NPYFa, A Chimeric Peptide of Met-Enkephalin, and NPFF Induces Tolerance-Free Analgesia.

Methionine-enkephalin-Arg-Phe is an endogenous amphiactive analgesic peptide. Neuropeptide FF, on the other hand, is reported for its role in opioid modulation and tolerance development. Based on these reports, in the present study we designed a chimeric peptide NPYFa (YGGFMKKKPQRFamide), having the Met-enkephalin (opioid) and PQRFamide sequence of neuropeptide FF, which can then target both the opioid and neuropeptide FF receptors. We hypothesized that the chimeric peptide so designed would have both analgesic properties and further aid in understanding of the role of neuropeptide FF in the development of opiate tolerance. Our studies indicated that NPYFa induced an early onset, potent, dose-dependent and prolonged antinociception. Additionally, antagonists (MOR, KOR, and DOR) pretreatment studies determined a KOR-mediated antinociception activity of the ligand. Further, in vitro binding studies using the Eu-GTP-γS binding assay on cell lines expressing opioid and NPFF receptors showed binding to both the opioid and neuropeptide FF receptors suggesting a multiple receptor binding character of NPYFa. Moreover, chronic (6 days) treatment with NPYFa exhibited an absence of tolerance development subsequent to its analgesia. The current study proposes NPYFa as a potent, long-acting antinociceptor lacking tolerance development as well as a probe to study opioid analgesia and the associated complex mechanisms of tolerance development.

Antinociceptive effect of [Met5]enkephalin semicarbazide is not affected by dipeptidyl carboxypeptidase-I.

Dipeptidyl carboxypeptidase-I is an enzyme involved in the biological degradation of enkephalins. It has been suggested that C-terminal amidation of enkephalins enhances their resistance to dipeptidyl carboxypeptidase-I-mediated biodegradation. In this study, a novel [Met5]enkephalin amide (MEA) analogue [Met5]enkephalin (ME)-semicarbazide synthesized by another laboratory in our group was assessed for its antinociceptive effects compared with ME-ethylamide, MEA and ME, using tail flick test. To protect the administered drugs from biodegradation, rats were pretreated with peptidase inhibitors including amastatin, phosphoramidon and captopril. Then captopril (dipeptidyl carboxypeptidase-I inhibitor) was deleted from the peptidase inhibitors' combination for evaluating in vivo resistance of the synthetic drugs to dipeptidyl carboxypeptidase-I. According to the results, ME-semicarbazide and MEA were resistant enough to dipeptidyl carboxypeptidase-I to exert their strong antinociception following intrathecal administration even in the absence of captopril, whereas the antinociceptive effects produced by ME-ethylamide (10 nmol) were abolished in rats not pretreated with captopril, indicating that significant amounts of the ME-ethylamide were degraded by dipeptidyl carboxypeptidase-I. Replacement of the amide moiety of MEA with semicarbazide provides a new ME derivative, with high analgesic effects as well as more resistance to dipeptidyl carboxypeptidase-I-mediated biodegradation.

[Pharmacodynamic properties of a combination of met-enkephalin and alpha 1-13 adrenocorticotropic hormone]. / Farmakodinamske karakteristike kombinacije met-enkephalin-a i alpha 1-13 adrenokortikotropnog hormona.

ENKORTEN is newly registered drug in Bosnia and Herzegovina. It consists of two peptide components: met-enkephalin and alpha 1-13-corticotropine (alpha-ACTH 1-13), previously called alpha-melanocyte-stimulating hormone-like (alpha-MSH-like) Met-enkephalin and alpha-MSH exhibited cytoprotective effects individually and statistically significant additive effect was registered when both peptides were applied in combination on the model of ethanol induced gastric lesions in rats. Combination has immunomodulatory effects. Method of selective immunomodulation with antigens and peptides in immunological mediated diseases and malignant tumors is directed towards long-term remission without so many adverse effects characteristic for immunosuppressive drugs. Adverse reactions registered for so long with ENKORTEN were mild, reversible and usually developed during and immediately after drug application.

Binding profile of the endogenous novel heptapeptide Met-enkephalin-Gly-tyr in zebrafish and rat brain.

