Preferential cerebrospinal fluid acetylcholinesterase inhibition by rivastigmine in humans.

This study sought to examine the feasibility of prolonged assessment of acetylcholinesterase (AChE) activity in the cerebrospinal fluid (CSF) of volunteers and to test the hypothesis that rivastigmine (ENA-713; Exelon, Novartis Pharma AG, Basel, Switzerland) selectively inhibits AChE in CSF in humans at a dose producing minimal inhibition of the peripheral enzyme. Lumbar CSF samples were collected continuously (0.1 mL x min(-1)) for 49 hours from eight healthy volunteers who took either placebo or a single oral dose of rivastigmine (3 mg). CSF specimens and samples of blood cells and blood plasma were analyzed at intervals for rivastigmine and its metabolite NAP 226-90 ([-] [3-([1-dimethylaminolethyl)-phenol]), erythrocyte AChE activity, CSF AChE activity, and plasma and CSF butyrylcholinesterase (BuChE) activity. Safety evaluations were performed 23 hours after drug dosing and at the end of the study. Evaluable data were obtained from six subjects. The mean time to maximal rivastigmine plasma concentration (tmax) was 0.83 +/- 0.26 hours, the mean maximal plasma concentration (Cmax) was 4.88 +/- 3.82 ng x mL(-1), the mean plasma area under the concentration versus time curve (AUC0-infinity) was 7.43 +/- 4.74 ng x hr x mL(-1), and the mean plasma t1/2 was 0.85 +/- 0.115 hours. The concentration of rivastigmine in CSF was lower than the quantification limit for assay (0.65 ng x mL(-1)), but NAP 226-90 reached a mean Cmax of 3.14 +/- 0.57 ng x mL(-1). Only minimal inhibition of erythrocyte AChE activity (approximately 3%) was observed. Inhibition of AChE in the CSF after rivastigmine administration was significantly greater than after placebo for up to 8.4 hours after the dose and was maximal (40%) at 2.4 hours. Plasma BuChE activity was significantly lower after rivastigmine than after placebo, but this was not clinically relevant. BuChE activity in CSF was significantly lower after rivastigmine than after placebo for up to 3.6 hours after dosing, but this difference was not sustained. This study confirms the feasibility of using continuous measurement of AChE activity in CSF over prolonged periods, that rivastigmine markedly inhibits CSF AChE after a single oral dose of 3 mg, and that the inhibition of central AChE is substantially greater than that of peripheral AChE or BuChE.

Clinical pharmacology of rivastigmine: a new-generation acetylcholinesterase inhibitor for the treatment of Alzheimer's disease.

Rivastigmine (ENA 713, or carbamoylatine) is an acetylcholinesterase (AChE) inhibitor with brain-region selectivity and a long duration of action. Both preclinical studies and studies in human volunteers have shown that rivastigmine induces substantially greater inhibition of AChE in the central nervous system (CNS) compartment than in the periphery (40% inhibition of central AChE compared with 10% inhibition of plasma butylcholinesterase in healthy volunteers). Moreover, rivastigmine preferentially inhibits the G1 enzymatic form of AChE, which predominates in the brains of patients with Alzheimer's disease (AD). Evidence from animal studies also suggests that rivastigmine is a more potent inhibitor of AChE in the cortex and hippocampus, the brain regions most affected by AD. Absorption of rivastigmine is rapid and almost complete (>96% of the administered dose). Extensive, saturable first-pass metabolism, however, leads to bioavailability of approximately 35% of the administered dose and nonlinear pharmacokinetics. The principal metabolite of rivastigmine has at least 10-fold lower activity against AChE compared with the parent drug. Rivastigmine is completely metabolized; the major route of elimination of the metabolites is renal. Although patients with AD demonstrate 30% to 50% higher plasma concentrations of rivastigmine and its principal metabolite than do healthy elderly patients, there is no evidence of drug accumulation, which is consistent with rivastigmine's short pharmacokinetic half-life. Distribution of rivastigmine into the CNS is extensive, and inhibition of AChE in the cerebrospinal fluid is detectable 1.2 hours after oral dosing in both healthy volunteers and patients with AD. Peak activity is reached somewhat more slowly in AD patients than in healthy subjects, and the inhibitory effects have a longer duration (6.0 vs 2.4 hours and 12.0 vs 8.5 hours, respectively). Rivastigmine is inactivated during the process of interacting with and inhibiting AChE, and, in contrast to other AChE inhibitors, the hepatic cytochrome P-450 (CYP-450) system is not involved in the metabolism of rivastigmine. This reduces its propensity to interact with drugs metabolized by specific CYP-450 isoenzymes. Consistent with rivastigmine's pharmacokinetic and pharmacodynamic profiles, Phase II and III trials have demonstrated that the drug is a well-tolerated and effective treatment for AD.

