Improved blood-brain barrier penetration and enhanced analgesia of an opioid peptide by glycosylation.

Neuropeptide pharmaceuticals have potential for the treatment of neurological disorders, but the blood-brain barrier (BBB) limits entry of peptides to the brain. Several strategies to improve brain delivery are currently under investigation, including glycosylation. In this study we investigated the effect of O-linked glycosylation on Ser(6) of a linear opioid peptide amide Tyr-D-Thr-Gly-Phe-Leu-Ser-NH(2) on metabolic stability, BBB transport, and analgesia. Peptide stability was studied in brain and serum from both rat and mouse by high-performance liquid chromatography. BBB transport properties were investigated by rat in situ perfusion. Tail-flick analgesia studies were performed on male ICR mice, injected i.v. with 100 microg of peptide ligand. Glycosylation of Ser(6) of the peptide led to a significant increase in enzymatic stability in both serum and brain. Glycosylation significantly increased the BBB permeability of the peptide from a value of 1.0 +/- 0.2 microl x min(-1) x g(-1) to 2.2 +/- 0.2 microl x min(-1) x g(-1) (p < 0.05), without significantly altering the initial volume of distribution. Analgesia studies showed that the glycosylated peptide gave a significantly improved analgesia after i.v. administration compared with nonglycosylated peptide. The improved analgesia profile shown by the glycosylated peptide is due in part to an improvement in bioavailability to the central nervous system. The bioavailability is increased by improving stability and transport into the brain.

O-Linked glycopeptides retain helicity in water.

A 17-residue O-linked glycopeptide model incorporating a central alpha-mannosyl serine residue, and its unglycosylated analog both demonstrate substantial helicity in water. The peptide sequence was derived from previous studies in which differences in overall helicity as a function of single amino acid substitutions were measured by circular dichroism (CD). The helical content was predicted by molecular modeling, and confirmed by CD and NMR. Moreover, the glycopeptide retained its helicity in the presence of SDS micelles, whereas the native peptide lost secondary structure in the presence of micelles. The inference is that the peptide sequence is a more important helix determinant than glycosylation per se.

Solid-phase synthesis of O-linked glycopeptide analogues of enkephalin.

The synthesis of 18 N-alpha-FMOC-amino acid glycosides for solid-phase glycopeptide assembly is reported. The glycosides were synthesized either from the corresponding O'Donnell Schiff bases or from N-alpha-FMOC-amino protected serine or threonine and the appropriate glycosyl bromide using Hanessian's modification of the Koenigs-Knorr reaction. Reaction rates of D-glycosyl bromides (e.g., acetobromoglucose) with the L- and D-forms of serine and threonine are distinctly different and can be rationalized in terms of the steric interactions within the two types of diastereomeric transition states for the D/L and D/D reactant pairs. The N-alpha-FMOC-protected glycosides [monosaccharides Xyl, Glc, Gal, Man, GlcNAc, and GalNAc; disaccharides Gal-beta(1-4)-Glc (lactose), Glc-beta(1-4)-Glc (cellobiose), and Gal-alpha(1-6)-Glc (melibiose)] were incorporated into 22 enkephalin glycopeptide analogues. These peptide opiates bearing the pharmacophore H-Tyr-c[DCys-Gly-Phe-DCys]- were designed to probe the significance of the glycoside moiety and the carbohydrate-peptide linkage region in blood-brain barrier (BBB) transport, opiate receptor binding, and analgesia.

Improved bioavailability to the brain of glycosylated Met-enkephalin analogs.

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),Ser(6),Gly(7)] enkephalin. The peptide was glycosylated on the Ser(6) 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 delta-opioid receptor, and mixed effect on binding to the mu-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.

Catalysis by amino acid-derived tetracoordinate complexes: enantioselective addition of dialkylzincs to aliphatic and aromatic aldehydes.

Me(2)Zn and Et(2)Zn added to aromatic and aliphatic aldehydes in the presence of 3 mol % of 2. (S)-1-Phenylethanol (91% ee) and (S)-1-phenylpropanol (86% ee) were synthesized from benzaldehyde and (S)-1-furan-2-yl-1-propanol (86% ee) from 2-furaldehyde. Nonanal and 3-phenylpropanal provided (S)-3-undecanol (96% ee) and (S)-1-phenyl-3-pentanol (94% ee). A solid-phase variant was effective with reduced ee's (e.g., 86% ee --> 79% ee) for (S)-1-phenylpropanol.