Zebrafish is considered a model organism, not only for the study of the biological functions of vertebrates but also as a tool to analyze the effects of some drugs or toxic agents. Five opioid precursor genes homologous to the mammalian opioid propeptide genes have recently been identified; one of these, the zebrafish proenkephalin, codes a novel heptapeptide, the Met-enkephalin-Gly-Tyr (MEGY). To analyze the pharmacological properties of this novel ligand, we have labeled it with tritium ([(3)H]MEGY). In addition, we have also synthesized two analogs: (d-Ala(2))-MEGY (Y-d-Ala-GFMGY) and (d-Ala(2), Val(5))-MEGY (Y-d-Ala-GFVGY). The binding profile of these three agents has been studied in zebrafish and rat brain membranes. [(3)H]MEGY presents one binding site in zebrafish, as well as in rat brain membranes, although it shows a slight higher affinity in zebrafish brain. The observed saturable binding is displaced by naloxone, thus confirming the opioid nature of the binding sites. Competition binding assays indicate that the methionine residue is essential for high-affinity binding of MEGY and probably of other peptidic agonists in zebrafish, whereas the change of a Gly for a d-Ala does not dramatically affect the ligand affinity. Our results show that the percentage of [(3)H]MEGY displaced by all the ligands studied is higher than 100%, thus inferring that naloxone (used to determine nonspecific binding) does not bind to all the sites labeled by [(3)H]MEGY. Therefore, we can deduct that some of the MEGY binding sites should not be considered classical opioid sites.

Metabolism and absorption enhancement of methionine enkephalin in human nasal epithelium.

The objective of this study was to investigate absorption enhancing approaches for systemic delivery of methionine enkephalin via the nose. Absorption promotion of methionine enkephalin in the presence of protease inhibitors (bestatin, puromycin) and absorption enhancers (glycocholate, dimethyl-beta-cyclodextrin) were investigated in human nasal epithelium. Co-administration of the peptide with protease inhibitors and absorption enhancers resulted in a remarkable increase in Met-Enk permeation (4- to 94-fold). The increase was proportional to transepithelial resistance reduction and permeation of paracellular marker dye. Perturbation of the epithelial tight junctions seen in vitro may not occur in vivo due to mucus protection and mucociliary clearance.

Transplacental transfer of the opioid growth factor, [Met(5)]-enkephalin, in rats.

Placental transfer of the pentapeptide [Met5]-enkephalin, known to function as a growth regulating factor and neuromodulatory agent, was studied in pregnant Sprague-Dawley rats. Using separation by reversed phase high-performance liquid chromatography, and analysis by derivative spectroscopy, [Met5]-enkephalin was detected in 20-day-old fetal tissue including brain, heart, lung, and kidney. Fetal tissues from pregnant rats given an injection of 40 mg/kg [Met5]-enkephalin on gestation day 20 had markedly elevated levels of peptide within 1 h, indicating the transplacental transfer of this opioid. [Met5]-enkephalin levels were increased from control samples at 1, 2, 4, and 14 h post-injection of peptide, but not at 24 h. Evaluation of breakdown products of [Met5]-enkephalin, along with the related peptide [Leu5]-enkephalin, revealed that elution times differed substantially from [Met5]-enkephalin. These data indicate that [Met5]-enkephalin is present in fetal organs, crosses the placenta, does not appear to be restrictive in organ specificity, and is sustained in fetal tissues at detectable levels for at least 14 h. Given that [Met5]-enkephalin tonically inhibits DNA synthesis in the fetus, these results raise the question of whether an elevated level of this peptide (either maternally or from the fetus) may be detrimental to cellular ontogeny in the fetus, and perhaps have long-term implications for postnatal development.

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.

Enzymatic and permeation barrier of [D-Ala(2)]-Met-enkephalinamide in the anterior membranes of the albino rabbit eye.

