Use of cholinesterase inhibitors in Alzheimer disease

According to the cholinergic theory of Alzheimer's disease, the cognitive failure depends on a deficit in acetylcholine. The study reported above examines the efficacy and tolerability of cholinesterase inhibitors, such as tetrahydroamino acridine (THA), for the management of this pathology.     

The results of the brain computer tomography and clinical picture in acute cholinesterase inhibitors poisoning

The aim of this study was to evaluate a morphological and functional status of the CNS in acute cholinesterase inhibitors (ChI) poisonings using the brain computer tomography (CT) and complex psychiatric examination. Under examination there were 59 cholinesterase inhibitors orally poisoned patients, treated at the Department of Clinical Toxicology in years 1984-1997, aged from 14 to 68 (mean 34.7 +/- 12.8) years. The examined group comprised 9 women (15.3%) and 50 men (84.7%). Between the 3rd and 7th day of hospitalisation a complex psychiatric examination was performed. The CNS damage was diagnosed when the point score from complex psychiatric examination was minimum 5 points. CT was performed between the 3rd and 10th day after the intoxication. Incorrect CT scans were found in 78% of poisoned patients. The most common lesion was generalised cortex atrophy and subcortex atrophy of the brain (73.9%), followed by isolated cortex (17.4%) and subcortex atrophy with simultaneous areas of low density in the subcortical nuclei (8.7%). The frequency of incorrect CT scans was statistically higher (p < 0.01) in the group of organophosphorous compounds poisoned patients compared to those poisoned with carbamates. The complex psychiatric examination revealed in 24 patients (40.7%) the differently intensified alterations, but the point score was not higher than 4. Unquestionable damage of the CNS was recognised in 34 patients (57.6%) of the ChI poisoned patients. The frequency of CNS changes detected in complex psychiatric examination was statistically higher (p < 0.01) in the group of the severely poisoned patients. The significantly higher frequency of pathological changes revealed by the brain CT was found in the group of patients with higher than 5 point score obtained from the complex psychiatric evaluation compared to those with score lower than 5 (p < 0.001).     

Comparative pharmacokinetics of four cholinesterase inhibitors in rats

Pharmacokinetics of a very short-acting, a short-acting and two long-acting cholinesterase (ChE) inhibitors, edrophonium, neostigmine, pyridostigmine and ambenonium, respectively, were compared to elucidate the major determinant of their pharmacokinetics. No dose-dependency in pharmacokinetic behavior was observed within the range of 2-10 mumol/kg for edrophonium, 0.5-2 mumol/kg for pyridostigmine, 0.1-0.5 mumol/kg for neostigmine and 0.3-3 mumol/kg for ambenonium, respectively. Neostigmine has the shortest elimination half-life, and edrophonium, pyridostigmine and ambenonium follow in that. Four ChE inhibitors have similar Vdss values within the range of 0.3-0.7 l/kg, which is similar to the muscle/plasma concentration ratio of these drugs. The liver or kidney to plasma concentration ratio of all ChE inhibitors at 20min after i.v. administration ranged from 5 to 15. Small distribution volumes estimated from the plasma concentration profiles may reflect the distribution to muscle and to the extracellular space of other organs/tissues, while the rapid disappearance of ChE inhibitors from plasma may reflect the concentrative uptake to the liver and kidney.     

Binding of amyloid beta-protein to intracellular brain proteins in rat and human

Amyloid beta-protein (Abeta), in its soluble form, is known to bind several circulatory proteins such as apolipoprotein (apo) E, apo J and transthyretin. However, the binding of Abeta to intracellular proteins has not been studied. We have developed an overlay assay to study Abeta binding to intracellular brain proteins. The supernatants from both rat and human brains were found to contain several proteins that bind to Abeta 1-40 and Abeta 1-42. No major difference was observed in the Abeta binding-proteins from brain supernatants of patients with Alzheimer's disease and normal age-matched controls. Binding studies using shorter amyloid beta-peptides and competitive overlay assays showed that the binding site of Abeta to brain proteins resides between 12-28 amino acid sequence of Abeta. The presence of several intracellular Abeta-binding (AbetaB) proteins suggests that these proteins may either protect Abeta from its fibrillization or alternatively promote Abeta polymerization. Identification of these proteins and their binding affinities for Abeta are needed to assess their potential role in the pathogenesis of Alzheimer's disease.     

