Inhibitor Report for: Galanthamine

Binge drinking and alcohol abuse are common during adolescence and cause both cognitive deficits and lasting cholinergic pathology in the adult basal forebrain. Acetylcholine is anti-inflammatory and studies using the preclinical adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2 day on/2 day off from postnatal day [P]25 to P54) model of human adolescent binge drinking report decreased basal forebrain cholinergic neurons (BFCNs) and induction of proinflammatory genes that persist long into adulthood. Recent studies link AIE-induced neuroimmune activation to cholinergic pathology, but the underlying mechanisms contributing to the persistent loss of BFCNs are unknown. We report that treatment with the cholinesterase inhibitor galantamine (4.0 mg/kg, i.p.) administered during AIE (i.e., P25-P54) or following the conclusion of AIE (i.e., P57-P72) recovered the persistent loss of cholinergic neuron phenotype markers (i.e., ChAT, TrkA, and p75(NTR)) and somal shrinkage of residual ChAT + neurons known to persist in AIE-exposed adults. Galantamine treatment also recovered the AIE-increased expression of the proinflammatory receptors TLR4 and RAGE, the endogenous TLR4/RAGE agonist HMGB1, and the transcription activation marker pNF-kappaB p65. Interestingly, we find BFCNs express TLR4 and RAGE, and that AIE treatment increased pNF-kappaB p65 expression in adult ChAT + IR neurons, consistent with intracellular HMGB1-TLR4/RAGE signaling within BFCNs. AIE increased epigenetic transcription silencing markers (i.e., H3K9me2 and H3K9me3) in the adult basal forebrain and H3K9me2 occupancy at cholinergic phenotype gene promoters (i.e., ChAT and TrkA). The finding of no AIE-induced changes in total basal forebrain NeuN + neurons with galantamine reversal of AIE-induced ChAT + neuron loss, TLR4/RAGE-pNF-kappaB p65 signals, and epigenetic transcription silencing markers suggests that AIE does not cause cell death, but rather the loss of the cholinergic phenotype. Together, these data suggest that AIE induces HMGB1-TLR4/RAGE-pNF-kappaB p65 signals, causing the loss of cholinergic phenotype (i.e., ChAT, TrkA, and p75(NTR)) through epigenetic histone transcription silencing that result in the loss of the BFCN phenotype that can be prevented and restored by galantamine.

The syntheses, the anticholinesterase activities and structure-activity relationships of homodimeric (3a-c) and heterodimeric (6a-c) alkylene linked bis-galanthamine are reported. Compounds 6b-c were found to be more potent than galanthamine and tacrine in inhibiting AChE.

Acetylcholinesterase (AChE) inhibitors from several chemical classes have been tested for the symptomatic treatment of Alzheimer's disease; however, the therapeutic success of these compounds has been limited. Recently, another AChE inhibitor, galanthamine hydrobromide (GAL), has shown increased clinical efficacy and safety. Using biochemical, behavioral and pharmacokinetic analyses, this report compares GAL with two of its analogs, 6-O-acetyl-6-O-demethylgalanthamine hydrochloride (P11012) and 6-O-demethyl-6-O[(adamantan-1-yl)-carbonyl]galanthamine hydrochloride (P11149), for their therapeutic potential. P11012 and P11149 were found to be potent, competitive and selective inhibitors of AChE, demonstrating central cholinergic activity, behavioral efficacy and safety. P11012 and P11149, though pharmacokinetic analyses, were shown to act as pro-drugs, yielding significant levels of 6-O-demethylgalanthamine. In vitro, 6-O-demethylgalanthamine was 10- to 20-fold more potent than GAL as an inhibitor of AChE, and it demonstrated greater selectivity for inhibition of AChE vs. butyrylcholinesterase. Like GAL, both P11012 and P11149 showed central cholinergic activity biochemically, by significantly inhibiting rat brain AChE; physiologically, by causing hypothermia; and behaviorally, by attenuating scopolamine-induced deficits in passive avoidance. In addition, GAL, P11012 and P11149 enhanced step-down passive avoidance, another measure of behavioral efficacy. By comparing efficacious doses with primary overt effects, P11012 and P11149 had better oral therapeutic indices than GAL. Oral pharmacokinetic analyses of GAL, P11012 and P11149 revealed differences. Although P11012 and P11149 exhibited similar area under the curve values, 191149 had slower, lower and more sustained concentration maximum levels. P11012 and GAL rapidly reached their concentration maximums, but GAL, in brain had the highest area under the curve and concentration maximum. Because of its composite profile, including duration of action, oral therapeutic index and pharmacokinetics, P11149 is considered the better therapeutic candidate for the treatment of Alzheimer's disease.

