Effect of Structural Modification on Absorption, Metabolism and Pharmacokinetics of alpha-Aminoxy Peptides

A series of novel α-aminoxy peptides have been recently developed, and showed a therapeutic potential for the treatment of CI- channel dysfunctional diseases. The present study aimed to investigate the pharmacokinetics of five structural related tx-aminoxy peptides (P1 to P5), and effect of structural modifications on their pharmacokinetic properties.;Three absorption models, including Caco-2 cell monolayer, Ussing chamber and in situ rat single-pass intestinal perfusion (SPIP) model, were used. The stability in gastrointestinal (GI) tract was determined using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The hepatic metabolism was investigated in rat liver subcellular fractions and human liver microsome. P1 and P5 were selected for pharmacokinetic study in rats.;P1 showed significantly low intestinal permeability due to P-gp-mediated efflux. Structural modifications resulted in alterations of transport mechanisms from P-gp-mediation (P1, P2) to multidrug resistance-associated protein (MRP)-mediation (P3), MRP plus breast cancer resistance protein (BCRP)-mediation (P4) active transport, or even passive paracellular diffusion (P5) without any efflux transporters involved. Comparing with P1, the absorbable permeability in Caco-2 monolayer increased to about 7-fold (P3), 4-fold (P4) and 11-fold (P5), respectively, and the absorption through intestine in SPIP model significantly increased to about 36-fold (P2), 42-fold (P3), 55-fold (P4) and 102-fold (P5), respectively. P1 was unstable in the GI tract with 41% degradation in SGF within 1 h and 47% degradation in SIF within 3 h. The other four peptides were much more stable than P1 with degradation less than 6% under the same incubated conditions. For the hepatic metabolism, about 31% of P1 was metabolized by rat liver S9 within 30 min, while the metabolic stability was significantly improved to 3.3-fold (P3), 2.9-fold (P4) and 7.5-fold (P5), respectively with no metabolism for P2. Their metabolism was mainly via oxidation catalyzed by CYP enzymes to form hydroxylated metabolites. After i.v. administration (5 mg/kg), both P1 and P5 was eliminated rapidly. P1 mainly distributed to liver and lung, while P5 to kidney and intestine. P1 was cleared mainly through metabolism via oxidation followed by sulphation (∼80% of the dose), while P5 was mainly eliminated as an intact form (∼53% of the dose). Oral bioavailability of P1 was low (0.36%) due to instability in the GI tract and poor intestinal absorption mediated by P-gp efflux transport. Oral bioavailability of P5 was improved to about 3-fold comparing with P1 but still low mainly because of poor intestinal absorption through passive diffusion and some unknown factor(s).;The present studies demonstrated that our rationale for the structural modifications of the designed cc-aminoxy peptides is an effective way to improve their intestinal absorption, gastrointestinal and metabolic stability, and appropriate pharmacokinetic properties. Our findings also provide scientific evidence to support further development of better α-aminoxy peptide cadidates with high oral bioavailability and appropriate pharmacokinetic properties.