Development and characterization of functionalized niosomes for brain targeting of dynorphin-B.

A niosomal formulation, functionalized with N-palmitoylglucosamine, was developed as potential brain targeted delivery system of dynorphin-B. In fact, this endogenous neuropeptide, selective agonist of k opioid receptors, is endowed with relevant pharmacological activities on the central nervous system, including a marked antinociceptive effect, but is unable to cross the blood brain barrier (BBB), thus requiring intracerebroventricular administration. Statistical design of experiments was utilized for a systematic evaluation of the influence of variations of the relative amounts of the components of the vesicle membrane (Span 60, cholesterol and SolulanC24) on vesicle mean diameter, polydispersity index and drug entrapment efficiency, chosen as the responses to optimize. A Scheffé simplex-centroid design was used to obtain the coefficients of the postulated mathematical model. The study of the response surface plots revealed that variations of the considered factors had different effects on the selected responses. The desirability function enabled for finding the optimal mixture composition, which represented the best compromise to simultaneously optimize all the three responses. The experimental values obtained with the optimized formulation were very similar to the predicted ones, proving the validity of the proposed regression model. The optimized niosomal formulation of dynorphin-B administered intravenously to mice (100mg/kg) showed a pronounced antinociceptive effect, significantly higher (P<0.05) than that given by i.v. administration of the simple solution of the peptide at the same concentration, proving its effectiveness in enabling the peptide brain delivery. These positive results suggest that the proposed approach could be successfully extended to other neuro-active peptides exerting a strong central action, even at low doses, but unable to cross the BBB.

Glucose and calcium ions may modulate the efficiency of bovine ß-casomorphin-7 permeability through a monolayer of Caco-2 cells.

Milk and dairy products provide a lot of valuable nutritive elements. They are also sources of biologically active peptides, including ß-casomorphins that manifest the properties of morphine. An activity of DPPIV seems to be most crucial factor decreasing the efficiency of the ß-casomorphin-7 (BCM7) transport. The increase of BCM7 concentration in blood may intensify symptoms of apparent life threatening events (ALTE), autism, schizophrenia, and allergy. This study aimed at identifying the influence of several selected substances on a transport efficiency of bovine BCM7 through an intestinal monolayer in a Caco-2 cell model system. Applying the ELISA method, the permeability coefficient of BCM7 through the Caco-2 monolayer was calculated. TEER values were used to evaluate the integrity of Caco-2 cell monolayers. An increase of glucose and Ca(2+) concentrations in the culture medium was accompanied by an increase of the BCM7 transport efficiency. The lowest permeability coefficients of BCM7 were observed for the membranes with high electrical resistances. The transport was enhanced in the presence of milk infant formulas, whereas no changes were observed when using µ-opioid receptor antagonist (casoxin-6). The results may be useful in understanding the pathogenesis of inflammation and food allergy in infants.

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

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

Inhibition of nociceptive responses after systemic administration of amidated kyotorphin.

BACKGROUND AND PURPOSE: Kyotorphin (KTP; L-Tyr-L-Arg), an endogenous neuropeptide, is potently analgesic when delivered directly to the central nervous system. Its weak analgesic effects after systemic administration have been explained by inability to cross the blood-brain barrier (BBB) and detract from the possible clinical use of KTP as an analgesic. In this study, we aimed to increase the lipophilicity of KTP by amidation and to evaluate the analgesic efficacy of a new KTP derivative (KTP-amide - KTP-NH(2) ). EXPERIMENTAL APPROACH: We synthesized KTP-NH(2) . This peptide was given systemically to assess its ability to cross the BBB. A wide range of pain models, including acute, sustained and chronic inflammatory and neuropathic pain, were used to characterize analgesic efficacies of KTP-NH(2) . Binding to opioid receptors and toxicity were also measured. KEY RESULTS: KTP-NH(2) , unlike its precursor KTP, was lipophilic and highly analgesic following systemic administration in several acute and chronic pain models, without inducing toxic effects or affecting motor responses and blood pressure. Binding to opioid receptors was minimal. KTP-NH(2) inhibited nociceptive responses of spinal neurons. Its analgesic effects were prevented by intrathecal or i.p. administration of naloxone. CONCLUSIONS AND IMPLICATIONS: Amidation allowed KTP to show good analgesic ability after systemic delivery in acute and chronic pain models. The indirect opioid-mediated actions of KTP-NH(2) may explain why this compound retained its analgesic effects although the usual side effects of opioids were absent, which is a desired feature in next-generation pain medications.