Plant biodiversity is an important source of compounds with medicinal properties. The alkaloid galanthamine, first isolated from Galanthus woronowii (Amaryllidaceae), is approved by the FDA for the palliative treatment of mild to moderate Alzheimer's disease due to its acetylcholinesterase (AChE) inhibitory activity. Obtaining this active pharmaceutical ingredient, still sourced on an industrial scale from the Amaryllidaceae species, is a challenge for pharmaceutical companies due to its low natural yield and the high cost of its synthesis. The aim of this work was to determine the alkaloid profile of three different Rauhia (Amaryllidaceae) species collected in Peru, and to assess the potential application of their extracts for the treatment of Alzheimer's disease. The alkaloids were identified by gas chromatography coupled to mass spectrometry (GC-MS), and the AChE inhibitory activity of the extracts was analyzed. Thirty compounds were quantified from the Rauhia species, the R. multiflora extract being the most interesting due to its high diversity of galanthamine-type structures. The R. multiflora extract was also the most active against AChE, with the half maximal inhibitory concentration (IC(50)) values of 0.17 +/- 0.02 microg.mL(-1) in comparison with the IC(50) values of 0.53 +/- 0.12 microg.mL(-1) for galanthamine, used as a reference. Computational experiments were carried out on the activity of the galanthamine-type alkaloids identified in R. multiflora toward five different human AChE structures. The simulation of the molecules 3-O-acetylgalanthamine, 3-O-acetylsanguinine, narwedine, and lycoraminone on the 4EY6 crystal structure theoretically showed a higher inhibition of hAChE and different interactions with the active site compared to galanthamine. In conclusion, the results of this first alkaloid profiling of the Rauhia species indicate that R. multiflora is an important natural source of galanthamine-type structures and could be used as a model for the development of biotechnological tools necessary to advance the sustainable production of galanthamine.

Binge drinking and alcohol abuse are common during adolescence and cause both cognitive deficits and lasting cholinergic pathology in the adult basal forebrain. Acetylcholine is anti-inflammatory and studies using the preclinical adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2 day on/2 day off from postnatal day [P]25 to P54) model of human adolescent binge drinking report decreased basal forebrain cholinergic neurons (BFCNs) and induction of proinflammatory genes that persist long into adulthood. Recent studies link AIE-induced neuroimmune activation to cholinergic pathology, but the underlying mechanisms contributing to the persistent loss of BFCNs are unknown. We report that treatment with the cholinesterase inhibitor galantamine (4.0 mg/kg, i.p.) administered during AIE (i.e., P25-P54) or following the conclusion of AIE (i.e., P57-P72) recovered the persistent loss of cholinergic neuron phenotype markers (i.e., ChAT, TrkA, and p75(NTR)) and somal shrinkage of residual ChAT + neurons known to persist in AIE-exposed adults. Galantamine treatment also recovered the AIE-increased expression of the proinflammatory receptors TLR4 and RAGE, the endogenous TLR4/RAGE agonist HMGB1, and the transcription activation marker pNF-kappaB p65. Interestingly, we find BFCNs express TLR4 and RAGE, and that AIE treatment increased pNF-kappaB p65 expression in adult ChAT + IR neurons, consistent with intracellular HMGB1-TLR4/RAGE signaling within BFCNs. AIE increased epigenetic transcription silencing markers (i.e., H3K9me2 and H3K9me3) in the adult basal forebrain and H3K9me2 occupancy at cholinergic phenotype gene promoters (i.e., ChAT and TrkA). The finding of no AIE-induced changes in total basal forebrain NeuN + neurons with galantamine reversal of AIE-induced ChAT + neuron loss, TLR4/RAGE-pNF-kappaB p65 signals, and epigenetic transcription silencing markers suggests that AIE does not cause cell death, but rather the loss of the cholinergic phenotype. Together, these data suggest that AIE induces HMGB1-TLR4/RAGE-pNF-kappaB p65 signals, causing the loss of cholinergic phenotype (i.e., ChAT, TrkA, and p75(NTR)) through epigenetic histone transcription silencing that result in the loss of the BFCN phenotype that can be prevented and restored by galantamine.

Three undescribed Amarylidaceae alkaloids, named gigantelline, gigantellinine and gigancrinine, were isolated from Crinum jagus (syn.=Crinum giganteum) collected in Senegal, together with the already known sanguinine, cherylline, lycorine, crinine, flexinine and the isoquinolinone derivative hippadine. Gigantelline, gigantellinine and gigancrinine were characterized as 4-(6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydro-isoquinolin-4-yl)-phenol, its 7-O-demethyl-5-hydroxy-4-methoxy derivative and 5,6a,7,7a,8a,9-hexahydro-6,9a-ethano[1,3]dioxolo[4,5-j]oxireno[2,3-b]phenanthridi n-9-ol, respectively, by using spectroscopic (1D and 2D (1)H and (13)C NMR and HRESIMS) and chemical methods. Their relative configuration was assigned by NOESY NMR spectra and NMR calculations, while the absolute configuration was assigned using electronic circular dichroism (ECD) experiments and calculations. Sanguinine, cherylline, crinine, flexinine, and the isoquinolinone hippadine, were isolated for the first time from C. jagus. Cherylline, gigantellinine, crinine, flexinine and sanguinine inhibited the activity of AChE in a dose-dependent manner, and inhibition by sanguinine was remarkably effective (IC50=1.83+/-0.01muM). Cherylline and hippadine showed weak cytotoxicity at 100muM.

