A new painkiller nanomedicine to bypass the blood-brain barrier and the use of morphine.

The clinical use of endogenous neuropeptides has historically been limited due to pharmacokinetic issues, including plasma stability and blood-brain barrier permeability. In this study, we show that the rapidly metabolized Leu-enkephalin (LENK) neuropeptide may become pharmacologically efficient owing to a simple conjugation with the lipid squalene (SQ). The corresponding LENK-SQ bioconjugates were synthesized using different chemical linkers in order to modulate the LENK release after their formulation into nanoparticles. This new SQ-based nanoformulation prevented rapid plasma degradation of LENK and conferred on the released neuropeptide a notable antihyperalgesic effect that lasted longer than after treatment with morphine in a rat model of inflammation (Hargreaves test). The biodistribution study as well as the use of brain-permeant and -impermeant opioid receptor antagonists indicated that LENK-SQ NPs act through peripherally located opioid receptors. This study represents a novel nanomedicine approach, allowing the specific delivery of LENK neuropeptide into inflamed tissues for pain control.

Tyr1-ψ[( Z)CF═CH]-Gly2 Fluorinated Peptidomimetic Improves Distribution and Metabolism Properties of Leu-Enkephalin.

Opioid peptides are key regulators in cellular and intercellular physiological responses, and could be therapeutically useful for modulating several pathological conditions. Unfortunately, the use of peptide-based agonists to target centrally located opioid receptors is limited by poor physicochemical (PC), distribution, metabolic, and pharmacokinetic (DMPK) properties that restrict penetration across the blood-brain barrier via passive diffusion. To address these problems, the present paper exploits fluorinated peptidomimetics to simultaneously modify PC and DMPK properties, thus facilitating entry into the central nervous system. As an initial example, the present paper exploited the Tyr1-ψ[( Z)CF═CH]-Gly2 peptidomimetic to improve PC druglike characteristics (computational), plasma and microsomal degradation, and systemic and CNS distribution of Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu). Thus, the fluoroalkene replacement transformed an instable in vitro tool compound into a stable and centrally distributed in vivo probe. In contrast, the Tyr1-ψ[CF3CH2-NH]-Gly2 peptidomimetic decreased stability by accelerating proteolysis at the Gly3-Phe4 position.

Nanoparticulate peptide delivery exclusively to the brain produces tolerance free analgesia.

The delivery of peptide drugs to the brain is challenging, principally due to the blood brain barrier and the low metabolic stability of peptides. Exclusive delivery to the brain with no peripheral exposure has hitherto not been demonstrated with brain quantification data. Here we show that polymer nanoparticles encapsulating leucine5-enkephalin hydrochloride (LENK) are able to transport LENK exclusively to the brain via the intranasal route, with no peripheral exposure and nanoparticle localisation is observed within the brain parenchyma. Animals dosed with LENK nanoparticles (NM0127) showed a strong anti-nociceptive response in multiple assays of evoked and on going pain whereas animals dosed intranasally with LENK alone were unresponsive. Animals did not develop tolerance to the anti-hyperalgesic activity of NM0127 and NM0127 was active in morphine tolerant animals. A microparticulate formulation of clustered nanoparticles was prepared to satisfy regulatory requirements for nasal dosage forms and the polymer nanoparticles alone were found to be biocompatible, via the nasal route, on chronic dosing.

Measurement of tissue azithromycin levels in self-collected vaginal swabs post treatment using liquid chromatography and tandem mass spectrometry (LC-MS/MS).

