Dual signaling of Wamide myoinhibitory peptides through a peptide-gated channel and a GPCR in Platynereis.

Neuropeptides commonly signal by metabotropic GPCRs. In some mollusks and cnidarians, RFamide neuropeptides mediate fast ionotropic signaling by peptide-gated ion channels that belong to the DEG/ENaC family. Here we describe a neuropeptide system with a dual mode of signaling by both a peptide-gated ion channel and a GPCR. We identified and characterized a peptide-gated channel in the marine annelid Platynereis dumerilii that is specifically activated by Wamide myoinhibitory peptides derived from the same proneuropeptide. The myoinhibitory peptide-gated ion channel (MGIC) belongs to the DEG/ENaC family and is paralogous to RFamide-gated ion channels. Platynereis myoinhibitory peptides also activate a previously described GPCR, MAG. We measured the potency of all Wamides on both MGIC and MAG and identified peptides that preferentially activate one or the other receptor. Analysis of a single-cell transcriptome resource indicates that MGIC and MAG signal in distinct target neurons. The identification of a Wamide-gated ion channel suggests that peptide-gated channels are more diverse and widespread in animals than previously appreciated. The possibility of neuropeptide signaling by both ionotropic and metabotropic receptors to different target cells in the same organism highlights an additional level of complexity in peptidergic signaling networks.-Schmidt, A., Bauknecht, P., Williams, E. A., Augustinowski, K., Gründer, S., Jékely, G. Dual signaling of Wamide myoinhibitory peptides through a peptide-gated channel and a GPCR in Platynereis.

A PEGylated analog of short-length Neuromedin U with potent anorectic and anti-obesity effects.

Neuromedin U (NMU) is a neuropeptide known to regulate food intake and energy homeostasis that is widely distributed in the gastrointestinal tract, hypothalamus, and pituitary. A short form of NMU, porcine NMU-8 has potent agonist activity for the receptors NMUR1 and NMUR2; however, its short half-life precludes its effective use in vivo. To address this limitation, we designed and synthesized NMU-8 analogs modified by polyethylene glycol (PEG) with a molecular weight of 30kDa (PEG30k) via a variety of linkers (i.e., ω-amino- and ω-imino-carboxylic acid linker). Integrated evaluation of NMUR1 and NMUR2 binding affinities in vitro and anorectic activity in mice revealed that the introduction of a linker with a rigid ring group, e.g., 2-(piperazin-1-yl)acetic acid (PipAc), yielded a highly potent anorectic peptide, PEG30k-PipAc-NMU-8 (14), possessing improved receptor binding affinity. Subsequent optimization of the molecular weight of the PEG moiety led to the discovery of a PEG20k conjugate (15), which exhibited significant anti-obesity effect upon once-daily subcutaneous administration in diet-induced obese mice with 10% and 22% body weight loss at doses of 10 and 30nmol/kg, respectively. In addition, 15 reduced the weights of the liver and adipose tissue in a dose-dependent manner and improved the plasma biochemical parameters, e.g., insulin, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and total cholesterol. Thus, our results suggest that 15 (NMU-0002), which showed potent and long-lasting biological profiles in vivo, represents a candidate peptide for investigating the central and peripheral actions of NMU and its potential for clinical use.

Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide.

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.

[Influence of membranes on alaptide permeation from hydrogels]. / Vplyv membrán na permeáciu alaptidu z hydrogélov.

