Engineered endomorphin-2 gene: A novel therapy for improving morphine reinstatement in CPP model of rats by using deficient adenovirus as the vector.

Optimal therapeutics to deal with high relapse rates when discontinued is urgent for opioid dependence treatments. Endogenous endomorphin-2 (EM2) level in the central nervous system (CNS) down-regulates obviously after sustained morphine exposure, which suggested that to up-regulate the EM2 level could be a novel method for reinstatement. But the clinical applications of EM2 through conventional administration are limited owing to its short half-life. In our study, we engineered an EM2 gene to achieve the sustained release of EM-2 in CNS by utilizing a signal peptide of mouse growth factor for out-secreting EM2 and a deficient adenovirus as the vector. By intrathecally injecting engineering EM2 gene, a sustained increase of EM2 concentration in the cerebral spinal fluid (CSF) was observed along with a reduction of CPP scores. Also, the activation of astrocytes was suppressed in the hippocampus. In summary, this study provides evidence and reference for using intraspinal gene therapy with a combination of mouse growth factor and EM2 to treat morphine reinstatement.

NYX-2925 Is a Novel NMDA Receptor-Specific Spirocyclic-ß-Lactam That Modulates Synaptic Plasticity Processes Associated with Learning and Memory.

Background: N-methyl-D-aspartate receptors are one member of a family of ionotropic glutamate receptors that play a pivotal role in synaptic plasticity processes associated with learning and have become attractive therapeutic targets for diseases such as depression, anxiety, schizophrenia, and neuropathic pain. NYX-2925 ((2S, 3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)butanamide) is one member of a spiro-ß-lactam-based chemical platform that mimics some of the dipyrrolidine structural features of rapastinel (formerly GLYX-13: threonine-proline-proline-threonine) and is distinct from known N-methyl-D-aspartate receptor agonists or antagonists such as D-cycloserine, ketamine, MK-801, kynurenic acid, or ifenprodil. Methods: The in vitro and in vivo pharmacological properties of NYX-2925 were examined. Results: NYX-2925 has a low potential for "off-target" activity, as it did not exhibit any significant affinity for a large panel of neuroactive receptors, including hERG receptors. NYX-2925 increased MK-801 binding to human N-methyl-D-aspartate receptor NR2A-D subtypes expressed in HEK cells and enhanced N-methyl-D-aspartate receptor current and long-term potentiation (LTP) in rat hippocampal slices (100-500 nM). Single dose ex vivo studies showed increased metaplasticity in a hippocampal LTP paradigm and structural plasticity 24 hours after administration (1 mg/kg p.o.). Significant learning enhancement in both novel object recognition and positive emotional learning paradigms were observed (0.01-1 mg/kg p.o.), and these effects were blocked by the N-methyl-D-aspartate receptor antagonist CPP. NYX-2925 does not show any addictive or sedative/ataxic side effects and has a therapeutic index of >1000. NYX-2925 (1 mg/kg p.o.) has a cerebrospinal fluid half-life of 1.2 hours with a Cmax of 44 nM at 1 hour. Conclusions: NYX-2925, like rapastinel, activates an NMDA receptor-mediated synaptic plasticity process and may have therapeutic potential for a variety of NMDA receptor-mediated central nervous system disorders.

[Inaugural psychotic events in multiple sclerosis?]. / Evènements psychotiques inauguraux de sclérose en plaques?

INTRODUCTION: Psychotic symptoms are not readily recognized in multiple sclerosis, especially at the beginning of the disease. METHODS: We report the cases of four patients who developed psychotic symptoms that led to the diagnosis of multiple sclerosis. We describe the psychiatric and neurological features, MRI findings, clinical outcome and treatment. RESULTS: Two patients developed persecutory delusions, one presented a manic episode and the fourth melancholia with catatonia. Mean age was 39 years (range 20-49 years). Two patients had a personal history, but none a familial history of psychiatric disease. Examination of the cerebrospinal fluid revealed an oligoclonal pattern in all patients. All patients fulfilled Barkhof's MRI criteria. Three have had brain MRI with injection during psychotic symptoms. In these three cases, a frontal lesion appeared. The patient with catatonia also had a new lesion in the cerebellum and in the brainstem. All patients needed a "psychiatric" treatment, including antipsychotics. The psychiatric event lasted three months for two patients and the two others experienced relapse. CONCLUSION: Acute psychiatric symptom may reveal multiple sclerosis at the beginning of the disease. Frontal lobe localization is suggested. We propose that a psychotic event may correspond to a multiple sclerosis event.

