Gt. Knipp et al., THE EFFECT OF BETA-TURN STRUCTURE ON THE PASSIVE DIFFUSION OF PEPTIDES ACROSS CACO-2 CELL MONOLAYERS, Pharmaceutical research, 14(10), 1997, pp. 1332-1340
Purpose. To investigate the relationships between the beta-turn struct
ure of a peptide and its passive diffusion across Caco-2 cell monolaye
rs, an in vitro model of the intestinal mucosa. Methods. Linear hydrop
hilic peptides (Ac-TyrProXaaZaaVal-NH2; Xaa = Gly, lie and Zaa = Asp,
Asn) and hydrophobic (Ac-Yaa-ProXaaIleVal-NH2; Yaa = Tyr, Phe and Xaa
= Gly, lie: and Ac-PheProXaane-NH2; Xaa = Gly, lie) peptides were synt
hesized and their effective permeability coefficients (P-eff) were det
ermined across Caco-2 cell monolayers. The lipophilicities of the pept
ides were estimated by measuring their partition coefficients (P-o/w b
etween 1-octanol and HBSS. Two-dimensional NMR (2D-NMR) spectroscopy a
nd circular dichroism (CD) spectroscopy was used to determine the solu
tion structures of these model peptides. Results. Using 2D-NMR spectro
scopy and CD spectroscopy, the hydrophilic Gly-containing peptides (Ac
-TyrProGlyZaaVal-NH2; Zaa = Asp, Asn) were shown to exhibit a higher d
egree of beta-turn structure in solution than the Ile-containing pepti
des (Ac-TyrProIleZaaVal-NH2; Zaa = Asp, Asn). CD spectroscopy was used
to show that the Gly-containing hydrophobic peptides (Ac-YaaProGlyIle
Val-NH2; Yaa = Tyr; Phe: and Ac-PheProGlyIle-NH2) exhibited a higher d
egree of beta-turn structure in solution than the lie-containing hydro
phobic peptides. The P-eff values of all four hydrophilic peptides acr
oss unperturbed Caco-2 cell monolayers were very low and no statistica
lly significant differences were observed between the Gly-and lie-cont
aining pentapeptides within either the Asp or Asn series. The P-eff va
lues for the hydrophobic Gly-containing peptides were significantly gr
eater than the P-eff values determined for their Lie-containing counte
rparts. The Gly-containing penta- and tetrapeptides in the Phe series,
which exhibited high permeation, were shown to be metabolically unsta
ble. In contrast, the Gly-and lie-containing pentapeptides in the Tyr
series and the lie-containing penta-and tetrapeptides in the Phe serie
s, which exhibited low permeation, were metabolically stable. Conclusi
ons. Hydrophobic peptides that exhibit significant beta-turn structure
in solution are more lipophilic as measured by log P-o/w and more rea
dily permeate Caco-2 cell monolayers via the transcellular route than
hydrophobic peptides that lack this type of solution structure. The ab
ility of these peptides to permeate Caco-2 cell monolayers via the tra
nscellular route also exposed them to metabolism, presumably by cytoso
lic endopeptidases. Similar secondary structural features in hydrophil
ic peptides do not appear to sufficiently alter the physicochemical pr
operties of the peptides so as to alter their paracellular flux throug
h unperturbed Caco-2 cell monolayers.