BACKGROUND: Azithromycin is recommended for the treatment of uncomplicated urogenital chlamydia infection although the standard 1gram dose sometimes fails to eradicate the infection (treatment failure). One hypothesis proposed for treatment failure has been insufficient levels of the antibiotic at the site of infection. We developed an assay using liquid chromatography and tandem mass spectrometry (LC-MS/MS) to measure azithromycin concentration in high-vaginal swabs and monitor how concentration changes over time following routine azithromycin treatment. METHODS: Azithromycin concentrations were measured in two groups of women either within the first 24h of taking a 1g dose (N = 11) or over 9 days (N = 10). Azithromycin concentrations were normalised to an internal standard (leucine enkephalin), and the bulk lipid species phosphatidylcholine [PC(34:1)], using an Agilent 6490 triple quadrupole instrument in positive ionisation mode. The abundances of azithromycin, PC(34:1), and leu-enkephalin were determined by multiple reaction monitoring and absolute levels of azithromycin estimated using standard curves prepared on vaginal specimens. RESULTS: Vaginal azithromycin concentrations of women were rapidly obtained after 5h post-treatment (mean concentration = 1031mcg/mg of lipid, range = 173-2693mcg/mg). In women followed for 9 days, peak concentrations were highest after day 2 (mean concentration = 2206mcg/mg, range = 721-5791mcg/mg), and remained high for at least 9 days with a mean concentration of 384mcg/mg (range = 139-1024mcg/mg) on day 9. CONCLUSION: Our study confirmed that a single 1g dose of azithromycin is rapidly absorbed and remains in the vagina at relatively high levels for at least a week, suggesting that poor antibiotic absorption is unlikely to be an explanation for treatment failure.

Chitosan amphiphile coating of peptide nanofibres reduces liver uptake and delivers the peptide to the brain on intravenous administration.

The clinical development of neuropeptides has been limited by a combination of the short plasma half-life of these drugs and their ultimate failure to permeate the blood brain barrier. Peptide nanofibres have been used to deliver peptides across the blood brain barrier and in this work we demonstrate that the polymer coating of peptide nanofibres further enhances peptide delivery to the brain via the intravenous route. Leucine(5)-enkephalin (LENK) nanofibres formed from the LENK ester prodrug - tyrosinyl(1)palmitate-leucine(5)-enkephalin (TPLENK) were coated with the polymer - N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ) and injected intravenously. Peptide brain delivery was enhanced because the GCPQ coating on the peptide prodrug nanofibres, specifically enables the peptide prodrug to escape liver uptake, avoid enzymatic degradation to non-active sequences and thus enjoy a longer plasma half life. Plasma half-life is increased 520%, liver AUC0-4 decreased by 54% and brain AUC0-4 increased by 47% as a result of the GCPQ coating. The increased brain levels of the GCPQ coated peptide prodrug nanofibres result in the pharmacological activity of the parent drug (LENK) being significantly increased. LENK itself is inactive on intravenous injection.

Delivery of analgesic peptides to the brain by nano-sized bolaamphiphilic vesicles made of monolayer membranes.

Inefficient drug delivery to the brain is a major obstacle for pharmacological management of brain diseases. We investigated the ability of bolavesicles - monolayer membrane vesicles self-assembled from synthetic bolaamphiphiles that contain two hydrophilic head groups at each end of a hydrophobic alkyl chain - to permeate the blood-brain barrier and to deliver the encapsulated materials into the brain. Cationic vesicles with encapsulated kyotorphin and leu-enkephalin (analgesic peptides) were prepared from the bolalipids GLH-19 and GLH-20 and studied for their analgesic effects in vivo in experimental mice. The objectives were to determine: (a) whether bolavesicles can efficiently encapsulate analgesic peptides, (b) whether bolavesicles can deliver these peptides to the brain in quantities sufficient for substantial analgesic effect, and to identify the bolavesicle formulation/s that provides the highest analgetic efficiency. The results indicate that the investigated bolavesicles can deliver analgesic peptides across the blood-brain barrier and release them in the brain in quantities sufficient to elicit efficient and prolonged analgesic activity. The analgesic effect is enhanced by using bolavesicles made from a mixture the bolas GLH-19 (that contains non-hydrolyzable acetylcholine head group) and GLH-20 (that contains hydrolysable acetylcholine head group) and by incorporating chitosan pendants into the formulation. The release of the encapsulated materials (the analgesic peptides kyotorphin and leu-enkephalin) appears to be dependent on the choline esterase (ChE) activity in the brain vs. other organs and tissues. Pretreatment of experimental animals with pyridostigmine (the BBB-impermeable ChE inhibitor) enhances the analgesic effects of the studied formulations. The developed formulations and the approach for their controlled decapsulation can serve as a useful modality for brain delivery of therapeutically-active compounds.

