Marion Wienrich

Treatment with the selective muscarinic ml agonist talsaclidine decreases cerebrospinal fluid levels of Aβ42 in patients with Alzheimer's disease

The amyloid p-peptides Aβ40 and Aβ42 are highly amyloidogenic constituents of brain Pamyloid plaques in Alzheimers disease (AD). Lowering their formation may be achieved by modulating the activities of proteases that cleave the amyloid precursor protein (AβPP), including α-β-, and γ-secretases. Talsaclidine is a functionally selective muscarinic ml agonist that stimulates non-amyloidogenic a-secretase processing in vitro. We compared cerebrospinal fluid (CSF) levels of Aβ40 and Aβ42 measured by ELISA before and at the end of 4 weeks of treatment with talsaclidine. The medication was administered in a double- blind, placebo-controlled, and randomized clinical study to 40 patients with AD. Talsaclidine (n=34) decreased CSF levels of Aβ42 by a median of 19% (p < 0.001) as compared to baseline. The mean diflerence between CSF levels of Aβ42 before and after treatment with talsaclidine (n=34) was -46.73 (SD) pg/ml as compared to 0.8 (SD)pg/ml with placebo (n=6) (p < 0.05). CSF levels of Aβ40 increased during treatement with placebo (n=6) while they remained stable during treatment with talsaclidine (n=31) (1.118±1.710 ng/ ml, and-0.170A0.967 ng/ml, respectively; p < 0.05). These data show that treatment with the ml agonist talsaclidine reduced Aβ peptides, and particularly Aβ42, in AD patients, suggesting it as a potential amyloid lowering therapy of AD.

Treatment with the Selective Muscarinic Agonist Talsaclidine Decreases Cerebrospinal Fluid Levels of Total Amyloid β‐Peptide in Patients with Alzheimer's Disease

Abstract: Brain amyloid load in Alzheimers disease (AD) is, at least in genetic forms, associated with overproduction of amyloid β‐peptides (Aβ). Thus, lowering Aβ production is a central therapeutic target in AD and may be achieved by modulating such key enzymes of amyloid precursor protein (APP) processing as β‐, γ‐, and α‐secretase activities. Talsaclidine is a selective muscarinic M1 agonist that stimulates the nonamyloidogenic α‐secretase pathway in model systems. Talsaclidine was administered double‐blind, placebo‐controlled, and randomized to 24 AD patients and cerebrospinal fluid (CSF) levels of total Aβ were quantitated before and after 4 weeks of drug treatment. We observed that talsaclidine decreases CSF levels of Aβ significantly over time within the treatment group (n=20) by a median of 16% as well as compared to placebo (n=4) by a median of 27%. We conclude that treatment with selective M1 agonists may reduce Aβ production and may thus be further evaluated as a potential amyloid‐lowering therapy of AD.

Pharmacodynamic profile of the M1 agonist talsaclidine in animals and man

In functional pharmacological assays, talsaclidine has been described as a functionally preferential M1 agonist with full intrinsic activity, and less pronounced effects at M2- and M3 receptors. In accordance with this, cholinomimetic central activation measured in rabbits by EEG recordings occurred at a 10 fold lower dose than that inducing predominantly M3-mediated side effects. This pharmacological profile is also reflected in the clinical situation: Both in healthy volunteers and in Alzheimer patients--unlike after unspecific receptor stimulation through cholinesterase inhibitors--the mainly M3-mediated gastrointestinal effects (like nausea and vomiting) were not dose-limiting. Rather, sweating and hypersalivation, mediated through muscarinic receptors, occurred dose-dependently and were finally dose-limiting. In contrast to talsaclidine, sabcomeline had a less pronounced functional M1 selectivity in pharmacological assays. This was also shown in anaesthetized guinea pigs where sabcomeline alone induced bronchoconstriction, and in the rabbit EEG where central activation and cholinergic side effects occurred in the same dose range. Neither drug, however, showed convincing improvement of cognitive functions in patients with mild-to-moderate Alzheimers disease. This asks for a reassessment of the muscarinic hypothesis for the treatment of this disease.