BACKGROUND: In Argentina, the Amaryllidaceae family (59 species) comprises a wide variety of genera, only a few species have been investigated as a potential source of cholinesterases inhibitors to treat Alzheimer disease (AD). PURPOSE: To study the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of the basic dichloromethane extracts (E) from Hieronymiella aurea, H. caletensis, H. clidanthoides, H. marginata, and H. speciosa species, as well as the isolated compounds from these plant extracts. STUDY DESIGN AND METHODS: AChE and BChE inhibitory activities were evaluated with the Ellman's spectrophotometric method. The alkaloids composition from the E was obtained by gas chromatography-mass spectrometry (GC-MS). The E were successively chromatographed on a silica gel column and permeated on Sephadex LH-20 column to afford the main alkaloids identified by means of spectroscopic data. Additionally, an in silico study was carried out. RESULTS: Nine known alkaloids were isolated from the E of five Hieronymiella species. Galanthamine was identified in all the species by GC-MS standing out H. caletensis with a relative abundance of 9.79% of the total ion current. Strong AChE (IC50=1.84 - 15.40microg/ml) and moderate BChE (IC50=23.74 - 136.40microg/ml) inhibitory activities were displayed by the extracts. Among the isolated alkaloids, only sanguinine and chlidanthine (galanthamine-type alkaloids) demonstrated inhibitory activity toward both enzymes. The QTAIM study suggests that sanguinine has the strongest affinity towards AChE, attributed to an additional interaction with Ser200 as well as stronger molecular interactions Glu199 and His440.These results allowed us to differentiate the molecular behavior in the active site among alkaloids possessing different in vitro inhibitory activities. CONCLUSION: Hieronymiella species growing in Argentina represent a rich and widespread source of galanthamine and others AChE and BChE inhibitors alkaloids. Additionally, the new trend towards the use of natural extracts as pharmaceuticals rather than pure drugs opens a pathway for the development of a phytomedicine derived from extracts of Hieronymiella spp.

Galantamine (Gnt) is a natural alkaloid inhibitor of acetylcholinesterase and is presently one of the most used drugs in the treatment against Alzheimer's disease during both the initial and intermediate stages. Among several natural Gnt derivatives, sanguinine (Sng) and lycoramine (Lyc) attract attention because of the way their subtle chemical differences from Gnt lead to drastic and opposite distinctions in inhibitory effects. However, to date there is no solved structure for these natural derivatives. In the present study, we applied computational modeling and free energy calculation methods to better elucidate the molecular basis of the subtle distinctions between these derivatives and Gnt. The results showed that differences in the mobility of the non-aromatic ring carried by the Lyc-like sp(2)-sp(3) modification display drastic conformational, vibrational, and entropic penalties at binding compared to Gnt. Additionally, the establishment of a stronger hydrogen bond network added enthalpic advantages for the linkage of the Sng-like methoxy-hydroxy substituted ligands. These results, which suggest an affinity ranking in agreement with that found in the literature, provided insights that are helpful for future planning and development of new anti-Alzheimer's disease drugs.

Acetylcholine is an excitatory neurotransmitter in the central nervous system that plays a key role in cognitive function, including learning and memory. Previous studies have shown that experimental traumatic brain injury (TBI) reduces cholinergic neurotransmission, decreases evoked release of acetylcholine, and alters cholinergic receptor levels. Galantamine (U.S. Food and Drug Administration approved for the treatment of vascular dementia and Alzheimer's disease) has been shown to inhibit acetylcholinesterase activity and allosterically potentiate nicotinic receptor signaling. We investigated whether acute administration of galantamine can reduce TBI pathology and improve cognitive function tested days after the termination of the drug treatment. Post-injury administration of galantamine was found to decrease TBI-triggered blood-brain barrier (BBB) permeability (tested 24 h post-injury), attenuate the loss of both GABAergic and newborn neurons in the ipsilateral hippocampus, and improve hippocampal function (tested 10 days after termination of the drug treatment). Specifically, significant improvements in the Morris water maze, novel object recognition, and context-specific fear memory tasks were observed in injured animals treated with galantamine. Although messenger RNAs for both M1 (Nos2, TLR4, and IL-12ss) and M2 (Arg1, CCL17, and Mcr1) microglial phenotypes were elevated post-TBI, galantamine treatment did not alter microglial polarization tested 24 h and 6 days post-injury. Taken together, these findings support the further investigation of galantamine as a treatment for TBI.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with memory impairment and cognitive deficit. Most of the drugs currently available for the treatment of AD are acetylcholinesterase (AChE) inhibitors. Plants of the Amaryllidaceae family are known to synthesize alkaloids, which have shown AChE inhibitory activity. Habranthus tubispathus and H. jamesonii are two Amaryllidaceae that can be found growing wild to the southwest of Buenos Aires in Argentina. Acetyl- and butyrylcholinesterase inhibition was observed for the extracts obtained from bulbs of H. tubispathus and bulbs and aerial parts of H. jamesonii. The strongest cholinesterase inhibition was observed for the alkaloid extract obtained from the aerial parts for H. jamesonii (AChE IC50 = 0.7 microg/mL; BChE IC50 = 6.7 microg/mL). The AChE inhibition observed for H. jamesonii could be explained by the presence of galanthamine and sanguinine, two potent AChE inhibitors. The levels of lycorine and hippeastidine, moderate AChE inhibitors, observed in the bulbs of H. tubispathus could be responsible for the significant AChE inhibition observed. The alkaloids present in these Amaryllidaceae were identified by means of GC-MS analysis. In the case of H. tubispathus, hippeastidine and 3-O-demethylhippeastidine, were isolated and completely characterized by 1H and 13C NMR spectroscopy.