Reversible lipidization for the oral delivery of leu-enkephalin.

The endogenous opioid peptide leu-enkephalin (ENK) was chemically modified by a method known as reversible aqueous lipidization (REAL) with a novel amine-reacting lipophilic dimethylmaleic anhydride analog, 3,4-bis(decylthiomethyl)-2,5-furandione. The binding affinity of the product, REAL-ENK, to opioid receptors was greatly reduced. This prodrug was stable in neutral and basic phosphate buffers but underwent rapid hydrolysis under acidic conditions in the presence of 50% acetonitrile. It also showed increased stability toward enzymatic degradations in various tissue preparations. The half-lives of REAL-ENK in mouse small intestinal mucosal homogenate and liver homogenate were 12 and 80 min, representing a 12- and 32-fold increase over those of ENK itself. In contrast to ENK (t(1/2) 6.7 min), REAL-ENK was stable in mouse plasma. More importantly, REAL-ENK produced significant and sustained antinociception mediated by peripheral opioid receptors in a rodent inflammatory pain model. Pharmacokinetic studies employing a radioimmunoassay (RIA) demonstrated that significantly higher and sustained plasma peptide levels were detected up to 24 h following the oral administration of REAL-ENK in normal mice. The peak concentration and area under the curve of oral REAL-ENK were 4.4 and 21 times higher than that of oral ENK. Our results indicate that like its disulfide-based counterpart, amine-based REAL may be an enabling technology which can be applied to enhance metabolic stability, increase oral absorption, and preserve and possibly prolong the pharmacological activity of peptide drugs.

Investigation of the route of absorption of lipid and sugar modified leu-enkephalin analogues and their enzymatic stability using the caco-2 cell monolayer system.

It has been demonstrated that conjugation of lipoamino acids or glucose units to the endogenous opioid peptide, Leu-enkephalin can significantly improve the peptide's metabolic stability and absorption across biological barriers. The purpose of this study was to investigate the possible involvement of specific carrier proteins in the absorption of these peptide conjugates. A series of lipo- glycol- and liposaccharide peptide conjugates were synthesised and examined using the Caco-2 monolayer assay for evidence of interaction with the human H(+)-coupled oligopeptide transporter (hPepT1), glucose transporters and the multidrug resistance efflux pump, p-glycoprotein. The investigation involved determining the apparent permeability of each compound in the absence of any inhibitors and comparing this to the apparent permeabilities of each compound in the presence of glycylsarcosine, glucose or vinblastine, respective inhibitors of the above mentioned transporters. None of the peptide conjugates were found to be substrates for p-glycoprotein. Of the six peptide conjugates examined, only the C-terminus glucose conjugate of Leu-enkephalin (Enk-glu) showed evidence of transport by both glucose transporters and hPepT1. In contrast, N-terminus conjugation of both lipids and sugars appeared to provide the greatest protection against enzymatic degradation.

In vitro evaluation of the potential of thiomers for the nasal administration of Leu-enkephalin.

It was the aim of this study to evaluate the potential of thiolated polycarbophil for the nasal administration of Leucine-enkephalin (Leu-enkephalin). The enzymatic degradation of Leu-enkephalin on freshly excised bovine nasal mucosa was analysed qualitatively via thin layer chromatography and quantitatively via high performance liquid chromatography (HPLC). The potential of thiolated polycarbophil gels to provide a sustained release for the therapeutic peptide was investigated via diffusion studies. Permeation studies were performed in Ussing-type diffusion chambers with freshly excised bovine nasal mucosa. Results demonstrated that Leu-enkephalin is mainly degraded by the cleavage of tyrosine from the N-terminus of the peptide. Within one hour more than 63.5 +/- 2% of this therapeutic peptide are degraded on the nasal mucosa. In the presence of 0.25% thiolated polycarbophil, this degradation process, however, could be significantly lowered. Diffusion studies demonstrated that Leu-enkephalin being incorporated in a 0.5% thiolated polycarbophil gel is sustained released out of it. The appearent permeability coefficient (P(app)) for Leu-enkephalin on the nasal mucosa was determined to be 1.9 +/- 1.2 x 10(-7) cm/sec. Furthermore, in the presence of 0.5% thiolated polycarbophil and 1% glutathione, which is used as permeation mediator for the thiomer, the uptake of Leu-enkephalin from the nasal mucosa was even 82-fold improved. According to these results thiolated polycarbophil might be a promising excipient for nasal administration of Leu-enkephalin.