Enhanced antinociceptive response to intracerebroventricular kyotorphin in Pept2 null mice.

L-Kyotorphin (L-KTP), an endogenous analgesic neuropeptide, is a substrate for aminopeptidases and a proton-coupled oligopeptide transporter, PEPT2. This study examined the CSF efflux, antinociceptive response, and hydrolysis kinetics in brain of L-KTP and its synthetic diastereomer D-kyotorphin (D-KTP) in wild-type and Pept2 null mice. CSF clearance of L-KTP was slower in Pept2 null mice than in wild-type animals, and this difference was reflected in greater L-KTP-induced analgesia in Pept2 null mice. Moreover, dose-response analyses showed that the ED50 of L-KTP in Pept2-deficient animals was one-fifth of the value observed in Pept2-competent animals (4 vs. 21 nmol for null vs. wild-type mice, respectively). In contrast, the ED50 of D-KTP was very similar between the two genotypes (9-10 nmol). Likewise, there was little difference between genotypes in slope factor or baseline effects of L-KTP and D-KTP. The enhanced antinociceptive response to L-KTP in Pept2 null mice could not be explained by differences in neuropeptide degradation as Vmax and Km values did not differ between genotypes. Our results demonstrate that PEPT2 can significantly impact the analgesic response to an endogenous neuropeptide by altering CSF (and presumably brain interstitial fluid) concentrations and that it may influence the disposition and response to exogenous peptide/mimetic substrates.

Stimulation of Na+/Cl--coupled opioid peptide transport system in SK-N-SH cells by L-kyotorphin, an endogenous substrate for H+-coupled peptide transporter PEPT2.

We have recently identified a Na+/Cl--coupled transport system in mammalian cells for endogenous and synthetic opioid peptides. This transport system does not transport dipeptides/tripeptides, but is stimulated by these small peptides. Here we investigated the influence of L-kyotorphin (L-Tyr-L-Arg), an endogenous dipeptide with opioid activity, on this transport system. The activity of the transport system, measured in SK-N-SH cells (a human neuronal cell line) with deltorphin II as a model substrate, was stimulated approximately 2.5-fold by L-kyotorphin, with half-maximal stimulation occurring at approximately 100 microM. The stimulation was associated primarily with an increase in the affinity for deltorphin II. The stimulation caused by L-kyotorphin was stereospecific; L-Tyr-D-Arg (D-kyotorphin) had minimal effect. The influence of L-kyotorphin was observed also in a different cell line which expressed the opioid peptide transport system. While L-kyotorphin is a stimulator of opioid peptide transport, it is a transportable substrate for the H+-coupled peptide transporter PEPT2, which is expressed widely in the brain. Since the activity of the opioid peptide transport system is modulated by extracellular L-kyotorphin and since PEPT2 is an important determinant of extracellular L-kyotorphin in the brain, the expression/activity of PEPT2 may be a critical factor in the modulation of opioidergic neurotransmission in vivo.

Uptake of radiolabeled morphiceptin and its analogs by experimental mammary adenocarcinoma: in vitro and in vivo studies.

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) and its analogs modified at position 3: [D-Phe(3)]morphiceptin, [D-ClPhe(3)]morphiceptin and [D-Cl(2)Phe(3)]morphiceptin were synthesized and labeled with [(125)I] or [(131)I]. Their binding to membranes isolated from experimental adenocarcinoma was examined in vitro with the use of a cross-linking assay followed by the Western blot technique. The radioactive complex had molecular weight of about 65 kDa and was detectable by anti-mu-opioid receptor polyclonal antibody. Expression of the mu-opioid receptor in mouse mammary adenocarcinoma was confirmed by reverse transcriptase-polymerase chain reaction. The binding studies showed the highest affinity and capacity for [D-Phe(3)]morphiceptin (K(d) 0.39 and B(max) 1112) and [D-ClPhe(3)]morphiceptin (K(d) 1.8 and B(max) 220). Morphiceptin and its D-Cl(2)Phe analog had significantly lower B(max) values (131 and 83, respectively). Biodistribution experiments in tumor-bearing C3H/Bi mice with the use of the (131)I-labeled peptides confirmed the results of our in vitro studies. The highest accumulation of radioactive peptides in the tumor tissue was also found for peptides with D-Phe and D-ClPhe.

