Anne-Lie Svensson

Cholinesterase inhibitors in the treatment of Alzheimer's disease: A comparison of tolerability and pharmacology

Cholinesterase inhibitors are currently the most established treatment strategy in Alzheimer’s disease. The treatment effect appears mainly to be symptomatic. Effects on progression of the disease following long term treatment, and possible neuroprotective effects, have been investigated. Delay until nursing home placement has been reported. Three cholinesterase inhibitors, tacrine, donepezil and rivastigmine, are in clinical use. Other cholinesterase inhibitors, such as galantamine (galanthamine), metrifonate, physostigmine, eptastigmine, are currently under clinical evaluation. So far the efficacy appears to be comparable between the various cholinesterase inhibitors; treatment for up to 6 months has produced an improvement in Alzheimer’s Disease Assessment Scale — Cognitive Subscale score (ADAS-cog) of between 1.8 and 4.9 in patients with Alzheimer’s disease.Tacrine, donepezil, galantamine and physostigmine are reversible inhibitors of acetylcholinesterase and butyrylcholinesterase, while metrifonate is considered to be an irreversible inhibitor and rivastigmine a pseudoirreversible inhibitor. Tacrine and physostigmine have lower bioavailability, 17 to 37% and 3 to 8%, respectively, than the other cholinesterase inhibitors such as rivastigmine, galantamine and donepezil (40 to 100%). The elimination half-life is considerably longer for donepezil (70 to 80h) in comparison to most of the other cholinesterase inhibitors (0.3 to 12h). Donepezil is therefore administered once daily in comparison to rivastigmine which is administered twice daily and tacrine which is administered 4 times daily.Simultaneous food intake lowers the plasma concentration of tacrine and reduces the adverse effects of rivastigmine. Drugs like theophylline and cimetidine have been reported to change the pharmacokinetics of tacrine and donepezil. In contrast, concomitant medication with various drugs with rivastigmine does not seem to cause any drug interactions in patients with Alzheimer’s disease. Tacrine, donepezil and galantamine are metabolised via the cytochrome P450 (CYP) liver enzymes. Active metabolites are known for tacrine and galantamine. Rivastigmine is not metabolised via CYP enzymes, but via esterases and is excreted in the urine.Tacrine is associated with hepatotoxicity while other cholinesterase inhibitors seem devoid this adverse effect. Increased liver enzyme values have been observed in 49% of patients with Alzheimer’s disease treated with tacrine. Rechallenge with tacrine reduces the incidence of elevated liver enzyme levels. Peripheral cholinergic adverse effects are common for the cholinesterase inhibitors, with a n incidence ranging between 7 to 30% For some cholinesterase inhibitors, such as rivastigmine, the cholinergic adverse effects such as nausea, vomiting, dizziness, diarrhoea and abdominal pain can be reduced by slowing the rate of dose titration.

Tacrine and donepezil attenuate the neurotoxic effect of Aβ(25-35) in rat PC12 cells

THE effect of the cholinesterase inhibitors tacrine and donepezil on Aβ(25–35)-induced toxicity was investigated in rat pheochromocytoma PC12 cells by measuring the mitochondrial activity. Tacrine and donepezil was found in clinical relevant concentrations (10−7 − 10−6M) to attenuate Aβ(25–35)-induced toxicity in PC12 cells. The neuroprotective effect of tacrine was blocked in the presence of the nicotinic antagonists mecamylamine (10−5 M) and tubocurarine (10−5 M), suggesting an interaction via nicotinic receptors. This study demonstrates that tacrine and donepezil can exert neuroprotective properties which might be of importance and contribute to the clinical efficacy of cholinesterase inhibitors in the treatment of Alzheimers disease.

Nicotinic receptors, muscarinic receptors and choline acetyltransferase activity in the temporal cortex of Alzheimer patients with differing apolipoprotein E genotypes

The number of nicotinic and muscarinic receptors and choline acetyltransferase (ChAT) activity were investigated in the temporal cortex of patients with Alzheimers disease (AD) with different apolipoprotein E (APOE) genotypes. A significant reduction in the ChAT activity (P < 0.001) and in the number of nicotinic receptors (P < 0.001) was observed in the temporal cortex of AD brains independent of APOE genotype. The number of muscarinic receptors were unchanged in AD brains compared to control in both epsilon 4 and epsilon 3 carriers. A significant negative correlation (P < 0.001) was observed in AD brains between the histopathological dementia score and ChAT activity, which was independent of the APOE genotype. In this study the presence of the APOE epsilon 4 allele was not related to specific deficits in cholinergic activity in the temporal cortex of AD brains.