Enzymatic and physical barrier properties of anterior ocular membranes were characterized. The permeation and metabolic degradation of [D-Ala(2)]-methionine enkephalinamide (DAMEA) in the albino rabbit cornea, conjunctiva and sclera were studied in vitro. DAMEA was administered with and without peptidase inhibitors bestatin (aminopeptidase inhibitor) and SCH 39370 (enkephalinase inhibitor). The modified Ussing chambers were used to study the peptide permeation and the samples were analyzed with a novel HPLC method using UV and EC detectors. Sclera was the most permeable membrane to DAMEA, while cornea was almost impermeable to DAMEA. Without inhibitors, the permeability coefficients of DAMEA were 2. 7x10(-8) cm/s, 3.1x10(-6) cm/s and 12.5x10(-6) cm/s in the cornea, conjunctiva and sclera, respectively. DAMEA was partly metabolized to tyrosine (Tyr) and tyrosine-D-alanine-glycine (Tyr-D-Ala-Gly). When inhibitors were co-administered with DAMEA, the corneal permeability of intact DAMEA increased 15 times, while conjunctival permeability increased 5.5 times and scleral permeability remained practically unaltered. The formation of metabolites decreased markedly, when the inhibitors were used. Interestingly, when the permeability of DAMEA was compared to permeabilities of polyethylene glycols in different membranes, the permeation was in the same range suggesting that DAMEA permeates through cornea via a paracellular pathway. Both enzymatic and physical barriers were more prominent in the cornea than in the conjunctiva and sclera. Non-corneal pathway of absorption and combined with inhibition of peptidases may be the most viable pathway for ocular peptide administration.

Peptide transport system-1 (PTS-1) for Tyr-MIF-1 and Met-enkephalin differs from the receptors for either.

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and Met-enkephalin share a saturable transport system (peptide transport system-1, PTS-1) across the blood-brain barrier but do not readily bind to each other's receptors. This information allows the unique opportunity to differentiate the transport protein(s) from the receptors for either peptide in brain endothelial cells. PTS-1 was studied in vitro by allowing radiolabeled Tyr-MIF-1 (125I-Tyr-MIF-1) to bind to the solubilized proteins of isolated murine brain microvessels in the presence or absence of potential inhibitors. Sephadex chromatography separated bound from free labeled peptide. The binding was saturable as shown by inhibition with increasing concentrations of unlabeled Tyr-MIF-1. 125I-Tyr-MIF-1 binding was not inhibited by an unrelated peptide or iodo-tyrosine. D-Tyr-MIF-1 had no effect, demonstrating the stereospecificity of the system. Met-enkephalin decreased the binding of 125I-Tyr-MIF-1 to 84.4% of total, whereas Leu-enkephalin was without effect. Agonists for the mu, delta, and kappa opiate receptors did not change the binding, indicating that the proteins which bound to 125I-Tyr-MIF-1 were not endogenous opiate receptors. The results indicate that, in vitro, Tyr-MIF-1 binds to brain microvessel proteins with characteristics similar to PTS-1.

First-pass metabolism of peptide drugs in rat perfused liver.

To elucidate the extent and mechanisms of the first-pass metabolism of peptide drugs in the liver after oral administration, a liver perfusion study was performed in rats using metkephamid, a stable analogue of methionine enkephalin, and thyrotropin-releasing hormone (TRH), as model peptides. The fraction of intact metkephamid recovered after single-pass constant perfusion through rat liver reached steady-state very quickly, and it was concluded that metkephamid was hydrolysed enzymatically at the surface of hepatocytes or endothelial cells of microvessels, or both, rather than being taken up by hepatocytes. The fraction of metkephamid recovered intact was approximately 40% under protein-free conditions but increased to 70-75% on addition of bovine serum albumin (BSA) to the perfusate. The fraction of metkephamid bound to BSA was approximately 50% under these conditions, implying that only the free fraction of metkephamid in the plasma was metabolized in the liver. Calculations based on the tube model showed that approximately 30-35% of metkephamid absorbed from the intestine undergoes first-pass metabolism before entering the systemic circulation in-vivo. In contrast, the fraction of TRH metabolized in the liver was less than 10%, indicating a remarkably low contribution of first-pass metabolism to the bioavailability of TRH. These results show that hepatic first-pass metabolism of metkephamid contributes to its low systemic bioavailability. After intestinal absorption free metkephamid is rapidly hydrolysed on the surface of hepatocytes or endothelial cells, rather than being taken up by hepatocytes. This information has important implications in the oral delivery of many kinds of peptide.