Conjugation with L-Glutamate for in vivo brain drug delivery.

In vitro studies have shown that conjugation of a model compound [p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD)] with L-Glu can improve D-MOD permeation through the bovine brain microvessel endothelial cell monolayers (Sakaeda et al., 2000). The transport of this D-MOD-L-Glu conjugate is facilitated by the L-Glu transport system. In this paper, we evaluate the in vivo brain delivery of model compounds (i.e. D-MOD, p-nitro-D-phenylalanine (p-nitro-D-Phe), 5,7-dichlorokynurenic acid (DCKA) and D-kyotorphin) and their L-Glu conjugates. DCKA was also conjugated with L-Asp and L-Gln amino acids. The analgesic activities of D-kyotorphin and its L-Glu conjugate were also evaluated. The results showed that the brain-to-plasma concentration ratio of D-MOD-L-Glu was higher than the D-MOD alone; however, the plasma concentration of both compounds were the same. The plasma concentration of p-nitro-D-Phe-L-Glu conjugate was higher than the parent p-nitro-D-Phe; however, the brain-to-plasma concentration ratio of p-nitro-D-Phe was higher than its conjugate. On the other hand, both DCKA and DCKA conjugates have a low brain-to-plasma concentration ratio due to their inability to cross the blood-brain barrier (BBB). The L-Asp and L-Glu conjugates of DCKA have elevated plasma concentrations relative to DCKA; however, the DCKA-L-Gln conjugate has the same plasma concentration as DCKA. For D-kyotorphin, both the parent and the L-Glu conjugate showed similar analgesic activity. In conclusion, conjugation of a non-permeable drug with L-Glu may improve the drug's brain delivery; however, this improvement may depend on the physicochemical and receptor binding properties of the conjugate.

Intestinal transport and metabolism of glucose-conjugated kyotorphin and cyclic kyotorphin: metabolic degradation is crucial to intestinal absorption of peptide drugs.

Intestinal transport and metabolism of modified kyotorphin (KTP) were studied in rats. Modified KTPs studied were C-terminally modified KTP with p-aminophenyl-beta-D-glucoside (KTP-pAPbeta glc), N-terminally modified KTP-pAPbeta glc with t-butyloxycarbonyl group (Boc-KTP-pAPbeta glc) and the N- and C-terminally modified KTP by cyclization (cyclic KTP). KTP-pAPbeta glc was metabolized at a similar rate to that of KTP, and did not appear on the serosal side. Although Boc-KTP-pAPbeta glc was also metabolized, it was more stable than KTP and appeared on the serosal side. Cyclic KTP was also quite stable and appeared on the serosal side. The modified KTPs were evaluated kinetically for absorption consisting of membrane transport and metabolism. Absorption clearance (CL(abs)) of cyclic KTP, Boc-KTP-pAPbeta glc and Boc-KTP was higher than that of KTP (0.247 microl/min/cm) (Mizuma et al., Biochim. Biophys. Acta 1335 (1997) 111-119), which is the theoretical maximum by complete inhibition of peptidase activity, indicating that derivatization of KTP increases the membrane permeability. Furthermore, the data clearly showed that the greater the metabolic clearance (CL(met)) of KTP and the KTP derivatives, the lower the absorption clearance (CL(abs)). These results and further simulation study led to the conclusion that metabolic degradation in the intestinal tissues is more critical than membrane permeability (transport) for oral delivery of peptide drugs. Based on the stability of cyclic KTP in serum, this appears to be a good candidate analgesic peptide drug.

Chitosans as nasal absorption enhancers of peptides: comparison between free amine chitosans and soluble salts.

