Robin Polt

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.

In Vivo Characterization of MMP-2200, a Mixed δ/μ Opioid Agonist, in Mice

We have previously reported the chemistry and antinociceptive properties of a series of glycosylated enkephalin analogs (glycopeptides) exhibiting approximately equal affinity and efficacy at δ opioid receptors (DORs) and μ opioid receptors (MORs). More detailed pharmacology of the lead glycopeptide MMP-2200 [H2N-Tyr-d-Thr-Gly-Phe-Leu-Ser-(O-β-d-lactose)-CONH2] is presented. MMP-2200 produced dose-related antinociception in the 55°C tail-flick assay after various routes of administration. The antinociceptive effects of MMP-2200 were blocked by pretreatment with the general opioid antagonist naloxone and partially blocked by the MOR-selective antagonist β-funaltrexamine and the DOR-selective antagonist naltrindole. The κ opioid receptor antagonist nor-binaltorphimine and the peripherally active opioid antagonist naloxone-methiodide were ineffective in blocking the antinociceptive effects of MMP-2200. At equi-antinociceptive doses, MMP-2200 produced significantly less stimulation of locomotor activity compared with morphine. Repeated administration of equivalent doses of morphine and MMP-2200 (twice daily for 3 days) produced antinociceptive tolerance (∼13- and 5-fold rightward shifts, respectively). In acute and chronic physical dependence assays, naloxone precipitated a more severe withdrawal in mice receiving morphine compared with equivalent doses of the glycopeptide. Both morphine and MMP-2200 inhibited respiration and gastrointestinal transit. In summary, MMP-2200 acts as a mixed DOR/MOR agonist in vivo, which may in part account for its high antinociceptive potency after systemic administration, as well as its decreased propensity to produce locomotor stimulation, tolerance, and physical dependence in mice, compared with the MOR-selective agonist morphine. For other measures (e.g., gastrointestinal transit and respiration), the significant MOR component may not allow differentiation from morphine.

Preparation of an electrophilic glycine cation equivalent and its reaction with heteroatom nucleophiles

Abstract A variety of heteroatom substituted Schiff base amino esters 4 - 7 are prepared either from the benzophenone imine of glycine ethyl ester ( 3 ) or by reaction of acetate 4 with heteroatom nucleophiles.

O-glycopeptides : a simple β-stereoselective glycosidation of serine and threonine via a favorable hydrogen bonding pattern

Glycosidation of serine and threonine is promoted by using an intramolecular hydrogen bond to the hydroxyl group

Glycosylation Improves the Central Effects of DAMGO

A series of μ‐agonist DAMGO analogs were synthesized and pharmacologically characterized to test the ‘biousian’ hypothesis of membrane hopping. DAMGO was altered by incorporating moieties of increasing water solubility into the C‐terminus via carboxamide and simple glycoside additions. The hydrophilic C‐terminal moieties were varied from glycinol in DAMGO (1) to l‐serine amide (2), l‐serine amide β‐d‐xyloside (3), l‐serine amide β‐d‐glucoside (4), and finally to l‐serine amide β‐lactoside (5). Opioid binding and mouse tail‐flick studies were performed. Antinociceptive potency (intravenous) increased, passing through a maximum (A50 ≈ 0.2 μmol/kg) for 2 and 3 as membrane affinity versus water solubility became optimal, and dropped off (A50 ≈ 1.0 μmol/kg) for 4 and 5 as water solubility dominated molecular behavior. Intravenous A50 values were plotted versus hydrodynamic values (glucose units, g.u.) for the glycoside moieties, or the hydrophilic/hydrophobic Connolly surface areas (A50 versus e−Awater/Alipid), and provided either a V‐shaped or a U‐shaped curve, as predicted by the ‘biousian’ hypothesis. The μ‐selective receptor profile was maintained (Ki’s = 0.66–1.3 nm) upon modifications at the C‐terminus. The optimal ‘degree of glycosylation’ for the DAMGO peptide message appears to be between 1.25 and 1.75 g.u. (hydrodynamic g.u.), or 0.75 and 0.90 in terms of the surface‐derived amphipathicity values.