Determination of transport in the Caco-2 cell assay of compounds varying in lipophilicity using LC-MS: enhanced transport of Leu-enkephalin analogues.

PURPOSE: To synthesize a number of analogues of Leu-enkephalin with different lipophilicities and to develop an LC-MS method for determining the Caco-2 cell permeability values of these compounds. METHODS: A number of sugar and sugar plus lipoamino acid analogues of Leu-enkephalin were synthesized by solid-phase and solution methods. An LC-MS method was developed for analyzing the Caco-2 cell assay samples and validated against the traditional method using radiolabelled compounds. RESULTS: A sensitive and specific LC-MS assay was developed. Standard curves were linear in the range of 0.025-5 microM. Apparent permeability values determined by LC-MS and liquid scintillation counter were identical, for both a hydrophilic drug, cephalexin and a lipophilic Leu-enkaphalin analogue. Caco-2 permeability values for the analogues of Leu-enkephalin were determined and it was found that attachment of sugar or sugar and lipoamino acid to the Leu-enkephalin peptide resulted in an increase in the apparent permeability values compared to the native peptide, which was not transported across the Caco-2 cell monolayers. CONCLUSIONS: A rapid, generic LC-MS method for analyzing a range of compounds was developed. Attachment of a sugar or sugar and lipoamino acid to Leu-enkephalin improves the apparent permeability across Caco-2 cell monolayers.

Peptidase activity on the surface of the porcine buccal mucosa.

Peptide drugs in buccal bioadhesive delivery systems are exposed to the surface of the buccal mucosa at high concentrations over long periods of time. The peptidase activity on the surface of the buccal mucosa has not been evaluated as a barrier to peptide buccal delivery. The in vitro stability of various synthetic substrates on the surface of intact porcine buccal mucosa was determined. No carboxypeptidase or dipeptidyl peptidase IV activity was detected on the buccal mucosa, while aminopeptidase N activity was detected using Leu-p-nitroanilide. No endopeptidase activity was observed towards the peptide substrates. Insulin and insulin B-chain were intact at the 2 h time point at 37 degrees C, while the percent of parent Leu-enkephalin remaining was 18+/-9 (mean+/-S.D., n=9). In the presence of aminopeptidase inhibitors, amastatin, sodium deoxycholate and EDTA, the degradation of Leu-enkephalin was dramatically reduced. This work suggests that the buccal route maybe advantageous for the delivery of peptides that are susceptible to such activities. The inclusion of aminopeptidase inhibitors in buccal bioadhesive delivery systems could improve buccal bioavailability of Leu-enkephalin. We suggest that compared with the existing in vitro metabolism methods, the analysis of peptide or protein metabolism on intact buccal mucosa could better predict the degradation of the drug as it crosses the tissue.

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.

In vitro skin penetration and degradation of peptides and their analysis using a kinetic model.

The main purpose of this study was to estimate the net percutaneous absorption of physiologically active peptides in vitro. The degradation of two peptides, Leu-enkephalin (Enk) and Tyr-Pro-Leu-Gly amide (TPLG), during skin penetration and on the dermal side following penetration, and the prevention of degradation by some protease inhibitors, were investigated using rat skin in vitro. In addition, these permeation and degradation data were analyzed using a kinetic model. These peptides were rapidly degraded in the receptor fluid of a Franz diffusion cell (rate constant: 0.977 h(-1) for Enk and 0.250 h(-1) for TPLG). The addition of phenylmethylsulfonyl fluoride (PMSF) and phenanthroline and the pretreatment of skin with these inhibitors prevented almost completely any degradation in the receptor fluid and skin, respectively. The pretreatment of skin with PMSF and phenanthroline had no effect on the penetration of dextran (1000 Da). The degradation rate constant during skin penetration, calculated from the difference in the penetration rate constants via pretreated and untreated skins, was also high (0.037 h(-1) for Enk and 0.050 h(-1) for TPLG). A kinetic model including an input rate (zero-order), the permeation rate across the viable skin (first-order) and the degradation rate in skin (first-order) was sufficient to describe the apparent steady-state flux of the peptides through skin. We have, thus, established a method for measuring the true flux of peptides across skin in vitro and a kinetic model which simply describes the skin penetration of peptides.

