Degradation peptides of secretin after storage in acid and neutral aqueous solutions

During the storage of secretin in acid and neutral aqueous solutions, five degradation peptides (A1, A2, A3, A4, A5) and one degradation peptide (N1) were produced, respectively. They were isolated in pure form by HPLC, and the intramolecular structures were studied by a combination of amino acid analysis, enzymatic digestions, HPLC, and Fab-mass spectroscopy. Although the degradation peptides are composed of the same amino acids as secretin after acid hydrolysis (except A1 and A4 which are cleavage products S16-27 and S4-27, respectively), reversed-phase HPLC analysis of their digestive fragments with trypsin and alpha-chymotrypsin are different from those of secretin. By Fab-mass spectroscopy, the m/z values for the S1-6 fragments obtained from secretin, A2, and A3 were 663, 663, and 645, respectively. When S1-6 from A2 was treated with aminopeptidase M, a fragment obtained was identical with the synthetic beta-aspartyl3 S3-6, as determined by HPLC. The A2 and N1 peptides are completely the same based on various chemical analyses. The A3 peptide can also be rapidly degraded to secretin and beta-aspartyl3 secretin. Consequently, A1 and A4 are concluded to be the cleavage peptides of secretin, S16-27 and S4-27, respectively, A2 and N1 are concluded to be beta-aspartyl3 secretin, and A3 is concluded to be aspartoyl3 secretin.     

D-Leu(1)] microcystin-LR, a new microcystin isoplated from waterbloom in a Canadian prairie lake.

A previously undescribed cyclic heptapeptide hepatotoxin was isolated from a cyanobacteria waterbloom collected in Pakowki Lake, Alberta, Canada (49 degrees 20'N and 110 degrees 55'W). The compound was characterized by amino acid analysis, ESIMS/CID/MS, (1)H and (13)C NMR, and UV spectroscopy. Structure of the new microcystin was assigned as [D-Leu(1)]microcystin-LR (1). The amino acid composition is the same as microcystin-LR (2) except for D-Leu and L-Leu in 1 instead of D-Ala and L-Leu in 2. This is the first microcystin identified, among the 64 known microcystins, that has both a D- and L-Leu amino acid. Toxicity as measured by the protein phosphatase inhibition activity of 1 is similar to microcystin-LR. The presence of microcystins in waterblooms from this lake is discussed in relation to the almost yearly bird mortalities that have occurred there since 1995.     

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.     

Effects of (D-Ala2,D-Leu5)enkephalinamide on isolated longitudinal muscle guinea pig and rat intestines

The effects of (D-Ala2,D-Leu5)enkephalinamide, a synthetic analogue of (Leu5) enkephalin, on the contractile activity of isolated segments from guinea-pig and rat colon and rectum were investigated and compared to the effects of natural (Leu5)enkephalin. It was found that the contractile effects of (D-Ala2,D-Leu5)enkephalinamide vary between species and according to the route of administration (application of single doses of cumulative application). (D-Ala2,D-Leu5)enkephalinamide was more potent than (Leu5)enkephalin. It is suggested that in the separate regions of the guinea-pig and rat intestines (D-Ala2,D-Leu5)enkephalinamide activates the same opiate receptors as (Leu5)enkephalin. The higher potency of (D-Ala2,D-Leu5)enkephalinamide could be due to its retarded enzyme degradation because of the replacement of Gly2 by D-Ala and of Leu5 by D-Leu. A selective activation of delta type opiate receptors by (D-Ala2, D-Leu5)enkephalinamide is also assumed.     

Comparison of the effectiveness of different opioid peptides in suppressing heroin withdrawal