Asymmetric syntheses of (-)-8-epi-swainsonine triacetate and (+)-1, 2-Di-epi-swainsonine. Carbonyl addition thwarted by an unprecedented aza-pinacol rearrangement.

Indolizidines (-)-8-epi-swainsonine triacetate and (+)-1, 2-di-epi-swainsonine were synthesized from the O'Donnell Schiff base ester 1 derived from D-serine. Reductive-alkenylation of 1 with (i)()Bu(5)Al(2)H/H(2)C=CHMgBr followed by substrate-directed dihydroxylation of the pendant allylic group with OsO(4), reduction of imine, and cyclization with Ph(3)P/CCl(4) gave the polyhydroxylated pyrrolidines 8a and 8b as advanced intermediates. Efficient protecting group manipulations converted pyrrolidines 8a and 8b to their corresponding partially protected analogues 10a and 10b, which upon Swern oxidation and diastereoselective Keck-type allylation with BF(3).Et(2)O afforded the required three-carbon homologues (10a, >20:1 de; 10b, 3.5:1 de). Use of the chelating Lewis acid MgBr(2) instead of BF(3).Et(2)O with 10a led to a novel aza-pinacol rearrangement and allylation at the alpha-carbon to yield amino alcohol 17, which is similar to a hydride migration in the biosynthetic pathway of indolizidine alkaloids. Subsequent hydroboration, cyclization, and deprotection furnished (-)-8-epi-swainsonine triacetate 15a and (+)-1,2-di-epi-swainsonine 16b in good overall yields (6.3% for 1 --> 15a, 13 steps, and 4.0% for 1 --> 16b, 14 steps).

Enkephalin glycopeptide analogues produce analgesia with reduced dependence liability.

Endogenous peptides (e.g. enkephalins) control many aspects of brain function, cognition, and perception. The use of these neuroactive peptides in diverse studies has led to an increased understanding of brain function. Unfortunately, the use of brain-derived peptides as pharmaceutical agents to alter brain chemistry in vivo has lagged because peptides do not readily penetrate the blood-brain barrier. Attachment of simple sugars to enkephalins increases their penetration of the blood-brain barrier and allows the resulting glycopeptide analogues to function effectively as drugs. The delta-selective glycosylated Leu-enkephalin amide 2, H(2)N-Tyr-D-Thr-Gly-Phe-Leu-Ser(beta-D-Glc)-CONH(2), produces analgesic effects similar to morphine, even when administered peripherally, yet possesses reduced dependence liability as indicated by naloxone-precipitated withdrawal studies. Similar glycopeptide-based pharmaceuticals hold forth the promise of pain relief with improved side-effect profiles over currently available opioid analgesics.

[New chemical method for synthesis of O-linked glycopeptides]. / Nowa chemiczna metoda syntezy O-polaczonych glikopeptydów.

The diphenylketone Shiff-base appeared to be a very good protection from amino groups of serine and threonine for glycosylation reaction. O-Glycosides of these amino acids with different monosaccharides, aminosugars and deoxysugars were obtained with excellent yield and very high stereoselectivity. Products of glycosylation reactions were used as building blocks for solid phase glycopeptide synthesis.

Stereoselective synthesis of O-serinyl/threoninyl-2-acetamido-2-deoxy-alpha- or beta-glycosides.

General glycosidation methodology has been developed which can selectively provide 2-acetamido-2-deoxy-alpha- or beta-glycosides of beta-hydroxy-alpha-amino acid derivatives [glucopyranoside-(8, 43), galactopyranoside- (9, 13), mannopyranoside- (10), lactoside analogs (11, 38) and 3-O-beta-galactopyranosyl-mannopyranoside (12)] stereoselectively in excellent yield from the highly nucleophilic alpha-imino esters (Schiff bases) of L-serine and L-threonine. Various glycosides were converted via their amino and acetamido derivatives to Fmoc-protected serinyl- or threoninyl-glycosides (24-28, 37, 41, 46) which are all suitable building blocks for the solid-phase synthesis of O-glycopeptides. Complete 1H- and 13C-NMR data are provided for all compounds.

