New Combined Eye Gel with Alpha-2-Beta Interferon.

The results of the development of combined eye gel with interferon alpha-2-beta are presented. Experimental samples of the gel based on different gelling agents were prepared and their biotechnological and technological characteristics (the absence of the cytotoxic effect, aggregation stability, osmotic activity, bioadhesion, and rheological parameters) were evaluated. The composition with hydroxyethyl cellulose, Natrosol 250HHX, in a concentration of 1.5% as a gelling agent showed the best results and the best one-year stability.

Hydrophilic interaction liquid chromatography for dalargin separation from its structural analogues and side products.

Retention behaviour of Dalargin and five peptide analogues of Leu-enkephalin, has been extensively studied by hydrophilic interaction liquid chromatography (HILIC) on a bare silica stationary phase (Atlantis® HILIC silica). The influence of buffer pH, ionic strength, and organic modifier content on peptide retentions was examined. Variation of organic modifier content (70-90% ACN) shows that, as expected, the most polar peptide, Dalargin, is the most retained. Moreover, at acidic pH, the retention mechanism for all the peptides studied seems to rely, mainly, on adsorption phenomenon. By varying the pswH buffer (between 4.4-7.5), we observed that the retention of all the peptides was mainly governed by their total number of charges, whatever the variation (increase or decrease) of their retention factor. At pswH 7.5, an increase of the cationic counter-ion concentration (NH4+) lead to a decrease of the retention factor of Dalargin, suggesting a weak cation exchange for this peptide. For the other peptides, the variation of the retention factors was negligible between 5-15mM. Above 15mM, the retention factors of all the peptides increased, probably due to an increase of the water layer thickness at the surface of the stationary phase. In the second part of the study, qualitative analysis of non-purified dalargin, resulting from solid-phase synthesis, was realized. Optimisation of the separation of the target peptide from its side products has been first performed with UV detection. Then, by coupling the HILIC column with ESI-MS, using the optimal separation conditions, it was possible to identify Dalargin and to propose the amino-acids sequence of its side-products.

Use of electrochemical oxidation and model peptides to study nucleophilic biological targets of reactive metabolites: the case of rimonabant.

Electrochemical oxidation of drug molecules is a useful tool to generate several different types of metabolites. In the present study we developed a model system involving electrochemical oxidation followed by characterization of the oxidation products and their propensity to modify peptides. The CB1 antagonist rimonabant was chosen as the model drug. Rimonabant has previously been shown to give high covalent binding to proteins in human liver microsomes and hepatocytes and the iminium ion and/or the corresponding aminoaldehyde formed via P450 mediated α-carbon oxidation of rimonabant was proposed to be a likely contributor. This proposal was based on the observation that levels of covalent binding were significantly reduced when iminium species were trapped as cyanide adducts but also following addition of methoxylamine expected to trap aldehydes. Incubation of electrochemically oxidized rimonabant with peptides resulted in peptide adducts to the N-terminal amine with a mass increment of 64 Da. The adducts were shown to contain an addition of C5H4 originating from the aminopiperidine moiety of rimonabant. Formation of the peptide adducts required further oxidation of the iminium ion to short-lived intermediates, such as dihydropyridinium species. In addition, the metabolites and peptide adducts generated in human liver microsomes were compared with those generated by electrochemistry. Interestingly, the same peptide modification was found when rimonabant was coincubated with one of the model peptides in microsomes. This clearly indicated that reactive metabolite(s) of rimonabant identical to electrochemically generated species are also present in the microsomal incubations. In summary, electrochemical oxidation combined with peptide trapping of reactive metabolites identified a previously unobserved bioactivation pathway of rimonabant that was not captured by traditional trapping agents and that may contribute to the in vitro covalent binding.

Development and in vivo characterization of a novel peptide drug delivery system providing extended plasma half life.

