In search of innovative therapeutics for neuropsychiatric disorders: the case of neurodegenerative diseases.

The recent medical literature highlights the lack of new drugs able to prevent or treat neurodegenerative diseases such as Alzheimer disease or Parkinson disease. Yet, the prevalence of these diseases is growing, related to increasing life expectancy, and is leading to a rise in their economic and social cost. At the same time, pharmaceutical companies are reducing or halting their investment in neuropharmacological research. Why have advances in basic neuroscience and our understanding of these diseases not allowed innovative discoveries in drug research? This review will try to explain this failure and suggest possible solutions: develop basic and clinical research but with the emphasis on translational and truly collaborative research; improve preclinical studies by developing more appropriate animal models, using new biomarkers and methodologies such as imaging suitable for clinical trials, providing worthwhile information on the ability of the drug to reach its intended target and induce significant pharmacological changes; build a new system of research management, based on stronger interdisciplinary relations between preclinical and clinical research and including the introduction of international precompetitive research between academic teams, start-up companies and pharmaceutical laboratories; hold early discussions with the regulatory authorities during preclinical studies and at the beginning of clinical trials in order to validate the methodological approaches; involve patients' associations in this new organization of research. These changes should help to ensure the discovery of effective treatments for these pathologies.

[Basal ganglia and psychiatric disorders: an experimental validation]. / Troubles psychiatriques et ganglions de la base : une validation expérimentale.

Abnormal movements and behavioral disorders are characteristic manifestations observed in certain neuropsychiatric diseases such as Tourette's syndrome or Huntington Disease. Together with brain imaging findings, the clinical data could suggest a relationship with basal ganglia dysfunction. In the first part of this review, we recall the anatomic relationships existing, via segregated cortico-cortical circuits, between these structures and the cortical areas having motor and cognitive or motivational-emotional attributes. This structure suggests that in pathologies like Parkinson's or Huntington disease cognitive and motivational-emotional disorders as well motor disturbances could be related to lesions or dysfunctions involving individual or combined zones of the basal ganglia. The second part of the paper focuses on a description of the various methodologies used to explore these relationships: behavioral, anatomic and brain imaging methods are used in non-human primate models in order to reproduce motor and behavioral disturbances and to determine the neuronal circuits involved. Microinjection of bicucullin into the external globus pallidus has been found to induce localized and reversible neuronal activation. Abnormal movements can be obtained from the motor territory of the external globus pallidus whereas hyperactivity with attentional deficit or stereotypies have been obtained from the associative or limbic territory of the same structure. In the striatum, the same pharmacological activation can induce either abnormal movements from motor and associative functional territories or behavioural changes with hyperactivity or, on the contrary, hypoactivity from associative functional territory with stereotyped behavior and sexual manifestations when the microinjections were done in the limbic striatum. Anatomic studies as well as brain imaging using PET confirm the involvement of segregated anatomic pathways through the basal ganglia in behavioral as well as motor disorders.

Impact on adenosine stress cardiac magnetic resonance for recanalisation and follow up of chronic total coronary occlusions.

OBJECTIVE: To evaluate the impact on cardiac magnetic resonance imaging (CMRI) with adenosine stress and delayed enhancement for indication and follow up after interventional recanalisation of chronic total coronary occlusions (CTOs). MATERIAL AND METHODS: Twenty consecutive patients (15 males; 5 females; mean age 65 years) with CTO verified by cardiac catheterisation referred to CMRI. Sixteen of them got CMRI before and after coronary recanalisation. Wall motion abnormalities (WMAs), first pass perfusion with adenosine and viability were assessed using a 1.5 T MR scanner (Sonata; Siemens). CMRI results were compared with clinical classifications, the results of cardiac catheterisation and follow up angiography. RESULTS: Sixteen patients had a successful recanalisation, 15 of the occluded coronary artery and one of collateral donor artery stenosis. After recanalisation all stress-induced progressive or new wall motion abnormalities (WMAs) of the corresponding segments and in the collateral donor territory (5 patients) and all adenosine induced perfusion defects (PD) or delay (12 patients) were regredient. 13/16 patients showed no transmural and one patient transmural delayed enhancement (DE) indicating myocardial scar. In 10/16 patients CSS grading of angina improved after recanalisation. CONCLUSION: After successful recanalisation of CTOs, patients with preinterventional stress-induced PDs and WMAs in viable myocardium did not display any signs of stress-induced ischemia postinterventionally. A comprehensive CMRI approach, including assessment of rest and stress WMAs, first pass perfusion and myocardial viability represents an important tool for the pre-interventional decision to recanalise CTOs and follow up.