Developmental regulation of AMPA-receptor properties in CA1 pyramidal neurons of rat hippocampus.

AMPA-receptor (AMPA-R) currents were recorded from CA1 pyramidal neurons in situ and after acute isolation from the hippocampus of 3- to 45-day-old rats. Membrane currents were analyzed by combining the patch clamp method with fast application techniques. The complete block of receptor currents by GYKI 53655 and the absence of modulation by Concanavalin A indicated that the cells exclusively expressed non-NMDA glutamate receptors of the AMPA subtype while functional kainate receptors could not be detected. The lowest sensitivity to kainate and NBQX was observed at postnatal day (p) 18. These changes might reflect a lower abundance of GluR1 at that developmental stage. A decrease of potentiation of receptor currents by cyclothiazide (CTZ), an acceleration of the recovery from CTZ potentiation and a faster and more complete desensitization of glutamate-evoked currents suggest an up-regulation of flop splice variants with increasing age. These functional data indicate that AMPA-R expression in CA1 pyramidal neurons varies during postnatal development which can be expected to influence the kinetics of synaptic transmission and the excitotoxic vulnerability as well.

Potassium currents in cultured cells of the rat retinal pigment epithelium

Whole-cell currents were investigated in cultured rat retinal pigment epithelial (RPE) cells. Two voltage-dependent conductances were discriminated. First, at potentials more positive than -30 mV, a time-dependent outward current was activated. Inhibition by Ba2+ (10 mM) and 4-aminopyridine (10 mM) indicated that this current was carried by potassium ions. This current showed no inactivation during 5 sec depolarizations. Second, an inward current, sensitive to Ba2+ (10 mM) and 4-aminopyridine (10 mM), was activated at potentials more negative than -70 mV. Under extra- and intracellular potassium-free conditions, both currents disappeared. In summary, cultured rat RPE cells expressed one potassium conductance similar to the delayed rectifier and one similar to the inward rectifier. The delayed rectifier expressed characteristics comparable with those known in mammalian species and different from those in non-mammalian species.

Extracellular matrix proteins as substrate modulate the pattern of calcium channel expression in cultured rat retinal pigment epithelial cells.

We investigated the effect of different culture substrates on the expression of membrane conductances for calcium in cultured rat retinal pigment epithelial (RPE) cells using the perforated patch technique and barium as charge carrier. In younger cultures (up to 12 days old) the RPE cells expressed L-type calcium channels, in older cultures (more than 12 days old) LVA-type channels. The LVA-type channels have been characterized as a tetrodotoxin sensitive Ca2+ channels. Coating the culture substrate with laminin, shifted the culture age for expression of LVA-type channels to 7 days. When collagen type 4 was used as substrate LVA-type channels and L-type channels were expressed simultaneously in 7 days old cultures. We concluded that proteins of the extracellular matrix which are known to enhance cell differentiation in culture, enhance the expression of LVA-type channels in RPE cells.

In vivo pharmacology of BIIR 561 CL, a novel combined antagonist of AMPA receptors and voltage‐dependent Na+ channels

Glutamate receptors of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) subtype and voltage‐gated Na+ channels are associated with diseases of the central nervous system characterized by neuronal over‐excitation as in epilepsy or cerebral ischaemia. In animal models, AMPA receptor antagonists and Na+ channel blockers provide protection in these conditions. Dimethyl‐{2‐[2‐(3‐phenyl‐[1,2,4]oxadiazol‐5‐yl)‐phenoxyl]‐ethyl}‐amine hydrochloride (BIIR 561 CL) combines both, AMPA receptor – and Na+ channel blocking properties in one molecule. Here, BIIR 561 CL was investigated in vivo. BIIR 561 CL protected mice against AMPA‐induced toxicity with an ED50 value of 4.5 mg kg−1 following subcutaneous (s.c.) administration. A 0.1% solution of BIIR 561 CL provided local anaesthesia in the corneal reflex test in rabbits. In mice, the compound prevented tonic seizures in the maximal electroshock (MES) model with an ED50 value of 3.0 mg kg−1 s.c. In amygdala‐kindled rats, BIIR 561 CL inhibited seizures at doses of 3 and 11 mg kg−1 following intraperitoneal (i.p.) injection. The data show that the combination of blocking AMPA receptor‐ and voltage‐gated Na+ channels in one molecule induces effective protection in animal models of neuronal over‐excitation.