Galanthamine-type alkaloids produced by plants of the Amaryllidaceae family are potent acetylcholinesterase inhibitors. One of them, galanthamine, has been marketed as a hydrobromide salt for the treatment of Alzheimer's disease. In the present work, gas chromatography with electron impact mass spectrometry (GC-EIMS) fragmentation of 12 reference compounds isolated from various amaryllidaceous plants and identified by spectroscopic methods (1D and 2D nuclear magnetic resonance, circular dichroism, high-resolution MS (HRMS) and EIMS) was studied by tandem mass spectrometry (GC-MS/MS) and accurate mass measurements (GC-HRMS). The studied compounds showed good peak shape and efficient GC separation with a GC-MS fragmentation pattern similar to that obtained by direct insertion probe. With the exception of galanthamine-N-oxide and N-formylnorgalanthamine, the galanthamine-type compounds showed abundant [M](+.) and [M-H](+) ions. A typical fragmentation pattern was also observed, depending on the substituents of the skeleton. Based on the fragmentation pathways of reference compounds, three other galanthamine-type alkaloids, including 3-O-(2'-butenoyl)sanguinine, which possesses a previously unelucidated structure, were identified in Leucojum aestivum ssp. pulchelum, a species endemic to the Balearic islands. GC-MS can be successfully applied to Amaryllidaceae plant samples in the routine screening for potentially new or known bioactive molecules, chemotaxonomy, biodiversity and identification of impurities in pharmaceutical substances.

Alkaloid extracts from 12 plant species of the families Amaryllidaceae, Fumariacae and Papaveraceae were studied with respect to their acetylcholinesterase inhibitory activity and alkaloid patterns. Fifty-three alkaloids were identified by GC-MS, including known acetylcholinesterase (AChE) inhibitors such as galanthamine, epigalanthamine, sanguinine and epinorgalanthamine in extracts of Amaryllidaceae plants and protopine in extracts of Fumariaceae and Papaveraceae plants. The galanthamine-containing extracts of the amaryllidaceous plants were found to be the most active while the extract of Corydalis bulbosa was the most active among the extracts of the tested plants from the Fumariaceae and Papaveraceae plants. TLC bioautographic assay, preparative TLC and GC-MS analysis were combined to identify the active compounds in the studied extracts. Galanthamine was isolated from the known AChE inhibitors in the extracts of Amaryllidaceae plants. Corydaline, bulbocapnine and stylopine were found to be active in the extracts of plant species of the families Fumariaceae and Papaveraceae. Available standards of deshydrocorydaline--a precursor of corydaline, corydaline and stylopine--were tested for AChE inhibitory activity. Deshydrocorydaline and corydaline showed potent inhibitory activity comparable with that of the positive control galanthamine.

Bifunctional derivatives of the alkaloid galanthamine, designed to interact with both the active site of the enzyme acetylcholinesterase (AChE) and its peripheral cation binding site, have been assayed with Torpedo californica AChE (TcAChE), and the three-dimensional structures of their complexes with the enzyme have been solved by X-ray crystallography. Differences were noted between the IC(50) values obtained for TcAChE and those for Electrophorus electricus AChE. These differences are ascribed to sequence differences in one or two residues lining the active-site gorge of the enzyme. The binding of one of the inhibitors disrupts the native conformation of one wall of the gorge, formed by the loop Trp279-Phe290. It is proposed that flexibility of this loop may permit the binding of inhibitors such as galanthamine, which are too bulky to penetrate the narrow neck of the gorge formed by Tyr121 and Phe330 as seen in the crystal structure.

Amaryllidaceous plants produce pharmacologically active alkaloids, galanthamine being the most interesting for its use in the treatment of Alzheimer's disease as a cholinesterase inhibitor. The aim of this work was to test 23 pure Amaryllidaceae alkaloids and 26 extracts from different species of the genus Narcissus for their acetylcholinesterase inhibitory activity using galanthamine as a reference. Only seven alkaloids, belonging to the galanthamine and lycorine skeleton types, exhibited such an effect, sanguinine being the most active, even more than galanthamine. All the extracts with the highest acetylcholinesterase inhibitory activity contained galanthamine except that of N. assoanus, a lycorine type alkaloid-bearing species.