The effectiveness of beta-endorphin, dynorphin-(1-13), dynorphin-(1-10) amide, alpha-neoendorphin and [D-Ala2,D-Leu5]enkephalin in suppressing withdrawal in heroin addicts was compared in this study. Groups of six patients were stabilized overnight in the hospital and were treated with either saline or peptide when withdrawal symptoms began to appear the following morning. Withdrawal was scored before and after treatment by the patient himself and an independent observer. Peptides were administered in a bolus dose of 60 micrograms/kg body weight. The patient, the observer and the physician who administered the injection were all blind to the nature of the compound given. All treatments, including those with saline, produced an overall reduction of withdrawal score. However, by statistical analysis, only treatments with beta-endorphin, [D-Ala2,D-Leu5]enkephalin and dynorphin-(1-13) were effective in producing a significant decrease of withdrawal symptoms. The length of relief brought about by the different peptides varied from less than an hour to a maximum of 5 h in one case. The average period of relief brought about by beta-endorphin, dynorphin-(1-13) and [D-Ala2,D-Leu5]enkephalin was 44, 46 and 60 min, respectively. Of the five peptides administered [D-Ala2,D-Leu5]enkephalin produced the largest number of side-effects.     

Opiate-like peptides. Part VIII. Methylamides and dimethylamides of [D-Leu5]-enkephalin and [D-Ala2, D-Leu5]-enkephalin. Synthesis and analgesic activity

Syntheses of [D-Ala2, D-Leu5]-enkephalin, methyl ester of [D-Ala2, D-Leu3]-enkephalin, methylamides and dimethylamides of [D-Leu5]-enkephalin and [D-Ala2, D-Leu5]-enkephalin are described together with their analgesic activity determined on the basis of four analgesic tests: the hot-plate method, the reaction to electric stimulus, the tail immersion test and the frequency of writhing syndrome test. The neurotoxicity was estimated by the rota-rod test. The most pronounced analgesic effect was induced by compound: [D-Ala2, D-Leu5]-enkephalin, [D-Ala2, D-Leu5-OMe]-enkephalin and [D-Ala2, D-Leu5-NMe2]-enkephalin. In the tail immersion test all analogs did not exhibit analgesic activity.     

Effects of amino acid chirality and the chemical linker on the cell permeation characteristics of cyclic prodrugs of opioid peptides

Previously, our laboratory showed that the oxymethyl-modified coumarinic acid (OMCA) cyclic prodrug of the opioid peptide DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) exhibited low permeation across both the intestinal mucosa and the blood-brain barrier (BBB). This low cell permeation arose from its strong substrate activity for efflux transporters in these biological barriers. In an attempt to determine whether the chirality of the amino acid asymmetric centers could influence the solution structure of the cyclic prodrugs and thus their substrate activities for efflux transporters, we synthesized cyclic prodrugs of the opioid peptides H-Tyr-Ala-Gly-Phe-D-Leu-OH ([Ala2,D-Leu5]-Enk), H-Tyr-D-Ala-Gly-Phe-Leu-OH ([D-Ala2,Leu5]-Enk), and H-Tyr-Ala-Gly-Phe-Leu-OH ([Ala2,Leu5]-Enk). In an attempt to determine whether the chemical linker (OMCA) bestowed efflux substrate activity on the cyclic prodrugs, we synthesized capped linear derivatives (acetylated on the N-terminal and amidated on the C-terminal end) of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk. The solution conformations of the cyclic prodrugs were determined by molecular dynamics simulations using two-dimensional NMR data. The physicochemical properties (molecular surface area, polar surface area, and cLogP) were estimated computationally using Sybyl. Cell permeation characteristics were assessed using Caco-2 cells in the presence and absence of known inhibitors of efflux transporters. Despite apparent differences in their solution conformations and their physicochemical properties, the cyclic prodrugs of DADLE, [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk all exhibited strong substrate activity for efflux transporters in Caco-2 cells. In contrast, the capped linear derivatives of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk exhibited very poor substrate activity for efflux transporters in Caco-2 cells. Therefore, the substrate activities of the cyclic prodrugs for efflux transporters in Caco-2 cells and in the intestinal mucosa and the BBB in vivo are most likely due to the chemical linker used to prepare these molecules and/or its effect on solution structures of the prodrugs.     

Properties of two toxins isolated from the blue-green alga Microcystis aeruginosa

Attempts were made to characterize the two toxins (P-1 and P-2) isolated from the blue-green alga Microcystis aeruginosa, by amino acid analysis, mass spectrometry, 1H- and 13C-NMR. P-2, the major toxin, had a molecular weight of 1044, and consisted of one molecule each of beta-methylaspartic acid, D-Glu, D-Ala, L-Arg, L-Tyr, N-methyldehydroalanine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda). P-1, with a molecular weight of 994, appeared to have almost the same composition except that it contained L-Leu instead of L-Tyr in P-2. Mass spectrometric studies, along with a negative ninhydrin reaction, indicated that each toxin was a cyclic peptide. P-2 showed an LD99 of 70 micrograms/kg mice when injected i.p. and [alpha]D24 of -72.42 degrees (c = 0.095 in methanol), and was decomposed at around 218 degrees C.     

