Synthesis and biological properties of gamma-glutamyl-dermorphin, a prodrug

Nimkar

The Journal of Peptide Research

et al. 1999

In an attempt to improve the membrane permeabilities of opioid peptides, we have synthesized cyclic prodrugs of [Leu5]-enkephalin and DADLE using a coumarinic acid or a phenylpropionic acid linker. The synthesis of the coumarinic acid- and phenylpropionic acid-based cyclic prodrugs followed similar strategies. Key intermediates were the compounds with the C-terminal amino acids of opioid peptides (L-Leu, [Leu5]-enkephalin; D-Leu, DADLE) attached to the phenol hydroxyl group and the remaining amino acids of the peptide linked via the N-terminal amino acid (L-Tyr) attached to the carboxylic acid groups of the prodrug moieties (coumarinic acid or propionic acid). Cyclization of these linear precursors gave the cyclic prodrugs in 30-50% yields. These cyclic prodrugs exhibited excellent transcellular permeation characteristics across Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. To correlate the cellular permeabilities of these cyclic prodrugs with their physicochemical properties, we calculated their Stokes-Einstein molecular radii from their diffusion coefficients which were determined by NMR and we determined their membrane interaction potentials using immobilized artificial membrane (IAM) column chromatography. The cyclic prodrugs exhibited molecular radii similar to those of the parent compounds, [Leu5]-enkephalin and DADLE. However, these cyclic prodrugs were shown to have much higher membrane interaction potentials than their corresponding opioid peptides. Therefore, the enhanced cellular permeation of the cyclic prodrugs is apparently due to the alteration of their lipophilicity and hydrogen bonding potential, but not their molecular sizes.

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confidence: 99%

Synthesis and evaluation of the physicochemical properties of esterase‐sensitive cyclic prodrugs of opioid peptides using coumarinic acid and phenylpropionic acid linkers

Nimkar

The Journal of Peptide Research

et al. 1999

“…The issue of metabolic lability has, for all practical purposes, been solved by medicinal chemists through the design of novel peptide bond bioisosteres to replace metabolically labile peptide bonds (Sawyer, 1995). Until recently, however, medicinal chemists have had less success in manipulating the structures of opioid peptides to achieve good BBB permeation while still retaining high affinity and selectivity for opioid receptors.Through prodrug strategies, some progress has been made recently in improving the BBB permeation characteristics of opioid peptides (Greene et al, 1996;Misicka et al, 1996;Patel et al, 1997;Prokai et al, 2000). For a prodrug strategy to be successful in delivering opioid peptides to the brain, the following criteria must be met: 1) the prodrug should have favorable physiochemical properties (e.g., hydrophobicity, low hydrogen-bonding potential, and no charge) for transcellular permeation across the BBB; 2) the prodrug should exhibit good "intrinsic" permeability across the BBB but not be a substrate for efflux transporters (e.g., P-gp); 3) the prodrug should be a good substrate for enzymes (e.g., esterases) in the brain that would catalyze its bioconversion to the opioid peptide but have less activity as a substrate for the same This research was supported by a grant from the U.S. Public Health Service (DA09315).…”

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confidence: 99%

“…Through prodrug strategies, some progress has been made recently in improving the BBB permeation characteristics of opioid peptides (Greene et al, 1996;Misicka et al, 1996;Patel et al, 1997;Prokai et al, 2000). For a prodrug strategy to be successful in delivering opioid peptides to the brain, the following criteria must be met: 1) the prodrug should have favorable physiochemical properties (e.g., hydrophobicity, low hydrogen-bonding potential, and no charge) for transcellular permeation across the BBB; 2) the prodrug should exhibit good "intrinsic" permeability across the BBB but not be a substrate for efflux transporters (e.g., P-gp); 3) the prodrug should be a good substrate for enzymes (e.g., esterases) in the brain that would catalyze its bioconversion to the opioid peptide but have less activity as a substrate for the same enzymes in other biological media/tissues or for other enzymes that could lead to nonproductive metabolism of the prodrug (e.g., endopeptidases); and 4) the prodrug should have a reasonable plasma half-life and not be rapidly metabolized in the blood compartment or cleared by the liver or kidney or be extensively protein-bound.…”

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confidence: 99%

In Vitro Stability and In Vivo Pharmacokinetic Studies of a Model Opioid Peptide, H-Tyr-d-Ala-Gly-Phe-d-Leu-OH (DADLE), and Its Cyclic Prodrugs