A regulatory role for protease-activated receptor-2 in motivational learning in rats.

Serine proteases such as tissue plasminogen activator (tPA), thrombin and neuropsin influence hippocampal plasticity involved in learning and memory by facilitating both synaptic remodelling and long-term potentiation. Given our previous findings that trypsin and its receptor, protease-activated receptor-2 (PAR2), are both highly expressed in pyramidal neurons of the hippocampus and that activation of PAR2 attenuates 'pathogenic' plasticity related to epilepsy, we wished to determine the role for PAR2 in normal, non-pathological hippocampal plasticity related to learning and memory. In a strain of rat that show high basal levels of anxiety, the Genetic Absence Epilepsy Rats from Strasbourg (GAERS), peripheral administration of the PAR2 peptide agonist, SLIGRL (1.5 mg/kg s.c.), induced distinct deficits in experience-dependent learning both in the test-retest paradigm of the elevated-plus maze and in the Morris water maze. In separate, conscious rats with indwelling intra-cerebroventricular cannulae, SLIGRL rapidly appeared in cerebrospinal fluid (CSF) following peripheral administration and had a half-life in CSF of approximately 25 min. These results suggest that activation of central PAR2 with brain accessible peptide agonists causes a temporary deficit in the formation and/or recollection of experience-dependent learning and memory.

How changes in the sequence of the peptide CLPFFD-NH2 can modify the conjugation and stability of gold nanoparticles and their affinity for beta-amyloid fibrils.

In a previous work, we studied the interaction of beta-amyloid fibrils (Abeta) with gold nanoparticles (AuNP) conjugated with the peptide CLPFFD-NH2. Here, we studied the effect of changing the residue sequence of the peptide CLPFFD-NH2 on the efficiency of conjugation to AuNP, the stability of the conjugates, and the affinity of the conjugates to the Abeta fibrils. We conjugated the AuNP with CLPFFD-NH 2 isomeric peptides (CDLPFF-NH2 and CLPDFF-NH2) and characterized the resulting conjugates with different techniques including UV-Vis, TEM, EELS, XPS, analysis of amino acids, agarose gel electrophoresis, and CD. In addition, we determined the proportion of AuNP bonded to the Abeta fibrils by ICP-MS. AuNP-CLPFFD-NH2 was the most stable of the conjugates and presented more affinity for Abeta fibrils with respect to the other conjugates and bare AuNP. These findings help to better understand the way peptide sequences affect conjugation and stability of AuNP and their interaction with Abeta fibrils. The peptide sequence, the steric effects, and the charge and disposition of hydrophilic and hydrophobic residues are crucial parameters when considering the design of AuNP peptide conjugates for biomedical applications.

In Vitro Potency and Preclinical Pharmacokinetic Comparison of All-D-Enantiomeric Peptides Developed for the Treatment of Alzheimer's Disease.

Diffusible amyloid-ß (Aß) oligomers are currently presumed to be the most cytotoxic Aß assembly and held responsible to trigger the pathogenesis of Alzheimer's disease (AD). Thus, Aß oligomers are a prominent target in AD drug development. Previously, we reported on our solely D-enantiomeric peptide D3 and its derivatives as AD drug candidates. Here, we compare one of the most promising D3 derivatives, ANK6, with its tandem version (tANK6), and its head-to-tail cyclized isoform (cANK6r). In vitro tests investigating the D-peptides' potencies to inhibit Aß aggregation, eliminate Aß oligomers, and reduce Aß-induced cytotoxicity revealed that all three D-peptides efficiently target Aß. Subsequent preclinical pharmacokinetic studies of the three all-D-peptides in wildtype mice showed promising blood-brain barrier permeability with cANK6r yielding the highest levels in brain. The peptides' potencies to lower Aß toxicity and their remarkable brain/plasma ratios make them promising AD drug candidates.