It was the aim of this study to develop a sustained parenteral peptide (DALCE) delivery system by the immobilization of DALCE to thiolated carboxymethyl dextran-cysteine (CMD-Cys) via disulfide bond formation. The resulting CMD-Cys-DALCE conjugate displayed a 22.6±7.9% (m/m) of DALCE (mean±S.D.; n=3). The conjugation of DALCE with CMD-Cys was confirmed by FTIR-ATR spectroscopy. In vitro release studies of conjugate CMD-Cys-DALCE in the presence of 2 µM/ml reduced glutathione (GSH) being also available in the plasma showed a sustained peptide release over a time period of 8 h, because of thiol/disulfide exchange reactions. For in vivo pharmacokinetic study, DALCE and CMD-Cys-DALCE were administered intravenously to male Sprague-Dawley rats at a dose of 1mg/kg. The AUC(0-8) (ng.min/ml) was determined to be 268848±924 and 40019±495 for CMD-Cys-DALCE and DALCE, respectively. The mean residence time (MRT) was determined to be 256±8 and 53.1±9.5 min for CMD-Cys-DALCE and for DALCE, respectively. CMD-Cys-DALCE showed a more than 5-fold increased elimination half-life (p<0.01), 3-fold decreased volume of distribution (p<0.01) and a 6.7-fold decreased plasma clearance rate (p<0.01) compared to DALCE. According to these findings, CMD-Cys-DALCE seems to act as prodrug by improving half-life and decreasing plasma clearance.

Induction of an in vitro reversible hypometabolism through chitosan-based nanoparticles.

OBJECTIVE: Chitosan-based nanoparticles (NPs) were prepared to promote intracellular sustained delivery of the synthetic delta opioid D-Ala(2)-D-Leu(5)-enkephalin (DADLE), prolonging peptide activity and inducing a safe and reversible hypometabolic state. MATERIALS AND METHODS: NPs were prepared by combining ionotropic gelation and ultrasonication treatment. NP uptake studies and the effects of encapsulated DADLE on HeLa cells proliferation were tested by transmission electron microscopy (TEM) analysis, by immuno-fluorescence and immuno-cytochemistry. RESULTS: DADLE-loaded NPs are produced with suitable characteristics, a satisfactory process yield (55.4% ± 2.4%) and encapsulation efficiency (64.6% ± 2.1%). NPs are effective in inducing a hypometabolic stasis at a 10(-4) M DADLE concentration. Moreover, as seen from the immunofluorescence study, the effect persists through the recovery period (72 h). Indeed, NPs labelled by anti-enkephalin antibody inside cell nucleus reassert that the in vivo release of the peptide can be prolonged with respect to the case of free peptide supply. CONCLUSION: The nanoparticulate drug delivery system described seems to be effective in inducing and prolonging a sort of hibernation-like state in the cells.

An ion pairing approach to increase the loading of hydrophilic and lipophilic drugs into PEGylated PLGA nanoparticles.

The aim of this study was to enhance the loading of dalargin (enkephalin derivatives) a hydrophilic drug and loperamide HCl (non-opiate antidiarrheal agent) a lipophilic drug candidates within PEGylated nanoparticles. A novel nanoencapsulation method based on the concept of s/o/w and ion pairing followed by solvent diffusion was adopted. The copolymers with three different mPEG densities (5%, 12% and 17%) were employed separately in combination with two different grades of dextran sulphate (DS) 5000 and 500,000 MW in the preparations. Nanoparticles prepared from copolymers with increasing mPEG densities, showed an insignificant (p>0.05) increasing trend of drug loading, this was however significantly increased when DS5000 was included in the preparations. The particle size remains unchanged after dalargin loading, with no significant (p>0.05) alteration in the neutral zeta potential compared to that of the preparations without DS5000. Considering that a dalargin ion pair could also have a neutral charge, it was not advisable to conclude its incorporation, as the size remain unchanged, which would otherwise increase if an ion pair was incorporated within the core of nanoparticles. Therefore, it was expected that a dalargin ion pair might be located outside the core as a separate particulate entity or reside in the hydrophilic shell of the nanoparticles. A loperamide HCl ion pair showed significant (p<0.05) increase in size when incorporated; at the same time it provided a neutral zeta potential despite adding negatively charged DS5000 in the preparation, hence it seemed encapsulated. Inclusion of DS500,000 in the preparation further increased the drug loading of dalargin and loperamide HCl. However, a significant (p<0.05) negative zeta potential was noted in both cases which suggested that excess charge was still available on the surface of nanoparticles which could trap further amounts of drug on the surface rather than inside the core of nanoparticles. During in vitro evaluation of drug loaded nanoparticles, dalargin released as quickly as free drug, when loperamide HCl showed almost burst free sustained release profile with respect to the release of their free drug solutions, suggested that ion pairing approach was more pronounced for loperamide HCl formulation.