Dose-dependent CSF acetylcholinesterase inhibition by SDZ ENA 713 in Alzheimer's disease.

INTRODUCTION: This study evaluates the activity of SDZ ENA 713, a centrally-selective acetylcholinesterase (AChE) inhibitor, in the cerebral spinal fluid (CSF) of patients with Alzheimer's disease (AD), and its relationship to central and peripheral pharmacokinetic parameters. METHODS: Eighteen AD patients were enrolled in this open-label, multiple-dose study. Patients were titrated in 1 mg bid/week increments to target doses of 1, 2, 3, 4, 5, or 6 mg bid SDZ ENA 713. After patients had been maintained at their target dose for at least 3 days, continuous CSF samples were obtained via a lumbar catheter for 12.5 h, beginning 0.5 h prior to the final dose of SDZ ENA 713. RESULTS: Dose-dependent inhibition of CSF AChE was significantly correlated (P < 0.05) with plasma drug and metabolite concentrations. The 6 mg bid treatment group showed a maximum mean inhibition of 62% at 5.6 h post-dose. CONCLUSION: Rapid, sustained, dose-dependent inhibition of CSF AChE suggests that SDZ ENA 713 has therapeutic potential in AD patients.

Plasma sulfoconjugated dopamine levels are normal in patients with autonomic failure.

Plasma levels of norepinephrine (NE), dihydroxyphenylglycol (DHPG), dihydroxyphenylalanine (DOPA), dopamine (DA), and dihydroxyphenylacetic acid (DOPAC)--all of which are free catechols--and sulfoconjugated DA (DASO4) were determined in 14 normal subjects and 18 patients with neurogenic orthostatic hypotension caused by either multiple system atrophy (MSA) (n = 11) or pure autonomic failure (n = 7). All free catechols were normal in patients with MSA, whereas NE, DHPG, DA, and DOPAC levels were significantly lower in patients with pure autonomic failure. The levels of DA-SO4, however, did not statistically differ among the three groups. The different plasma levels of free catechols in MSA and pure autonomic failure are consistent with the view that peripheral sympathetic neurons are relatively preserved in MSA, whereas they are severely affected in pure autonomic failure. Because DASO4 does not appear to be affected in pure autonomic failure, it appears likely that this metabolite is derived mainly from non-neural sources, such as the gastrointestinal tract, rather than from the sympathoadrenomedullary system.

Electrophysiological tests of autonomic function in patients with idiopathic autonomic failure syndromes.

Three electrophysiological tests of autonomic function were performed in patients with autonomic nervous system dysfunction to define test sensitivities and specificities. The skin sympathetic response, Valsalva ratio, and heart rate variation with deep breathing were studied in 10 patients with multiple system atrophy (MSA) and in 7 patients with pure (also called progressive or primary) autonomic failure (PAF); control subjects were 17 normal individuals of similar age. Thirteen patients had abnormal skin sympathetic responses, and 16 had abnormal Valsalva ratios. Fourteen patients had an abnormal variation of the heart rate with deep breathing. Taking the three tests together, binary logistic regression for distinguishing between patients and normal subjects correctly classified 91% of the 33 individuals for whom there were complete data with sensitivity of 88% and specificity of 94%. However, only 69% of the patients could be correctly classified by a logistic regression for discriminating between MSA and PAF. Electromyography (EMG) studies showed that 7 of 8 patients with MSA but only 2 of 7 patients with PAF (both multiparous women) had denervation of the rectal sphincter muscle. The EMG study is, therefore, valuable in men, but has a high false positive rate in women, probably because of pudendal nerve injury from parturition.

