Improved bioavailability to the brain of glycosylated Met-enkephalin analogs

doi:10.1016/S0006-8993(00)02794-3

Brain Research

Volume 881, Issue 1, 20 October 2000, Pages 37-46

https://doi.org/10.1016/S0006-8993(00)02794-3 Get rights and content

Abstract

The blood–brain barrier prevents the entry of many potentially therapeutic peptide drugs to the brain. Glycosylation has shown potential as a methodology for improving delivery to the CNS. Previous studies have shown improved bioavailability and improved centrally mediated analgesia of glycosylated opioids. In this study we investigate the effect of glycosylation on the cyclic opioid peptide [d-Cys^2,5,Ser⁶,Gly⁷] enkephalin. The peptide was glycosylated on the Ser⁶ via an O-linkage with various sugar moieties and alignments. The peptides were then investigated for receptor binding, physiochemical attributes, in situ brain uptake in female Sprague–Dawley rats and antinociception in male ICR mice. Glycosylation resulted in a slight decrease in affinity to the δ-opioid receptor, and mixed effect on binding to the μ-opioid receptor. There was a significant decrease in lipophilicity resulting from glycosylation and a slight reduction in binding to bovine serum albumin. In situ perfusion showed that brain uptake was improved by up to 98% for several of the glycosylated peptides, and the nociceptive profiles of the peptides, in general, followed the rank order of peptide entry to the brain with up to a 39-fold increase in A.U.C.

Introduction

Endogenous peptides play an important role in the maintenance of homeostasis by their roles as hormones and neurotransmitters in both the periphery and CNS. Despite this there has been limited use of peptides for treatment of neurological disorders, due to their poor biodistribution to the brain. Many peptides either have poor metabolic stability or cannot cross the blood–brain barrier (BBB). The BBB is situated at the level of the endothelial cells of the brain microvascular capillaries [43]. BBB endothelial cells are connected by tight cellular junctions, which give a high electrical resistance of 2000 Ω cm², to compare to 3–30 Ω cm² in peripheral vessels (see Ref. [24] for review). The BBB endothelial cells have a low number of vesicles [10], indicating reduced vesicular transport. The BBB also lacks fenestrations and is ensheathed by astrocytic endfeet, which provide autocrine factors to maintain BBB function [2]. The BBB also acts as a metabolic barrier possessing a number of enzymes including aminopeptidase A [7], aminopeptidase M [14], and angiotensin-converting enzyme [12] that are known to degrade peptides.

The BBB plays an important role in brain homeostasis and thus a number of transport systems are present which enable substances to enter the brain. Highly lipophilic compounds can enter the brain via diffusion. Specific transport systems have been reported for amino acids [35], glucose [20] and iron transferrin [23]. Several saturable transport systems have also been reported for peptides both influx and efflux. Influx (blood-to-brain) systems include Leu-enkephalin [61], [62], arginine vasopressin [60] and [d-penicillamine^2,5] enkephalin [17], [49], [54]. Several efflux systems (brain-to-blood) have been characterized, including a carrier-mediated efflux system for small N-Tyr peptides such as N-Tyr-MIF-1 [4] and the ATP binding cassette efflux pump P-glycoprotein (Pgp) [47], [48].

A number of methodologies have been used to improve delivery of peptides to the brain (see Ref. [16] for review), including improving lipophilicity, making the peptide more cationic, targeting transporters and glycosylation.

Glycosylation of peptides has been shown to improve bioavailability by increasing metabolic stability [41], and attenuating in vivo clearance [19]. Proteins that have been glycated by Amidori rearrangement have been shown to have an increased distribution to the central nervous system (CNS) [38]. Deltorphin C (an opioid peptide) when given peripherally does not elicit any analgesia; however, the glycosylated analog of Deltorphin C was analgesic when given peripherally with an ED₅₀ (median analgesic dose) of 96.4 μmol kg⁻¹[50]. Receptor binding studies indicated that this improved analgesia was not due to an increased affinity to either δ or μ opioid receptors [50].