The 3D structure of a complex of the anti-Alzheimer drug galanthamine with Torpedo californica acetylcholinesterase is reported. Galanthamine, a tertiary alkaloid extracted from several species of Amarylidacae, is so far the only drug that shows a dual activity, being both an acetylcholinesterase inhibitor and an allosteric potentiator of the nicotinic response induced by acetylcholine and competitive agonists. The X-ray structure, at 2.5A resolution, shows an unexpected orientation of the ligand within the active site, as well as unusual protein-ligand interactions. The inhibitor binds at the base of the active site gorge, interacting with both the acyl-binding pocket and the principal quaternary ammonium-binding site. However, the tertiary amine group of galanthamine does not directly interact with Trp84. A docking study using the program AUTODOCK correctly predicts the orientation of galanthamine in the active site. The docked lowest-energy structure has a root mean square deviation of 0.5A with respect to the corresponding crystal structure of the complex. The observed binding mode explains the affinities of a series of structural analogs of galanthamine and provides a rational basis for structure-based drug design of synthetic derivatives with improved pharmacological properties. Proteins 2001;42:182-191.

The syntheses, the anticholinesterase activities and structure-activity relationships of homodimeric (3a-c) and heterodimeric (6a-c) alkylene linked bis-galanthamine are reported. Compounds 6b-c were found to be more potent than galanthamine and tacrine in inhibiting AChE.

Galanthamine proved effective in symptomatic treatment of senile dementia of Alzheimer's type. The aim of this study was to elucidate the metabolism of galanthamine. Two novel metabolites of galanthamine have been isolated from the urine of eight young men after single doses of 10-15 mg. Some 19.8% of the doses were excreted as O-demethylgalanthamine glucuronide, 5% as N-demethylgalanthamine, 25.1% as galanthamine, and 0.8% as epigalanthamine. After coadministration of quinidine hydrogen sulfate, which inhibits cytochrome P450 2D6 (CYP2D6) selectively, O-demethylgalanthamine glucuronide was highly diminished in urine. In vitro, human liver microsomes metabolized galanthamine to O-demethylgalanthamine with Vmax 5.2 nmol/mg protein/h and Km 187 microM. Ki of quinidine to inhibit O-demethylation was 28 nM. To inhibit cholinesterases, O-demethylgalanthamine was 10-fold more selective for acetylcholinesterase (AChE) versus butyrylcholinesterase (BCHE) than galanthamine. After glucuronidation, O-demethylgalanthamine failed to inhibit AChE and BCHE. N-Demethylgalanthamine inhibited cholinesterases less potently than galanthamine.

Alzheimer's disease (AD) is characterized in the brain by the deposition of amyloid protein outside the neuron, resulting in the formation of plaques, and inside the neuron with neurofibrillary tangles. It is not yet known what causes these pathologic changes, although age and genetics are major risk factors. The cholinesterase inhibitors tacrine and donepezil block acetylcholinesterase and therefore preserve the neurotransmitter acetylcholine. Three other investigational cholinesterase inhibitors are rivastigmine, metrifonate, and galanthamine. Existing therapies being studied for use in AD include vitamin E, estrogen preparations, and anti-inflammatory agents. The physician's role is to care for both the AD patient and the family, which are profoundly affected by this disease.

(-)-Galanthamine (GAL), an alkaloid from the flower, the common snowdrop (Galanthus nivalis), shows anticholinesterase activity. This property has made GAL the target of research as to its effectiveness in the treatment of Alzheimer's disease. We have solved the X-ray crystal structure of GAL bound in the active site of Torpedo californica acetylcholinesterase (TcAChE) to 2.3 A resolution. The inhibitor binds at the base of the active site gorge of TcAChE, interacting with both the choline-binding site (Trp-84) and the acyl-binding pocket (Phe-288, Phe-290). The tertiary amine group of GAL does not interact closely with Trp-84; rather, the double bond of its cyclohexene ring stacks against the indole ring. The tertiary amine appears to make a non-conventional hydrogen bond, via its N-methyl group, to Asp-72, near the top of the gorge. The hydroxyl group of the inhibitor makes a strong hydrogen bond (2.7 A) with Glu-199. The relatively tight binding of GAL to TcAChE appears to arise from a number of moderate to weak interactions with the protein, coupled to a low entropy cost for binding due to the rigid nature of the inhibitor.

Different cholinomimetics are used in conditions of CNS acetylcholine (ACh) deficit. In this study, we examined the effect of the acetylcholinesterase inhibitor galanthamine in a prolonged alcohol intake model of ACh deficit in male Wistar rats. After 16 weeks of alcohol intake and a 2-week pause, rats administered galanthamine (2.5 mg/kg/day i.p.) showed an improved speed of learning and short-term memory in the shuttle box test as compared to the saline-injected alcoholic group (p < 0.05). Four weeks later, significant improvement in the passive avoidance memory of alcoholic galanthamine-treated rats was noted in the eight-arm radial maze (14 day test duration) as compared to the saline-injected alcoholic group (p < 0.05). During the first week in the shuttle box test, the nonalcoholic galanthamine-treated animals exhibited significantly impaired performance as compared to the untreated nonalcoholic control, while four weeks later, in the eight-arm radial maze, both groups did not differ. Our results show that galanthamine improves the speed of learning, short-term memory and spatial orientation of rats in conditions of prolonged alcohol intake.

Galanthamine is a third-generation cholinesterase inhibitor used against Alzheimer's disease. New analytical methods for the determination of galanthamine in pharmaceutical preparatives and biological fluids, such as urine and serum, were developed. An experimental design and artificial neural network approach were used for method optimization. Thirty-five ppb of galanthamine were determined in serum samples (with addition of 10 mM magnesium chloride and using solid-phase preconcentration).