Biochemical and pharmacologic assessment of autonomic function.

Autonomic failure produces distinct pathophysiological abnormalities that differ according to the site and nature of the lesion(s). Although the anatomic organization and processes mediating chemical neurotransmission in the autonomic nervous system facilitate clinical investigation, limited access to the central compartment hampers evaluation of central neurotransmitter metabolism and neuropeptide function. As illustrated in the discussions of noradrenergic and cholinergic function, several indirect strategies have been used to assess the biochemical and neuropharmacologic consequences of autonomic dysfunction. The methods validated in patients with autonomic failure can be applied to investigate autonomic function in other clinical disorders including Parkinson's disease. The results of such studies may help to guide therapy and develop improved strategies for managing those patients with autonomic insufficiency.

Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease.

Some cases of Alzheimer's disease are inherited as an autosomal dominant trait. Genetic linkage studies have mapped a locus (AD3) associated with susceptibility to a very aggressive form of Alzheimer's disease to chromosome 14q24.3. We have defined a minimal cosegregating region containing the AD3 gene, and isolated at least 19 different transcripts encoded within this region. One of these transcripts (S182) corresponds to a novel gene whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein. Five different missense mutations have been found that cosegregate with early-onset familial Alzheimer's disease. Because these changes occurred in conserved domains of this gene, and are not present in normal controls, they are likely to be causative of AD3.

Effects of the cholinomimetic SDZ ENS-163 on scopolamine-induced cognitive impairment in humans.

Scopolamine-induced cognitive impairment was used in healthy men to evaluate the central nervous system activity of the new cholinomimetic SDZ ENS-163. Eighteen subjects were treated in a crossover design with oral placebo/intravenous saline, 50 mg of oral SDZ ENS-163/intravenous saline, oral placebo/0.4 mg of intravenous scopolamine, and 50 mg of oral SDZ ENS-163/0.4 mg of intravenous scopolamine. The administration of placebo with scopolamine caused significant cognitive impairment, as assessed by the Computerized Neuropsychological Test Battery (CNTB), and also decreased salivation and heart rate. In contrast, SDZ ENS-163 with saline had no effect on CNTB scores, increased salivation, and increased heart rate. Despite the observed cholinomimetic effects of SDZ ENS-163 when administered with saline, the changes in CNTB scores, heart rate, and salivation were indistinguishable between placebo/scopolamine and SDZ ENS-163/scopolamine. Thus, 50 mg of oral SDZ ENS-163 has cholinomimetic activity in normal men, but this dose is insufficient to reverse the muscarinic effects of 0.4 mg of intravenous scopolamine.

Alzheimer's disease cerebrospinal fluid antibodies display selectivity for microglia. Investigations with cell cultures and human cortical biopsies.

Previous investigations demonstrated that the cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients contains antibodies that recognize specific neuronal populations in the adult rat central nervous system (CNS). These findings suggest a pathogenic role for immunological aberrations in this disorder. To determine if antibodies may provide a means to differentially diagnose the dementias, CSF from a diversified dementia population was screened against the developing rat CNS and a cell culture system. Markings produced by AD CSF were distinctly different from those of vascular dementias (VAD) against the developing rat CNS. More importantly, some AD CSF recognized amoeboid microglia. The recognition of amoeboid microglia by antibodies in AD CSF is particularly interesting since these cells proliferate in response to nervous system disease and also engulf debris. A cell culture technique was developed to allow the rapid screening of CSF antibodies. Patient CSF produced five different types of markings in the cell culture: microglia, glioblasts, fibers, nonspecific, or negative. Correlations with these structures and the diagnosis of four different dementia populations revealed that, in comparison to the other groups, AD CSF displayed remarkable selectivity toward microglial cells. Cortical biopsies from patients suspected to have AD were incubated with the patient's own CSF and that of confirmed AD patients. Both CSF samples recognized microglial cells in the patient's cortical biopsy. The same CSF samples incubated against normal human cortical autopsy or a biopsy from a 3-mo-old child displayed negative immunoreactivity. These three approaches suggest that the presence of CSF microglial antibodies may be a means to distinguish AD patients from other dementias. The results add further support to the widely growing concept that inflammation and similar immune mechanisms may contribute to AD pathogenesis.