Metabolic effects of nigrostriatal denervation in basal ganglia.

In the past, functional changes in the circuitry of the basal ganglia that occur in Parkinson's disease were primarily analyzed with electrophysiological and 2-deoxyglucose measurements. The increased activity of the subthalamic nucleus (STN) observed has been attributed to a reduction in inhibition mediated by the external segment of the globus pallidus (GPe), secondary to the loss of dopaminergic-neuron influence on D2-receptor-bearing striato-pallidal neurons. More recently, in situ hybridization studies of cytochrome oxidase subunit I have confirmed the overactivity of the STN in the parkinsonian state. In addition, this technique has provided evidence that the change in STN activity is owing not only to decreased inhibition from the GPe but to hyperactivity of excitatory inputs from the parafascicular nucleus of the thalamus and the pedunculopontine nucleus in the brainstem.

Five isoenzymes of protein kinase C are expressed in normal and STZ-diabetic rat hepatocytes: effect of phorbol 12-myristate 13-acetate.

Using isoenzyme-specific antisera, five Protein Kinase Cs (PKCs) were detected in cytosol and membrane hepatocytes from normal rats: PKC alpha (80 kDa), PKC beta II (40, 50, 55, 85 kDa), PKC delta (74, 76 kDa), PKC epsilon (95 kDa), PKC zeta (65, 70 kDa). STZ-diabetes induced a lower expression of the five PKCs, a higher localization in the cytosol, a preferential expression of PKC delta as the 76 kDa phosphorylated species and a decreased kinase activity towards Histone III-S. A 1 microM phorbol 12-myristate 13-acetate (PMA) incubation induced similar translocation to the membrane of PKCs alpha, native 85 kDa beta II and epsilon. The 74 kDa PKC delta was switched to the 76 kDa species, the normal form in STZ-diabetic cells. The truncated PKC beta II and PKC epsilon were unchanged.

Kinetic evidence of a surface membraneous step during the endocytosic process of 3H-labelled asialoorosomucoid and its alteration in diabetic mellitus rats.

The apparent internalization rate constant of asialoorosomucoid in normal and diabetic hepatocytes was determined using different experimental processes, either following a synchronous wave of prebound ligand or a continuous flux ligand endocytosis, either alone or simultaneously. In continuous flux conditions, no difference between normal and diabetic hepatocytes appeared (k = 0.15 +/- 0.04 and 0.11 +/- 0.02 min-1, respectively). In contrast, in the one-turn endocytosis of prebound ligand, k was lower for diabetic hepatocytes than for normal ones whether it was measured alone (0.20 +/- 0.03 and 0.59 +/- 0.09 min-1, respectively) or simultaneously with a continuous flux of unlabelled ligand (0.25 +/- 0.03 and 0.70 +/- 0.08 min-1, respectively). These differences are attributed to an impediment or a delay in the preclustering of receptors in coated pits at the cell surface of diabetic cells.

Effect of phenobarbital on the oligosaccharide structure of rat alpha-acid glycoprotein.