(1-Amino-2-propenyl) phosphonic acid, an inhibitor of alanine racemase and D-alanine:D-alanine ligase

DL-(1-Amino-2-propenyl)phosphonic acid was synthesized through the sequential oxidation, sulfoxide elimination, and deprotection of diphenyl [1-[(benzyloxycarbonyl)amino]-3-(phenylthio)propyl] phosphonate. This analogue of vinylglycine is a strong inhibitor of the alanine racemases from Pseudomonas aeruginosa and Streptococcus faecalis and of the D-Ala:D-Ala ligase from this latter species. This molecule is ineffective against the whole bacterial cells. Unlike vinylglycine, this unsaturated phosphonate does not inhibit the following mammalian enzymes: aspartate aminotransferase, alanine aminotransferase, D-amino acid oxidase, which indicates its specificity. Thus, its incorporation in a peptide structure could induce interesting antimicrobial properties.     

Effect of [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin on cholinergic and noradrenergic neurotransmission in isolated atria

In rabbit isolated atria, [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.1-3 microM) inhibited responses to cholinergic nerve stimulation in a concentration-dependent manner without affecting responses to exogenous acetylcholine. The inhibitory effect was blocked by the opiate receptor antagonist naloxone (1 microM). In rabbit atria in which the transmitter acetylcholine stores had been radioactively labelled by preincubating the tissue in [3H]choline, tetrodotoxin (100 ng/ml) significantly (P less than 0.001) blocked the stimulation-induced (2 Hz for 3 min) release of radioactivity. Both [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.3 and 1 microM) significantly decreased stimulation-induced radioactivity release and their effects were blocked by naloxone (1 microM). In rat isolated atria, [D-Ala2,Met5]enkephalinamide and [D-Ala2,D-Leu5]enkephalin (0.3-3 microM) inhibited responses to cholinergic nerve stimulation without affecting responses to exogenous acetylcholine. The inhibitory effect was blocked by naloxone (1 microM). In guinea-pig isolated atria, responses to cholinergic nerve stimulation were unaffected by the enkephalin analogues. In rabbit, rat and guinea-pig isolated atria, responses to noradrenergic nerve stimulation and exogenous noradrenaline were unaffected by the enkephalin analogues.     

Factors that restrict the cell permeation of cyclic prodrugs of an opioid peptide, part 4: Characterization of the biopharmaceutical and physicochemical properties of two new cyclic prodrugs designed to be stable to oxidative metabolism by cytochrome P-450 enzymes in the intestinal mucosa

The biopharmaceutical and physicochemical properties of two new cyclic prodrugs (CA-[cychexalanine (Cha(4)), D-Leu(5) ]-Enkephalin (Enk) and coumarinic acid (CA)-[Cha(4), D-Ala(5)]-Enk) of opioid peptides that were designed to be stable to oxidative metabolism by cytochrome P-450 enzymes in the intestinal mucosa are described in this paper. Two-dimensional nuclear magnetic resonance studies and molecular dynamics simulations showed that these cyclic prodrugs exhibit unique solution conformations (i.e., type I β-turns), which are favorable for transcellular permeation. The calculated molecular surface areas and cLog P values confirmed that these new cyclic prodrugs are more lipophilic than linear opioid peptides and, thus, they should exhibit better transcellular permeation characteristics. However, Caco-2 cell permeation studies showed that the cyclic prodrugs were substrates for apically polarized efflux transporters (e.g., P-glycoprotein, which significantly limited their transcellular permeation). Permeability studies using an in situ rat intestinal perfusion model confirmed the poor intestinal permeation characteristics of CA-[Cha(4), D-Leu(5) ]-Enk and CA-[Cha(4), D-Ala(5)]-Enk as well as the stability of these two new cyclic prodrugs of opioid peptides to oxidative metabolism. In conclusion, these data clearly show that oral absorption of cyclic prodrugs of opioid peptides can only be achieved by designing molecules devoid of substrate activity for both cytochrome P-450 enzymes and efflux transporters in the intestinal mucosa.     