The paper evaluates the liberation of alaptide from gels through various types of permeable membranes. The gels were prepared on the basis of three different polymers (3% chitosan; 2.5% hydroxypropyl cellulose; 3% hydroxyethyl cellulose) in different concentrations with additions of humectants (5 %; 15% propylene glycol and 10% glycerol) and the preserving agent, 0.3% Sepicide HBR with 1% alaptide, and finally without the drug. The permeation of the drug from gels into the acceptor solution was evaluated with the use of the following membranes: the hydrophilic membrane from Chemosvit, the chicken skin, the stripped snakeskin, and the wall of the small intestine. The measurements showed that the highest percentage of the drug penetrated through the small intestine, a smaller percentage through the chicken skin, and the smallest amount through the snakeskin. Rheological properties of the prepared hydrogels were evaluated as well. The pseudoplastic flow was only confirmed for the hydrogel prepared on the basis of hydroxypropyl cellulose. An utterly opposite situation was with the hydrogels prepared on the basis of chitosan and hydroxyethyl cellulose. They showed a significant thixotropic character and the degree of thixotropy increased with time. Based on the results of the pH measurement, the samples prepared on the basis of chitosan and hydroxypropyl cellulose have been shown to be inconvenient because they reached a lower pH and had a potential of causing skin irritation. The hydroxyethyl cellulose hydrogel matched the physiological values of skin pH even after 14 days since its preparation.

Novel vasoconstrictor formulation to enhance intranasal targeting of neuropeptide therapeutics to the central nervous system.

The intranasal route of drug administration is noninvasive, convenient, and rapidly targets therapeutics to the central nervous system (CNS) using olfactory and trigeminal neural pathways connecting the nasal passages to the brain. The purpose of this research was to enhance intranasal drug targeting to the CNS by incorporating a vasoconstrictor [phenylephrine (PHE)] into nasal formulations containing therapeutic neuropeptides [hypocretin-1 (HC) or the dipeptide L-Tyr-D-Arg (D-KTP)]. Concentrations in CNS tissues, peripheral tissues, and blood were determined at 30 min following intravenous or intranasal administration of (125)I-labeled neuropeptides with and without PHE. Compared with intranasal controls, inclusion of 1% PHE in nasal formulations significantly reduced absorption into the blood for HC (65% reduction) and D-KTP (56% reduction), whereas it significantly increased deposition into the olfactory epithelium by approximately 3-fold for both. PHE (1%) significantly increased delivery to the olfactory bulbs for HC (2.1-fold) and D-KTP (3.0-fold), whereas it significantly reduced concentrations in the trigeminal nerve for HC (65% reduction) and D-KTP (39% reduction) and in most remaining brain regions by approximately 50% for both. The dramatic reduction in blood concentrations with PHE contributed to brain-to-blood concentration ratios that were significantly increased for HC throughout the brain (1.6-6.8-fold) compared with intranasal controls. For D-KTP, 1% PHE significantly increased ratios only in the olfactory bulbs (5.3-fold). With a 5% PHE formulation, D-KTP ratios were significantly increased to additional brain areas (1.5-16-fold). Vasoconstrictor nasal formulations may have particular relevance for CNS therapeutics with adverse side effects where it would be advantageous to limit systemic exposure.

The intestinal barrier in lepidopteran larvae: permeability of the peritrophic membrane and of the midgut epithelium to two biologically active peptides.

Endogenous peptide regulators of insect physiology and development are presently being considered as potential biopesticides, but their efficacy by oral delivery cannot be easily anticipated because of the limited information on how the insect gut barrier handles these kind of molecules. We investigated, in Bombyx mori larvae, the permeability properties of the two components of the intestinal barrier, the peritrophic membrane (PM) and the midgut epithelium, separately isolated and perfused in conventional Ussing chambers. The PM discriminated compounds of different dimensions but was easily crossed by two small peptides recently proposed as bioinsecticides, the neuropeptide proctolin and Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF), although their flux values indicated that the permeability was highly affected by their steric conformation. To date, there is very little functional data available on how peptides cross the insect intestinal epithelium, but it has been speculated that peptides could reach the haemocoel through the paracellular pathway. We characterized the permeability properties of this route to a number of organic molecules, showing that B. mori septate junction was highly selective to both the dimension and the charge of the permeant compound. Confocal images of whole-mount midguts incubated with rhodamine(rh)-proctolin or fluorescein isothiocyanate (FITC)-Aea-TMOF added to the mucosal side of the epithelium, revealed that rh-proctolin did not enter the cell and crossed the midgut only by the paracellular pathway, while FITC-Aea-TMOF did cross the cell apical membrane, permeating also through the transcellular route.