Distribution of six transplasma membrane NADH-dehydrogenases in rat brain tissue

Transplasma membrane redox plays a significant role in cellular activation and growth. Six isoenzymes could be prepared from purified rat brain synaptic plasma membrane. Polyclonal antibodies have been prepared against six transplasma membrane oxydoreductases (PMO-I to PMO-VI) and the tissue distribution of the various iso-enzymes have been investigated in adult rat brains by means of immunohistochemistry. PMO-I is densely observed in layers I, IV and V of the parietal cortex, in CA1 of the hippocampus (except for the molecular layer), in the caudate putamen, in the dorsal, granular and ventral parts of the auditory nuclei, in some loci of the vestibular nuclei as well as in the deep cerebellar nucleus and in the granular layer of the cerebellar cortex. PMO-II is mainly located in the polymorphic layer of the dentate gyrus and in the deep cerebellar nucleus and in the granular layer of the cerebellar cortex. PMO-III is abundant in the piriform cortex, in the pyramidal layers of both CA1 and CA2, in the diagonal band of the basal ganglia, in the supraoptic nucleus and in various loci of the magnetocellular paraventricular nucleus of the hippothalamus as well as in the vestibular nuclei from the brain stem. In addition PMO-III is also densely present in motor nuclei (oculomotor, facial, hypoglossal and ambiguus nuclei), in the reticular formation and in the deep cerebellar nucleus as well as in the Purkinje layer of the cerebellar cortex. PMO-IV has a similar location but is less abundant in the vestibular nuclei of the sensory brain stem and in the motor nucleus. PMO-V in contrast is poorly present in most brain areas compared to the other iso-enzymes, apart of the Purkinje layer of the cerebellar cortex. Finally PMO-VI is mainly present in the oriens layer and in the stratum radiatum of the hippocampus formation, in the supraoptic and lateral magnocellular nucleus of the hypothalamus, in the mesencephalic trigeminal nucleus, in the ventral auditory nucleus and in the facial nucleus of the brain stem as well as in red nucleus of the reticular formation and in the Purkinje layer of the cerebellar cortex. These data show that the iso-enzymes are located in specific brain nuclei. The significance of the results in respect to the yet very poorly defined function of PMO's is discussed.     

Transplanted neural tissue develops connections with host rat brain

Superior collicular fragments transplanted from fetal to newborn rat brains develop complex internal organization and receive visual afferents from the host providing they lie sufficiently close to the host visual pathways. This system allows investigation in vivo of special affinities between cells of the mammalian central nervous system.     

Toxicodynamic analysis of cardiac effects induced by four cholinesterase inhibitors in rats

The cardiac effect of edrophonium (2-20 mumol kg-1), pyridostigmine (0.5-5 mumol kg-1), neostigmine (0.05-0.5 mumol kg-1) and ambenonium (0.02-0.3 mumol kg-1) was investigated after intravenous administration to rats. For pyridostigmine and neostigmine, the heart rate decreased in a dose-dependent manner, and then gradually recovered to the basal level at about 10 min. Rapid decrease of heart rate was observed after edrophonium and ambenonium administration, and rapid recovery to the basal level within 1 min. For ambenonium, a dose-dependent tachycardiac response was observed. The time-course of heart rate change was analysed by the effect-compartment model. Significant correlation was observed between bradycardiac EC50 values obtained by effect-compartment model analysis and inhibitory constant (Ki) to acetylcholinesterase in-vitro, suggesting that the bradycardiac response was induced by inhibition of this enzyme and following elevation of acetylcholine concentration in the synaptic elect. On the other hand, the tachycardiac EC50 values of edrophonium and ambenonium based on the effect-compartment model analysis were similar to dissociation constants (Kd) of these drugs to muscarinic receptors in-vitro, suggesting that the tachycardiac activity of these drugs may be associated with antagonistic activity to postsynaptic muscarinic receptors. We conclude that, clinically, edrophonium and ambenonium are safer drugs than pyridostigmine and neostigmine, at least as regards muscarinic side-effects, including bradycardia.     

Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain

Several compounds of relatively rigid molecular structure have been found to exert strong blockade of monoamine uptake by synaptosomal preparations of rat corpus striatum (dopamine and serotonin) and hypothalamus (norepinephrine). These include CP-24,441 (1R, 4S-N-methyl-4-phenyl-1,2,3,4-tetrahydro-1-naphthylamine), EXP-561 (4-phenylbicyclo[2.2.2]octan-1-amine), nomifensine and nefopam. The well-defined molecular geometry of the potent inhibitor EXP-561 is a fundamental structural/conformational requirement for uptake blocking activity for the large family of phenylbutylamine- and phenoxypropylamine-related inhibitors. The tubular configuration of EXP-561 may be the most appropriate for blocking serotonin uptake. The requisite conformation for blocking dopamine uptake appears to be defined by the combination resulting from superimposition of the CP-24,441 and nomifensine structures. The conformation defined by the combination resulting from superimposition of the CP-24-441 and desipramine structures is apparently optimal for blocking norepinephrine uptake. The conformational requirements for diphenylpropylamine-related uptake blockers may be defined by the rigid compound CP-39,332 (N-methyl-4-phenyl-1,2,3,4-tetrahydro-2-naphthylamine). The actual potency of any given inhibitor is probably modulated by additional structural and stereochemical factors.     