Analysis of the c-FOS gene on chromosome 14 and the promoter of the amyloid precursor protein gene in familial Alzheimer's disease.

The c-FOS gene product, a putative transacting transcriptional regulator of the amyloid precursor protein (APP) gene, is a candidate locus for the familial Alzheimer's disease (FAD) mutation on chromosome 14 (FAD14). In light of this functional relationship, we investigated the nucleotide sequence and segregation of c-FOS and the nucleotide sequence of the 5' APP promoter. Single-stranded conformational polymorphisms (SSCPs) in the c-FOS gene revealed that c-FOS closely cosegregates with the FAD14 gene but does not show allelic association with FAD. A conservative third-position T-->C mutation was demonstrated in exon 2 (codon 84) of c-FOS, and a C-->G substitution was detected at -209 bp in the 5' promoter of APP. Neither were unique to FAD and are unlikely to be pathogenic or secondary modifiers of the FAD phenotype. We conclude that the c-FOS open reading frame is probably not the site of the FAD14 locus, but we cannot exclude the existence of modifier loci on chromosome 21.

Cerebrospinal fluid microglial antibodies: potential diagnostic markers for immune mechanisms in Alzheimer's disease.

Hallmark lesions of Alzheimer's disease (AD) are filled with reactive immunocompetent microglia, suggesting that immunological aberrations may participate in the pathophysiology of this disorder. Microglia may participate in the initial stages of neurodegeneration before the onset of dementia. If immune mediated processes are closely linked to neuronal breakdown it would be of importance to have a reliable means to detect these processes. Serum and cerebrospinal fluid (CSF) antibodies are discussed as such potential sources. The serendipitous use of the developing rat central nervous system (CNS) to screen CSF antibrain antibodies produced some unexpected findings. Firstly, CSF antibodies of AD and other dementia patients recognized distinctly different neuronal structures in the developing rat brain. Secondly, some AD CSF recognized fiber networks whereas others recognized amoeboid microglial cells. The same AD CSF which recognized amoeboid microglia cells also specifically marked activated microglia and neural macrophages in experimentally induced lesions. AD CSF microglial antibodies appear to be significant in view of the increasing association between microglia and neurogenerative processes in AD. In addition, CSF microglial antibodies are present in numerous at-risk descendants of familial AD patients. Some have subsequently developed the disorder. These findings together with the fact that microglial antibodies are usually found in the early stages of AD suggest that AD CSF microglial antibodies could be of value in detecting neurodegenerative processes before the onset of dementia. These findings add further support to the concept that inflammation and similar immune mechanisms may contribute to AD pathogenesis.

Serum- and bradykinin-induced calcium transients in familial Alzheimer's fibroblasts.

The calcium-sensitive photoprotein, aequorin, was used to examine serum- and bradykinin-induced transient increases in free cytosolic calcium ions in skin fibroblasts from 10 individuals with early onset familial AD (FAD), including four who were biopsied before their clinical symptoms would allow a diagnosis of AD, 2 individuals with late onset FAD, 8 at-risk but nonsymptomatic individuals, and 13 controls. The data show that (a) among controls, the peaks of the calcium transients increase in height as a function of donor age; (b) transients induced by 10% serum, 10 nM bradykinin (BK) or 100 nM BK were generally lower in FAD fibroblasts, including those from donors in the early stages of the disease, than in age-matched control cells; (c) such transients are reduced in cells from a proportion of the nonsymptomatic, at-risk individuals. Thus, serum- and BK-induced calcium transients are reduced in fibroblasts from both early and more advanced stage FAD donors and perhaps even from donors who are presymptomatic carriers of the defective gene. The data also suggest that changes in calcium transients in FAD fibroblasts neither mimic nor exaggerate the effects of normal aging.

Olfactory dysfunction in the Shy-Drager syndrome.