Comparison and validation of drug loading parameters of PEGylated nanoparticles purified by a diafiltration centrifugal device and tangential flow filtration.

This article describes drug loading validation of nanoparticles. Ultracentrifugation was avoided because of problems arising from small-sized particles. Ultrafiltration was adopted in two different modes followed by monitoring of polyvinyl alcohol (PVA), dextran sulfate (DS), and loperamide HCl contents. Diafiltration centrifugation removed all PVA at the fourth cycle and provided significantly (p = .000, .017) higher drug loading values compared with tangential flow filtration (TFF). This was due to residual PVA associated with the nanoparticles. TFF enabled satisfactory dry weight recovery (101.66 +/- 4.45%, n = 3) of nanoparticles during extended purification. Indirect drug loading (from the purification curve) was not significantly different (p = .450, .487) to the direct drug loading values. Encapsulation parameters were obtained from the purification curve once quantitative estimation of the all formulation components was established.

Development of a chitosan-based nanoparticle formulation for delivery of a hydrophilic hexapeptide, dalargin.

Nanoparticle based delivery systems can offer opportunities for targeting, controlled release, and enhanced stability of their drug, protein, or gene therapy payload. This study investigated the use of chitosan in combination with the ionic additives sulfobutyl-ether-7-beta-cyclodextrin (SB-CD) or SB-CD/dextran sulfate (SB-CD/DS) mixture in comparison with chitosan: DS in the formulation of nanoparticles incorporating the hexapeptide dalargin. The physical characteristics (particle size, zeta potential), entrapment and loading efficiency, and release of dalargin were quantified. It was demonstrated that anionic cyclodextrin, SB-CD, can be used in complex coacervation with chitosan, with and without the presence of DS, to form nanoparticles. The presence of SB-CD or DS in the nanoparticle formulation and the weight ratio of chitosan to anionic additive(s) influenced the physical properties of the nanoparticles and their ability to carry dalargin. In addition, the particle size of nanoparticles was also affected by the molecular weight of chitosan and DS. The use of either DS or SB-CD/DS mixture produced chitosan nanoparticles with small particle size, high dalargin entrapment efficiency, enhanced peptide stability, and sustained release characteristics.

Effect of formulation factors on incorporation of the hydrophilic peptide dalargin into PLGA and mPEG-PLGA nanoparticles.

The objective of this study was to examine formulation factors that influence the incorporation of the hydrophilic peptide dalargin into poly(D, L-lactic-co-glycolic acid) (PLGA) and methoxy-polyethylene glycol (mPEG)-PLGA nanoparticles. In particular, the effect of ionic additives and nanoparticle method of preparation on the incorporation of dalargin and resultant nanoparticle properties was investigated. Biodegradable nanoparticles were prepared from mPEG-PLGA and PLGA by both solvent evaporation and solvent diffusion methods with inclusion of ionic additives of dextran sulphate (DS), sulfobutyl ether-beta-cyclodextrin (SB-CD), or sodium dodecyl sulfate (SDS). The resultant nanoparticles were analyzed for their mean particle size and size distribution, zeta-potential, peptide loading, yield, and morphology. The inclusion of ionic additives in the nanoparticle formulation significantly influenced dalargin entrapment efficiency (EE). For example, with the PLGA/SDS formulation EE increased from 13.3% to 91.2% and from 4.1% to 68.6% with the solvent diffusion and evaporation methods, respectively. The inclusion of ionic surfactant SDS has also lead to the formation of smaller size of nanoparticles. Isothermal titration microcalorimetry revealed a strong interaction between dalargin and DS, medium level interaction with SDS, and weak interaction with SB-CD. The results of this study suggest that a strong ionic interaction between peptides and additives may lead to enhanced peptide incorporation but also increased particle size. Intermediate ionic interaction, especially when it is associated with the formation of reversed micelles in a hydrophobic polymer solution, could be used to enhance the incorporation of hydrophilic peptides in PLGA and mPEG-PLGA nanoparticles.