Nasal delivery of peptides: an in vitro cell culture model for the investigation of transport and metabolism in human nasal epithelium.

We investigated the transport- and metabolism properties of three peptides in monolayers of human nasal epithelial cells. The effective permeability coefficients of thyrotropin-releasing hormone, met-enkephalin and human recombinant insulin were found to be 4.5, 4.4 and 0.4 x 10(-7) cm/s, respectively. The permeability was inversely proportional to the molecular weight and one order of magnitude lower than in excised nasal mucosa of rabbits. The metabolic cleavage of thyrotropin-releasing hormone (TRH) to the free acid by cytosolic prolyl-endopeptidase was also detected in human nasal cell monolayers, suggesting that ca. 10% of the total amount of TRH is transported via a transcellular pathway. Met-enkephalin is a substrate for aminopeptidases, located on the apical membrane of nasal epithelial cells. Metabolites and enzyme activity are comparable with literature data. Our studies demonstrate that not only morphological, but also functional properties of human nasal epithelial cells are preserved under in vitro conditions. Such a cell culture model based on human nasal cells could be beneficial for the characterization of peptide transport on a cellular level and for investigation of the absorption enhancer mechanism. Further studies are necessary, however, to establish correlations between in vitro permeabilities in cell cultures and nasal drug absorption in animals and humans.

Gradient high-performance liquid chromatographic analysis of enkephalin peptides, their metabolites, and enzyme inhibitors using combined ultraviolet and electrochemical detection II. Application to ocular permeability studies in vitro.

A gradient HPLC method with combined ultraviolet (UV) and electrochemical detection (ED) was used to study the ocular permeability of [D-Ala2]-methionine enkephalinamide (MEA) in vitro. Coulometric ED was selective for MEA and its tyrosine-containing metabolites with quantitation limits between 20 and 60 nM (1.0-3.0 pmol per 50-microl injection), whereas UV detection at 205 nm allowed the determination of several aromatic metabolites and enzyme inhibitors with quantitation limits between 40 and 500 nM (2.0-25.0 pmol). This method was capable of detecting permeability of MEA and metabolite formation in the cornea and conjunctiva in vitro. Furthermore, effects of aminopeptidase inhibitor bestatin and enkephalinase inhibitor SCH 39370 on permeation and metabolism of MEA could be determined.

P-Glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: the influence of culturing conditions and drug exposure on P-gp expression levels.

The influence of cell culture conditions and previous drug exposure on P-glycoprotein (P-gp) expression levels in Caco-2 cells was determined. In this study, the expression of P-gp is demonstrated (i) visually by confocal laser scanning microscopy (CLSM), (ii) functionally by transport studies with substrates of the efflux pump, and (iii) quantitatively by flow cytometry (FCM) analysis using specific monoclonal antibodies (anti P-gp MRK 16 as an external antibody and P-GlycoCheck C219 as an internal antibody). Trypsinization of the cells after reaching confluence led to a decrease of P-gp expression levels, while trypsinization before reaching confluence led to an increase after long-term cultivation. Culturing the cells on polycarbonate filters did not elicit a significant change of P-gp expression over time in culture, whereas in plastic flasks (polystyrene) a decrease was detected. Using CLSM a strong fluorescence on the apical side of Caco-2 cell monolayers was observed, as a result of incubation with MRK 16 as primary and IgG Cy5 as secondary antibody. Previous drug exposure of the cells showed that verapamil, celiprolol, and vinblastine induced the P-gp expression, while metkephamid (MKA) decreased the P-gp expression level as compared to the control. Permeation studies consolidated the theory that P-gp is expressed in the Caco-2 cells examined. For talinolol and MKA, a higher transport from basolateral to apical side than from apical to basolateral could be measured. Incubation of the cell monolayer with MRK 16 reduced the secretion process to the apical side, but did not influence [3H]mannitol flux. Caco-2 cells seem to be a suitable cell line model for P-gp-mediated secretion studies. However, the variability of the P-gp expression requires careful control when this model is to be used in quantitative structure/secretion studies.