Developing drugs that can cross the blood-brain barrier: applications to Alzheimer's disease.

Development of therapeutics for the central nervous system is one of the most challenging areas in drug development. This is primarily because, in addition to all of the other complications one faces in developing new drugs targeting peripheral sites, one must also negotiate the blood-brain barrier (BBB). There are dozens of strategies to overcome the obstacle of the BBB, but many of these are bound to fail, barring extreme serendipity, because they are based on an inaccurate or incomplete picture of the BBB. This article therefore starts with a brief review of the BBB as it pertains to drug development. It then examines some examples of the delivery of drugs to the central nervous system that are relevant to Alzheimer's disease, placing emphasis on peptides, antibodies, and antisense oligonucleotides.

Transepithelial flux of an allatostatin and analogs across the anterior midgut of Manduca sexta larvae in vitro.

The transepithelial flux of cydiastatin 4 and analogs across flat sheet preparations of the anterior midgut of larvae of the tobacco hawkmoth moth, Manduca sexta, was investigated using a combination of reversed-phase high-performance liquid chromatography (RP-HPLC), enzyme-linked immunosorbent assay (ELISA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The lumen to hemolymph (L-H) flux of cydiastatin 4 was dose and time-dependent, with a maximum rate of flux of c. 178 pmol/cm2/h) measured after a 60-min incubation with 100 micromol/l of peptide in the lumen bathing fluid. The rates of flux, L-H and H-L, across the isolated gut preparations were not significantly different. These data suggest that uptake across the anterior midgut of larval M. sexta is via a paracellular route. Cydiastatin 4 was modified to incorporate a hexanoic acid (Hex) moiety at the N-terminus, the N-terminus extended with 5 P residues and/or the substitution of G7 with Fmoc-1-amino-cyclopropylcarboxylic acid (Acpc). The incorporation of hexanoic acid enhanced the uptake of these amphiphilic analogs compared to the native peptide. Analogs were also more resistant to enzymes in hemolymph and gut preparations from larval M. sexta. A modified N-terminus gave protection against aminopeptidase-like activity and incorporation of Acpc inhibited endopeptidase-like activity. Although analogs were stable in the hemolymph, they were susceptible to amidase-like activity in the gut, which appears to convert the C-terminal amide group to a free carboxylic acid, identified by an increase in 1 mass unit of the peptide analog.

Distribution of 26RFa binding sites and GPR103 mRNA in the central nervous system of the rat.

The novel RFamide peptide 26RFa, the endogenous ligand of the orphan receptor GPR103, affects food intake, locomotion, and activity of the gonadotropic axis. However, little is known regarding the localization of 26RFa receptors. The present report provides the first detailed mapping of 26RFa binding sites and GPR103 mRNA in the rat central nervous system (CNS). 26RFa binding sites were widely distributed in the brain and spinal cord, whereas the expression of GPR103 mRNA was more discrete, notably in the midbrain, the pons, and the medulla oblongata, suggesting that 26RFa can bind to a receptor(s) other than GPR103. Competition experiments confirmed that 26RFa interacts with an RFamide peptide receptor distinct from GPR103 that may be NPFF2. High densities of 26RFa binding sites were observed in olfactory, hypothalamic, and brainstem nuclei involved in the control of feeding behavior, including the piriform cortex, the ventromedial and dorsomedial hypothalamic nuclei, the paraventricular nucleus, the arcuate nucleus, the lateral hypothalamic area, and the nucleus of the solitary tract. The preoptic and anterior hypothalamic areas were also enriched with 26RFa recognition sites, supporting a physiological role of the neuropeptide in the regulation of the gonadotropic axis. A high density of 26RFa binding sites was detected in regions of the CNS involved in the processing of pain, such as the dorsal horn of the spinal cord and the parafascicular thalamic nucleus. The wide distribution of 26RFa binding sites suggests that 26RFa has multiple functions in the CNS that are mediated by at least two distinct receptors.