A total of three free amine chitosans (CS J, CS L and CS H) and two soluble chitosan salts (CS G and CS HCl) were evaluated for their efficacy and safety as nasal absorption enhancers of peptides based on in situ nasal perfusion and subacute histological evaluation in rat. At 0.5% w/v, all chitosans were effective in enhancing the nasal absorption of [D-Arg(2)]-Kyotorphin, an enzymatically stable opioid dipeptide. The enhancing effect of the free amine chitosans increased as the pH was decreased from 6.0 to 4.0 (P<0.05). However, the pH effect was not significant for the two chitosan salts (P0.05), suggesting that their adjuvant activity may be less pH-dependent than the free amine form. CS J and CS G were subsequently selected for further studies. At only 0.02% w/v, their enhancing effect was already significant and comparable to that of 5% w/v hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Both chitosans at 0.1% caused minimal release of total protein and phosphorus from the rat nasal mucosa, with the values similar to that of 5% HP-beta-CD. At 0. 5% the two chitosans also stimulated smaller release of lactate dehydrogenase, an intracellular enzyme used as marker of nasal membrane damage, than 1.25% dimethyl-beta-cyclodextrin. Morphological evaluation of the rat nasal mucosa following 2-week daily administration indicated that the two chitosans (1.0%) produced only mild to moderate irritation. In conclusion, both the free amine and the acid salt forms of chitosans are effective in enhancing the nasal absorption of [D-Arg(2)]-Kyotorphin and have potential for further studies as a safe and effective nasal absorption enhancer of peptide drugs.

Optimizing oral absorption of peptides using prodrug strategies.

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

Nanoparticle technology for delivery of drugs across the blood-brain barrier.

The Leu-enkephalin dalargin and the Met-enkephalin kyotorphin normally do not cross the blood-brain barrier (BBB) when given systemically. To transport these neuropeptides across the BBB they were adsorbed onto the surface of poly(butylcyanoacrylate) nanoparticles (NPs) and the NPs were coated with polysorbate 80. Central analgesia was measured by the hot plate test in mice. The antidepressant amitriptyline, which normally penetrates the BBB, was used to examine the versatility of the NP method. The concentration of amitriptyline in serum and brain of mice was determined by a gas chromatographic method. Furthermore, NPs were fabricated with different stabilizers. After the adsorption of the peptides on polysorbate 85-stabilized NPs, analgesia was noted after intravenous application when NPs were not coated. The amitriptyline level was significantly enhanced in brain when the substance was adsorbed onto the NP and coated or when the particles were stabilized with polysorbate 85.

Preference of Peyer's patches to jejunal epithelium for intestinal absorption of oligopeptides, tyrosylglycylglycine and D-kyotorphin.

The intestinal absorption of oligopeptides, peptidase-degradable tyrosylglycylglycine (TGG) and peptidase-resistant L-tyrosyl-D-arginine (D-kyotorphin, D-KTP) across Peyer's patches (PP) in rabbit intestine were studied using an Ussing-type chamber and in situ closed perfusion methods. The clearance for the serosal appearance of intact TGG across PP by the Ussing-type chamber method was a little higher than that across the jejunal epithelium (JE). Meanwhile, the in situ closed perfusion experiment showed that the clearance for the plasma appearance of intact TGG across PP was about 10 times that of JE. Furthermore, it was shown that the clearance for the plasma appearance of D-KTP in PP was about twice that in JE by the in situ closed perfusion method, indicating that the membrane permeability of PP was higher than that of JE. Therefore, these results indicate that PP had less metabolic peptidase activity than JE, and that the PP was a suitable site for the intestinal absorption of oligopeptides, especially peptidase-degradable peptides.

Intestinal transport and metabolism of analgesic dipeptide, kyotorphin: rate-limiting factor in intestinal absorption of peptide as drug.