Stereospecificity of amino acid side chains in deltorphin defines binding to opioid receptors.

A series of individual D-amino acid replacement analogues of deltorphin A, several of which were in combination with a His4 deletion, were used to probe alterations of side-chain orientation on peptide binding parameters with rat brain opioid receptors. Peptides with D-amino acids in residues 1, 3, and 5 exhibited diminished affinities primarily for delta receptors (88-1200-fold) with selectivity decreasing by factors of 13-64-fold relative to deltorphin A (Ki delta = 0.45 nM; Ki mu/Ki delta = 764): the aromatic side chains Tyr1 and Phe3, which lie in the N-terminal "message" domain and the aryl side chain of Leu5 in the C-terminal "address" domain, appear to play essential roles in conferring high delta affinity and selectivity. Although D-His4 only decreased delta affinity by 6-fold and selectivity by a factor of 4, His appears to be involved as an integral component of both domains: [des-His4]deltorphin A and [des-His4] analogues containing consecutive D-amino acid replacements in the remaining residues exhibited weak binding to delta receptors and poor delta selectivity. Substitution of D-Met2 in deltorphin A by D-Ala or D-Nle decreased delta selectivities 3-6-fold through an elevation in mu affinities; however, the converse replacement, D-Met for D-Ala2 in deltorphin B, diminished beta selectivity by an order of magnitude only through the loss in delta affinity. The data show that the high delta affinity and selectivity of deltorphins correlate with and require a strict stereospecificity of the amino acid residue side chains.     

Studies on peptides

The heptacosapeptide amide corresponding to the entire amino acid sequence of chicken gastrin-releasing peptide (cGRP) was synthesized similarly to the synthesis of porcine GRP by assembling six peptide fragments followed by depro-tection with 1 M trifluoromethanesulfonic acid-thioanisole in TFA. A new carboxyl-activating reagent, thiazolidine-2-thione, was preferentially adopted for preparation of necessary fragments. The synthetic cGRP, purified by ion-exchange chromatography, followed by partition chromatography, was active as the synthetic porcine GRP, when plasma immunoreactive gastrin level was examined in rats. No obvious difference was observed when synthetic and natural cGRP preparations were compared by HPLC, immunochemical property and biological activity in dogs.     

Opiate-receptor interactions on single locus coeruleus neurones

Intracellular recordings were made from neurones of the rat locus coeruleus (LC) which were located in a slice of pons superfused in vitro. Opioid agonists and antagonists were applied by adding them to the superfusing solution; normorphine and enkephalin analogues were also applied by ejecting a few nanoliters of a solution which contained the drugs from a pipette situated above the tissue slice. Opioid agonists hyperpolarized LC neurones. This has been shown previously to result from an increase in the membrane potassium conductance. The lowest concentration of normorphine which was effective was 30 nM, the EC50 was 1 microM, and the maximum effect was observed with 30 microM. The irreversible antagonist beta-funaltrexamine (beta-FNA) was used to estimate the dissociation equilibrium constants; these ranged from 9-16 microM for normorphine and [Met5]enkephalin and was about 2 microM for [D-Ala2,D-Leu5]enkephalin. beta-FNA also blocked the hyperpolarization caused by [D-Ala2,D-Leu5]enkephalin, ethylketacyclazocine, and [D-Ser2,D-Leu5] enkephalin-Thr. Naloxone reversibly antagonized the hyperpolarizations caused by normorphine and [D-Ala2,D-Leu5]enkephalin, with a dissociation equilibrium constant of 2 nM. It is suggested that the opioid hyperpolarization of LC neurones is mediated by a receptor having a high affinity for naloxone, previously termed a mu-receptor. The affinity of this receptor for normorphine appears to be 3 to 4 orders or magnitude lower than its affinity for naloxone.     