Effects of peptidase inhibitors on the enkephalin-induced anti-nociception in rats.

The intra-third-ventricular (i.t.v.) administration of [Met5]-enkephalin (enk) to rats pretreated i.t.v. with three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon, inhibited the tail-flick response. The enk-induced inhibition was augmented by increasing the doses of the three PIs, with the maximum inhibition being attained at the doses of 10 nmol each. The enk-induced inhibition in rats pretreated with any combination of two PIs, however, were markedly smaller than that in rats pretreated with all three PIs, indicating that three kinds of enzymes all played important roles in the inactivation of enk. The inhibitory effect of enk on the tail-flick response in rats pretreated with the three PIs at doses of 10 nmol each was approximately tenfold higher than that of morphine. The relative anti-nociceptive potencies of enk and morphine were similar to the relative inhibitory potencies obtained previously with the isolated guinea pig ileum pretreated with the three PIs, indicating that the hydrolysis of the i.t.v. administered enk was largely prevented by the three PIs. However, the magnitude of the enk-induced inhibition in rats pretreated s.c. with the three PIs indicated that the hydrolysis of enk injected i.t.v. was not largely prevented by the s.c. administration of three PIs at doses up to 10 micromol each/kg.

Gastrointestinal absorption of peptide drug: quantitative evaluation of the degradation and the permeation of metkephamid in rat small intestine.

The intestinal absorption of metkephamid (MKA), an analog of natural [Met]enkephalin, was investigated by means of vascular perfusion of the rat small intestine. Most of the MKA administered to the jejunal loop was degraded in the lumen by enzymatic hydrolysis, whereas only 0.3 to 1.2% of it was absorbed into the vascular flow. This means that enzymatic degradation is a major barrier against the intestinal absorption of MKA. The absorption of MKA was divided into two steps, degradation and permeation, and is expressed as clearance from the intestine. The degradation clearance (CLd) of MKA was 60- to 200-fold higher than the permeation clearance (CLp), indicating the rapid hydrolysis of MKA before absorption. The absorbed fraction of MKA increased with increasing luminal MKA concentration, mainly due to an increase in CLp rather than a decrease in CLd. MKA was degraded not only before absorption but also during permeation across the intestinal epithelium. Three kinds of enzyme inhibitors were co-administered with MKA into the intestinal loop. Puromycin, an aminopeptidase M inhibitor, markedly enhanced MKA absorption by both decreasing CLd and increasing CLp, indicating the predominant role of this enzyme in MKA degradation. Bestatin, another aminopeptidase M inhibitor, also effectively suppressed the degradation of MKA before absorption, whereas it only slightly increased CLp. It was further found that bestatin was less effective in inhibiting MKA hydrolysis during permeation. Thiorphan, an enkephalinase inhibitor, had no effect on MKA absorption.

Colonic absorption and bioavailability of the pentapeptide metkephamid in the rat.

The concept of delivering systemically active peptide drugs to the colon in order to improve their oral absorption requires reasonable peptide permeability of the large intestinal wall and stability against the activity of the colonic microflora. In addition, the role of hepatic extraction needs to be addressed. In this study the absorption of the pentapeptide metkephamid following single pass perfusion of rat ascending colon was investigated by monitoring its disappearance from the large intestine and simultaneous appearance in the portal vein, the hepatic vein and the aorta. In addition its stability against colonic microflora was tested in vitro using pig caecal contents. Metkephamid was absorbed from the large intestine and appeared in the blood circulation; peptide concentrations in the portal vein increased over-proportionally with increasing perfusate concentrations (0.1-4.6 mmol/L) from 0.19 microgram/mL +/- 0.12 (SD, n = 7) to 31.6 micrograms/mL + 20.65 (SD, n = 4), respectively, and thus suggesting a saturable transport or metabolism. Concentrations in the hepatic vein were significantly lower than in the portal vein, hepatic extraction ratios were 0.35 +/- 0.14, 0.61 +/- 0.18 and 0.62 +/- 0.28 (SD, n = 4) for 0.1, 0.5 and 1.0 mM metkephamid perfusate concentrations, respectively. In the anaerobic colon metabolism model the degradation half-life of the peptide was 14.9 hours, thus, indicating relative stability in the bacterial environment of the colon. The results of the present study encourage further investigations on colonic delivery of peptide drugs.