Acute blood-brain barrier permeabilization in rats after systemic Phoneutria nigriventer venom.

A highly controlled transport of substances at the interface between blood and brain characterizes the blood-brain barrier (BBB), fundamental for maintenance of the homeostasis of the cerebral milieu. In this study, we investigated the time course (15 min, 1, 2, and 5 h) of BBB opening induced by intravenous (i.v.) injection of Phoneutria nigriventer spider venom (PNV) using quantitative and morphological approaches on cerebellum and hippocampus vessels for assessment of BBB permeability. The results showed vasogenic edema and tracer extravasation faster and severalfold higher in hippocampus than in cerebellum. Reactive astrocytes with swollen perivascular end-feet processes were found only in cerebellum. An immediate and total degradation of laminin in capillaries occurred resulting in the disappearance of the basement membrane. In medium-sized vessels, this effect was less prominent. The changes were transient, with cerebellum in general presenting a faster recovery. However, at 5 h laminin was overexpressed, principally in hippocampus. The rapid and abrupt shift of laminin expression in capillaries (at 15 min) coincided with the immediate and severe signs of intoxication shown by the animals, but not with the peak of leakage of vessels and vasogenic edema, which occurred later (1-2 h). The findings suggest a complex regulatory mechanism, since the extension of BBB impairment caused by PNV depends on the region of the SNC, and on the vessels types. It is suggested that the components of the BBB (gliovascular unit) have a critical role in these differences. P. nigriventer venom can be a useful tool to explore the mechanisms of BBB.

The pharmacokinetics of the conopeptide contulakin-G (CGX-1160) after intrathecal administration: an analysis of data from studies in beagles.

BACKGROUND: The synthetic peptide agent Contulakin-G (CGX-1160), isolated from the toxin of the snail Conus geographus, produces significant analgesia in animals. Its peptide structure requires intrathecal administration for effectiveness, therefore we determined the intrathecal pharmacokinetics of CGX-1160 after bolus dose and multiple day infusions to beagles. METHODS: For the bolus dose study, eight animals received a dose ranging from 16.7 to 1000 nmol under isoflurane anesthesia. Cerebral spinal fluid sampling for drug assay occurred up to 24 h. For the multiple day infusion study, three animals received infusions of 10, 40, and 160 microg/h respectively for 24 h at each rate. Cerebral spinal fluid sampling occurred during the infusion rate and the washout period after the 72 h of cumulative drug delivery. Data from the two study designs were modeled separately using NONMEM. RESULTS: The results showed a biexponential disposition profile for both experiments with a rapid rate constant that was an order of magnitude greater than the slow rate constant. The bolus results showed a nonlinear dependence of the slow rate constant on administered dose due to the large bolus range used in the study. CONCLUSION: These data, coupled with clinical pharmacology results, provide a basis for determining appropriate dosing strategies to achieve therapeutic intrathecal concentrations of Contulakin-G.

Manufacturing of a Secretoneurin Drug Delivery System with Self-Assembled Protamine Nanoparticles by Titration.