We have recently shown that the administration of phenobarbital to rats leads to an increased serum alpha 1-acid glycoprotein content with alterations in the relative proportion of the sugar moiety. Therefore, alpha 1-acid glycoprotein was purified from normal (alpha 1-acid glycoproteine N) and phenobarbital-treated rats (alpha 1-acid glycoprotein PB) Glycans were separated by AX-10 chromatography and analysed by gas chromatography. It appears that, compared to alpha 1-acid glycoprotein N, alpha 1-acid glycoprotein PB had a higher carbohydrate content (31.7% compared to 26%) and a non-negligible amount of neutral oligosaccharide (12.2% compared to 1.3%). No tetrasialyl oligosaccharides in alpha 1-acid glycoprotein PB were detected, whereas their relative proportion in alpha 1-acid glycoprotein N was 27%.

Alterations in the carbohydrate moiety of alpha-1-acid glycoprotein purified from human cirrhotic ascitic fluid.

The carbohydrate analysis of alpha 1-AGPc purified from cirrhotic ascitic fluid was performed by immunoaffinity chromatography. It showed a large increase in the fucosyl molar ratio and sugar content (47%). The molar ratio of the oligosaccharides which were released by hydrazinolysis and fractionated by high-performance liquid chromatography confirms the marked increase in fucosyl residues in each fraction. A shift towards fractions with a high degree of branching was also observed. Moreover, the studies of sugar molar ratios and methylation of the tetrasialylated fraction indicated the simultaneous presence of sialyl and fucosyl residues on one of the outer branches.

Measurement of skin vasoconstrictor response in healthy subjects.

OBJECTIVES: Laser Doppler flowmetry enables non-invasive quantification of skin blood flow and its sympathetically mediated change. Four different maneuvers were performed in 60 healthy subjects aged 20-78 years to investigate their variability, reproducibility and to determine the influence of gender, age and height. MATERIAL AND METHODS: Skin blood flow was measured on the pulp of both index fingers using laser Doppler flowmetry. Vasoconstriction was induced by deep inspiratory gasp, arm dependency, acoustic stimulation and a modified cold pressor test. RESULTS: More than 95% of normal subjects showed a vasoconstrictor response to cold pressure test and 100% to inspiratory gasp. In all other maneuvers vasoconstrictor response was less reliable. The magnitude of vasoconstrictor responses decreased with age in all maneuvers, while latencies remained unchanged. Only during inspiratory gasp men showed more pronounced vasoconstrictor response compared with women. Body height influenced latencies if peripheral stimuli were applied like in the cold pressor and arm dependency tests. CONCLUSIONS: Inspiratory gasp and the modified cold pressor test were found to be more suitable maneuvers for routine clinical testing than arm dependency and acoustic stimulation. Normal data also for side differences are provided as a base for routine clinical testing in systemic and unilateral neurological disorders.

[Pharmacology, an innovative factor in therapeutic research]. / La pharmacologie, facteur d'innovation en recherche thérapeutique.

As members of the pharmacology training group set up by the committee of pharmacological science of the French Academy of Pharmacy, we examine the situation of pharmacology in drug discovery. Today, it is obvious that by integrating genome sequencing, cellular and molecular biology, and bioinformatics, pharmacology has become a cross-disciplinary science. Pharmacologists must become knowledgeable in a wide range of domains, using the major points in each to direct them towards the discovery and development of new therapeutic agents. It is also clear that pharmacology remains a major factor in the different steps of drug discovery, from the molecular and cellular stages, to clinical and pharmaceutical developments.

Kinetic analysis of epidermal growth factor endocytosis in rat hepatocytes. Effects of diabetes.