Behavioral Pharmacology of the μ/δ Opioid Glycopeptide MMP2200 in Rhesus Monkeys

H2N-Tyr-d-Thr-Gly-Phe-Leu-Ser-(O-β-d-lactose)-CONH2 (MMP2200) is a novel glycopeptide opioid agonist with similar affinities for μ and δ receptors. Glycosylation promoted brain penetration and production of centrally mediated behavioral effects in mice; however, it is unknown whether the magnitude of enhanced brain penetration is sufficient to permit central mediation of drug effects and production of synergistic μ/δ antinociceptive interactions after systemic administration in primates. To address this issue, the present study compared the effects of MMP2200 and the μ-agonist morphine in four behavioral procedures in rhesus monkeys. In an assay of thermal nociception, morphine (1.0–5.6 mg/kg) produced dose-dependent antinociception, whereas MMP2200 (10–56 mg/kg) was ineffective. In an assay of capsaicin-induced thermal allodynia, both morphine (0.01–1.0 mg/kg) and MMP2200 (0.032–3.2 mg/kg) produced dose-dependent antiallodynic effects. MMP2200-induced antiallodynia was blocked by the moderately μ-selective antagonist naltrexone (0.01 mg/kg), the δ-selective antagonist naltrindole (1.0 mg/kg), and the peripherally selective opioid antagonist quaternary naltrexone (0.32 mg/kg). In an assay of schedule-controlled behavior, both morphine (0.01–1.0 mg/kg) and MMP2200 (10–56 mg/kg) decreased response rates. Morphine effects were antagonized by naltrexone (0.001–0.01 mg/kg); however, the effects of MMP2200 were not antagonized by either naltrexone (0.01 mg/kg) or naltrindole (1.0 mg/kg). In an assay of drug self-administration, morphine (0.0032–0.32 mg/kg/injection) produced reinforcing effects, whereas MMP2200 (0.032–0.32 mg/kg/injection) did not. These results suggest that systemically administered MMP2200 acted as a peripheral, μ/δ-opioid agonist with limited distribution to the central nervous system in rhesus monkeys. These results also suggest the existence of species differences in the pharmacokinetics and brain penetration of glycopeptides.

Opioid glycopeptide analgesics derived from endogenous enkephalins and endorphins

Over the past two decades, potent and selective analgesics have been developed from endogenous opioid peptides. Glycosylation provides an important means of modulating interaction with biological membranes, which greatly affects the pharmacodynamics and pharmacokinetics of the resulting glycopeptide analogues. Furthermore, manipulation of the membrane affinity allows penetration of cellular barriers that block efficient drug distribution, including the blood-brain barrier. Extremely potent and selective opiate agonists have been developed from endogenous peptides, some of which show great promise as drug candidates.

Stereoselective synthesis of O-serinyl/threoninyl-2-acetamido-2-deoxy-α- or β-glycosides

Abstract General glycosidation methodology has been developed which can selectively provide 2-acetamido-2-deoxy-α- or β-glycosides of β-hydroxy-α-amino acid derivatives [glucopyranoside-( 8, 43 ), galactopyranoside- ( 9, 13 ), mannopyranoside- ( 10 ), lactoside analogs ( 11, 38 ) and 3- O - β -galactopyranosyl-mannopyranoside ( 12 )] stereoselectively in excellent yield from the highly nucleophilic α-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 1 H- and 13 C-NMR data are provided for all compounds.

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

Abstract Glycosylation provides an effective means of enhancing penetration of the blood–brain barrier by pharmacologically active peptides. Glycosylated enkephalin analogues demonstrate much greater analgesic effects than their unglycosylated counterparts when administered peripherally. The solution conformations of glycopeptide enkephalin analogues with the sequences H-Tyr-c-[ d -Cys-Gly-Phe- d -Cys]-Ser(β-O-Glcp)-Gly-NH2, 2, and H-Tyr-c-[ d -Cys-Gly-Phe- d -Cys]-Ser(α-O-Glcp)-Gly-NH2, 3, have been determined by NMR and molecular modeling, and were compared to the unglycosylated peptide H-Tyr-c-[ d -Cys-Gly-Phe- d -Cys]-Ser-Gly-NH2, 1, to determine the impact of glycosylation on peptide conformation. The only observed conformational effects were on the residue of attachment, Ser6, and on the adjacent Gly7-amide. This has important implications in peptide-based drug design in that strategically placed glycosylation can improve transport without destruction of the receptor selectivity of a pre-existing non-glycosylated peptide pharmacophore.

β-Amino alcohols from amino acids: Chelation control via schiff bases.

Abstract Sequential addition of iBu 2 AIH and RLi or RMgX to Schiff base esters derived from amino acids provides a simple route to β-amino alcohols. The reaction procedes without racemization, and with high threo selectivity. Several representative sphingosines are synthesized.