Since therapeutic peptides and oligonucleotides are gathering interests as active pharmaceutical ingredients (APIs), nanoparticulate drug delivery systems are becoming of great importance. Thereby, the possibility to design drug delivery systems according to the therapeutic needs of APIs enhances clinical implementation. Over the last years, the focus of our group was laid on protamine-oligonucleotide-nanoparticles (so called proticles), however, the possibility to modify the size, zeta potential or loading efficiencies was limited. Therefore, at the present study we integrated a stepwise addition of protamine (titration) into the formation process of proticles loaded with the angiogenic neuropeptide secretoneurin (SN). A particle size around 130 nm was determined when proticles were assembled by the commonly used protamine addition at once. Through application of the protamine titration process it was possible to modify and adjust the particle size between approx. 120 and 1200 nm (dependent on mass ratio) without influencing the SN loading capacity. Dynamic light scattering pointed out that the difference in particle size was most probably the result of a secondary aggregation. Initially-formed particles of early stages in the titration process aggregated towards bigger assemblies. Atomic-force-microscopy images also revealed differences in morphology along with different particle size. In contrast, the SN loading was only influenced by the applied mass ratio, where a slight saturation effect was observable. Up to 65% of deployed SN could be imbedded into the proticle matrix. An in-vivo biodistribution study (i.m.) showed a retarded distribution of SN from the site of injection after the application of a SN-proticle formulation. Further, it was demonstrated that SN loaded proticles can be successfully freeze-dried and resuspended afterwards. To conclude, the integration of the protamine titration process offers new possibilities for the formulation of proticles in order to address key parameters of drug delivery systems as size, API loading or modified drug release.

Phase IA Clinical Trial Evaluating the Tolerability, Pharmacokinetics, and Analgesic Efficacy of an Intrathecally Administered Neurotensin A Analogue in Central Neuropathic Pain Following Spinal Cord Injury.

We evaluated CGX-1160 in a Phase Ia clinical trial to determine the safety of escalating doses in patients with central neuropathic pain following spinal cord injury (SCI). Our secondary objective was to detect a trend toward analgesic efficacy. Four subjects received 3 consecutive escalating doses of CGX-1160 starting at 25 µg/h over 6 hours until 2 consecutive subjects experienced any adverse effect; 2 of the 4 subjects received 2 sequences of 3 consecutive dose escalations. Maximum tolerated dose was defined by the development of diarrhea (900 µg/h over 6 hours). Cerebrospinal fluid (CSF) and blood were collected for pharmacokinetic (PK) evaluation. The CSF concentration-versus-time data fit to a biexponential PK model, showing a rapid redistribution phase followed by a significantly slower terminal elimination phase. Incorporating an effect site delay into the model improved the fit to the data (concentration producing 50% of the maximum effect [C50 ], 58.7 ug/mL at the site of drug effect). Maximal reduction from the baseline pain intensity was 63%. In summary, CGX-1160 was generally well tolerated when administered intrathecally at doses up to 1000 µg/h. Peak analgesic effect occurred after the peak intrathecal concentration, indicating the presence of an effect site compartment to the PK model to represent the concentration and effect profiles for this unique compound.

Distribution in Rats Internal Organs of Intraperitoneally Given 125I-Labeled Heptapeptide [2-8]-Leucopyrokinin ([2-8]-LPK), a Truncated Analog of Insect Neuropeptide Leucopyrokinin.

BACKGROUND: It was previously found that synthetic, insect-derived octapeptide leucopyrokinin (LPK) applied directly into the lateral brain ventricle induced a significant antinociceptive effect in rats. Its synthetic truncated analog heptapeptide [2-8]-leucopyrokinin displayed a stronger antinociceptive effect in comparison to native LPK. Moreover it was previously found a high accumulation of these both 125I-labeled peptides in adrenals, as well as in hypothalamus and in hippocampus of rats brain. OBJECTIVES: The aim of the present study was to assess the distribution of 125I-labeled [2-8]-leucopyrokinin in rats' internal organs an in several parts of the brain after peripheral - intraperitoneal (i.p.) administration. MATERIAL AND METHODS: The study was performed on male Wistar rats. A synthetic [2-8]-leucopyrokinin ([2-8]-LPK) was iodinated with Na125I. On the day of experiment a solution of 125I-[2-8]-LPK was i.p. injected and the next after 1 and 24 h animals were sacrificed by decapitation. Radioactivity levels in samples of parts of the brain and of internal organs were determined by counter Gamma Auto Count. RESULTS: A uniform, low accumulation 125I-[2-8]-LPK was found in evaluated samples of the brain and in internal organs. CONCLUSIONS: The results of the present study indicate a weak penetration into the brain and internal organs of intraperitoneally applied 125I-[2-8]-LPK in rats and correspond with previously determined weak biological effects of i.p. injected LPK and [2-8]-LPK.