Intestinal transport and metabolism of kyotorphin (KTP) were studied in rat everted small intestine. KTP on the mucosal side was metabolized completely within 60 min, and any amounts of KTP were not detected on the serosal side. On the other hand, [D-Arg2]-KTP (D-KTP) was stable on the mucosal side to appear on the serosal side. However, N-t-butoxycarbonyl-KTP (Boc-KTP), which was metabolized on the mucosal side faster than KTP, appeared on the serosal side. In intestinal homogenate, KTP was metabolized, and the metabolic clearance (CL(met)) was decreased by peptidase inhibitors, bestatin, o-phenanthrolin and tryptophan hydroxamate. In the presence of these peptidase inhibitors, the absorption clearance (CL(abs)) of KTP was increased. The less the CL(met) of KTP was, the more the CL(abs) of KTP was. Meanwhile, Boc-KTP in intestinal homogenate was stable even in the absence of peptidase inhibitors. The CL(abs) of Boc-KTP was constant irrespective of the stability on the mucosal side. Kinetic analysis by the metabolic inhibition model indicated that the stabilization of KTP in the intestinal tissue could increase the CL(abs) up to 0.247 microl/min per cm, which was as much as the CL(abs) of stable D-KTP. These results led to the conclusion that rate-limiting process in intestinal absorption of KTP is metabolic degradation in intestinal tissue during the absorption.

Body temperature and analgesic effects of selective mu and kappa opioid receptor agonists microdialyzed into rat brain.

Opioids administered by i.c.v. injection produce body temperature (Tb) changes and analgesic responses in rats. The present study was undertaken to investigate the effects on Tb and analgesia of highly selective mu and kappa opioid receptor agonists and antagonists delivered directly into the preoptic anterior hypothalamus (POAH) and periaqueductal gray (PAG) by the intracerebral microdialysis method. Microdialyzed into the POAH, the mu receptor agonist Tyr-Pro-N-MePhe-D-Pro-NH2 induced dose-related hyperthermia that could be prevented or antagonized by the mu receptor antagonist cyclic D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 or by naloxone, but not by the kappa receptor antagonist nor-binaltorphimine. The kappa receptor agonist dynorphin A(1-17), microdialyzed into the POAH, induced dose-related hypothermia that was prevented or antagonized by nor-binaltorphimine but not cyclic D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2. Neither Tyr-Pro-N-MePhe-D-Pro-NH2 nor dynorphin A(1-17) microdialyzed into the PAG produced significant changes in Tb. However, these agonists microdialyzed into the PAG produced analgesic responses that did not occur after administration into the POAH. These results support the hypothesis that the hyperthermic response to opioids is mediated by the mu receptor and the hypothermic response is mediated by the kappa receptor in rats. The POAH is a primary functional area in Tb, but not in analgesic, responses to opioids, whereas the PAG is a sensitive area for analgesic, but not for Tb, responses to opioids.

Neurofisiología del dolor / Pain neurophysiology

El estudio de la neurofisiología del dolor ha permitido, a través del tiempo, poder contar con substancias y técnicas que permitan su control, en beneficio del enfermo. Esto es de especial importancia en el campo de la Oncología, en que muchas veces por el carácter de la lesión, lo único que se puede ofrecer al enfermo es una sobrevida libre del dolor. Siempre resulta algo engorroso el estudio de las vías neuroanatómicas, en especial de las vías del dolor, ya que están permanentemente en revisión a la luz de las investigaciones, tanto del ámbito neurofisiológico como clínico-quirúrgico. En este artículo se da una visión global y sistemática de los fenómenos que desencadenan el dolor, su transmisión hasta el nivel central, su integración y el control que el propio organismo tiende frente al dolor

Los péptidos opioides en la integración sensorial y los trastornos perceptuales del dolor en la médula espinal / Opioid peptides in pain sensorial integration and perceptual dysfunction in the spinal cord

Se revisan las evidencias de la localización anatómica y el papel de los péptidos opioides en el procesamiento de la información sensorial nociceptiva y no nociceptiva en la médula espinal. En particular se analizan algunos de los mecanismos que se constituyen per se, en generadores de estados complejos de nocicepción , como la alodinia, es decir estímulos sensoriales no nociceptivos que producen dolor. se propone un modelo experimental con el cual se obtienen resultados prolongados (más de 2 h.) de actividad unicelular, de neuronas registradas en el asta dorsal de la médula espinal de la rata íntegra y anestesiada (uretano, 1500mg/kg). La preparación permite la indentificación de las neuronas registradas, por medio de la activación directa de su campo sensorial. Los cambios en la codificación sensorial se inducen mediante la infiltración subcutánea de carragenina (200 µl, al 1 por ciento) en el mismo campo. Los resultados muestran un incremento de la frecuencia de disparo de las neuronas, que en situación control responden sólo a estimulación táctil suave o al movimiento del pelo. Este incremento es lo que consideramos dolor, dado que se revirtió con la administración de naloxona (1 mg/kg iv) incrementó la frecuencia, después de 80 min. de infiltrada la carragenina. Se puede concluir que con el abordaje experimental presentado se han obtenido datos que reproducen el fenómeno de la alodinia y que éste se encuentra mediado por el sistema opioide