Spectrum of the mu, delta- and kappa-binding sites in homogenates of rat brain

1 In homogenates of rat brain, the binding characteristics of tritiated opiates and opioid peptides were examined and the relative capacities of mu-, delta- and kappa-binding sites of the opiate receptor determined by saturation analysis.2 In competition experiments, binding of the selective mu-ligand [(3)H]-[D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin at the mu-site was displaced by [D-Ala(2),D-Leu(5)]enkephalin with rather low affinity (K(I) = 12.6 nM) and more readily by the ketazocine-like compounds (-)-ethylketazocine (K(I) = 3.1 nM) and (-)-bremazocine (K(I) = 0.32 nM), which also displaced the binding of [(3)H]-[D-Ala(2),D-Leu(5)]enkephalin from the delta-site. In contrast, the binding to the kappa-site was easily displaced by ethylketazocine (1.0 nM) and bremazocine (0.37 nM) but not by the mu-ligand [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin (K(I) = 2000-3000 nM) or the delta-ligand [D-Ala(2),D-Leu(5)]enkephalin (K(I) > 20,000 nM).3 The dissociation equilibrium constant (K(D)) and the binding capacity (pmol/g) of the mu-binding site were determined with the selective mu-ligand [(3)H]-[D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin. For the delta-site, [(3)H]-[D-Ala(2),D-Leu(5)]enkephalin was used in the presence of unlabelled [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin in order to suppress cross-reactivity to the mu-binding site. For the estimation of kappa-binding, [(3)H]-(+/-)-ethylketazocine or [(3)H]-(-)-bremazocine were used in the presence of unlabelled mu- and delta-ligands for the suppression of cross-reactivities to the mu- and delta-binding sites.4 In rat brain the capacity of the mu-binding site was 7.3 pmol/g brain, that of the delta-binding site 6.7 pmol/g brain and that of the kappa-binding site 2.0 pmol/g brain. Thus, the kappa-binding site had the lowest value whereas in the guinea-pig brain the capacity of the mu-binding site was lower than that of the delta- or kappa-binding site.     

Use of a Fundamental Approach to Spray-Drying Formulation Design to Facilitate the Development of Multi-Component Dry Powder Aerosols for Respiratory Drug Delivery

Purpose

A fundamental approach incorporating current theoretical models into aerosol formulation design potentially reduces experimental work for complex formulations. A D-amino acid mixture containing D-Leucine (D-Leu), D-Methionine, D-Tryptophan, and D-Tyrosine was selected as a model formulation for this approach.

Methods

Formulation design targets were set, with the aim of producing a highly dispersible D-amino acid aerosol. Particle formation theory and a spray dryer process model were applied with boundary conditions to the design targets, resulting in a priori predictions of particle morphology and necessary spray dryer process parameters. Two formulations containing 60% w/w trehalose, 30% w/w D-Leu, and 10% w/w remaining D-amino acids were manufactured.

Results

The design targets were met. The formulations had rugose and hollow particles, caused by deformation of a crystalline D-Leu shell while trehalose remained amorphous, as predicted by particle formation theory. D-Leu acts as a dispersibility enhancer, ensuring that both formulations: 1) delivered over 40% of the loaded dose into the in vitro lung region, and 2) achieved desired values of lung airway surface liquid concentrations based on lung deposition simulations.

Conclusions

Theoretical models were applied to successfully achieve complex formulations with design challenges a priori. No further iterations to the design process were required.     

Effect of [D-Ala2,MePhe4,Gly-ol5]enkephalin and [D-Ala2,D-Leu5]enkephalin on ion and amino acid transport in rabbit ileum

The selective mu-opioid agonist [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) (10 microM) reduced the short circuit (Isc) and the L-valine induced increase of the transepithelial potential difference and Isc(delta Vms and delta Isc) measured in-vitro in rabbit ileum, with a mechanism antagonized by naloxone (1 microM). [D-Ala2,D-Leu5]enkephalin (DADLE) (10 microM) had no significant effect on the transepithelial potential difference (Vms), Isc, delta Vms and delta Isc. In the ileum deprived of the serosa and muscolaris, DAGO reduced the delta Vms and delta Isc, but not the Vms and Isc, suggesting localization of the receptors responsible for this latter effect in the myenteric plexus and/or the muscularis mucosae. These preliminary results suggest that in the rabbit ileum opioids influence electrolyte and amino acid transport and these effects may be at least partly mediated by mu-receptors.     

