Effect of guanidino modification and proline substitution on the in vitro stability and blood–brain barrier permeability of endomorphin II

doi:10.1002/jps.10202

Journal of Pharmaceutical Sciences

Volume 91, Issue 10, October 2002, Pages 2140-2149

https://doi.org/10.1002/jps.10202 Get rights and content

Abstract

Endomorphin II (ENDII), an endogenous ligand for the μ‐opioid receptor, was investigated as a possible analgesic with fewer side effects than morphine. To improve CNS entry of END II, structural modification was also examined to determine whether Pro⁴ substitution and cationization affected physico‐chemical characteristics, blood–brain barrier (BBB) transport, and analgesic profile. END II and its Pro⁴‐substituted analog, Morphiceptin (MOR), were cationized by guanidino (GU)‐addition. MOR was seven times less lipophilic than END II, whereas GU‐addition decreased lipophilicity of both peptides. MOR did not affect in vitro BBB permeability; however, GU‐addition increased permeability of MOR by 31%. MOR decreased protein binding by 23% compared to END II, whereas GU‐addition increased protein binding of both peptides by 71 and 113%, respectively. MOR increased brain t_1/2 compared to END II. GU‐addition significantly increased t_1/2 of MOR and END II in both brain (sixfold and 10‐fold, respectively) and serum (over 10‐fold). Pro⁴‐substitution and GU‐addition enhanced the in vivo analgesia profiles of i.v. administered END II and MOR, but decreased i.c.v. analgesia profiles. This study demonstrates Pro⁴‐substitution decreases protein binding and enhances brain stability while cationization enhances both brain and serum stability with variable effects on BBB permeability. The analgesic profiles show that both Pro⁴‐substitution and cationization enhance i.v. analgesia and thus, are promising structural modifications for the development of successful opioid drugs. © 2002 Wiley‐Liss Inc. and the American Pharmaceutical Association J Pharm Sci 91:2140–2149, 2002

Section snippets

INTRODUCTION

Morphine and most clinically useful opioids bind to the μ‐opioid receptor where they elicit potent analgesia.¹,² However these compounds, while effective as analgesics, are limited by numerous opioid‐related side effects, including respiratory depression, constipation, and dependence.³ Recently, endomorphins have been suggested to be an endogenous ligand for the μ‐opioid receptor due to their central nervous system (CNS) distribution, and high specificity and affinity for the μ‐opioid receptor.⁴

Radioisotopes/Reagents

Na¹²⁵I (2175 Ci/mmol) and [¹⁴C] sucrose 462 mCi/mmol were purchased from Dupont NEN Research Products (Boston, MA) and ICN Pharmaceutics, Inc. (Irvine, CA), respectively. All other chemicals, unless otherwise stated, were purchased from Sigma Chemical Corp. (St. Louis, MO).

Peptide Synthesis

Peptides were synthesized by an “in solution, step‐by‐step” method, starting with proline or phenylalanine amides, respectively. To C‐terminal fragments respective Boc‐amino acids were coupled using dicyclohexylcarbodiimide

Octanol/Buffer Distribution

Lipophilicity of the iodinated peptides was expressed as octanol/buffer distribution coefficients (D) (Table 2). END II partitioned into the octanol phase significantly (p < 0.01) more than MOR. Addition of GU to END II and MOR significantly decreased the D, indicating a decrease in lipophilicity.

BBMEC Uptake

In vitro analysis of uptake for the iodinated peptides was determined by a unidirectional rate constant, K_cell (Table 2). No significant difference was shown between any of the test compounds for K_cell

DISCUSSION

A major factor limiting the delivery of peptide drugs to the CNS is the blood–brain barrier. In this study, we investigated Pro⁴ substitution and cationization of END II as a method to improve BBB transport and peptide stability. Octanol/buffer coefficient studies assess the lipophilicity of a compound and are an important factor when studying endothelial cell permeability.³² Banks and Kastin³³ showed in a study of 18 peptides that octanol/buffer coefficients were the best predictor of membrane

Acknowledgements

This work was supported by NIH Grants DA06037, DA11271, and NS 39592.

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Research ArticlesEffect of guanidino modification and proline substitution on the in vitro stability and blood–brain barrier permeability of endomorphin II

Abstract

Section snippets

INTRODUCTION

Radioisotopes/Reagents

Peptide Synthesis

Octanol/Buffer Distribution

BBMEC Uptake

DISCUSSION

Acknowledgements

Trends Neurosci

Peptides

Peptides

FEBS Lett

J Biol Chem

Methods Enzymol

Life Sci

J Biol Chem

Brain Res Bull

Peptides

Life Sci

J Chromatogr A

Pharmacological mechanisms of opioid analgesics

Clin Neuropharmacol

Enkephalin analog prodrugs: Assessment of in vitro conversion, enzyme cleavage characterization and blood–brain barrier permeability

J Pharmacol Exp Ther

A potent and selective endogenous agonist for the mu‐opiate receptor

Nature

Endogenous opioid peptides: Multiple agonists and receptors

Nature

Dynorphin‐(1–13), an extraordinarily potent opioid peptide

Proc Natl Acad Sci USA

Assessment of stereoselectivity of trimethylphenylalanine analogues of delta‐opioid [D‐Pen(2),D‐Pen(5)]‐enkephalin

J Neurochem

Peptidases, peptides, and the mammalian blood–brain barrier

J Neurochem

The blood–brain barrier: principles for targeting peptides and drugs to the central nervous system

J Pharm Pharmacol

Proline motifs in peptides and their biological processing

FASEB J

Research Articles
Effect of guanidino modification and proline substitution on the in vitro stability and blood–brain barrier permeability of endomorphin II