Cloning and tissue distribution of two new potassium channel alpha-subunits from rat brain

The release of excitatory amino acids from Schwann cell cultures in the rat was monitored using high-performance liquid chromatography. The basal concentration of glutamate and aspartate was 33 +/- 4 nM (mean +/- S.E.M., n = 12) and 8 +/- 1 nM (mean +/- S.E.M., n = 12), respectively. ATP (100 microM) caused a receptor-mediated increase in release of glutamate and aspartate from Schwann cell cultures. Bath application of adenosine (100 microM) was without effect on release of excitatory amino acids suggesting involvement of P2 receptors. Suramin, a competitive antagonist at P2 receptors, prevented the response to ATP. The release of excitatory amino acids evoked by ATP was not abolished in calcium-depleted saline. Pretreatment of the Schwann cultures with 50 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetracetic acid-acetoxymethyl ester (BAPTA-AM) abolished the effect of ATP. ATP-evoked release of glutamate from cultured Schwann cells was significantly reduced by thapsigargin (1 microM), an inhibitor of Ca(2+)-ATPase of the Ca2+ pump of internal stores. U73122, a selective inhibitor of receptor-coupled phospholipase C-dependent processes, abolished stimulatory effect of ATP suggesting that ATP's action is mediated through an inositol 1,4,5-triphosphate-sensitive calcium store. The action of ATP was not blocked by L-trans-pyrrolidine-2,4-dicarboxylate, an inhibitor of the electrogenic glutamate transporter, nor was it blocked in Na(+)-free medium, and glutamate release was not stimulated by a depolarizing stimulus, suggesting that ATP-evoked release of glutamate from Schwann cells is not due to the reversal of the glutamate uptake. An anion transport blocker, furosemide, reduced ATP-induced glutamate release. These results suggest that ATP-stimulated glutamate and aspartate release from Schwann cells may be through a calcium-dependent furosemide-sensitive mechanism.     

Antisense inhibition of butyrylcholinesterase gene expression predicts adverse hematopoietic consequences to cholinesterase inhibitors

1. To investigate the possibility that cholinesterase inhibitors may cause adverse hematopoietic effects, we employed antisense oligodeoxynucleotides selectively inhibiting butyrylcholinesterase gene expression (AS-BCHE). Complementary sense (S) oligonucleotides served as controls. 2. In primary bone marrow cell cultures grown with interleukin 3 (IL-3), AS-BCHE but not S-BCHE reduced growth of megakaryocyte colony-forming units (CFU-MK) in a dose-dependent manner at the micromolar range. 3. In cultures grown with IL-3, transferrin, and erythropoietin (Epo), cell counts increased up to twofold, yet colony counts (CFU-GEMM) remained unchanged under AS-BCHE treatment. 4. Electrophoretic measurements of DNA ladder as an apoptotic index revealed that the above oligonucleotide effects were not due to nonspecific induction of programmed cell death. 5. Differential cell counts demonstrated increased myeloidogenesis and reduced levels of early megakaryocytes in CFU-GEMM under AS-BCHE, suggesting requirement of the BuChE protein for megakaryopoiesis. 6. In vivo injection of AS-BCHE reduced BCHE mRNA levels in both young and mature megakaryocytes for as long as 20 days, as shown by in situ hybridization. 7. Ex vivo growth of primary bone marrow cells revealed a twofold reduction in CFU-MK colonies grown from the AS-BCHE- but not the S-BCHE-injected mice, 15 days posttreatment. 8. These findings demonstrate that deficient butyrylcholinesterase expression, and hence interference with this enzyme's activity through treatment with or exposure to cholinesterase inhibitors, may cause hematopoietic differences in treated patients.     

Transplantation of brain tissue in the brain of adult rats

Brain tissues obtained from rat embryos were transplanted in the forebrain and/or cerebellum of the adult rats. The transplants survived, grew and achieved normal cellular and cytoarchitectural differentiation. They had become anatomically integrated with the host brain. The animals did not show any obviously detectable abnormal behavior or pathology of the brain. The transplants survived as long as the animals did suggesting that they had become a part and parcel of the host brain.     

