SURPRISINGLY HIGH RESISTANCE OF Aβ OLIGOMER ELIMINATING ALL-D-ENANTIOMERIC PEPTIDES AGAINST METABOLIZATION BY ENZYMES CONTAINED IN THE GASTROINTESTINAL TRACT, BLOOD, LIVER AND BRAIN

Abstract

Previously, we have developed an Aβ oligomer eliminating compound consisting solely of D-enantiomeric amino acid residues. Based on this lead compound we developed the derivative Pri-002 that is currently in clinical phase I. Both D-peptides improved cognition in various transgenic Alzheimer's disease mouse models even after oral administration and have an oral bioavailability of more than 50%. To further investigate the underlying properties and to predict the safety for administration in humans, we examined in vitro the resistance of the lead compound and Pri-002 against metabolization in media simulating the human gastrointestinal tract, blood and liver in comparison to their corresponding L-enantiomeric mirror images. It was also tested if Pri-002 acts as substrate for the human D-amino acid oxidase (hDAAO) mainly contained in the kidney, liver and brain. We additionally examined, if Pri-002 has any inhibitory effects on the enzymes contained in these experiments. For the stability and inhibition tests in simulated gastric and intestinal fluid, human plasma and human liver microsomes, the peptides were incubated in these media and potential metabolization was followed by RP-HPLC. Additional inhibition assays regarding the main human CYP isoforms contained in liver microsomes were performed by monitoring isoform-specific probe substrate metabolites with LC-MS/MS or fluorescence. The hDAAO substrate and inhibition tests with Pri-002 were performed using a coupled enzyme assay. The D-peptides showed high stability in all investigated media in comparison to their L-enantiomeric mirror images. To summarize only the most surprising results, the D-peptides remained stable in simulated intestinal fluid for 24 hours, while the L-peptides were completely metabolized within seconds. Indeed, human plasma and human liver microsomes metabolized the L-peptides several hundred times faster than the D-peptides. Pri-002 did neither act as substrate for hDAAO nor did it have any inhibitory effects on any of the investigated enzymes. This surprisingly high stability allows oral administration of the drug candidate and may also explain the absence of adverse side effects even at high doses due to low levels of potential biologically active metabolites during treatment. In conclusion, all-D-enantiomeric peptides represent a promising new class of drug candidate compounds, especially for oral administration.