This review starts with an historical background of the pharmacological development of tacrine almost fifty years ago (1949). Tacrine is the first drug to be tested, clinically, on a large scale and to be registered (1993) for treatment of Alzheimer's disease. For the first time, clinical results of four second generation cholinesterase inhibitors (ChEI) (donepezil, ENA 713, eptastigmine and metrifonate) are reviewed and compared with other ChEI such as tacrine, physostigmine and galanthamine. Data based on more than 6000 patients show that second generation drugs are well tolerated and show evidence of clinical efficacy. Differences are mainly due to frequency of side effects, number of drop outs and percentage of improved patients. These results also demonstrate the presence of clinical efficacy for all ChEI tested so far. Clinical mechanism of action, levels of efficacy and differences among various ChEI are discussed. Future potential indications are suggested. The present data indicate that optimization of effects prolongation and maintenance of clinical gains will depend on further knowledge of the compounds pharmacodynamic properties.

New galanthamine derivatives, especially bis-interacting ligands 3-5 and 7-9 were prepared in order to interact with the catalytic and the peripheral sites of acetylcholinesterase (AChE). The synthesis, the anticholinesterase activities, and the structure-activity relationships of bis-interacting ligands are reported. Compounds 4d-e were found to be more potent than galanthamine and tacrine in inhibiting AChE.

Galanthamine, an alkaloid present in the Amaryllidaceae is currently undergoing clinical trials for the treatment of Alzheimer's. Common daffodils, Narcissus spp., contain galanthamine and other alkaloids. Four commercial Narcissus cultivars were evaluated as potential sources of galanthamine. Planting depths, planting densities, bulb size or flower bud removal did not affect galanthamine content

Alzheimer's disease is a progressive neurodegenerative disorder primarily manifesting as a loss of memory. Senile plaques and neurofibrillary tangles are the major histopathological alteration in the brain of Alzheimer's disease patients. A considerable deficiency of cholinergic neurons is a consistent finding in Alzheimer's disease. Therefore, many therapeutic strategies to augment cerebral concentration of acetylcholine such as cholinergic precursors, cholinergic receptor agonists, cholinesterase inhibitors and acetylcholine release modulators have been evaluated in Alzheimer's disease. Although cholinesterase inhibitors such as tacrine and galanthamine offer modest clinical benefits, other cholinergic agents have proved to be of limited therapeutic value. Efforts to enhance monoaminergic neurotransmission have also been largely disappointing. Therefore, emphasis is not being put on the use of combination of two class of drugs. Moreover, use of therapeutic agents based on the putative pathogenic etiology of the disease such as excitotoxicity, amyloidosis, aluminium accumulation, inflammatory mechanisms and free radical production is being evaluated. Desferrioxamine, non-steroidal anti-inflammatory drugs, prednisone, dapsone, vitamin E and idebenone are some such agents that are currently under investigation for the preventive or palliative effect in Alzheimer's disease. Neurotrophic factors such as nerve growth factor, brain derived neurotrophic factor and epidermal growth factor have shown promising results in animal studies. However, novel methods for delivering these molecules into the brain required to be developed before launching their clinical trials in man.

Acetylcholinesterase (AChE) inhibitors from several chemical classes have been tested for the symptomatic treatment of Alzheimer's disease; however, the therapeutic success of these compounds has been limited. Recently, another AChE inhibitor, galanthamine hydrobromide (GAL), has shown increased clinical efficacy and safety. Using biochemical, behavioral and pharmacokinetic analyses, this report compares GAL with two of its analogs, 6-O-acetyl-6-O-demethylgalanthamine hydrochloride (P11012) and 6-O-demethyl-6-O[(adamantan-1-yl)-carbonyl]galanthamine hydrochloride (P11149), for their therapeutic potential. P11012 and P11149 were found to be potent, competitive and selective inhibitors of AChE, demonstrating central cholinergic activity, behavioral efficacy and safety. P11012 and P11149, though pharmacokinetic analyses, were shown to act as pro-drugs, yielding significant levels of 6-O-demethylgalanthamine. In vitro, 6-O-demethylgalanthamine was 10- to 20-fold more potent than GAL as an inhibitor of AChE, and it demonstrated greater selectivity for inhibition of AChE vs. butyrylcholinesterase. Like GAL, both P11012 and P11149 showed central cholinergic activity biochemically, by significantly inhibiting rat brain AChE; physiologically, by causing hypothermia; and behaviorally, by attenuating scopolamine-induced deficits in passive avoidance. In addition, GAL, P11012 and P11149 enhanced step-down passive avoidance, another measure of behavioral efficacy. By comparing efficacious doses with primary overt effects, P11012 and P11149 had better oral therapeutic indices than GAL. Oral pharmacokinetic analyses of GAL, P11012 and P11149 revealed differences. Although P11012 and P11149 exhibited similar area under the curve values, 191149 had slower, lower and more sustained concentration maximum levels. P11012 and GAL rapidly reached their concentration maximums, but GAL, in brain had the highest area under the curve and concentration maximum. Because of its composite profile, including duration of action, oral therapeutic index and pharmacokinetics, P11149 is considered the better therapeutic candidate for the treatment of Alzheimer's disease.