In the present study we followed the different steps of epidermal growth factor receptor (EGF-R) endocytosis in freshly isolated rat hepatocytes. Hepatocytes exhibit two classes of surface EGF receptors consisting of approximately 5,000 high-affinity sites (Kd = 15 pM) and 166,000 low-affinity sites (Kd = 670 pM). Binding of labeled EGF to hepatocytes permeabilized by digitonin shows that 75% of the total EGF-R are localized at the cell surface. At 37 degrees C, hepatocytes continuously internalized and degraded EGF in spite of a down-regulation of cell surface receptors. The internalization rate constants measured as a function of a range 125I-EGF concentrations (0.01 - 5 nM) involving various degrees of EGF-R occupancy show superimposable curves. This indicates that the specific internalization rate of EGF-R complex is independent of receptor occupancy. Streptozotocin-induced diabetes reduces the number of low-affinity EGF-R to 50,000 and produces a complete loss of high-affinity sites. The dynamics of 125I-EGF endocytosis show that diabetic hepatocytes fail to down-regulate the surface EGF-R efficiently although the constant rate of internalization is not modified. Decreased down-regulation of EGF-R together with enhanced EGF endocytosis suggest a greater efficiency in EGF-R recycling in diabetic rat hepatocytes.

Increased affinity to concanavalin A and enhanced secretion of alpha 1-acid glycoprotein by hepatocytes isolated from turpentine-treated rats.

Hepatocytes were isolated from adult rats at various times after subcutaneous injection of turpentine (1 ml). The affinity to concanavalin A (Con A) of alpha 1-acid glycoprotein (AGP) and the intracellular content and rate of secretion of AGP and albumin were evaluated over a period of 19 days. Inflamed hepatocytes secreted mainly the Con A-reactive form of AGP whereas control hepatocytes secreted a higher amount of the Con A-non-reactive form. The intracellular content and rate of secretion of AGP by inflamed hepatocytes increased markedly whereas those of albumin decreased. However, when the residence time (ratio of intracellular content to rate of secretion) was evaluated, it appeared that the efficiency of secretion of both proteins was higher than in control hepatocytes. The changes in the affinity of AGP to Con A and in the secretion of AGP and albumin were reversible. These findings indicate that acute inflammation leads to posttranslational alterations during the intracellular transit of these secretory proteins.

Study of hepatic binding protein activity in jejuno-ileal by-passed rat hepatocytes.

The kinetic constants of internalization of asialoorosomucoid were determined for normal and jejuno-ileal by-passed rat hepatocytes. In by-passed rats the maximum velocity of asialoorosomucoid internalization is decreased 3-fold, without any modification of apparant constant of internalization. Moreover, the rate constant of internalization was the same in the two groups of rats. These data suggest that the process of asialoorosomucoid internalization is not altered in by-passed hepatocytes and that the decrease of maximal velocity is only due to a decrease of total uptake receptor number.

[Relationship between the structure and activity of a histamine-blocking serum glycoprotein]. / Relation entre structure et activité biologique d'une glycoprotéine sérique histamino-bloquante

A plasmatic glycoprotein is submitted to a mild periodate oxydation and its pharmacological activity is studied. This glycoprotein contains much N acetyl Neuraminic Acid (NANA = 15 p. cent), and it reduces the biological activity of histamine on smooth muscle such as guinea pig ileum. See article. We also identify the 8 NANA and 7 NANA derivaties. Th only 8 carbon derivative is obtained when about one mole of m-periodate is consumed for one mole of NANA. The 7 carbon derivative appears as soon as the consumption of a second mole leads ta a second cleavage. These results prove that the oxydation islimited to the sole N acetyl neuraminic acid and more precisely to the lateral polyhydroxylic chain. Under these conditions, pharmacological activity gradually decreases, it disappears as soon as the lateral polyhydroxylic chain is completely cut off.

Effect of polyisobutylcyanoacrylate nanoparticles and lipofectin loaded with oligonucleotides on cell viability and PKC alpha neosynthesis in HepG2 cells.