Identification of D-Amino Acids in Light Exposed mAb Formulations

Purpose

We previously demonstrated that D-amino acids can form as a result of photo-irradiation of a monoclonal antibody (mAb) at both λ?=?254 nm and λ?>?295 nm (λ_max?=?305 nm), likely via reversible hydrogen transfer reactions of intermediary thiyl radicals. Here, we investigate the role of various excipients (sucrose, glucose, L-Arg, L-Met and L-Leu) on D-amino acid formation, and specifically the distribution of D-amino acids in mAb monomers and aggregates present after light exposure.

Methods

The mAb-containing formulations were photo-irradiated at λ?=?254 nm and λ_max?=?305 nm, followed by fractionation of aggregate and monomer fractions using size exclusion chromatography. These aggregate and monomer fractions were subjected to hydrolysis and subsequent amino acid analysis.

Results

Both aggregate and monomer fractions collected from all formulations showed the formation of D-Glu and D-Val, whereas the formation of D-Ala was limited to the aggregate fraction collected from an L-Arg-containing formulation. Interestingly, quantitative analysis revealed higher yields of D-amino acids in the L-Arg-containing formulation.

Conclusions

Generally, D-amino acids accumulated to similar extents in monomers and aggregates.

     

Combined effect of oleic acid and polyethylene glycol 200 on buccal permeation of [D-ala2, D-leu5]enkephalin from a cubic phase of glyceryl monooleate

The combined use of the lipophilic permeation enhancer, oleic acid together with polyethylene glycol 200 (PEG 200) as a co-enhancer and incorporated into the cubic liquid crystalline phase of glyceryl monooleate was investigated in the ex vivo buccal permeation of [D-Ala2, D-Leu5]enkephalin (DADLE) through porcine buccal mucosa mounted in a Franz cell. The addition of oleic acid (1%) and PEG 200 (1-10%) did not change the intact appearance of the cubic phase. PEG 200 increased the aqueous solubility of oleic acid incorporated into the cubic phase and hence promoted the transport of oleic acid into the porcine buccal mucosa. The solubilising effect of PEG 200 on oleic acid incorporated into the cubic phase was dependent on the PEG 200 concentration but was non-linear. The buccal permeation flux of DADLE significantly increased when 5% (P<0.01) or 10% (P<0.001) of PEG 200 was co-administered with 1% oleic acid compared with the cubic phase containing 1% oleic acid alone. The present results suggest that PEG 200 enhances the action of the lipophilic permeation enhancer oleic acid and that the combination of oleic acid and PEG 200 as a co-enhancer can be a useful tool to improve the membrane permeability in the buccal delivery of peptide drugs using a cubic liquid crystalline phase of glyceryl monooleate and water.     

Characterization of the kappa-subtype of the opiate receptor in the guinea-pig brain.

1. In homogenates of guinea-pig brain, the characteristics of the binding of [3H]-ethylketazocine, an agonist for the putative kappa-receptor, were determined by estimation of the affinity and capacity of binding, by competitive inhibition for the binding site by unlabelled ligands and by selective protection of the binding site from alkylation by phenoxybenzamine. 2. At 25 degrees C the maximum number of binding sites for [3H]-ethylketazocine was about 14 pmol/g fresh brain, of which about 50% were high affinity sites. 3. In competition experiments, the high affinity binding of [3H]-ethylketazocine to the kappa-binding site was readily displaced by several kappa-agonists but not by the selective mu-ligand, D-Ala2, MePhe4, Gly-ol5-enkephalin or the selective delta ligand, D-Ala2, D-Leu5-enkephalin. In contrast, the kappa-agonists tested so far exhibit a high degree of cross-reactivity with the mu-binding site but somewhat less with the sigma-binding site. Similar specificities were observed in protection experiments. 4. The approximate proportions of the three subtypes of opiate receptor in the guinea-pig brain are 25% mu-binding sites, 45% delta-binding sites and 30% kappa-binding sites. 5. The endogenous opioids, Met-enkephalin, Leu-enkephalin and porcine beta-endorphin have only a low affinity for the kappa-binding site.