Transport of low and high molecular peptides across rabbit Peyer's patches.

The permeability of peptides across rabbit jejunal epithelium (JE) and Peyer's patches (PP) was compared. Kyotorphin (L-tyrosyl-L-arginine) was almost completely hydrolyzed during its membrane transport in both PP and JE, but [D-Arg2]Kyotorphin (L-tyrosyl-D-arginine) was less hydrolyzed in PP than in JE. Since the permeability of intact [D-Arg2]Kyotorphin was almost equal in PP and JE, no superiority of PP to JE was found for dipeptide transport. More intact fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (FITC-BSA) and concanavalin A (FITC-Con A) were transported in PP than in JE. At both absorption sites, the transport of the intact FITC-Con A was superior to that of the intact FITC-BSA. Colchicine significantly reduced the total transport of the intact and degradation forms of both peptides and the reduction ratio was greater in PP than in JE. Accordingly, it was suggested that PP can be used as prominent absorption sites for polypeptides since they have lower peptidase activity and higher endocytosis activity than JE.

Behavioral effects of systemically administered mu and kappa opioid agonists in the squirrel monkey: peptides versus alkaloids.

This study compared the effects of receptor-selective peptide and nonpeptide opioid agonists administered intramuscularly to squirrel monkeys responding under a fixed-interval 3-min schedule of stimulus termination. The mu opioid receptor agonist morphine (0.1-3.0 mg/kg) increased response rate at low doses and decreased it and quarter-life at higher doses. [D-Ala2,N-Me-Phe4,Gly-ol]Enkephalin (DAMGO; 0.3-3.0 mg/kg) reduced quarter-life at the highest dose. The kappa dose. The kappa opioid receptor agonist U50,488H (0.1-1.0 mg/kg) elevated response rate transiently and dose-dependently decreased quarter-life. Dynorphin A(1-13) (0.3-10 mg/kg), a purported endogenous ligand of the kappa opioid receptor, decreased response rate slightly but significantly at 3.0 mg/kg and had no effect on quarter-life. Thus, the behavior of squirrel monkeys was affected by systemically administered peptide as well as by nonpeptide opioid drugs. The two alkaloids were much more effective than the two peptides, presumably because of greater ability to penetrate the blood-brain barrier. Quarter-life was often a more sensitive measure of drug effects than was response rate.

The intestinal uptake of "enzymatically-stable" peptide drugs in rats as influenced by D-glucose in situ.

In previous in situ and in vivo rat perfusion studies, the intestinal absorption of several low molecular weight drugs was increased by the presence of luminal D-glucose. The intent of this study was to determine the potential of this fed-state effect to improve the intestinal uptake of poorly permeable, small peptide and peptide-like drugs. Jejunal wall permeabilities (Pw*) of di-(D-kyotorphin), tri-(cephradine), hexa-(growth hormone releasing peptide, GHRP-6) and octa-(octreotide, a somatostatin analogue) peptides and corresponding net water fluxes were determined in rats using an in situ single-pass perfusion technique. Glucose was shown to enhance the uptake of the smaller (di- and tri-) peptides but not the larger peptides despite the fact that glucose elicited a significant net water absorption with each of the four peptide drugs. It is concluded that glucose enhances jejunal permeabilities of smaller peptides by solvent drag and the enhancement is limited in situ by peptide molecular size. The studies with nonmetabolizable 3-O-methylglucose suggest that the augmentation of the proton gradient across the transmucosal membrane by glucose contributes to the carrier-mediated transport observed with the smaller peptides.