Binding characteristics of a potent AMPA receptor antagonist [3H]Ro 48-8587 in rat brain

A new AMPA receptor antagonist, Ro 48-8587, was characterized pharmacologically in vitro. It is highly potent and selective for AMPA receptors as shown by its effects on [3H]AMPA, [3H] kainate, and [3H] MK-801 binding to rat brain membranes and on AMPA- or NMDA-induced depolarization in rat cortical wedges. [3H]Ro 48-8587 bound with a high affinity (KD = 3 nM) to a single population of binding sites with a Bmax of 1 pmol/mg of protein in rat whole brain membranes. [3H]Ro 48-8587 binding to rat whole brain membranes was inhibited by several compounds with the following rank order of potency: Ro 48-8587 > 6-nitro-7-sulphamoylbenzo[f] quinoxaline-2,3-dione (NBQX) > YM 90K > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > quisqualate > AMPA > glutamate > kainate > NMDA. The distribution and abundance of specific binding sites (approximately 95% of total) in sections of rat CNS, revealed by quantitative receptor radioautography and image analysis, indicated a very discrete localization. Highest binding values were observed in cortical layers (binding in layers 1 and 2 > binding in layers 3-6), hippocampal formation, striatum, dorsal septum, reticular thalamic nucleus, cerebellar molecular layer, and spinal cord dorsal horn. At 1 nM, the values for specific binding were highest in the cortical layers 1 and 2 and lowest in the brainstem (approximately 2.6 and 0.4 pmol/mg of protein, respectively). Ro 48-8587 is a potent and selective AMPA receptor antagonist with improved binding characteristics (higher affinity, selectivity, and specific binding) compared with those previously reported.     

Transplantation of brain tissue in the brain of rat. I. Growth characteristics of neocortical transplants from embryos of different ages

Differential growth of neural transplants as related to the age of the donor embryos was investigated in this study. Neocortical tissue of constant volume, obtained from embryos of 15, 16, 17, 18, 19, 20, and 21 days' gestational age, was transplanted into the cerebellum of 10-day-old rats. The fully grown transplants were analyzed quantitatively and qualitatively 90 days after transplantation. The ultimate volume of the transplants and the estimated total number of neurons in them followed a gradient in relation to the age of the donor embryos. At one extreme, the neural transplants from 15-day-old embryos grew very large, showing a 21-fold increase in size, and at the other extreme, those from 21-day-old embryos grew less than two-fold in volume. These differences were determined by the developmental history of the transplants. Neural tissue obtained from 15-day-old embryos contained predominantly neuroepithelial cells which continued to proliferate even after transplantation. This resulted in the large size of these transplants. At the other extreme, neural tissue from 21-day-old embryos contained predominantly preformed neuroblasts, and they simply differentiated afte transplantation. Due to this, the transplants were small in size. Neural tissues obtained from other embryos of different gestational ages between these two extremes contained neuroepithelial cells and preformed neuroblasts in differential ratios. The number of neuroepithelial cells in the transplants and their differential proliferative activity after transplantation, and the number of neuroblasts present, determined the differential sizes of these transplants. In histological preparations, all transplants were seen to contain normal-looking and well-differentiated neurons, and normal-looking neuropil. The transplants were integrated with the host brain, in that there was neither any gap nor any scar tissue between the transplants and the host neural tissue surrounding them. Neither the transplants nor the host brains showed any pathological reaction or neoplastic growth.     

Quantitation of acetazolamide in rat plasma, brain tissue and cerebrospinal fluid by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method for the analysis of acetazolamide (AZ) in rat blood (plasma/serum, whole blood and serum ultrafiltrate), brain tissue and cerebrospinal fluid (CSF) was described. Quantitative extraction of AZ with ethyl acetate from both buffered plasma and brain tissue homogenate (pH 8.0) was achieved. Each extract was evaporated to dryness and the residue was chromatographed on a reversed-phase column. CSF was directly analysed without extraction step. The limits of detection were 0.05 microgram ml-1 for plasma, 0.02 microgram g-1 for brain tissue and 0.004 microgram ml-1 for CSF. Calibration curves were linear over the working ranges of 0.1-100 micrograms ml-1 for plasma, 0.05-50 micrograms g-1 for brain tissue and 0.025-50 micrograms ml-1 for CSF. The reproducibility of AZ assay in the rat biologic media indicated very low relative standard deviations (RSDs). The recoveries of AZ added to plasma and brain tissue were more than 96% with an RSD of less than 5%. The present method was applied to studies of plasma concentration profiles of the drug after administration and its distribution into central nervous system.     

The use of pupillometry for the timely diagnosis of the intoxication by cholinesterase inhibitors

The article deals with diagnostic potential of new generation pupillometers having hi-tech units and using up-to-date methods of information processing. Those pupillometers proved to be effective for express diagnosis of intoxications.