Glycopeptide enkephalin analogues produce analgesia in mice: evidence for penetration of the blood-brain barrier.

Most peptides have not proved useful as neuroactive drugs because they are blocked by the blood-brain barrier and do not reach their receptors within the brain. Intraperitoneally administered L-serinyl beta-D-glucoside analogues of [Met5]enkephalin (glycopeptides) have been shown to be transported across the blood-brain barrier to bind with targeted mu- and delta-opioid receptors in the mouse brain. The opioid nature of the binding has been demonstrated with intracerebroventricularly administered naloxone. Paradoxically, glucosylation decreases the lipophilicity of the peptides while promoting transport across the lipophilic endothelial layer. It is suggested that glucose transporter GLUT-1 is responsible for the transport of the peptide message. Profound and long-lasting analgesia has been observed in mice (tail-flick and hot-plate assays) with two of the glycopeptide analogues when administered intraperitoneally.

Determination and metabolism of dithiol chelating agents. XV. The meso-2,3-dimercaptosuccinic acid-cysteine (1:2) mixed disulfide, a major urinary metabolite of DMSA in the human, increases the urinary excretion of lead in the rat.

Meso-2,3-dimercaptosuccinic acid (DMSA) in humans is an effective p.o. therapeutically useful chelating agent of Pb. In humans given DMSA p.o., the major urinary metabolite is DMSA-cysteine (1:2) mixed disulfide. In order to determine its efficacy in mobilizing Pb and increasing urinary Pb excretion, the mixed disulfide was given to rats treated previously with Pb acetate. The mixed disulfide was as effective as DMSA in increasing the urinary excretion of Pb and mobilizing Pb from the kidney. DMSA, however, appears to be superior for mobilizing Pb from the liver and the brain. After the mixed disulfide was given s.c. to rats, DMSA was found in the blood and urine. Twenty-four hours after administration, 0.7% of the administered mixed disulfide was found in the urine as DMSA, indicating the mixed disulfide can be reduced to DMSA. The mixed disulfide was also reduced in vitro to DMSA during incubation with rat blood. Although in the rat the DMSA-cysteine (1:2) mixed disulfide mobilized Pb from the kidney, increased the urinary excretion of Pb and was to some extent reduced to DMSA, its fate and pharmacological properties in the human, where it is found after DMSA administration, are unknown.

Assessment of an in vitro blood-brain barrier model using several [Met5]enkephalin opioid analogs.

Confluent monolayers of primary and continuous passaged cultures of bovine brain microvessel endothelial cells (BMEC) have been suggested to model the blood-brain barrier (BBB). Increased lipophilicity has been previously suggested to increase BBB penetration. The intent of this study was to examine the effect that structural modifications of the [Met5]enkephalin analog DPDPE had on lipophilicity and passage across the BMEC. The BMEC consisted of a monolayer of confluent primary BMEC grown on polycarbonate (10 microns) filters. Permeability coefficients were calculated on the basis of the diffusion of peptides across the BMEC in a Side-Bi-Side diffusion chamber. Lipophilicity of the peptides examined was determined by using reversed-phase HPLC and calculating the capacity factor (k). Diffusion across the BMEC (for all peptides examined) was linear from 15 to 120 min; therefore, these time points were used to calculate permeability coefficients. Permeability coefficients ranged from 14.34 to 92.00 cm/min (x 10(-4), with [rho-ClPhe4,4']biphalin the highest. Analysis of variance coupled with the Newman-Keuls test showed significantly greater (P < .01) passage of select peptide analogs across the BMEC, including [rho-ClPhe4,4']biphalin, [rho-ClPhe4]DPDPE and reduced DPDPE. Interestingly, upon passage across the confluent monolayer, reduced DPDPE was converted to cyclized DPDPE. Calculated HPLC capacity factors ranged from 3.82 to 12.50. The most lipophilic peptide (highest) examined was acetylated Phe0-DPDPE. Analysis of the regression line of permeability coefficients plotted against capacity factors yielded a correlation coefficient of 0.745 (P < .01). The data provided in this study offer strong evidence that increasing peptide lipophilicity enhances passage across the BMEC.(ABSTRACT TRUNCATED AT 250 WORDS)