The aim of the present study was to evaluate the inhibitory effect on protein kinase C alpha (PKC alpha) neosynthesis of antisense oligonucleotides delivered by two types of carriers. First, PKC alpha antisense oligonucleotides were associated with polyisobutylcyanoacrylate (PIBCA) nanoparticles pre-coated with cetyltrimethyl ammonium bromide (CTAB), a hydrophobic cation. Adsorption of oligonucleotides onto PIBCA nanoparticles was shown to be a saturating process. From these studies, it was possible to identify two types of particles: positively and negatively charged. Secondly, Lipofectin was used as another carrier system. These systems were incubated with HepG2 cells. Toxicity was evaluated by the MTT assay, and PKC alpha neosynthesis was determined by Western blots in conditions where nanoparticles and Lipofectin were not inducing cytotoxicity. It was observed that both mismatch and antisense oligonucleotides induced an inhibition of PKC alpha neosynthesis when loaded onto cationic or anionic nanoparticles as well as when complexed to cationic liposomes (Lipofectin). This non-specific effect was only observed in the phase of PKC alpha neosynthesis when the cells were first depleted in PKC alpha by phorbol 12-myristate beta-acetate (12-PMA) and in the absence of serum. These results strongly suggest that delivery systems, PIBCA nanoparticles or Lipofectin, containing a positively charged component (CTAB or cationic lipids), are able to induce a perturbation in the intracellular metabolic activity. In conclusion, it was shown that the commonly used strategy of oligonucleotides targeting with cationic non-viral vectors may display non-specific effects which can lead to artifactual results.

Comparative effects of diabetes and monensin on the lectin asialoglycoprotein receptor: biosynthesis, structure and function in rats.

The hepatic asialoglycoprotein receptor is the first studied mammalian lectin. Modulations in vivo by diabetes and in vitro by the carboxylic ionophore monensin gave rise to similar apparent alterations on its biosynthesis, structure and ligand binding capacity. In normal rats, the receptor (whether purified by ligand or antibody-affinity chromatography) presented a similar pattern in SDS-PAGE analysis, with a major 42-kDa band and two minor ones (49 and 52-54 kDa). In diabetic rats, a new 38-kDa band appeared, but only after antibody-affinity purification. In vitro biosynthesis of the receptor by normal hepatocytes in the presence of 35S-methionine showed that this 38-kDa band was present at the end of a 30-min pulse but decreased during a 180-min chase, in association with an increase in the major 42-kDa band. In diabetic cells, this evolution was retarded. Using a 30-min pulse followed by a 120-min chase in the presence of 100 microM monensin, we showed that this carboxylic ionophore had similar effects on diabetes, leading to a delay in the maturation process of the 42-kDa band and the persistent emergence of the 38-kDa species. Allowing incubation in the presence of 25 or 100 microM monensin, we observed a decrease in the number of ligand binding sites both at the surface (40%) and within the cell (28%). In hepatocytes from diabetic rats, monensin showed no additional effect on the partial diabetes-induced inactivation.

An immunochemical procedure to evaluate the degree of desialylation of alpha 1-acid glycoprotein in rat serum.

When radial immunodiffusion (RID) and electroimmunodiffusion (EID) were used for the determination of rat alpha 1-acid glycoprotein (alpha 1-AGP) a significant discrepancy in the results was encountered depending on the degree of sialylation. When alpha 1-AGP was desialylated, the amounts estimated by EID were much lower than those actually present as assayed by the RID method. The relationship between the percentage of desialylation of alpha 1-AGP and the percentage of its underestimation by EID relative to RID was determined and a calibration curve was plotted to evaluate the degree of desialylation of rat alpha 1-AGP. When compared to other procedures (rat membrane inhibition assay and isoelectrofocusing), the proposed method was easier to perform and allowed the specific evaluation of the degree of undersialylation of the glycoprotein.

Chronic blockade of D2 but not D1 dopamine receptors facilitates behavioural responses to endogenous enkephalins, protected by kelatorphan, administered in the accumbens in rats.