Effect of peptidases at the blood brain barrier on the permeability of enkephalin.

The blood brain barrier (BBB) presents an enzymatic barrier to the passage of peptides, from blood to brain. The studies presented here used a well established in vitro model of the BBB to measure the presence of peptidases and the permeability of two opioid peptides. The in vitro BBB model consisted of confluent monolayers of bovine brain microvessel endothelial cells (BMECs). Enkephalin metabolizing enzymes, total aminopeptidase, aminopeptidase M (APM), angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) activities were measured in BMEC monolayers. The effect of specific inhibitors of APM, ACE and NEP on the permeability of [Met5]enkephalin (Met-Enk) and a conformationally constrained and enzymatically stable analog, DPDPE, also was determined. High levels of membrane-associated enzyme activity were measured for total aminopeptidase, APM and ACE. Interestingly, the permeability coefficient of Met-Enk was increased 4-fold in the presence of specific inhibitors of APM and ACE. Low levels of NEP activity were measured in BMEC monolayers and inhibition of NEP had no effect on Met-Enk permeability. The permeability coefficient for DPDPE was not increased with enzyme inhibitors but was 4-fold greater than Met-Enk alone. In the presence of APM or ACE inhibitors, there was no difference in the permeability of DPDPE and Met-Enk. These experiments demonstrate the presence of specific peptidases in BMECs and that the presence of inhibitors to Met-Enk inactivating peptidases significantly increased permeability of this biologically active peptide.

Oral absorption of peptides: the effect of absorption site and enzyme inhibition on the systemic availability of metkephamid.

In this study the intestinal degradation and absorption of a synthetic pentapeptide, metkephamid, were investigated in the rat by determination of its wall permeabilities in the small and large intestine and the extent and mechanism of its intestinal degradation. The peptide was metabolized in the gut wall through contact with membrane-bound enzymes in the brush border membrane. The extent of metabolic inactivation depended on the intestinal segment investigated and decreased in the axial direction. No metabolism was found in the colon. The dimensionless wall permeabilities (Pw*), determined by single-pass perfusion, were also site dependent. Pw* was highest in the ileum [1.91 +/- 0.24, (SE); n = 4], followed by the jejunum (1.64 +/- 0.34; n = 4) and the colon (0.67 +/- 0.38; n = 4). Based on the permeability data alone and under the assumption of no presystemic metabolism, complete bioavailability would be predicted for metkephamid. However, following oral administration, the mean absolute bioavailability was only 0.22 +/- 0.065% (n = 3), indicating the overall dominance of degradation in the absorption process. Thus future strategies in oral peptide delivery should focus on increasing the stability of the peptide in the intestine by modifying the peptide structure and/or delivering the compound to an intestinal segment showing little or no enzymatic degradation.

Metabolism and transport of the pentapeptide metkephamid by brush-border membrane vesicles of rat intestine.

Intestinal metabolism and transport of the pentapeptide metkephamid (Tyr-D-Ala-Gly-Phe-N-Me-Met-NH2) were studied using isolated brush-border membranes from the rat. Analysis of the metabolic fragments of enzymatic hydrolysis revealed that cleavage of the N-terminal peptide bond leads to the formation of tyrosine and a tetrapeptide D-Ala-Gly-Phe-N-Me-Met-NH2. The inactivation was due to aminopeptidase N activity and could be inhibited by peptidase inhibitors puromycin, bacitracin and certain dipeptides. Transport studies demonstrated uptake of the intact pentapeptide into the intravesicular space of the vesicles. The transport was a first-order process; no participation of known intestinal peptide carrier systems in the transport of metkephamid could be shown. Modelling of simultaneous metabolism and transport kinetics suggests strategies to improve the fraction absorbed of a peptide by either decreasing its affinity to the metabolizing enzymes (increase Km) or decreasing the concentration of the metabolizing enzymes e.g. by delivering the peptide to an absorption site with reduced enzymatic activity (decrease Vmax) or increasing its absorption velocity.

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)