Mutation of tyrosine 318 (Y318F) in the delta-opioid receptor attenuates tyrosine phosphorylation, agonist-dependent receptor internalization, and mitogen-activated protein kinase activation.

Opioid receptors are known for their ability to activate diverse second messenger systems. Previously, we showed that selective delta-opioid agonists were able to induce the rapid tyrosine phosphorylation of delta-opioid receptors (delta-ORs) through Src. Src-dependent tyrosine phosphorylation of delta-ORs appears to be important for activation of the mitogen-activated protein kinase cascade and for receptor sequestration into clathrin-coated endosomes, as the Src antagonist, PP1, inhibited both. In an attempt to clarify the role of tyrosine phosphorylation in delta-OR signalling and regulation, we constructed a mutant receptor in which the tyrosine located in the conserved NPXXY motif of the C-terminus was replaced by a phenylalanine (Y318F-delta-OR). Mutation of Y318 resulted in a receptor that was comparable to the wild type in its expression level in HEK-293 cells and in its affinity for opioid ligands. Both receptors showed effective coupling to G proteins and were capable of inhibiting forskolin-stimulated cAMP accumulation with similar potencies. However, the mutant receptor was able to stimulate (35)S-GTPgammaS binding with a lower EC(50) than the wild type receptor. The stimulation of tyrosine phosphorylation in delta-ORs by [D-Thr(2)]-Leu-enkephalin-Thr (DTLET) was significantly less in cells expressing the Y318F-delta-OR than in cells expressing the wild type. In addition, both rapid receptor internalization and down-regulation were markedly attenuated in the mutant. Finally, the mutant receptor was unable to induce a robust activation of the MAPK pathway, suggesting that tyrosine phosphorylation of the delta-OR protein is important for this signalling pathway. These findings implicate tyrosine phosphorylation of Y318 in receptor signalling and agonist-mediated regulation.

TR146 cells grown on filters as a model of human buccal epithelium: V. Enzyme activity of the TR146 cell culture model, human buccal epithelium and porcine buccal epithelium, and permeability of leu-enkephalin.

The objective of the present study was to characterise the TR146 cell culture model as an in vitro model of human buccal mucosa with respect to the enzyme activity in the tissues. For this purpose, the contents of aminopeptidase, carboxypeptidase and esterase in homogenate supernatants of the TR146 cell culture model, and human and porcine buccal epithelium were compared. The esterase activity in the intact cell culture model and in the porcine buccal mucosa was compared. Further, the TR146 cell culture model was used to study the permeability rate and metabolism of leu-enkephalin. The activity of the three enzymes in the TR146 homogenate supernatants was in the same range as the activity in homogenate supernatants of human buccal epithelium. In the TR146 cell culture model, the activity of aminopeptidase (13.70+/-2.10 nmol/min per mg protein) was approx. four times the activity of carboxypeptidase (3.73+/-0.53 nmol/min per mg protein), whereas the level of esterase activity was significantly higher (223.39+/-69.82 nmol/min per mg protein). In the TR146 cell culture model, the apical esterase activity was found significantly higher than the basal activity, and found comparable to the porcine buccal mucosa. However, the esterase activity on the serosal side of the porcine buccal mucosa was higher than in the TR146 cell culture model. Approx. 1.5% of leu-enkephalin permeated the TR146 cell layers within 5 h (P(app) 7.38+/-0.83x10(-7) cm/s) and approx. 77% of intact peptide was still present in the donor phase after 5 h. The present study suggests that the TR146 cell culture model is a valuable in vitro model for permeability and metabolism studies with enzymatically labile drugs, such as leu-enkephalin, intended for buccal drug delivery.

