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.

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.

Effect of recombinant adenovirus coding for endomorphin-2 on neuropathic pain in rats.

OBJECTIVE: To construct a transgene expressing human endomorphin-2 by linking the signal peptide of mouse nerve growth factor (PN) to a human endomorphin-2 DNA sequence containing a short linker recognized by the protease FURIN and test the analgesic effect of endomorphin-2 on neuropathic pain. METHODS: The transgene was inserted into the cosmid pAxCAwt to generate PN-EM-2-pAxCAwt. The recombinant adenovirus Ad-PNEM2 was packaged and propagated in HEK293 cells. After the Ad-PNEM2-infected NIH3T3 cells had been cultured, protein expression was examined by immunofluorescence and ELISA. A CCI rat model was constructed and the Ad-PNEM2 was administered intrathecally. The rats' pain thresholds (PWL) were measured and the presence of endomorphin-2 in the cerebrospinal fluid was confirmed through ELISA. RESULTS: The Ad-PNEM2 expressed endomorphin-2 smoothly and abundantly in NIH3T3 cells at a significantly higher rate than the viral control (P<0.01) or blank control (P<0.01). The expressed endomorphin-2 was mainly observed in the cytoplasm. The concentration of endomorphin-2 in the cerebrospinal fluid increased 1 day after injection and peaked between 7 and 14 days after injection. After injection, PWL approached normal levels in the operated study group. No significant change was observed in the control groups. There was a significant correlation between PWL and endomorphin-2 level (r = 0.944, P<0.001). CONCLUSION: The constructed human endomorphin-2 transgene was expressed effectively, and endomorphin-2 expressed by the recombinant adenovirus altered the threshold to thermal stimulus and showed good analgesic effect.

[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.

Intrathecally injected Ile-Pro-Ile, an inhibitor of membrane ectoenzyme dipeptidyl peptidase IV, is antihyperalgesic in rats by switching the enzyme from hydrolase to synthase functional mode to generate endomorphin 2.

We have found recently that membrane-bound dipeptidyl peptidase IV (DPP-IV) generated extracellularly immunoreactive endomorphin-2 from Tyr-Pro precursor in a depolarisation-sensitive manner in rat isolated L4,5 dorsal root ganglia when the enzyme was switched to synthase mode by the hydrolase inhibitor Ile-Pro-Ile. Presently, we induced hyperalgesia in rats by injecting carrageenan into the right hindpaw and measured the reduction in nociceptive threshold (hyperalgesia) to pressure (Randall-Selitto test). The hyperalgesia, peaking at 180 min after injection, was fully reversed by intrathecal administration of 30 nmol/rat Ile-Pro-Ile. The antihyperalgesic action was antagonized by s.c. naloxone (1 mg/kg) and intrathecally injected specific antiserum to endomorphin-2 indicating that the opioid receptor-mediated effect was produced by an endogenously generated endomorphin-2-like immunoreactive substance. Intrathecal Ile-Pro-Ile was ineffective as an analgesic in the acute nociceptive test such as the rat tail-flick, whereas endomorphin-2 (EC(50)=13.3 nmol/rat), endomorphin-1 (6.8 nmol/rat), morphine (0.11 nmol/rat) and DAMGO (0.0059 nmol/rat) exerted opioid receptor-mediated analgesia given by the same route. We concluded that carrageenan-induced C-fiber barrage (wind-up) may create ideal conditions for the de novo synthesis of endomorphin-2 in rat spinal cord dorsal horns if the DPP-IV enzyme is switched to the synthase functional mode by Ile-Pro-Ile.

Plasma and cerebrospinal fluid pharmacokinetics of tasidotin (ILX-651) and its metabolites in non-human primates.

PURPOSE: To evaluate the plasma and cerebrospinal fluid (CSF) pharmacokinetics and CSF penetration of tasidotin and metabolites in a nonhuman primate model. METHODS: Tasidotin 0.75 mg/kg was administered intravenously. The plasma and CSF concentrations of tasidotin and its metabolites were determined. Pharmacokinetic parameters were estimated using model-independent and model-dependent methods. RESULTS: The mean (+/-SD) CSF:plasma AUC ratio for tasidotin was 1.1 +/- 0.4. For tasidotin, tasidotin-C-carboxylate and desprolyl-tasidotin-C-carboxylate the plasma AUCs (mean +/- SD) were 30 +/- 10, 54 +/- 19 and 12 +/- 2 microM min, and apparent plasma half-lives were 27 +/- 4, 229 +/- 73 and 100 +/- 29 min. The plasma clearance of tasidotin was 44 +/- 14 ml/min/kg. The CSF AUC and half-life of tasidotin was 28 +/- 10 microM min and 96 +/- 40 min. The model-dependent plasma clearance was 35 ml/min/kg for tasidotin and 2 ml/min/kg for tasidotin-C-carboxylate. CONCLUSIONS: Tasidotin penetrates into the CSF well and further evaluation of its activity in the treatment of central nervous system malignancies should be considered.

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.

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.

[Neurohumoral induction of structural and functional compensatory reorganization of the damaged brain]. / Neirogumoralnaia induktsiia strukturno-funktsionalnoi kompensatornoi reorfanizatsii povrezhdennogo mozga.

A correlation has been found between the time course of the biological activity of specific oligo- and polypeptide factors in cerebrospinal fluid and the effectiveness of motor function recovery. The fluid as a donor material accelerates the compensatory process in recipient animals with unilateral neocortical damage. The procedure stimulates the formation process of connections of the centrally denervated semisegments of the spinal cord with the somatosensory cortex of the intact brain hemisphere. Endolumbar introduction of cerebrospinal fluid from reconvalescents induced the regress of symptoms of central motor disorders in patients with stroke sequelae.

CSF-plasma relationships for DSIP and some other neuropeptides.

The relationships between CSF and plasma hormonal levels of several peptides were studied in the same samples of simultaneously obtained plasma and CSF. A significant correlation existed between CSF and plasma levels of DSIP as well as gastrin. Preliminary results also showed a correlation between CSF and plasma levels of NT, but not VIP or calcitonin. CSF/plasma ratios and the effect of BBB disruption also varied from peptide to peptide. These diverse CSF/plasma relationships are not easily explained by models of nonspecific passage but may indicate individual systems or axes that could be involved in the central effects of peripherally administered peptides.