Galanthamine is a selective acetylcholinesterase inhibitor which has shown potential for the treatment of Alzheimer's disease. Galanthamine is selective for acetylcholinesterase versus butyrylcholinesterase; however, the drug produces greater enzyme inhibition in human erythrocytes than in human brain tissue. Galanthamine attenuates drug-and lesion-induced cognitive deficits in animal models of learning and memory. Preliminary results in patients with Alzheimer's disease have reported galanthamine to be associated with a reduction in cognitive deterioration on some neuropsychiatric rating scales. Nausea and vomiting are the most commonly reported adverse effects; liver toxicity has not been reported to date.

The results of application of cholinesterase inhibitors, aminostigmin and galantamin, for treatment of acute poisoning with cyclodol, dimedrol, and solutan of moderately grave condition are presented. Aminostigmin was shown to exhibit the more pronounced stable and universal effect. The experiments in animals showed that aminostigmine affected peripheral and central M-cholinoreactive structures and conjugated with them more actively than galantamin. Aminostigmin, but not galantamin increases the rate of dopamine circulation and content of cyclic guanozinemonophosphate in frontal brain of rats, and this effect is exhibited even under the conditions of N-cholinoreceptor blockade with amizyl.

Galanthamine is an alkaloid found in the bulbs of snowdrops and several Amaryllidaceae plants. At submicromolar concentrations, it inhibits acetylcholinesterase activity, but it is much less potent against butyrylcholinesterase activity. Galanthamine has been used in anaesthetics to reverse neuromuscular paralysis by tubocurarine-like muscle relaxants, but it is a tertiary amine that gets into the brain to cause central effects. Galanthamine is being studied as a possible therapeutic agent in Alzheimer's disease because of its central cholinergic effects. Positive effects have been demonstrated in several learning and memory tests in animals.

We studied the effect of intravenous injection of the cholinesterase inhibitor galanthamine (GAL) in doses from 0.025 to 5.0 mg/kg on electrically evoked field potentials in rat visual cortex. In all the experiments the amplitude of late components of evoked potentials was significantly reduced, while early components remained unaffected. These findings indicate that cortical cells are inhibited by acetylcholine (ACh). Furthermore, combined application of a muscarinic receptor blocker (atropine) and GAL reliably suppressed the effects of galanthamine. These observations suggest that ACh-induced inhibition may be mediated by activation of GABAergic interneurones that possess muscarinic receptors.

The acetylcholine esterase inhibitor (-)-physostigmine has been shown to act as agonist on nicotinic acetylcholine receptors from muscle and brain, by binding to sites on the alpha-polypeptide that are distinct from those for the natural transmitter acetylcholine (Schrder et al., 1994). In the present report we show that (-)-physostigmine, galanthamine, and the morphine derivative codeine activate single-channel currents in outside-out patches excised from clonal rat pheochromocytoma (PC12) cells. Although several lines of evidence demonstrate that the three alkaloids act on the same channels as acetylcholine, the competitive nicotinic antagonist methyllycaconitine only inhibited channel activation by acetylcholine but not by (-)-physostigmine, galanthamine or codeine. In contrast, the monoclonal antibody FK1, which competitively inhibits (-)-physostigmine binding to nicotinic acetylcholine receptors, did not affect channel activation by acetylcholine but inhibited activation by (-)-physostigmine, galanthamine and codeine. The three alkaloids therefore act via binding sites distinct from those for acetylcholine, in a 'noncompetitive' fashion. The potency of (-)-physostigmine and related compounds to act as a noncompetitive agonist is unrelated to the level of acetylcholine esterase inhibition induced by these drugs. (-)-Physostigmine, galanthamine and codeine do not evoke sizable whole-cell currents, which is due to the combined effects of low open-channel probability, slow onset and slow inactivation of response. In contrast, they sensitize PC12 cell nicotinic receptors in their submaximal response to acetylcholine. While the abundance of nicotinic acetylcholine receptor isoforms expressed in PC12 cells excludes identification of specific nicotinic acetylcholine receptor subtypes that interact with noncompetitive agonists, the identical patterns of single-channel current amplitudes observed with acetylcholine and with noncompetitive agonists suggested that all PC12 cell nicotinic acetylcholine receptor subtypes that respond to acetylcholine also respond to noncompetitive agonist. The action of noncompetitive agonists therefore seems to be highly conserved between nicotinic acetylcholine receptor subtypes, in agreement with the high level of structural conservation in the sequence region harboring major elements of this site.

Experiments on non-inbred albino mice have demonstrated that aminostigmine is an active reversible centrally active cholinesterase inhibitor close to the properties of physostigmine, but greatly superior to it in its action duration. Clinical examinations of healthy volunteers and patients have shown that aminostigmine-induced inhibition of cholinesterase activity persists 6 hours. The agent have been found to be more highly effective in treating cholin blocker-induced intoxications than galanthamine, which manifests itself in its greater stability of the therapeutical effect achieved and in its higher ability to prevent cardiovascular events occurring in intoxication.