Development of neuropeptide analogs capable of traversing the integument: A case study using diapause hormone analogs in Helicoverpa zea.

Diapause hormone and its analogs terminate pupal diapause in Helicoverpa zea when injected, but if such agents are to be used as effective diapause disruptors it will be essential to develop simple techniques for administering active compounds that can exert their effect by penetrating the insect epidermis. In the current study, we used two molecules previously shown to have high diapause-terminating activity as lead molecules to rationally design and synthesize new amphiphilic compounds with modified hydrophobic components. An assay for diapause termination identified 13 active compounds with EC50's ranging from 0.9 to 46.0 pmol per pupa. Three compounds, Decyl-1963, Dodecyl-1967, and Heptyl-1965, selected from the 13 compounds most active in breaking diapause following injection, also successfully prevented newly-formed pupae from entering diapause when applied topically. These compounds feature straight-chain, aliphatic hydrocarbons from 7 to 12 carbons in length; DH analogs with either a short-chain length of 4 or an aromatic phenethyl group failed to act topically. Compared to a high diapause incidence of 80-90% in controls, diapause incidence in pupae receiving a 10 nmole topical application of Decyl-1963, Dodecyl-1967, or Heptyl-1965 dropped to 30-45%. Decyl-1963 and Dodecyl-1967 also remained effective when topically applied at the 1 nmole level. These results suggest the feasibility of developing DH agonists that can be applied topically and suggest the identity of new lead molecules for development of additional topically-active DH analogs. The ability to penetrate the insect epidermis and/or midgut lining is critical if such agents are to be considered for future use as pest management tools.

The role for IGF-1-derived small neuropeptides as a therapeutic target for neurological disorders.

INTRODUCTION: Exogenous IGF-1 protects the brain from ischemic injury and improves function. However, its clinical application to neurological disorders is limited by its large molecular size, poor central uptake and mitogenic potential. AREAS COVERED: In this review, the authors have discussed the efficacy, pharmacokinetics and mechanisms of IGF-1 derivatives on protecting acute brain injury, preventing memory impairment and improving recovery from neurological degenerative conditions evaluated in various animal models. We have included natural metabolites of IGF-1, glycine-proline-glutamate (GPE), cleaved from N-terminal IGF-1 and cyclic glycine-proline (cGP) as well as the structural analogues of GPE and cGP, glycine-2-methyl-proline-glutamate and cyclo-l-glycyl-l-2-allylproline, respectively. In addition, the regulatory role for cGP in bioavailability of IGF-1 has also been discussed. EXPERT OPINION: These small neuropeptides provide effective neuroprotection by offering an improved pharmacokinetic profile and more practical route of administration compared with IGF-1 administration. Developing modified neuropeptides to overcome the limitations of their endogenous counterparts represents a novel strategy of pharmaceutical discovery for neurological disorders. The mechanism of action may involve a regulation of IGF-1 bioavailability.

[Pharmacokinetic and pharmacodynamic synergism between neuropeptides and lithium in the neurotrophic and neuroprotective action of cerebrolysin].

OBJECTIVE: To study the synergism between neuropeptides and lithium ions. MATERIAL AND METHODS: An experimental model of stroke (chronic bilateral occlusion of the common carotid arteries in rats), neuronal culture studies, histomorphological analyses, determination of micronutrient profile of brain substrates were used. RESULTS: A complex of experimental studies revealed that the effect of cerebrolysin is influenced by the synergism between lithium ions and the neuropeptide contentof this drug. Pharmacokinetic synergism promotes the accumulation of lithium in brain tissues during cerebrolysin treatment. The existence of the pharmacokinetic synergism is evident from the potentiation of neuroprotective effects of the drug under the action of lithium ions established in the model of stroke. CONCLUSION: Lithium ions potentiate neuroprotective effects of cerebrolysin.

