Effects of Amino Acid Chirality and the Chemical Linker on the Cell Permeation Characteristics of Cyclic Prodrugs of Opioid Peptides

Abstract

Previously, our laboratory showed that the oxymethyl-modified coumarinic acid (OMCA) cyclic prodrug of the opioid peptide DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) exhibited low permeation across both the intestinal mucosa and the blood-brain barrier (BBB). This low cell permeation arose from its strong substrate activity for efflux transporters in these biological barriers. In an attempt to determine whether the chirality of the amino acid asymmetric centers could influence the solution structure of the cyclic prodrugs and thus their substrate activities for efflux transporters, we synthesized cyclic prodrugs of the opioid peptides H-Tyr-Ala-Gly-Phe-D-Leu-OH ([Ala2,D-Leu5]-Enk), H-Tyr-D-Ala-Gly-Phe-Leu-OH ([D-Ala2,Leu5]-Enk), and H-Tyr-Ala-Gly-Phe-Leu-OH ([Ala2,Leu5]-Enk). In an attempt to determine whether the chemical linker (OMCA) bestowed efflux substrate activity on the cyclic prodrugs, we synthesized capped linear derivatives (acetylated on the N-terminal and amidated on the C-terminal end) of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk. The solution conformations of the cyclic prodrugs were determined by molecular dynamics simulations using two-dimensional NMR data. The physicochemical properties (molecular surface area, polar surface area, and cLogP) were estimated computationally using Sybyl. Cell permeation characteristics were assessed using Caco-2 cells in the presence and absence of known inhibitors of efflux transporters. Despite apparent differences in their solution conformations and their physicochemical properties, the cyclic prodrugs of DADLE, [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk all exhibited strong substrate activity for efflux transporters in Caco-2 cells. In contrast, the capped linear derivatives of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk exhibited very poor substrate activity for efflux transporters in Caco-2 cells. Therefore, the substrate activities of the cyclic prodrugs for efflux transporters in Caco-2 cells and in the intestinal mucosa and the BBB in vivo are most likely due to the chemical linker used to prepare these molecules and/or its effect on solution structures of the prodrugs.

Cyclization-activated Prodrugs

“…Intramolecular cyclization has been demonstrated to improve biological properties of bioactive peptides [28][29][30][31][32][33][34][35][36][37][38], in many cases allowing one to improve selectivity for a given receptor and/or metabolic stability [39][40][41]. Cyclic peptides can be divided into homodetic and heterodetic, the first being obtained by formation of an intramolecular peptide (amide) bond, whereas heterodetic refers to all cyclic peptides where the intramolecular bond newly formed is other than an amide (e.g., lactone, ether, thioether and, most commonly, disulphide bridges).…”

Cyclic Opioid Peptides

“…[Ala 2 ,DLeu 5 ]-ENK, [DAla 2 ,Leu 5 ]-ENK, and [Ala 2 ,Leu 5 ]-ENK prodrugs were cyclized from the N- and C-termini using oxymethyl coumarinic acid (OMCA) as a linker to determine the impact of Ala 2 and Leu 5 chirality on peptide conformation, physiochemical properties, and cell penetration [104]. These cyclic analogues interacted with efflux transporters unlike a series of linear variants which did not contain OMCA, but were instead N-acetylated and C-amidated.…”

Significant differences in the disposition of cyclic prodrugs of opioid peptides in rats and guinea pigs following IV administration

“…The cyclic prodrugs OMCA‐DADLE (>95%) and OMCA‐[ D ‐Ala 2 ,Leu 5 ]‐Enk (>90%), their internal standard CA‐[Leu 5 ]‐Enk (coumarinic acid‐based cyclic prodrug of [Leu 5 ]‐enkephalin), and the capped peptide Ac‐DADLE‐NH 2 were synthesized in our laboratory following procedures reported elsewhere 13,22,24. DADLE, its internal standard [Leu 5 ]‐Enk, diethyl p ‐nitrophenyl phosphate (paraoxon, approx.…”

“…In an attempt to overcome these problems, our laboratory recently explored how modifications in the chirality of specific amino acid residues (Ala 2 and Leu 5 ) in the peptide portion of oxymethyl‐modified coumarinic acid (OMCA)‐based cyclic prodrugs of the opioid peptide [Ala 2 ,Leu 5 ]‐Enk (H‐Tyr‐Ala‐Gly‐Phe‐Leu‐OH) affected their membrane permeabilities and their bioconversion by esterases 22,23. Although we have observed differences in the solution structures of these cyclic prodrugs and their calculated physicochemical properties (e.g., polar surface area), no significant differences in the transport characteristics of these cyclic prodrugs were observed.…”