Chronic Ethanol Treatment Decreases [3H]Epibatidine and [3H]Nicotine Binding and Differentially Regulates mRNA Levels of Nicotinic Acetylcholine Receptor Subunits Expressed in M10 and SH‐SY5Y Neuroblastoma Cells

Abstract: For a study of the underlying mechanisms of a possible interaction between ethanol and nicotinic receptors during ethanol dependence, the aim of this work was to investigate the effect of chronic ethanol exposure on nicotinic receptor subtypes in a transfected fibroblast cell line (M10 cells) stably expressing α4β2 nicotinic receptor subtype and an SH‐SY5Y neuroblastoma cell line expressing α3, α5, α7, β2, and β4 nicotinic acetylcholine receptor (nAChR) subunits. A significant dose‐related decrease (−30–80%) in number of [3H]nicotine binding sites was observed in ethanol‐treated (25–240 mM) compared with untreated M10 cells. Similarly, 4‐day treatment with ethanol in concentrations relevant to chronic alcoholism (100 mM) decreased the number of nicotinic receptor binding sites in the SH‐SY5Y cells when measured using [3H]epibatidine. When M10 cells were chronically treated with nicotine, ethanol partly inhibited the up‐regulation of nicotinic receptors when present in the cells together with nicotine. Chronic treatment for 4 days with 100 mM ethanol significantly decreased the mRNA level for the α3 nAChR subunit (−39%), while the mRNA levels for the α7 (+30%) and α4 (+22%) subunits were significantly increased. Chronic ethanol treatment did not affect the mRNA levels for the β2 nAChR subunit. Changes in the levels of nAChR protein and mRNA may have adaptive significance and be involved in the development of dependence, tolerance, and addiction to chronic ethanol and nicotine exposure. They also may be targets for therapeutic strategies in the treatment of ethanol and nicotine dependence.

Upregulation of neuronal nicotinic receptor subunits α4, β2, and α7 in transgenic mice overexpressing human acetylcholinesterase

Neuronal nicotinic receptor binding sites as well as mRNA levels encoding for subunits α4, β2, and α7 were analysed in 3-mo-old transgenic mice generated with a neuronal overexpression of human acetylcholinesterase and in age-matched controls. The acetylcholinesterase transgenic mice display progressive cognitive impairment in spatial learning and memory. We here report a significantly increased [3H]epibatidine and [125I]αbungarotoxin binding in the cortex and the caudate putamen of these mice. Quantitative in situ hybridization showed significant upregulation of mRNA corresponding to the nicotinic receptor subunits α4, β2, and α7 in various brain regions in the transgenic mice compared to nontransgenic controls. Our results suggest that disruption of balanced cholinergic transmission by constitutive overexpression of acetylcholinesterase is accompanied by variable upregulation of several nicotinic receptor subtypes, in particular these associated with cholinergic terminals participating in compensatory response.

Tacrine interacts with an allosteric activator site on α4β2 nAChRs in M10 cells

The effect of chronic treatment with the cholinesterase inhibitor tacrine on α4β2 nicotinic acetylcholine receptors (nAChRs) was investigated in a transfected fibro-blast cell line, M10. Tacrine significantly increased (+46%; 5 ± 10−8 to 10−5 M) and decreased (-74%; 2 ± 10−5 to 10−4 M) the number of nAChRs in the M10 cells in a concentration-dependent manner when using [3H]cyti-sine as labelled ligand. The mRNA levels for α4 or β2 nAChR subunits remained unchanged following the treatment. The tacrine-induced increase in number of nAChRs was significantly blocked by the antagonist mecamylamine (10−4 M), while tubocurarine (10−4 M) had no effect. Neither of the antagonists prevented the decrease in the number of nAChRs obtained at the higher concentration of tacrine. Similar to nicotine, tacrine (5 ± 10−5 M) decreased the turnover rate of nAChRs, which might indicate neuroprotective properties. This study demonstrates that tacrine interacts with two sites on nAChRs, where activation of the non-competitive allosteric site might contribute to the clinical efficacy of tacrine treatment in AD patients.