Yang¹,

Chen²,

Borchardt³

2002

J Pharmacol Exp Ther

In vitro stability and in vivo pharmacokinetic studies of a model opioid peptide, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE), and its cyclic prodrugs (acyloxyalkoxy-based cyclic prodrug of DADLE, coumarinic acid-based cyclic prodrug of DADLE, and oxymethyl-modified coumarinic acid-based cyclic prodrug of DADLE) were conducted. The enzymatic stability of DADLE and its prodrugs in various biological media was determined at 37°C in the presence and absence of paraoxon, a known esterase inhibitor. The prodrugs exhibited metabolic stability to exo-and endopeptidases, and esterase-catalyzed bioconversion of the prodrugs to DADLE was observed. For pharmacokinetic studies in rats, various biological samples (blood, bile, urine, and brain) were collected after i.v. administration of DADLE and its prodrugs. The samples were analyzed by highperformance liquid chromatography with tandem mass spectrometric detection, and the conversion from the prodrugs to intermediates to DADLE was monitored. The prodrugs exhibited similar pharmacokinetic properties and showed improved stability compared with DADLE in rat blood. This increased stability led to higher plasma concentrations of DADLE after i.v. administration of the prodrugs compared with i.v. administration of DADLE alone. In terms of elimination pathways, metabolism by endopeptidases was the major route for DADLE elimination, whereas rapid biliary excretion was the major route of elimination for the prodrugs. The rapid elimination of the prodrugs by the liver and the formation of stable intermediates after esterase hydrolysis limited the bioconversion efficiencies of the prodrugs to DADLE after i.v. administration. The substrate activity of the prodrugs for efflux transporters (e.g., Pglycoprotein) in the blood-brain barrier significantly restricted their access to the brain.The delivery of opioid peptides to the brain has presented significant challenges to pharmaceutical scientists due to the poor biopharmaceutical properties of the peptides. These include their lability to metabolism by exo-and endopeptidases and their low permeation across the blood-brain barrier (BBB) (Fricker and Drewe, 1996;Pauletti et al., 1996aPauletti et al., , 1997Prokai, 1998). The issue of metabolic lability has, for all practical purposes, been solved by medicinal chemists through the design of novel peptide bond bioisosteres to replace metabolically labile peptide bonds (Sawyer, 1995). Until recently, however, medicinal chemists have had less success in manipulating the structures of opioid peptides to achieve good BBB permeation while still retaining high affinity and selectivity for opioid receptors.Through prodrug strategies, some progress has been made recently in improving the BBB permeation characteristics of opioid peptides (Greene et al., 1996;Misicka et al., 1996;Patel et al., 1997;Prokai et al., 2000). For a prodrug strategy to be successful in delivering opioid peptides to the brain, the following criteria must be met: 1) the prodrug should have favorable physiochemical properties...

“…Therefore, by adjusting the pH of the reaction mixture, we can make GGT catalyze the transpeptidation reaction selectively. ␥-Glutamyl compounds are very attractive, because (i) compounds that are not very soluble in water become much more soluble with ␥-glutamylization (5); (ii) the ␥-glutamyl linkage is resistant to peptidases in serum, and some ␥-glutamyl compounds can possibly be used as prodrugs specific for the organs that express GGT (8,10,18,30); and (iii) some ␥-glutamyl compounds taste good and thus can be used as food additives (21,22). We have already developed and reported efficient methods for synthesizing various ␥-Lglutamyl compounds using Escherichia coli 21,22,26).…”

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confidence: 99%

Use of Bacterial γ-Glutamyltranspeptidase for Enzymatic Synthesis of γ-d-Glutamyl Compounds

Suzuki

Izuka

Minami

et al. 2003

Appl Environ Microbiol

An enzymatic method for synthesizing various ␥-D-glutamyl compounds efficiently and stereospecifically involving bacterial ␥-glutamyltranspeptidase (EC 2.3.2.2) with D-glutamine as a ␥-glutamyl donor was developed. With D-glutamine as a ␥-glutamyl donor instead of L-glutamine in ␥-glutamyltaurine synthesis, byproducts such as ␥-glutamylglutamine and ␥-glutamyl-␥-glutamyltaurine were not synthesized and the yield of ␥-glutamyltaurine dramatically increased from 25 to 71%. It was also shown that the purification could be simplified without these ␥-glutamyl by-products. The possibility of synthesizing various ␥-D-glutamyl compounds was also shown.␥-Glutamyltranspeptidase (GGT) (EC 2.3.2.2) catalyzes the hydrolysis of ␥-glutamyl compounds such as glutathione and the transfer of their ␥-glutamyl moieties to other amino acids and peptides (27). The reactions catalyzed by GGT proceed via a ␥-glutamyl enzyme intermediate involving the hydroxyl group of . If the intermediate is subjected to nucleophilic attack by water, the reaction is hydrolytic, releasing glutamic acid. If the intermediate is subjected to nucleophilic attack by amino acids and peptides, the reaction is a transpeptidation yielding new ␥-glutamyl compounds. By employing various ␥-glutamyl acceptors, we can synthesize various ␥-glutamyl compounds using GGT. The pH optima for these reactions are very different (25). Therefore, by adjusting the pH of the reaction mixture, we can make GGT catalyze the transpeptidation reaction selectively. ␥-Glutamyl compounds are very attractive, because (i) compounds that are not very soluble in water become much more soluble with ␥-glutamylization (5); (ii) the ␥-glutamyl linkage is resistant to peptidases in serum, and some ␥-glutamyl compounds can possibly be used as prodrugs specific for the organs that express GGT (8,10,18,30); and (iii) some ␥-glutamyl compounds taste good and thus can be used as food additives (21,22). We have already developed and reported efficient methods for synthesizing various ␥-Lglutamyl compounds using Escherichia coli 21,22,26). The characteristics of our methods are as follows. (i) L-Glutamine, which is less expensive than glutathione, can be used as a ␥-glutamyl donor. (ii) No energy source, such as ATP, is required because GGT is a transferase and not a synthetase. (iii) Since E. coli GGT exhibits a broad substrate specificity for ␥-glutamyl acceptors, various ␥-glutamyl compounds can be synthesized. (iv) Since E. coli GGT can be purified from overproducing strains by means of a simple twostep method (1, 23), a large amount of GGT is readily available.During the development of a method for the enzymatic production of various ␥-glutamyl compounds, we found that the yield of a ␥-glutamyl compound with a ␥-glutamyl acceptor such as taurine was not very high. This was mainly because nonnegligible amounts of by-products, such as ␥-glutamylglutamine and ␥-glutamyl-␥-glutamyltaurine, were formed (26). It has been reported that GGT cannot utilize D-amino acids as ␥-glutamyl acceptors (27-29). Th...