Liquid chromatography-mass spectrometry assay for quantification of Gluten Exorphin B5 in cerebrospinal fluid.

A sensitive, precise and accurate method for the quantification of the alimentary opioid peptide Gluten Exorphin B5 (GE-B5, Tyr-Gly-Gly-Trp-Leu) in cerebrospinal fluid (CSF) was developed using liquid chromatography-mass spectrometry (LC-MS). Aliquots (10 microL) of sheep CSF were injected into a LC-MS instrument equipped with a reversed-phase C12 column at a flow rate of 250 microL/min. The mobile phase consisted of Eluent A water with 0.01% acetic acid as an ion-pairing reagent, and Eluent B acetonitrile. The LC-MS system was programmed to divert column flow to waste for 3.5 min after injection, after which time flow was directed into the mass spectrometer that operated in positive ion mode. DADLE (Tyr-D-Ala-Gly-Phe-D-Leu) was used as Internal Standard. No significant interfering peaks were detected at the retention times of GE-B5 in CSF blanks. The calibration curves were linear in the range of 0.39-78.00 ng/mL. The lower limit of detection and the lower limit of quantitation values for GE-B5 in CSF were established at 0.30 and 0.78 ng/mL, respectively. The intra-day and inter-day precision values were <12% relative standard deviation. The intra-day and inter-day accuracy were 99.46-100.86% and 98.95-100.02%, respectively. Recovery of GE-B5 in CSF samples was greater than 80%. Stability studies indicate that GE-B5 in CSF undergoes significant degradation (>55% after 600 min), which is reduced by the addition of protease inhibitors. This is the first reported method for the quantification of GE-B5 in CSF.

Quantification of Gluten Exorphin A5 in cerebrospinal fluid by liquid chromatography-mass spectrometry.

In the present work, for the first time, a method for the quantification of the alimentary opioid peptide Gluten Exorphin A5 (GE-A5; Gly-Tyr-Tyr-Pro-Thr) in cerebrospinal fluid (CSF) was developed. Aliquots (5 microL) of CSF were injected into a liquid chromatography-mass spectrometry (LC-MS) instrument equipped with a reversed-phase C18 column at a flow-rate of 0.4 mL/min. The mobile phase consisted of Eluent A water with 0.6% acetic acid as an ion-pairing reagent and Eluent B acetonitrile/methanol (75:25, v/v). The LC-MS system was programmed to divert column flow to waste for 4 min after injection, after which time flow was directed into the mass spectrometer that operated in positive ion mode. No significant interfering peaks were detected at the retention times of GE-A5 in CSF blanks. The lower limit of detection and the lower limit of quantitation values for GE-A5 in CSF were established at 0.60 and 1.50 ng/mL, respectively. The intra- and inter-day precision values were <5% relative standard deviation. The intra- and inter-day accuracy were 99.6-102.8% and 100.0-101.9%, respectively. The reported assay employs extremely small volumes of CSF, thus allowing the analysis of GE-A5 from both small and large animal models.

Effects of morphine and endomorphins on the polysynaptic reflex in the isolated rat spinal cord.