It has previously been shown that kelatorphan, (R)-3-(N-hydroxycarboxamido-2-benzyl-propanoyl)-L-alanine, a mixed inhibitor of the catabolism of enkephalins, injected into the nucleus accumbens, induced a dose-dependent hyperlocomotion in rats. In this study, the consequence of chronic treatment with sulpiride, a selective D2 dopamine receptor antagonist, SCH 23390, a selective D1 dopamine receptor antagonist, or haloperidol, a nonspecific but preferential D2 receptor antagonist, on the behavioural response induced by acute administration of kelatorphan into the accumbens, has been investigated in rats. The drug SCH 23390 did not modify the behavioural response to kelatorphan, whereas sulpiride and haloperidol induced an increase which was maximal in the third week after the beginning of treatment, a period corresponding to the appearance of the antipsychotic effect of the neuroleptics. This facilitation was reversed by prior administration of the delta-selective antagonist, ICI 174864. These results suggest that the phasic activity of enkephalinergic neurones of the nucleus accumbens and the associated behavioural hyperactivity are facilitated after chronic blockade of the D2 but not the D1 subtypes of dopamine receptor.

Effects of intrasubthalamic injection of dopamine receptor agonists on subthalamic neurons in normal and 6-hydroxydopamine-lesioned rats: an electrophysiological and c-Fos study.

Subthalamic neuronal activity is controlled by a dopaminergic innervation, which may act via D1 and D2 dopamine receptors. This study investigates the effect of apomorphine and the selective D1 and D2 agonists, SKF 82958 and quinpirole respectively, in normal and 6-hydroxydopamine-lesioned rats. The effect of microinjection of these drugs into the subthalamic nucleus was assessed by recording unit activity and the expression of the c-Fos-immunoreactive protein in the subthalamic nucleus. Dopaminergic agonists reduced the discharge rate and did not induce c-Fos expression in the normal rat. Apomorphine and quinpirole increased the discharge rate and induced a strong expression of c-Fos-like immunoreactive proteins, whereas SKF 82958 induced a decrease of the discharge rate and a slight expression of c-Fos in 6-hydroxydopamine-lesioned rats. The striking contrast in the changes obtained with apomorphine and quinpirole in normal and 6-hydroxydopamine-lesioned rats is discussed in relation to a hyperexpression of D2 dopaminergic receptors on the GABAergic terminals into the subthalamic nucleus. These results show that, in normal rats, dopamine agonists exert an inhibitory control on subthalamic neurons via D1 and D2 receptors. However, in 6-hydroxydopamine-lesioned rats, the hyperactivity of subthalamic neurons is also reduced by D1 receptor agonist but not by D2 dopamine agonists. This last result points out one aspect of the complex mechanisms underlying the physiopathology of Parkinson's disease.

Electrophysiological study of the excitatory parafascicular projection to the subthalamic nucleus and evidence for ipsi- and contralateral controls.

The activity of subthalamic neurons was recorded extracellularly in anaesthetized rats after stimulation, inhibition or lesioning of the parafascicular nucleus. Electrical stimulation of the parafascicular nucleus evoked a complex response with two excitatory phases. The first response was correlated with a monosynaptically-driven excitation via a parafascicular input to the subthalamic nucleus. Since the second phase was observed even when the early excitation was not recorded and was eliminated by lesion of the globus pallidus, we suggest that it is not generated by a mechanism intrinsic to the subthalamic nucleus and is due to a disinhibitory effect originating from the globus pallidus. Microinjection of carbachol into the parafascicular nucleus enhanced by 119% the discharge rate of the neurons in the ipsilateral subthalamic nucleus and that of muscimol decreased the discharge rate by 91%. Opposite changes, a decrease of the discharge rate of 49% after microinjection of carbachol and an increase of 47% after muscimol, occurred in the contralateral subthalamic nucleus. In contrast to the above results, the unilateral excitotoxic lesion of the parafascicular nucleus, performed one week before recording, decreased the discharge rate by 69% of the ipsilateral subthalamic nucleus neurons and by 34% that of the contralateral neurons. We suggest that the parafascicular input to the subthalamic nucleus is an excitatory pathway which can tonically drive the neuronal activity in this structure. The opposite changes recorded in the ipsi- and contralateral subthalamic nucleus during unilateral microinjection of excitatory or inhibitory drugs in the parafascicular nucleus emphasize the importance of this thalamic structure in the bilateral regulation of basal ganglia activity via the subthalamic nucleus.