Glycosylated analogs of Met-enkephalin have also been investigated. The cyclic Met-enkephalin analog [d-Cys^2,5,Ser⁶,Gly⁷] enkephalin (LSZ 916 in this study, see Table 1) does not elicit analgesia when given peripherally (i.p.) [39]; however when administered into the lateral ventricle (i.c.v.) gave favorable analgesia [39]. The glycosylated analog [d-Cys^2,5,Ser⁶-β-d-glucose,Gly⁷] enkephalin (LSZ 1025, see Table 1) gave a robust and prolonged analgesia when given peripherally, which could be blocked centrally but not peripherally by naloxone [38]. Despite this, no clear evidence has been provided to date that improved analgesia of glycosylated analgesics is due to improved BBB penetration.

In this study, we investigate the effect of glycosylation on BBB penetration of the [d-Cys^2,5,Ser⁶,Gly⁷] enkephalin family of peptides, using physiochemical parameters and in situ brain perfusion studies.

Section snippets

Materials

Unless otherwise stated all chemicals were supplied by Sigma (St. Louis, MO). The ¹²⁵I was purchased as Na¹²⁵I from NEN (Boston, MA).

Synthesis of peptides

Fmoc-amino acid glycosides were synthesized from acetobromo glycosyl donors with N-diphenylmethylene Schiff base aglycones using Hannesssian’s modification of the Koenigs–Knorr reaction [21], employing silver triflate promotion and conversion to glycopeptide amenable protection [40]. These included l-Ser-O-α-2,3,4,6-tetra-O-acetyl-d-glucopyranosyl-FMOC-amino acid,

Receptor binding

Table 2 shows the receptor binding data for the five peptide drugs. It can be seen that glycosylation reduces the affinity of the peptide for the δ-receptor, with the largest effect seen for the two β-d-glucose-containing peptides (LSZ 1025 and SAM 1025). In contrast, addition of a sugar moiety has a mixed effect on μ receptor affinity. Two peptides (SAM 1025 and SAM 1040) had improved affinity and two (LSZ 1025 and LSZ 62) had slightly reduced affinity.

Octanol/saline distribution (D)

Table 3 shows the mean±S.E.M.

Discussion

In this study, we have investigated receptor binding, lipophilicity, brain uptake and analgesia for glycosylated analogs of the opioid peptide ([d-Cys^2,5,Ser⁶,Gly⁷] enkephalin. Both receptor binding and in situ studies were carried out in female rats, while analgesia studies were performed on male mice. Differences in both sex and species have previously been reported for opioid response. The predominate difference for sex has been linked to estrus cycle [32]. Studies have also shown a larger

Acknowledgements

This work was funded by NIH grants DA 11271 and DA 06284.

References (62)

F.E Arthur et al.
Astrocyte-mediated induction of tight junctions in brain capillary endothelium: an efficient in vitro model
Dev. Brain Res.
(1987)
W.A Banks et al.
A brain-to-blood carrier-mediated transport system for small N-tyrosinated peptides
Pharmacol. Biochem. Behav.
(1984)
W.A Banks et al.
Peptides and the blood–brain barrier: lipophilicity as a predictor of permeability
Brain Res. Bull.
(1985)
H.H Bausback et al.
Angiotensin metabolism by cerebrovascular aminopeptidase A
Biochem. Pharmacol.
(1988)
A.E Bolton
Comparative methods for the radiolabeling of peptides
Methods Enzymol.
(1986)
M.S Brownfield et al.
Differential distribution of immunoreactive angiotensin and angiotensin-converting enzyme in rat brain
Neuroscience
(1982)
L Churchill et al.
Metabolism of opioid peptides by cerebrovascular aminopeptidase M
Biochem. Biophys. Acta.
(1987)
T.P Davis et al.
High performance liquid chromatographic analysis of in vitro central neuropeptide processing
J. Chromatogr.
(1985)
R.D Egleton et al.
Bioavailability and transport of peptides and peptide drugs into the brain
Peptides
(1997)
R.D Egleton et al.
Transport of the δ-opioid receptor agonist [d-penicillamine2,5] enkephalin involves transcytosis
J. Pharm. Sci.
(1999)

C Fillebeen et al.

Receptor-mediated transcytosis of lactoferrin through the blood–brain barrier

J. Biol. Chem.

(1999)

S Hanessian et al.

Chemistry of the glycoside linkage. An efficient synthesis of 1,2-trans-disaccharides

Carbohydrate Res.

(1977)

H.C Jones et al.

The development of ion regulation at the blood–brain barrier

E Kaiser et al.

Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides

Anal. Biochem.

(1970)

C.T Kriss et al.