At the spinal level, mu-opioids exert their actions on nociceptive primary afferent neurons both pre- and postsynaptically. In the present study, we used an in vitro isolated neonatal rat (11-15 days old) spinal cord preparation to examine the effects of morphine and the endogenous mu-opioid ligands endomorphin-1 (EM-1) and endomorphin-2 (EM-2) on the polysynaptic reflex (PSR) of dorsal root-ventral root (DR-VR) reflex. The actions of mu-opioids on spinal nociception were investigated by quantification of the firing frequency and the mean amplitude of the PSR evoked by stimuli with 20 x threshold intensity. EM-1 decreased the mean amplitude of PSR, whereas EM-2 and morphine decreased the firing frequency. The pattern of the effects elicited by morphine was the same as that for EM-2, except at high concentration. Naloxonazine, a selective mu(1) opioid receptor antagonist, had no significant effect on PSR by itself, but blocked the inhibition of PSR firing frequency or amplitude induced by EM-1, -2 and morphine. This may suggest that EM-1, EM-2 and morphine modulate spinal nociception differently and act mainly at the mu(1)-opioid receptors. Although they all act via mu(1)-opioid receptors, their different effects on the PSR may suggest the existence of different subtypes of the mu(1)-opioid receptor. The present data is also consistent with a further hypothesis, namely, that morphine and EM-2 activate a subtype of mu(1)-opioid receptor presynaptically, while EM-1 acts mainly through another subtype postsynaptically. However, since other reports indicate that EM-2, but not EM-1, could stimulate the release of enkephalins or dynorphin, presynaptic delta and kappa receptors may be also involved indirectly in the different regulation by mu-opioids at the spinal level.

Antinociceptive effects of endomorphin-2: suppression of substance P release in the inflammatory pain model rat.

Endomorphin-2 (EM2) and Substance P (SP) exert suppressive and facilitative influences upon nociception, respectively. Although EM2 and SP were often co-expressed in single neurons in dorsal root ganglion (DRG), it is still unknown if and how the nociception-suppressive influences of EM2 might be exerted upon nociception-facilitative effects of SP in the DRG neurons. We examined these issues in the inflammatory pain model rats produced by subcutaneous injection of the complete Freund's adjuvant into the hind paw. The paw withdrawal threshold for mechanical allodynia was measured. Changes of EM2 and SP release were estimated by measuring intrathecal levels of EM2 and SP through in vivo microdialysis analysis of cerebrospinal fluid. The mechanical allodynia was dose-dependently attenuated by intrathecal injection of EM2 or a neurokinin-1 receptor antagonist, and facilitated by intrathecal injection of SP or a mu-opioid receptor (MOR) antagonist. Importantly, intrathecal level of SP was found to be lowered by intrathecal injection of EM2. Morphologically, colocalization of EM2-, MOR- and SP-immunoreactivity in single DRG neurons was observed by immunofluorescent histochemistry, and co-expression of EM2 and SP in large, dense-cored presynaptic vesicles in primary afferents, as well as localization of MOR on pre- and postsynaptic membrane in spinal dorsal horn, was also confirmed electron miscroscopically. Thus, the results indicated that analgesic influences of EM2 upon inflammatory pain might be exerted through suppression of SP release, supporting the assumptions that binding of EM2 to presynaptic MOR might induce such effects.

Identification of neuropeptide FF-related peptides in human cerebrospinal fluid by mass spectrometry.

Several neuropeptide FF (NPFF)-related peptides, known as modulators of the opioid system, have been previously characterized in bovine and rodent brain. Reverse-phase high pressure liquid chromatography (HPLC) fractions of a human with normal pressure hydrocephalus cerebrospinal fluid (CSF), co-migrating with NPFF-related synthetic peptides, were characterized by capillary HPLC coupled on-line to nanospray ion trap tandem mass spectrometry. Two peptides present in the pro-NPFF(A) precursor, NPAF (AGEGLNSQFWSLAAPQRF-NH2) and NPSF (SLAAPQRF-NH2), were identified. The monitoring of NPFF-related peptides in human CSF can be helpful to understand their roles in pain sensitivity.

Distribution and characterization of enterostatin-like immunoreactivity in human cerebrospinal fluid.

Enterostatins belong to a family of peptides (e.g., Val-Pro-Asp-Pro-Arg, VPDPR; Ala-Pro-Gly-Pro-Arg, APGPR; and Val-Pro-Gly-Pro-Arg, VPGPR) derived from the tryptic cleavage of amino-terminal pentapeptide from procolipase. Pharmacologic studies have suggested a role for these peptides in appetite regulation and insulin secretion. Studies into the distribution of enterostatins or the role of endogenous peptides have not been possible until now due to the lack of a suitable method for assay. Using two polyclonal antibodies raised against VPDPR and APGPR and different chromatographic methods, we have examined the nature and distribution of enterostatin-like immunoreactivity in human cerebrospinal fluid. The results reported here show for the first time the presence of enterostatin-like immunoreactivity in the human cerebrospinal fluid. Further characterization of cerebrospinal fluid enterostatin-like immunoreactivity revealed that it is not due to APGPR, VPGPR, or VPDPR but to another peptide similar to VPDPR.