Effect of dalargin on DNA synthesis in the gastric mucosa of albino rats.

3H-thymidine autoradiography and chemiluminescence study demonstrated pronounced effect of dalargin on the state of the gastric mucosa in albino rats. Dalargin stimulated DNA synthesis in the epithelium of the gastric mucosa and increased buffer capacity of its antiradical and antioxidant systems. Dalargin analogue not containing arginine ([D-Ala2]-leu-enkephalin) had little effect on these parameters. NO synthase inhibitor L-NAME abolished the effects of dalargin on DNA synthesis in the gastric mucosa. Our results suggest that the NO system plays an important role in the effect of dalargin on the gastric mucosa.

Diastereomeric differentiation of peptides with CuII and FeII complexation in an ion trap mass spectrometer.

Complexation by transition metal ions (CuII and FeII) was successfully used to differentiate the diastereomeric YAGFL, YDAGFL and Y(D)AGF(D)L pentapeptides by electrospray ionization-ion trap mass spectrometry in the positive ion mode using low-energy collision conditions. This distinction was allowed by the stereochemical effects due to the (D)Leu/(L)Leu and the (D)Ala/(L)Ala residues yielding various steric interactions which direct relative dissociation rate constants of the binary [(M - H) + MeII]+ complexes (Me = Cu or Fe) subjected to low-energy, collision-induced dissociation processes. The interpretation of the collision-induced dissociation spectra obtained from the diastereomeric cationized peptides allowed the location of the deprotonated site(s), leading to the postulation of ion structures and fragmentation pathways for both the [(M - H) + CuII]+ and [(M - H) + FeII]+ complexes, which differed significantly. With CuII, consecutive fragmentations, initiated by the decarboxylation at C-terminus, were favored relative to sequence product ions. On the other hand, with FeII, competitive fragmentations resulting in abundant sequence product ions and significant internal losses were preferred. This could be explained by different localizations of the negative charge, which directs the orientation of both the [(M - H) + CuII]+ and [(M - H) + FeII]+ binary complexes fragmentations. Indeed, the free negative charge of the [(M - H) + CuII]+ ions was mainly located at one oxygen atom: either at the C-terminal carboxylic group or, to a minor extent, at the Tyr phenol group (i.e. zwitterionic forms). On the other hand, the negative charge of the [(M - H) + FeII]+ ions was mainly located at one of the nitrogen atoms of the peptide backbone and coordinated to FeII (i.e. salt non-zwitterionic form).Moreover, this study reveals the particular behavior of CuII reduced to CuI, which promotes radical losses not observed from the peptide-FeII complexes. Finally, this study shows the analytical potentialities of the complexation of transition metal ions with peptides providing structural information complementary to that obtained from low-energy, collision-induced dissociation processes of protonated or deprotonated peptides.

Stability of oxymethyl-modified coumarinic acid cyclic prodrugs of diastereomeric opioid peptides in biological media from various animal species including human.

In vitro stability studies of oxymethyl-modified coumarinic acid (OMCA) cyclic prodrugs of the diastereomeric opioid peptides DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), [Ala2,D-Leu5]-Enk (H-Tyr-Ala-Gly-Phe-D-Leu-OH), [D-Ala2,Leu5]-Enk (H-Tyr-D-Ala-Gly-Phe-Leu-OH), and [Ala2,Leu5]-Enk (H-Tyr-Ala-Gly-Phe-Leu-OH) were conducted to evaluate how the chirality of specific amino acid residues (Ala2 and Leu5) in the peptide portion affects their bioconversion by esterases. The stability studies were conducted at 37 degrees C in plasma and tissue homogenates (liver and brain) from five animal species (rat, mouse, canine, guinea pig, and hamster) and human in an attempt to identify an animal species that had a "prodrug bioconversion profile" comparable to that of humans. Initially, the total esterase activity in these biological media was measured using p-nitrophenyl butyrate (PNPB) as a substrate. By repeating this activity assay in the presence of paraoxon, a potent esterase B inhibitor, it was possible to estimate the relative amounts of esterases B and esterases A/C in a biological sample. Stability studies of the cyclic prodrugs were carried out under identical conditions, that is, in the presence and absence of paraoxon. Significant differences in the rates of hydrolysis of the cyclic prodrugs were observed, particularly between cyclic prodrugs with differences in the chirality of the amino acid on the C-terminus of the peptide portion, for example, L-amino acids at the C-terminus hydrolyzed more rapidly than D-amino acids. This stereoselective hydrolysis was independent of the animal species but tended to be more pronounced in brain and liver homogenates compared to plasma. Increased esterase specific activity, as measured by PNPB, in the biological media did not necessarily correlate with increased bioconversion rates of the cyclic prodrugs. The enzymatic stability profiles of the cyclic prodrugs in biological media from canine and guinea pig most closely resembled the profiles from human biological media. Therefore, canine and guinea pig appear to be the most relevant animal models for conducting pharmacokinetic studies on these cyclic prodrugs of opioid peptides.

