevant factors in the development of ␥-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the A␤ rise, including the "incomplete processing" and endocytic models, are discussed.Evidence suggests that the amyloid-␤ (A␤) 9 peptide plays a key role in Alzheimer disease. A␤ is generated by proteolytic processing of the amyloid-␤ precursor protein (APP) through consecutive cleavages by the ␤-site APP-cleaving enzyme (BACE) and ␥-secretase. APP is cleaved by BACE to form a ␤-secretase-derived C-terminal fragment of APP (␤CTF), which undergoes further cleavage by ␥-secretase to form A␤. In addition, APP is cleaved by ␣-secretase to form ␣CTF, which undergoes ␥-secretase cleavage to produce an N-terminally truncated form of A␤ known as "p3." Using the conventional amino acid numbering of A␤, BACE cleavage leads to A␤ peptides with N-terminal ends at positions 1 and 11, whereas ␣-secretase leads to p3 peptides with an N-terminal end at position 17. Cleavage of ␤CTF and ␣CTF by ␥-secretase produces a mixture of different C termini in the resulting A␤ and p3 peptides. For example, the predominant ␥-secretase cleavage of ␤CTFs at position 40 produces A␤-(1-40) and A␤-(11-40), whereas other ␥-secretase cleavage sites produce a range of less abundant A␤ peptides, such as the disease-associated A␤-(1-42) (1, 2).

Multiple endogenous compounds have been proposed as candidate biomarkers to monitor organic anion transporting polypeptide (OATP) function in preclinical species or humans. Previously, we demonstrated that coproporphyrins (CPs) I and III are appropriate clinical markers to evaluate OATP inhibition and recapitulate clinical drug-drug interactions (DDIs). In the present study, we investigated bile acids (BAs) dehydroepiandrosterone sulfate (DHEAS), hexadecanedioate (HDA), and tetradecanedioate (TDA) in plasma as endogenous probes for OATP inhibition and compared these candidate probes to CPs. All probes were determined in samples from a single study that examined their behavior and their association with rosuvastatin (RSV) pharmacokinetics after administration of an OATP inhibitor rifampin (RIF) in healthy subjects. Among endogenous probes examined, RIF significantly increased maximum plasma concentration () and area under the concentration-time curve (AUC) of fatty acids HDA and TDA by 2.2- to 3.2-fold. For the 13 bile acids in plasma examined, no statistically significant changes were detected between treatments. Changes in plasma DHEAS did not correlate with OATP1B inhibition by RIF. On the basis of the magnitude of effects for the endogenous compounds that demonstrated significant changes from baseline over interindividual variations, the overall rank order for the AUC change was found to be CP I > CP III > HDA ≈ TDA ≈ RSV > > BAs. Collectively, these results reconfirmed that CPs are novel biomarkers suitable for clinical use. In addition, HDA and TDA are useful for OATP functional assessment. Since these endogenous markers can be monitored in conjunction with pharmacokinetics analysis, the CPs and fatty acid dicarboxylates, either alone or in combination, offer promise of earlier diagnosis and risk stratification for OATP-mediated DDIs.