Neuropeptide FF-like immunoreactivity in human cerebrospinal fluid of chronic pain patients and healthy controls.

Neuropeptide FF (NPFF) is a neuropeptide with some antiopioid characters found in several mammalian species. In human brain it might be an important pain-regulating peptide. Using a specific and sensitive radioimmunoassay we found a mean concentration of NPFF in human cerebrospinal fluid (CSF) of healthy volunteers of 1.6 +/- 1.1 pg/ml (n = 19) and in chronic pain (CPD) patients of 1.4 +/- 1.2 pg/ml (n = 16). The NPFF concentrations in CSF and plasma did not correlate. There was no difference in the NPFF concentrations in CSF and plasma between CPD patients and healthy controls. NPFF in CPD patients did not correlate significally with any pain characteristic. This study provides evidence for the presence of NPFF in human brain, but does not support the hypothesis that chronic pain is a consequence of elevated production of NPFF.

Radioimmunoassay of delta sleep-inducing peptide using an iodinated p-hydroxyphenylpropionic acid derivative as tracer.

A highly sensitive radioimmunoassay for delta sleep-inducing peptide (DSIP) has been developed. A p-hydroxyphenylpropionic acid conjugate of DSIP was used for radioiodination. Using reversed-phase high performance liquid chromatography the labelled DSIP derivative was isolated in a high yield and with a high specific activity. The assay allows measurement of DSIP-like material in body fluids with a minimum detectable concentration of 0.1 ng/ml standard DSIP (10 pg/tube).

Pro-xenopsin(s) in vesicles of mammalian brain, liver, stomach and intestine is apparently released into blood and cerebral spinal fluid.

Mammalian pro-xenopsins (proXP), proteins (such as alpha-coatomer) that yield XP-related peptides when digested by pepsin-related proteases, are ubiquitously distributed in rats, with highest concentrations in liver and gastrointestinal tissues. Here, the cellular and subcellular distributions of canine and rat proXP were determined in brain, liver, stomach and intestine. Elutriation and percoll density centrifugation of collagenase-dispersed cells demonstrated that proXP was primarily associated with hepatocytes in liver, chief and parietal cells in stomach and endocrine/exocrine cells in intestine. When fragmented cells were subjected to differential centrifugation, congruent with85% of proXP was associated with particulate fractions and only congruent with15% was cytosolic. Sucrose-gradient centrifugation of crude mitochondrial preparations (P2 pellets) for liver, stomach and intestine demonstrated that proXP was localized to vesicles (density, congruent with1.19; size, 80-400 micrometer), which contained material of variable electron density. In isotonic homogenates of brain, proXP migrated primarily with synaptosomes (density, congruent with1. 15) which contained vesicles (size, 50-100 micrometer). During HPLC-sizing and ion exchange chromatography, proXP gave at least three components, the major one being an anionic 140-kDa protein. ProXP-like activity was found in human and rat blood, human cerebral spinal fluid and in contents of the gastrointestinal lumen. These results are consistent with the idea that these vesicle-associated protein(s) could be released during endocrine and/or exocrine secretion and serve as precursors to XP-related peptides.

Investigation of inhibition angiotensin-converting enzyme (ACE) activity and opioid activity of two hemorphins, LVV-hemorphin-5 and VV-hemorphin-5, isolated from a defined peptic hydrolysate of bovine hemoglobin.