Double-coated poly (butylcynanoacrylate) nanoparticulate delivery systems for brain targeting of dalargin via oral administration.

The aim of this study is to evaluate oral administration of poly (butylcyanoacrylate) nanoparticulate delivery systems (PBCA-NDSs), double-coated with Tween 80 and poly (ethylene) glycol (PEG) 20000 for brain delivery of hexapeptide dalargin, an anti-nociceptive peptide that does not cross blood-brain barrier (BBB) by itself. Studies have proven the brain uptake of Tween 80 overcoated nanoparticles after intravenous administration, but studies for brain delivery of nanoparticles after oral administration had been limited due to reduced bioavailability of nanoparticles and extensive degradation of the peptide and/or nanoparticles by gastrointestinal enzymes. To address this problem, dalargin-loaded PBCA-NDS were successively double-coated with Tween 80 and PEG 20000 in varied concentrations of up to 2% each. Measurement of in vivo central anti-nociceptive effect of dalargin along with a dose response curve was obtained by the tail flick test following the oral administration of PBCA-NDSs to mice. Results from the tail flick test indicated that significant dalargin-induced analgesia was observed from PBCA-NDSs with double-coating of Tween and PEG in comparison with single-coating of either Tween or PEG. Hence, it could be concluded that surface coated PBCA-NDS can be used successfully for brain targeting of dalargin or other peptides administered orally. However, further studies are required to elucidate the exact transport mechanism of PBCA-NDSs from gastrointestinal tract to brain.

Delta opioid peptide (D-Ala 2, D-Leu 5) enkephalin: linking hibernation and neuroprotection.

Hibernation is a potential protective strategy for the peripheral, as well as for the central nervous system. A protein factor termed hibernation induction trigger (HIT) was found to induce hibernation in summer-active ground squirrels. Purification of HIT yielded an 88-kD peptide that is enriched in winter hibernators. Partial sequence of the 88-kD protein indicates that it may be related to the inhibitor of metalloproteinase. Using opioid receptor antagonists to elucidate the mechanisms of HIT, it was found that HIT targeted the delta opioid receptors. Indeed, delta opioid (D-Ala 2, D-Leu 5) enkephalin (DADLE) was shown to induce hibernation. Specifically, HIT and DADLE were found to prolong survival of peripheral organs, such as the lung, the heart, liver, and kidney preserved en bloc or as a single preparation. In addition, DADLE has been recently demonstrated to promote survival of neurons in the central nervous system. Exposure to DADLE dose-dependently enhanced cell viability of cultured primary rat fetal dopaminergic cells. Subsequent transplantation of these DADLE-treated dopaminergic cells into the Parkinsonian rat brain resulted in a two-fold increase in surviving grafted cells. Interestingly, delivery of DADLE alone protected against dopaminergic depletion in a rodent model of Parkinson s disease. Similarly, DADLE blocked and reversed the dopaminergic terminal damage induced by methamphetamine (METH). Such neuroprotective effects of DADLE against METH neurotoxicity was accompanied by attenuation of mRNA expressions of a tumor necrosis factor p53 and an immediate early gene c-fos. In parallel to these beneficial effects of DADLE on the dopaminergic system, DADLE also ameliorated the neuronal damage induced by ischemia-reperfusion following a transient middle cerebral artery occlusion. In vitro replication of this ischemia cell death by serum-deprivation of PC12 cells revealed that DADLE exerted neuroprotection in a naltrexone-sensitive manner. These results taken together suggest that DADLE stands as a novel therapeutic agent. In this review paper, we present laboratory evidence supporting the use of DADLE for protection of peripheral and central nervous system.