Degradation kinetics of leucine5-enkephalin by plasma samples from healthy controls and various patient populations: in vitro drug effects.

Incubation of [3H]tyrosine leucine5-enkephalin with platelet-poor human plasma (final concentration 1 x 10(-8) M; 1:9 ratio to Trizma base buffer, pH 7.4) resulted in rapid and complete peptide degradation in each of the subjects studied, with more than 95% of the initial labeled tyrosine consistently recovered as the free amino acid (< or =30 minutes). Essentially, and irrespective of the incubation time (1-180 minutes), tyrosine was the only Leu metabolite formed; we were unable to identify significant amounts (> or =3%) of any other possible labeled or nonlabeled Leu degradation fragments. Neither gender (64 men and 20 women; samples tested individually), age (men, 23-70; women, 25-65 years), nor the subjects' medical condition appeared to make a significant difference in either the t1/2 of Leu elimination, the initial velocity of this reaction (x +/- SD, median, minimum and maximum of 12.0 +/- 0.9, 12.0, and 10.6-13.7 minutes; 1.2 +/- 0.3, 1.1, and 0.6-2.0 pg/min, respectively), or in the Km and Vmax values for aminopeptidase Leu degradation (x +/- SD; 0.81 +/- 0.01 mM and 14.30 +/- 1.17 micromol/L/min, respectively). Subjects were diagnosed as chronic schizophrenics (n = 15), polydrug abusers including alcohol (n = 9) and polydrug abusers excluding alcohol (n = 8), chronic alcoholics (n = 12), and migraineurs (n = 10) during or outside an acute migraine episode; for comparison we used a group of gender-matched (20 men and 10 women), age-comparable, drug-free, healthy volunteers. Differences in plasma storage time or repeated sample freezing and thawing failed to alter significantly any of these kinetic parameters of Leu metabolism or to change the identity and/or relative ratio of the products formed. The Leu degradation rate was pH and temperature dependent (optimum, 7.4 and 37 degrees C, respectively). Leu degradation was strongly and similarly inhibited by puromycin, bacitracin, and bestatin (IC50 [+/- SD] of 1.4 +/- 0.2 micromol/L) and to a lesser extent by various L-tyrosine-containing Leu fragments. The kinetics of this reaction was not significantly affected by either thiorphan, N-carboxyphenylmethyl leucine, or any other of a number of monoamine neurotransmitters, substances of abuse, nonsteroidal anti-inflammatory agents, and miscellaneous compounds tested (concentration up to 10(-4) mol/L).

Development of novel lipophilic derivatives of DADLE (leucine enkephalin analogue): intestinal permeability characateristics of DADLE derivatives in rats.

PURPOSE: The objective of this study is to examine the intestinal permeability of novel lipophilic derivatives of DADLE (Tyr-D-Ala-Gly-Phe-D-Leu), an enkephalin analogue, using isolated rat intestinal membranes. METHODS: The novel lipophilic derivatives of DADLE were synthesized by chemical modification with various fatty acids at the C terminus. The pharmacological activities of these DADLE derivatives were assessed by a hot plate test. The intestinal permeability of these derivatives was estimated by the in vitro Ussing chamber method. RESULTS: We obtained four different DADLE derivatives including acetyl-DADLE (DADLE-C2), butyryl-DADLE (DADLE-C4), caproyl-DADLE (DADLE-C6), and caprylyl-DADLE (DADLE-C8). All the derivatives of DADLE had at least 75% of the activity of native DADLE, suggesting that chemical modification of DADLE at the C terminus did not markedly affect its pharmacological activity. These DADLE derivatives were more stable than native DADLE in jejunal and colonic homogenates. A "bell-shaped" profile was observed between the apparent permeability coefficients (Papp) of DADLE derivatives and lipophilicity. In particular, DADLE-C4 had the greatest permeability characteristics across the intestinal membrane of the acyl derivatives studied in this experiment. The permeability of DADLE-C4 across the jejunal membrane was further improved in the presence of puromycin, amastatin, and sodium glycocholate (NaGC), all at a concentration of 0.5 mM. CONCLUSIONS: We suggest that the combination of chemical modification with butyric acid and the application of a protease inhibitor are effective for improving the absorption of DADLE across the intestinal membrane.