Chronic delivery of α-melanocyte-stimulating hormone in rat hypothalamus using albumin-alginate microparticles: effects on food intake and body weight.

Chronic delivery of neuropeptides in the brain is a useful experimental approach to study their long-term effects on various biological parameters. In this work, we tested albumin-alginate microparticles, as a potential delivery system, to study if continuous release in the hypothalamus of α-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, may result in a long-term decrease in food intake and body weight. The 2-week release of α-MSH from peptide-loaded particles was confirmed by an in vitro assay. Then, daily food intake and body weight were studied for 18 days in rats injected bilaterally into the paraventricular hypothalamic nucleus with particles loaded or not with α-MSH. A decrease in body weight gain, persisting throughout the study, was found in rats injected with α-MSH-charged particles as compared with rats receiving non-charged particles and with rats injected with the same dose of α-MSH in solution. Food intake was significantly decreased for 3 days in rats receiving α-MSH-loaded particles and it was not followed by the feeding rebound effect which appears after food restriction. The presence of α-MSH-loaded particles in the hypothalamus was confirmed by immunohistochemistry. In conclusion, our study validates albumin-alginate microparticles as a new carrier system for long-term delivery of neuropeptides in the brain and demonstrates that chronic delivery of α-MSH in the hypothalamus results in a prolonged suppression of food intake and a decrease of body weight gain in rats.

Orexin-A, an hypothalamic peptide with analgesic properties.

The hypothalamic peptide orexin-A and the orexin-1 receptor are localized in areas of the brain and spinal cord associated with nociceptive processing. In the present study, localization was confirmed in the spinal cord and demonstrated in the dorsal root ganglion for both orexin-A and the orexin-1 receptor. The link with nociception was extended when orexin-A was shown to be analgesic when given i.v. but not s.c. in mouse and rat models of nociception and hyperalgesia. The efficacy of orexin-A was similar to that of morphine in the 50 degrees C hotplate test and the carrageenan-induced thermal hyperalgesia test. However, involvement of the opiate system in these effects was ruled out as they were blocked by the orexin-1 receptor antagonist SB-334867 but not naloxone. Orexin-1 receptor antagonists had no effect in acute nociceptive tests but under particular inflammatory conditions were pro-hyperalgesic, suggesting a tonic inhibitory orexin drive in these circumstances. These data demonstrate that the orexinergic system has a potential role in the modulation of nociceptive transmission.

Distribution and effects of pituitary adenylate cyclase activating peptide in cat and human lower oesophageal sphincter.

1. The localization, tissue concentrations, and effects of pituitary adenylate cyclase activating peptide (PACAP) 27 and 38 were investigated in cat and human lower oesophageal sphincter (LOS), and compared with those of vasoactive intestinal peptide (VIP) and helospectin. 2. PACAP-immunoreactive nerve structures were found in the cat and human LOS, with an abundance in the circular smooth muscle layer. PACAP 27-immunoreactivity was often co-localized with VIP-immunoreactivity. 3. In cat tissue, PACAP (PACAP 27 plus PACAP 38) concentrations were 50 fold lower than VIP concentrations; in human tissue they were 10 fold lower. 4. PACAP 27, PACAP 38, helospectin I, and VIP induced concentration-dependent relaxations in circular smooth muscle preparations from cat and human LOS. The order of potency was: VIP > helospectin I > or = PACAP 27 > PACAP 38. NG-nitro-L-arginine, scopolamine, or apamin, did not influence the relaxant effects of PACAP 27 or VIP. 5. In cat preparations, both cyclic AMP and cyclic GMP levels were increased after exposure to PACAP 27 and helospectin I, whereas exposure to VIP was followed by an increase in cyclic AMP levels only. In human preparations, there was an increase in cyclic AMP levels without any change in cyclic GMP levels. 6. These results suggest that in the cat and human LOS, PACAP 27 and VIP can occur within the same nerve structures. PACAP 27 has a potent relaxant action, but its functional importance has to be established.