The effect of substitution patterns on the release rates of opioid peptides DADLE and [Leu(5)]-enkephalin from coumarin prodrug moieties.

A coumarin-based prodrug system has been developed in our laboratory for the preparation of esterase-sensitive prodrugs of amines, peptides, and peptidomimetics. The drug release rates from this prodrug system were found to be dependent on the structural features of the drug moiety. The effect of the phenyl ring substitutions on the release kinetics of such prodrugs of model amines was examined recently and it was found that appropriately positioned alkyl substituents on the phenyl ring could help to facilitate the release. Aimed at further understanding the structure-release rate relationship of the coumarin-based cyclic prodrugs, we synthesized and examined a series of substituted coumarinic acid derivatives of opioid peptides, DADLE, and [Leu(5)]-enkephalin.

Improved brain uptake and pharmacological activity of dalargin using a peptide-vector-mediated strategy.

The blood-brain barrier restricts the passage of substances into the brain. Neuropeptides, such as enkephalins, cannot be delivered into the brain when given systemically because of this barrier. Therefore, there is a need to develop efficient transport systems to deliver these drugs to the brain. Recently, we have demonstrated that conjugation of doxorubicin or penicillin to peptide vectors significantly enhances their brain uptake. In this study, we have conjugated the enkephalin analog dalargin with two different peptide vectors, SynB1 and SynB3, to improve its brain delivery and its pharmacological effect. We show by in situ brain perfusion that vectorization markedly enhances the brain uptake of dalargin. We also show using the hot-plate model that this enhancement in brain uptake results in a significant improvement in the observed antinociceptive effect of dalargin. These results support the usefulness of peptide-mediated strategies for improving the availability and efficacy of central nervous system drugs.

New development in the tritium labelling of peptides and proteins using solid catalytic isotopic exchange with spillover-tritium.

The mechanism of the reaction of high temperature solid state catalytic isotope exchange (HSCIE) of hydrogen in peptides with spillover-tritium at 140-180 degrees C was analyzed. This reaction was used for preparing [(3)H]enkephalins such as [(3)H]DALG with specific activity of 138 Ci/mmol and [(3)H]LENK with specific activity of 120 Ci/mmol at 180 degrees C. The analogues of [(3)H]ACTG(4-10) with specific activity of 80 Ci/mmol, [(3)H]zervamicin IIB with specific activity of 70 Ci/mmol and [(3)H]conotoxin G1 with specific activity 35 Ci/mmol were produced. The obtained preparations completely retained their biological activity. [(3)H]Peptide analysis using (3)H NMR spectroscopy on a Varian UNITY-600 spectrometer at 640 MHz was carried out. The reaction ability of amino fragments in HSCIE was shown to depend both of their structures and on the availability and the mobility of the peptide chain. The reaction of HSCIE with the beta-galactosidase from Termoanaerobacter ethanolicus was studied. The selected HSCIE conditions allow to prepare [(3)H] beta-galactosidase with specific activity of 1440 Ci/mmol and completely retained its the enzymatic activity.

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.

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 degrees 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 high-performance 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., P-glycoprotein) in the blood-brain barrier significantly restricted their access to the brain.

Characterization of the efflux transporter(s) responsible for restricting intestinal mucosa permeation of an acyloxyalkoxy-based cyclic prodrug of the opioid peptide DADLE.

