Page - (000320) - in Biomedical Chemistry: Current Trends and Developments

was thus observed for the α-carbon methylated analogs. When the cis 3-phenylproline derivative was incorporated into the N- terminal part of H-Phe-Phe-NH2 replacing phenylalanine (38 and 39), the binding affinity decreased 4 and 6 times, respectively. A possible reason for this loss in affinity is that these compounds may have problems adopting an optimal binding conformation because of the introduced rigidification provided by the proline analogue. Replacement of the C-terminal phenylalanine by the cis 3-phenylproline moiety in the S,S,S configuration (40) resulted in a more potent ligand than H-Phe- Phe-NH2. This rigidification also increased the half-life by 7-fold (cf. 26 and 40). β-Methylation of the C-terminal phenylalanine gave compounds 42 and 43, with lower binding affinity than H-Phe-Phe-NH2. When comparing the metabolic stability in all the diastereomeric pairs (38 vs. 39, 40 vs. 41, and 42 vs. 43), the natural S,S,S configuration was more easily metabolized. Incorporation of d-amino acids is a known strategy to improve metabolic stabilization (Humphrey, 1986; Veber, 1985). The physiochemical properties of all the synthesized compounds were further explored by evaluation of their intestinal epithelial permeability. This was determined from transport rates across a Caco-2 cell monolayer, and is expressed as the apparent permeability coefficient (Papp) (Hubatsch, 2007). A good relationship between the permeability across the Caco-2 monolayer and the extent of absorption in vivo has been reported (Stewart, 1995). Each compound was investigated in the apical to basolateral (a–b) and basolateral to apical (b–a) direction. One of several efflux transporters present in the Caco-2 cells is PgP. Measurement of the efflux (the ba/ab ratio) can thus indicate whether or not the compounds are substrates for PgP. Since uptake transporters can enhance absorption, the possibility of the peptides being actively transported was studied in Chinese hamster ovary (CHO) cells stably transfected with the PepT1 transporter (CHO)- PepT1, using CHO-K1 cells as the control. The results are expressed as pmol mg-1 of protein/min. For peptides being actively transported the PeptT1/K1 ratio should be greater than one. Uptake and permeability are