Galanthamine, physostigmine and 9-amino-1,2,3,4-tetrahydroacridine (tacrine) were evaluated as inhibitors of human acetylcholinesterase activity from samples of postmortem human brain, fresh brain cortex biopsies and human erythrocytes. Acetylcholinesterase activity was most effectively inhibited in all tissues by physostigmine, followed by tacrine and galanthamine. The respective inhibitor concentrations exerting a half maximal effect (IC50) on acetylcholinesterase in postmortem human brain frontal cortex were 14 nmol/l, 1.0 mumol/l and 3.2 mumol/l versus 15 nmol/l, 1.1 mumol/l and 2.8 mumol/l in the hippocampus region. In addition, the inhibition of acetylcholinesterase by galanthamine was similar in postmortem brain and brain cortical biopsies from patients submitted to brain-tumour removal, indicating that postmortem changes up to 28 h after death probably did not influence the measurement of acetylcholinesterase inhibition. While physostigmine and tacrine acted equally on acetylcholinesterase from different sources, galanthamine was 10-fold less potent in inhibiting the enzyme activity from human brain that from human erythrocytes. Comparison with issues from mice revealed that galanthamine was selectively more potent in suppressing acetylcholinesterase in human erythrocytes. The results are discussed in the light of pharmacokinetic data, and conclusions are drawn for further clinical studies.

Catalytic properties of human blood erythrocyte acetylcholinesterase and horse blood serum butyrylcholinesterase immobilized and nonimmobilized in the gelatin membrane have been comparatively studied. Cholinesterase immobilization induces an increase in the Michaelis constant value and a decrease in the maximum rate value in reactions of enzymic hydrolysis of thiocholine ethers, but exerts no effect on these kinetic parameters in case of enzymic hydrolysis of indophenylacetate. The effect of reversible inhibitors: galanthamine, N-methyl-4-piperidinyl benzylate and 1,2,3,4-tetrahydro-9-aminoacridine (tacrine), as well as of irreversible inhibitors: O-ethyl-O-(4-nitrophenyl)ethyl phosphonate (armin), diisopropyl fluorophosphate (DFP), O,O-diethyl-O-(4-nitrophenyl) phosphate (paraoxon) and O,O-dimethyl-O-(2,2-dichlorovinyl) phosphate (DDVP) on immobilized cholinesterases is weaker as compared with the effect on nonimmobilized enzymes. The results obtained are discussed for the effect of immobilization on the catalytically active enzyme surface.

We investigated the inhibition of human cholinesterases by galanthamine, an alkaloid of the common snowdrop (galanthus nivalis). In vitro, the compound showed potent enzyme inhibition and 50-fold selectivity for acetylcholinesterase (EC 3.1.1.7) as opposed to butyrylcholinesterase (EC 3.1.1.8). There was no difference between enzyme inhibition by galanthamine in whole blood and separated fractions of plasma and erythrocytes. We conclude that galanthamine does not accumulate in large amounts in red blood cells. In vivo, administration of galanthamine in a healthy volunteer and in a patient who underwent long-term treatment confirmed the selectivity of galanthamine for acetylcholinesterase.

The pharmacokinetics of the long-acting anticholinesterase drug, galanthamine, were investigated in eight patients. After i.v. injection of 0.3 mg kg-1, the decrease in the serum concentration of galanthamine followed a biexponential curve. The serum concentration decreased rapidly from 543 +/- 47 ng ml-1 to 128 +/- 14 ng ml-1 between 2 and 30 min with a T1/2 alpha of 6.42 +/- 2.15 min, and then declined more slowly with a T1/2 beta of 264 +/- 28 min. Total serum clearance of galanthamine amounted to 5.37 +/- 0.87 ml min-1 kg-1, and the renal clearance was 1.36 +/- 0.10 ml min-1 kg-1. The cumulative urinary excretion of galanthamine between 0 and 48 h after injection amounted to 28.0 +/- 5.4% of the administered dose. The biliary excretion of galanthamine during 24 h amounted to 0.2 +/- 0.1% of the dose. There was no evidence of glucuronide or sulphate conjugation of galanthamine.

Miniature end-plate currents (MEPC) in rat diaphragm were studied with voltage-clamp technique when synaptic acetylcholinesterase (AChE) was inhibited with different concentrations of galanthamine. The MEPC amplitude and time course were increased progressively with galanthamine concentrations in the range of 3.16 X 10(-8) - 10(-6) g/ml. The decay of MEPC was always exponential. The input resistance of muscle fibres increased. Galanthamine (10(-5) g/ml) produced a curare-like action: the amplitude and duration of MEPC were less as compared with those at galanthamine concentration 10(-6) g/ml, the decay of MEPC became biphasic. During washing out of the drug, the duration of MEPC began to increase and then to diminish, returning to the initial value 3 hours later. The decay of MEPC became exponential. A positive correlation was found between half-decay time and amplitude of MEPC both in the presence and in the absence of anticholinesterase. It is supposed that the functional role of synaptic AChE in limiting the postsynaptic effect of acetylcholine is not so significant as it is usually considered, therefore it is possible to use the parameters of MEPC for the estimation of functional AChE activity.