Biphasic effect of tacrine on acetylcholine release in rat brain via M1 and M2 receptors

Rat cortical synaptosomes preloaded with [3H]choline were superfused and stimulated with K+ in order to investigate the effect of the cholinesterase inhibitor tacrine on the in vitro release of acetylcholine (ACh). Tacrine was found to biphasically both increase (10(-6) and 5 x 10(-6) M) and decrease (10(-5)-10(-4) M) the release of ACh in a concentration-dependent manner. The facilitatory effect of tacrine was prevented by atropine and the M1 antagonist pirenzepine, whereas the inhibitory effect induced by tacrine was blocked by atropine and the M2 antagonist AF-DX 116. These results indicate that tacrine causes a biphasic effect on K+ stimulated ACh release in the brain via M1 and M2 muscarinic receptors. The tacrine induced enhancement of the ACh release occurs at clinical relevant tacrine concentrations and might therefore be of importance for the treatment outcome of Alzheimers disease.

Neuronal nicotinic and muscarinic receptor subtypes at different ages of transgenic mice overexpressing human acetylcholinesterase

Subtypes of nicotinic (alpha4 and alpha7) as well as muscarinic (M1 and M2) receptor binding sites were quantified in the brain of transgenic mice overexpressing human acetylcholinesterase (AChE) at different ages using selective radioligands. A significant increase in [(3)H]cytisine (alpha4) binding was found in the cortex and striatum of AChE transgenic (hAChE-Tg) mice from 3 days to 12 months of age in comparison to non-transgenic mice. In addition a significant increase in [(3)H]AF-DX-384 (M2) binding was found in the striatum of hAChE-Tg mice at 3 months of age compared to controls. No major alteration was observed in the [(125)I]alpha-bungarotoxin (alpha7) or the [(3)H]pirenzepine (M1) binding sites. The persistent increase in alpha4 and M2 receptor binding sites in hAChE-Tg mice suggests that these receptor subtypes may play an important role in compensatory mechanisms facilitating the impaired cholinergic neurotransmission in hAChE-Tg.

The Large Hydrophilic Loop of Presenilin 1 Is Important for Regulating γ-Secretase Complex Assembly and Dictating the Amyloid β Peptide (Aβ) Profile without Affecting Notch Processing

γ-Secretase is an enzyme complex that mediates both Notch signaling and β-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid β peptide (Aβ), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of γ-secretase gives rise to Aβ peptides of different lengths, where Aβ42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320–374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of γ-secretase, for γ-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, γ-secretase complex formation, and had a differential effect on Aβ-peptide production. Although the production of Aβ38, Aβ39, and Aβ40 was severely impaired, the effect on Aβ42 was affected to a lesser extent, implying that the production of the AD-related Aβ42 peptide is separate from the production of the Aβ38, Aβ39, and Aβ40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the ϵ/S3 and γ sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different Aβ peptides without affecting Notch processing, two parameters of significance when considering γ-secretase as a target for pharmaceutical intervention in AD.

Minor contribution of presenilin 2 for γ-secretase activity in mouse embryonic fibroblasts and adult mouse brain.

γ-Secretase plays an important function in the development of Alzheimer disease, since it participates in the production of the toxic amyloid β-peptide (Aβ) from the amyloid precursor protein (APP). Besides APP, γ-secretase cleaves many other substrates resulting in adverse side effects when γ-secretase inhibitors are used in clinical trials. γ-Secretase is a membrane bound protein complex consisting of at least four subunits, presenilin (PS), nicastrin, Aph-1 and Pen-2. PS and Aph-1 exist as different homologs (PS1/PS2 and Aph-1a/Aph-1b, respectively), which generates a variation in complex composition. PS1 and PS2 appears to have distinct roles since PS1 is essential during embryonic development whereas PS2 deficient mice are viable with a mild phenotype. The molecular mechanism behind this diversity is, however, largely unknown. In order to investigate whether PS1 and PS2 show different substrate specificity, we used PS1 or PS2 deficient mouse embryonic fibroblasts to study the processing on the γ-secretase substrates APP, Notch, N-cadherin, and ephrinB. We found that whereas depletion of PS1 severely affected the cleavage of all substrates, the effect of PS2 depletion was minor. In addition, less PS2 was found in active γ-secretase complexes. We also studied the effect of PS2 depletion in adult mouse brain and, in concordance with the results from the mouse embryonic fibroblasts, PS2 deficiency did not alter the cleavage of the two most important substrates, APP and Notch. In summary, this study shows that the contribution of PS2 on γ-secretase activity is of less importance, explaining the mild phenotype of PS2-deficient mice.