Two peptides, LVV-hemorphin-5 and VV-hemorphin-5, were isolated from a defined peptic bovine hemoglobin hydrolysate by reversed-phase HPLC. These peptides were identified as 31-38 and 32-38 fragments of beta chain of bovine hemoglobin. Their inhibitory activity towards angiotensin-converting enzyme and opioid potency were determined. Since their amino acid sequences show close homology with spinorphin, which is found in human cerebrospinal fluid and in the bovine spinal cord, the possible physiological role in vivo of these peptides was discussed.

Delta-sleep inducing peptide in cerebrospinal fluid from schizophrenics, depressives and healthy volunteers.

CSF levels of DSIP was measured in healthy volunteers and in hospitalized patients with schizophrenia or major depressive disorders. Both patient groups had significantly lower CSF levels of DSIP than the healthy volunteers. There was a tendency for lower CSF levels of DSIP in female compared to male schizophrenics. Neuroleptic treatment did not significantly affect those levels. The levels of DSIP in CSF tended to be inversely correlated to the sleep disturbancy scores in the depressed patients.

The small sodium-channel blocking factor in the cerebrospinal fluid of multiple sclerosis patients is probably an oligopeptide.

An endogenous factor that is able to reduce the fast transient sodium current of excitable cells has been reported to exist in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. This was confirmed with nine clinically definite MS patients in the acute relapse. In order to purify and chemically identify the factor, microconcentration and gel filtration high-performance liquid chromatography (HPLC) were applied. After each purification step the activity-containing fraction was determined using a biological assay. With all CSFs the activity was contained in the fraction corresponding to 600-800 Da molecular weight, indicating that the factor is chemically homogeneous. The biological activity of the CSF specimens was not correlated to the laboratory CSF data; however, it was correlated to the area under the 210 nm UV light absorption peak in the corresponding chromatogram, i.e. the 600-800 Da MW fraction. As the factor was degradable by acid hydrolysis and a carboxypeptidase, it is suggested that it might be a small peptide.

Delta sleep-inducing peptide crosses the blood-brain-barrier in dogs: some correlations with protein binding.

To determine if the nonapeptide delta sleep-inducing peptide (DSIP) and some of its analogs can enter the cerebrospinal fluid (CSF) from the peripheral circulation, dogs anesthetized with sodium pentobarbital were injected with a 100 micrograms/kg bolus of DSIP, desTrp1-DSIP, or D-Ala3-DSIP. Using an antibody that is highly specific for these nonapeptides, we found significant increases in CSF after IV injection of each. Column chromatography of CSF withdrawn after an IV bolus of DSIP showed the increase in immunoreactivity to be due to DSIP and desTrp1-DSIP. Chromatography of radioactivity appearing in the CSF after an IV bolus of 125I-N-Tyr-DSIP showed co-elution with the intact labeled peptide. The CSF/plasma ratios for DSIP and 125I-N-Tyr-DSIP were higher than that for tritiated insulin injected IV. It was also shown that DSIP is reversibly bound to a large molecule in dog plasma and CSF, and that the binding is greater in blood than in CSF. The binding protein demonstrates some specificity for DSIP, binding a smaller percentage of D-Ala3-DSIP, and it appears that it is probably free DSIP that crosses the blood-brain-barrier. Although non-specific crossing cannot be ruled out, the results presented here are also consistent with a specific saturable process for DSIP.

DSIP occurs in free form in mammalian plasma, human CSF and urine.

Although delta-sleep inducing peptide was isolated and characterized several years ago, no definitive evidence has been presented for the natural existence of the free peptide. Several attempts at the partial characterization of DSIP-like immunoreactivity (DSIP-LI) have indicated that a small part of the total immunoreactivity is probably present as the free nonapeptide. Using gel chromatography (Sephadex G-100) and subsequent high performance liquid chromatography on rabbit, human, rat and dog plasma, we now show a distinct peak of DSIP-LI that has the same elution position as synthetic DSIP. Free DSIP was also found in human CSF, whereas in human urine most of the small molecular weight DSIP-LI eluted at a position corresponding to DSIP-P, the phosphorylated analog of DSIP. A newly developed antibody recognizing primarily small molecular weight DSIP-LI was used in a modified, rapid assay to facilitate demonstration of the natural occurrence of free DSIP.