PURPOSE: To elucidate the efflux transporter(s) responsible for restricting the permeation of an acyloxyalkoxy-based cyclic prodrug of the opioid peptide DADLE (AD) through Caco-2 cell monolayers. METHODS: The cellular permeation characteristics of AD were investigated using Caco-2 cells, Madin-Darby canine kidney wild-type II cells (MDCK-WT), MDCK cells transfected with the human MDR1 gene (MDCK-MDR1), and MDCK cells transfected with the human MRP2 gene (MDCK-MRP2). These cells were grown as monolayers onto microporous membranes. The disappearance of AD from the donor side and its appearance on the receiver side were monitored by high-performance liquid chromatography. The substrate activity of AD for P-glycoprotein (P-gp) was determined using GF120918, a known P-gp specific inhibitor. The substrate activity of AD for MRP2 was determined by using cyclosporin A, a known MRP2 and P-gp inhibitor. RESULTS: In Caco-2 cells, the ratio of the apparent permeability coefficients (Papp) of AD flux measured in the basolateral (BL) to apical (AP) direction vs. the flux in the AP-to-BL direction (Papp BL-to-AP/ Papp AP-to-BL) was 99. In the presence of 2 microM GF120918 or 25 microM cyclosporin A. the Papp BL-to-AP/Papp AP-to-BL ratio was decreased to 11. In MDCK-WT, MDCK-MDR1, and MDCK-MRP2 cells, the Papp BL-to-AP/Papp AP-to-BL ratios of AD were 4.7, 10, and 5.8, respectively. A mixture of GF120918 (2 microM) and cyclosporin A (25 microM) decreased the Papp BL-to-AP/Papp AP-to-BL ratios of AD in MDCK-WT, MDCK-MDR1, and MDCK-MRP2 cells to 1.2,1.8, and 2.3, respectively. CONCLUSIONS: These data suggest that AD is a much better substrate for P-gp than MRP2 and that the restricted permeation of this cyclic prodrug in Caco-2 cells and in the intestinal mucosa probably is due primarily to its substrate activity for P-gp.

Characterization of the efflux transporter(s) responsible for restricting intestinal mucosa permeation of the coumarinic acid-based cyclic prodrug of the opioid peptide DADLE.

PURPOSE: To elucidate the efflux transporter(s) responsible for restricting the permeation of a coumarinic acid-based cyclic prodrug of the opioid peptide DADLE (CD) thorough Caco-2 cell monolayers. METHODS: The cellular permeability characteristics of CD were investigated using Caco-2 cells, Madin-Darby canine kidney-wild type II cells (MDCK-WT). MDCK cells transfected with the human MDR1 gene (MDCK-MDR1), and MDCK cells transfected with human MRP2 gene (MDCK-MRP2). These cells were grown as monolayers onto microporous membranes. The disappearance from the donor side and appearance on the receiver side of CD were monitored by HPLC. The substrate activity of CD for P-gp was determined by using GF120918. a known P-gp specific inhibitor. The substrate activity of CD for MRP2 was determined by using cyclosporin A (CsA), a known MRP2 and P-gp inhibitor. RESULTS: In Caco-2 cells, the ratio of the apparent permeability coefficients (Papp) of CD flux in the basolateral (BL) to apical (AP) direction vs. the flux in the AP-to-BL direction (Papp-BL-to-AP/Papp AP-to-BL) was 71. In the presence of GF120918 (2 microM), the Papp BL-to-AP/Papp AP-to-BL ratio was decreased to 16. In the presence of CsA (25 microM), the ratio was decreased to 5.6. In MDCK-WT. MDCK-MDR1, and MDCK-MRP2 cells, the Papp BL-AP/Papp AP-to-BL ratios of CD were 13, 35, and 22, respectively. CsA (25 microM) greatly decreased the Papp BL-P-AP/Papp AP-to-BL ratios in MDCK-WT and MDCK-MDR1 cells to 1.5 and 3.2, respectively. However, in MDCK-MRP2 cells. CsA (25 microM) decreased the ratio only to 11. A mixture of GF120918 (2 microM) and CsA (25 microM) decreased the Papp BL-to-AP/Papp AP-to-BL ratios of CD in MDCK-WT, MDCK-MDR1, and MDCK-MRP2 cells to 1.4, 2.7, and 5.4. respectively. CONCLUSIONS: These data suggest that CD is a good substrate for both P-gp and MRP2 and that the restricted permeation of this cyclic prodrug in Caco-2 cells and in the intestinal mucosa is probably due to its substrate activities for both of these efflux transporters.