The aetiology of the Shy-Drager syndrome (multiple system atrophy) is unknown. We reported previously a preliminary association between environmental-occupational risk factors and Shy-Drager syndrome. To further investigate this relationship, we evaluated olfactory function in eight patients in different stages of disease. When the eight patients' olfactory function was compared with 203 age- and sex-matched controls using a self-administered olfactory test, seven scored below the 39th percentile of this population. Five of the eight patients had total anosmia or microsmia. Additional studies will be required to elucidate the significance of this abnormal clinical observation.

Cerebellar excitatory and inhibitory amino acid receptors in multiple system atrophy.

We studied excitatory and inhibitory amino acid binding sites autoradiographically in control and multiple system atrophy (MSA) cerebella. Within the dentate nucleus (DN) of MSA specimens, we found a significant increase in the level of GABAA, benzodiazepine, and metabotropic binding sites compared with controls. In the granule cell layer, kainate, N-methyl-D-aspartate, and GABAA binding sites were all decreased significantly in MSA specimens compared with controls. In the molecular layer of MSA cerebellum, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding sites were decreased significantly compared with controls. Cerebellar cortical binding site decreases are likely due to Purkinje and granule cell loss. The increase of binding site levels in DN of MSA specimens may represent receptor up-regulation reflecting loss of descending inhibitory Purkinje cell and ascending excitatory afferents to the DN.

Linkage of familial Alzheimer disease to chromosome 14 in two large early-onset pedigrees: effects of marker allele frequencies on lod scores.

Alzheimer disease (AD) is a devastating neurodegenerative disease leading to global dementia. In addition to sporadic forms of AD, familial forms (FAD) have been recognized. Mutations in the amyloid precursor protein (APP) gene on chromosome (CHR) 21 have been shown to cause early-onset AD in a small number of pedigrees. Recently, linkage to markers on CHR 14 has been established in several early-onset FAD pedigrees. We now report lod scores for CHR 14 markers in two large early-onset FAD pedigrees. Pairwise linkage analysis suggested that in these pedigrees the mutation is tightly linked to the loci D14S43 and D14S53. However, assumptions regarding marker allele frequencies had a major and often unpredictable effect on calculated lod scores. Therefore, caution needs to be exercised when single pedigrees are analyzed with marker allele frequencies determined from the literature or from a pool of spouses.

Genetic evidence for a novel familial Alzheimer's disease locus on chromosome 14.

Familial Alzheimer's disease (FAD) has been shown to be genetically heterogeneous, with a very small proportion of early onset pedigrees being associated with mutations in the amyloid precursor protein (APP) gene on chromosome 21, and some late onset pedigrees showing associations with markers on chromosome 19. We now provide evidence for a major early onset FAD locus on the long arm of chromosome 14 near the markers D14S43 and D14S53 (multipoint lod score z = 23.4) and suggest that the inheritance of FAD may be more complex than had initially been suspected.

Studies of activated microglial cells and macrophages using Alzheimer's disease cerebrospinal fluid in adult rats with experimentally induced lesions.

Previous investigations have shown that cerebrospinal fluid from Alzheimer's disease patients contains antibodies that recognize the amoeboid microglia--a nascent and active form of microglia in the developing rat brain [McRae et al. (1991) Neuroscience 41, 739-752]. The present study extended this to show that the same cerebrospinal fluid from Alzheimer's disease patients also labeled the activated microglia and macrophages induced experimentally in adult central nervous system. Thus, in the spinal cord, activated microglia were elicited following the destruction of the motor neurons by the toxic lectin, Ricinus communis agglutinin, injected into the sciatic nerve. The activated microglia which were closely associated with the soma of the degenerating neurons were intensely immunostained with the cerebrospinal fluid from Alzheimer's disease patients. The labeling pattern was comparable to some known monoclonal antibodies including OX-42, OX-18 and OX-6 that mark microglia. The microglia cells on the contralateral normal side remained unstained. In the cerebrum, activated microglia and neural macrophages were induced following an epidural application of the excitotoxin, kainic acid or cryolesion. Immunoelectron microscopy of these cells showed that the immunoreactivity was localized at the plasma membrane and its derivatives suggesting that these are the sites where the antigens are associated. The results obtained in this investigation suggest that these experimental models may be a means to gain further insight to antigens recognized by antibodies in the cerebrospinal fluid of Alzheimer's disease patients.