Chemical and enzymatic stability as well as transport properties of a Leu-enkephalin analogue and ester prodrugs thereof.

The Leu-enkephalin analogue (Tyr-D-Ala-Gly-Phe-Leu-NH(2)) was synthesized together with three esters prodrugs hereof. The prodrugs synthesized were the O-acetyl, O-propionyl and O-pivaloyl99%) and in good yields (60-75%). The chemical and enzymatic stability of the prodrugs has been investigated in detail. The prodrugs studied are quite chemically stable and the degradation of the prodrugs follows the pattern previously shown for similar esters (U-shaped pH-profile; maximal stability at pH 4-5). The prodrugs are degraded quantitatively in plasma to the parent peptide with half-lives in the range 2.9 min-2.6 h. Type B esterases were shown to be involved in the degradation as the half-lives increased in the presence of paraoxon. No significant stabilization was seen in 10% porcine gut homogenate. Half-lives in the same order were seen for the analogue and the prodrugs in pure Leucine aminopeptidase solution. The analogue was stable in Carboxypeptidase A solution whereas a faster degradation of the prodrugs was seen in this media. Furthermore the transport properties of the compounds has been studied. A P(app) value of 0.284x10(-6) cm/s for the analogue was obtained for the transport across Caco-2 cell monolayers in the BL-AP direction. The P(app) values were increased by a factor of 2, 7 and 18 for the acetyl-, propionyl- and pivaloylprodrug. The increase could be explained by higher lipofilicities of the prodrugs compared to the analogue.

Optimizing oral absorption of peptides using prodrug strategies.

Prodrug strategies applied to peptides have tended to focus on modification of a single functional group (e.g., N-terminal end). Recently, our laboratory introduced the concept of making cyclic prodrugs of peptides as a way to modify their physicochemical properties sufficiently to allow them to permeate biological barriers (i.e., intestinal mucosa). This cyclization strategy required the development of new 'chemical linkers,' including an acyloxyalkoxy linker, a phenylpropionic acid linker, and a coumarinic acid linker. All three chemical linkers were designed to be susceptible to esterase metabolism (slow step), leading to a cascade of chemical reactions (fast steps) that result in release of the peptide. These cyclic prodrug strategies have been applied to opioid peptides in an attempt to stabilize them to metabolism and/or improve their intestinal mucosal permeation. Specifically, we prepared acyloxyalkoxy-, phenylpropionic acid- and coumarinic acid-based cyclic prodrugs of [Leu(5)]-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) and its metabolically stable analog DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) and determined their metabolic and biopharmaceutical properties. The cyclic prodrugs of these opioid peptides were shown to have: (i) favorable physicochemical properties (e.g., increased lipophilicity) for membrane permeation; (ii) unique solution structures (e.g., beta-turns) that reduce their hydrogen bonding potential; and (iii) metabolic stability to exo- and endopeptidases. The cell membrane permeation characteristics of [Leu(5)]-enkephalin, DADLE and the cyclic peptide prodrugs were evaluated using Caco-2 cell monolayers, a cell culture model of the intestinal mucosa. The phenylpropionic acid- and coumarinic acid-based cyclic prodrugs of [Leu(5)]-enkephalin and DADLE were shown to have significantly better cell permeation characteristics than the parent opioid peptides. Furthermore, these cyclic prodrugs were shown to be transcellular permeants (in contrast to the opioid peptides, which are paracellular permeants), and were not substrates for polarized efflux systems. Surprisingly, the acyloxyalkoxy-based prodrugs of [Leu(5)]-enkephalin and DADLE were shown to exhibit very low permeation through Caco-2 cell monolayers, which could be attributed to their substrate activity for efflux systems.