TY - JOUR
T1 - Enhancing Oral Bioavailability of Cyclic RGD Hexa-peptides by the Lipophilic Prodrug Charge Masking Approach
T2 - Redirection of Peptide Intestinal Permeability from a Paracellular to Transcellular Pathway
AU - Schumacher-Klinger, Adi
AU - Fanous, Joseph
AU - Merzbach, Shira
AU - Weinmüller, Michael
AU - Reichart, Florian
AU - Räder, Andreas F.B.
AU - Gitlin-Domagalska, Agata
AU - Gilon, Chaim
AU - Kessler, Horst
AU - Hoffman, Amnon
N1 - Publisher Copyright:
Copyright © 2018 American Chemical Society.
PY - 2018/8/6
Y1 - 2018/8/6
N2 - Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junctions (paracellular pathway) in the intestine. This permeability mechanism restricts the magnitude of their oral absorption and bioavailability. We hypothesized that concealing the hydrophilic residues of the peptide using the lipophilic prodrug charge masking approach (LPCM) can improve the bioavailability of hydrophilic peptides. To test this hypothesis, a cyclic N-methylated hexapeptide containing Arg-Gly-Asp (RGD) and its prodrug derivatives, masking the Arg and Asp charged side chains, were synthesized. The library was evaluated for intestinal permeability in vitro using the Caco-2 model. Further investigation of metabolic stability ex vivo models in rat plasma, brush border membrane vesicles (BBMVs), and isolated CYP3A4 microsomes and pharmacokinetic studies was performed on a selected peptide and its prodrug (peptide 12). The parent drug analogues were found to have a low permeability rate in vitro, corresponding to atenolol, a marker for paracellular permeability. Moreover, palmitoyl carnitine increased the P app of peptide 12 by 4-fold, indicating paracellular permeability. The P app of the prodrug derivatives was much higher than that of their parent peptides. For instance, the P app of the prodrug 12P was 20-fold higher than the P app of peptide 12 in the apical to basolateral (AB) direction. Whereas the permeability in the opposite direction (BA of the Caco-2 model) was significantly faster than the P app AB, indicating the involvement of an efflux system. These results were corroborated when verapamil, a P-gp inhibitor, was added to the Caco-2 model and increased the P app AB of prodrug 12P by 3-fold. The prodrug 12P was stable in the BBMVs environment, yet degraded quickly (less than 5 min) in the plasma into the parent peptide 12. Pharmacokinetic studies in rats showed an increase in the bioavailability of peptide 12 > 70-fold (from 0.58 ± 0.11% to 43.8 ± 14.9%) after applying the LPCM method to peptide 12 and converting it to the prodrug 12P. To conclude, the LPCM approach converted the absorption mechanism of the polar peptides from a paracellular to transcellular pathway that tremendously affects their oral bioavailability. The LPCM method provides a solution for the poor bioavailability of RGD cyclohexapeptides and paves the way for other active hydrophilic and charged peptides with poor oral bioavailability.
AB - Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junctions (paracellular pathway) in the intestine. This permeability mechanism restricts the magnitude of their oral absorption and bioavailability. We hypothesized that concealing the hydrophilic residues of the peptide using the lipophilic prodrug charge masking approach (LPCM) can improve the bioavailability of hydrophilic peptides. To test this hypothesis, a cyclic N-methylated hexapeptide containing Arg-Gly-Asp (RGD) and its prodrug derivatives, masking the Arg and Asp charged side chains, were synthesized. The library was evaluated for intestinal permeability in vitro using the Caco-2 model. Further investigation of metabolic stability ex vivo models in rat plasma, brush border membrane vesicles (BBMVs), and isolated CYP3A4 microsomes and pharmacokinetic studies was performed on a selected peptide and its prodrug (peptide 12). The parent drug analogues were found to have a low permeability rate in vitro, corresponding to atenolol, a marker for paracellular permeability. Moreover, palmitoyl carnitine increased the P app of peptide 12 by 4-fold, indicating paracellular permeability. The P app of the prodrug derivatives was much higher than that of their parent peptides. For instance, the P app of the prodrug 12P was 20-fold higher than the P app of peptide 12 in the apical to basolateral (AB) direction. Whereas the permeability in the opposite direction (BA of the Caco-2 model) was significantly faster than the P app AB, indicating the involvement of an efflux system. These results were corroborated when verapamil, a P-gp inhibitor, was added to the Caco-2 model and increased the P app AB of prodrug 12P by 3-fold. The prodrug 12P was stable in the BBMVs environment, yet degraded quickly (less than 5 min) in the plasma into the parent peptide 12. Pharmacokinetic studies in rats showed an increase in the bioavailability of peptide 12 > 70-fold (from 0.58 ± 0.11% to 43.8 ± 14.9%) after applying the LPCM method to peptide 12 and converting it to the prodrug 12P. To conclude, the LPCM approach converted the absorption mechanism of the polar peptides from a paracellular to transcellular pathway that tremendously affects their oral bioavailability. The LPCM method provides a solution for the poor bioavailability of RGD cyclohexapeptides and paves the way for other active hydrophilic and charged peptides with poor oral bioavailability.
KW - RGD integrin inhibitor
KW - enterocytes
KW - intestinal permeability
KW - metabolic stability
KW - oral bioavailability
KW - peptides
KW - pharmacokinetics
KW - prodrug
UR - http://www.scopus.com/inward/record.url?scp=85049747568&partnerID=8YFLogxK
U2 - 10.1021/acs.molpharmaceut.8b00466
DO - 10.1021/acs.molpharmaceut.8b00466
M3 - Article
C2 - 29976060
AN - SCOPUS:85049747568
SN - 1543-8384
VL - 15
SP - 3468
EP - 3477
JO - Molecular Pharmaceutics
JF - Molecular Pharmaceutics
IS - 8
ER -