Enkephalin-based drug design: conformational analysis of O-linked glycopeptides by NMR and molecular modelling

Tetrahedron Asymmetry

(2000)

C Leloup et al.

Glucose transporter 2 (Glut2): expression in specific brain nuclei

Brain Res.

(1994)

O.H Lowry et al.

Protein measurement with the folin phenol reagent

J. Biol. Chem.

(1951)

J.S Mogil et al.

Sex differences in thermal nociception and morphine antinociception in rodents depend on phenotype

Neurosci. Biobehav. Rev.

(2000)

J Plendl et al.

Quantitative differences in neoglycoprotein binding for vascular endothelial cells from porcine brain, ovary and testis in vitro

Microvasc. Res.

(1995)

J.F Poduslo et al.

Glycation increases the permeability of proteins across the blood–nerve and blood–brain barriers

Mol. Brain Res.

(1994)

J.E Preston et al.

Permeability of the developing blood–brain barrier to ¹⁴C-mannitol using the rat in situ brain perfusion technique

Dev. Brain Res.

(1995)

H Rink

Solid-phase synthesis of protected peptide fragments using a trialkyoxy-diphenyl-methyl ester resin

Tetrahedron Lett.

(1987)

P Rivaille et al.

Synthesis of LH-RH using a new phenolic polymer as solid support and ‘BOP’ reagent for fragment coupling

Tetrahedron

(1980)

B.C Yoburn et al.

Species differences in mu- and delta-opioid receptors

Eur. J. Pharmacol.

(1991)

B.V Zlokovic et al.

Transport, uptake and metabolism of blood-borne vasopressin by the blood–brain barrier

Brain Res.

(1992)

B.V Zlokovic et al.

Kinetics of arginine-vasopressin uptake at the blood–brain barrier

Biochim. Biophys. Acta

(1990)

T.J Abbruscato et al.

Blood-to-CNS entry and stability of biphalin, a unique double-enkephalin analog, and its halogenated derivatives

J. Pharmacol. Exp. Ther.

(1996)

W.A Banks et al.

Blood to brain and brain to blood passage of native horseradish peroxidase, wheat germ agglutinin, and albumin: pharmacokinetic and morphological assessments

J. Neurochem.

(1994)

W.A Banks et al.

HIV-1 protein gp120 crosses the blood–brain barrier: role of adsorptive endocytosis

Life Sci.

(1997)

R.G Blasberg et al.

Transport of α-aminoisobutyric acid across brain capillary and cellular membranes

J. Cereb. Blood Flow Metab.

(1983)

M.W Brightman et al.

Junctions between intimately apposed cell membranes in the vertebrate brain

J. Cell Biol.

(1969)

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Research reportImproved bioavailability to the brain of glycosylated Met-enkephalin analogs

Abstract

Introduction

Section snippets

Materials

Synthesis of peptides

Receptor binding

Octanol/saline distribution (D)

Discussion

Acknowledgements

Dev. Brain Res.

Pharmacol. Biochem. Behav.

Brain Res. Bull.

Biochem. Pharmacol.

Methods Enzymol.

Neuroscience

Biochem. Biophys. Acta.

J. Chromatogr.

Peptides

J. Pharm. Sci.

J. Biol. Chem.

Carbohydrate Res.

Anal. Biochem.

Tetrahedron Asymmetry

Brain Res.

J. Biol. Chem.

Neurosci. Biobehav. Rev.

Microvasc. Res.

Mol. Brain Res.

Dev. Brain Res.

Tetrahedron Lett.

Tetrahedron

Eur. J. Pharmacol.

Brain Res.

Biochim. Biophys. Acta

Blood-to-CNS entry and stability of biphalin, a unique double-enkephalin analog, and its halogenated derivatives

J. Pharmacol. Exp. Ther.

Blood to brain and brain to blood passage of native horseradish peroxidase, wheat germ agglutinin, and albumin: pharmacokinetic and morphological assessments

J. Neurochem.

HIV-1 protein gp120 crosses the blood–brain barrier: role of adsorptive endocytosis

Life Sci.

Transport of α-aminoisobutyric acid across brain capillary and cellular membranes

J. Cereb. Blood Flow Metab.

Junctions between intimately apposed cell membranes in the vertebrate brain

J. Cell Biol.

Research report
Improved bioavailability to the brain of glycosylated Met-enkephalin analogs