Interactions of the dye Evans Blue and GYKI 52466, a 2,3-benzodiazepine, with (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in cultured rat cortical neurons: electrophysiological evidence for at least two different binding sites for non-competitive antagonists

The effects of the dye Evans Blue and GYKI 52466, a 2,3-benzodiazepine, on (S)- alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors in primary cultured rat cortical neurons were investigated using the patch-clamp technique. Evans Blue and GYKI 52466 reduced the currents induced by the application of 100 microM kainate with IC50 values of 10.6 +/- 1.4 microM and 12.1 +/- 0.4 microM, respectively. In contrast to the similar potencies of the two compounds, their kinetics of block were quite different with those of Evans Blue being more complex. The on-, as well as the off-reaction of the block by GYKI 52466 could be described by single exponential functions, whereas two different time-constants for binding and one time-constant for the unbinding of Evans Blue were found. The block of AMPA receptors by Evans Blue was not completely reversible under the experimental conditions applied in this study. GYKI 52466 was not able to augment the recovery after inhibiting AMPA receptors with Evans Blue. Moreover, preapplication of a high concentration of GYKI 52466 did not prevent the inhibition of AMPA receptors by Evans Blue. We therefore conclude that GYKI 52466 and Evans Blue bind to two different sites at AMPA receptors in primary cultured cortical neurons.

The AMPA receptor/Na(+) channel blocker BIIR 561 CL is protective in a model of global cerebral ischaemia.

In this study, we investigated whether the novel neuroprotective compound dimethyl-[2-[2-(3-phenyl-[1,2,4]oxadiazol-5-yl)-phenoxy]-ethyl]-amine hydrochloride, BIIR 561 CL, a combined non-competitive antagonist of AMPA receptors and blocker of voltage-gated Na+ channels, is protective in a rat model of severe global ischaemia. BIIR 561 CL administered immediately after 10 min of ischaemia (occlusion of both carotid arteries plus reduction of arterial blood pressure to 38-40 mm Hg) significantly reduced hippocampal damage at 4 x 26.8 mg/kg (subcutaneous injections). The competitive AMPA receptor antagonist 2,3-dihydro-6-nitro-7-sulfamoyl-benz(F)quinoxaline, NBQX, was used as a reference compound and was protective at 3x30 mg/kg (intraperitoneal and/or subcutaneous administration). BIIR 561 CL significantly reduced the ischaemia-induced premature mortality from 33.6% in the controls to 14.3%, whereas NBQX treatment had no statistically significant effect.Thus, BIIR 561 CL could be shown to reduce hippocampal damage and premature mortality in a model of severe global ischaemia. A compound with these properties might be an interesting candidate for the treatment of disorders related to global cerebral ischaemia in man.

Altered Regulation of Ion Channels in Cultured Retinal Pigment Epithelial Cells From Rcs Rats

The Royal College of Surgeon (RCS) rat suffers from an inherited retinal degeneration because their retinal pigment epithelium (RPE) is unable to phagocytose shed photoreceptor outer membranes (1,2,3,4). The genetical cause leading to this functional defect is unknown. The phagocytosis of photoreceptor outer membranes is regulated by the calcium/inositolphosphate second messenger system (5,6,7). Generation of inositolphosphates represents the “on” signal (6) whereas an increase of the cytosolic free calcium represents the “off” signal for phagocytosis (5). The RPE cells from RCS rats are able to bind shed photoreceptor membranes but are unable to ingest these membranes (1,2,3,4). Thus, RPE cells express receptors for photoreceptor membranes but the second messenger system seems to be unable to initialize the ingestion of bound membranes (7). Several lines of evidence point to a altered second messenger metabolism in RPE cells of RCS rats. Abnormal inositol phosphate generation (8), a reduced cAMP generation (9), changed protein phosphorylation (10) and, in consequence, a changed growth factor responsiveness have been reported (11,12).