WO1999024830A1

WO1999024830A1 - Method for diagnosing alzheimer's disease

Info

Publication number: WO1999024830A1
Application number: PCT/US1998/023198
Authority: WO
Inventors: Jeffrey K. Aronson; Asita H. De Silva; David G. Grahame-Smith
Original assignee: Bristol-Myers Squibb Company
Priority date: 1997-11-10
Filing date: 1998-11-02
Publication date: 1999-05-20
Also published as: AU750582B2; EP1031034A4; AU1371699A; CA2308371A1; EP1031034A1; JP2001523002A

Abstract

A method is provided for diagnosing Alzheimer's disease using human blood platelets wherein the presence or absence of functioning calcium-dependent potassium channels in blood platelets are determined by employing potassium channel blockers such as apamin or charybdotoxin, the absence of functioning calcium-dependent potassium channels indicating a positive diagnosis for Alzheimer's disease.

Description

METHOD FOR DIAGNOSING ALZHEIMER'S DISEASE

Field of the Invention The present invention relates to a method for diagnosing Alzheimer's disease by detecting the presence or absence of functioning calcium-dependent potassium channels in human blood platelets, the absence of such potassium channels indicating a positive diagnosis of Alzheimer's disease.

Background of the Invention Alzheimer's disease is a progressive neurodegenerative disorder which causes irreversible damage to brain cells leading to dementia and ultimately death. It is characterized by formation of amyloid plaques and neurofibrillary tangles in the brain. Currently, it is primarily diagnosed by exclusion of other known causes of dementia. Diagnosis at an early stage prior to irreversible changes is practically non-existent. In order for a therapeutic intervention to be significantly effective, it will have to be administered very early on prior to irreversible changes .

Accordingly, a non-invasive diagnostic test for early diagnosis of Alzheimer's disease would be a most welcomed addition to the diagnostician's armamentarium.

Calcium-dependent potassium channels have been found to be implicated with Alzheimer's disease. Abnormalities of potassium (K⁺) channel function have been reported in cultured cells in Alzheimer's disease (AD)¹. Depending upon the single channel conductance, a calcium-dependent potassium (K_a) channel is termed high- conductance or maxi-K (100-250 picosiemens (pS) ) , intermediate-conductance (18-50 pS) , or low-conductance (10-14 pS)K_Ca ²- The high conductance Kc is present in neurons, cardiac cells and various types of smooth muscles³. The intermediate-conductance channel has been shown to be present in red blood cells⁴, and in smooth muscle⁵. The low-conductance channel is present in a variety of cell types⁶.

The most important tools to distinguish between low- and high-conductance Kc are the toxins apamin⁷ , and charybdotoxin⁸.

Atwal, footnote 2 at page 581, points out that "while charybdotoxin specifically blocks maxi-K, apamin is a potent blocker of the low conductance Kc_a. However, in certain tissues, for example, rat brain, charybdotoxin may block Kc_a of all three types."

Mahaut-Smith^8a discloses that blood platelets contain a 30pS conductance charybdotoxin-sensitive channel.

It is also known that iberiotoxin specifically inhibits maxi-K channels (Elelvez et al^8b) .

A 113 pS K⁺ channel sensitive to tetraethylammonium has been described as being absent or not functional in cultured fibroblasts from patients with AD⁹, and this defect was mimicked in normal fibroblasts by the addition of amyloid beta-protein (Aβ)¹⁰, which is also plentiful in platelets¹¹ _' ¹². However, tetraethylammonium is not a selective inhibitor of K⁺ channels, and so the pharmacological identity of the abnormal channel in cultured fibroblasts is not clear. U.S. Patent No. 5,580,748 to Alkon et al (issued

December 3, 1996) discloses a method for the diagnosis of Alzheimer's disease using human cells such as fibroblasts, buccal mucosal cells, neurons, and blood cells such as erythrocytes , lymphocytes and lymphoblastoid cells, wherein the absence of a functional 133 pS potassium channel in the test cells indicates the presence of Alzheimer's disease. Tetraethylammonium is employed as a potassium channel blocker to aid in detecting the presence of the functioning 113 pS potassium channel. Description of the Invention In accordance with the present invention, a method is provided for diagnosing Alzheimer's disease which includes the steps of (a) obtaining a sample of platelets from a human subject, and (b) detecting the presence or absence in such platelets of functioning calcium- dependent potassium (Kc_a) channels, the absence of such functioning Kc_a channel indicating a positive diagnosis for Alzheimer's disease.

Detection of the absence of the functioning calcium- dependent potassium channel will be indicated by lack of inhibition by a potassium channel blocker which has the ability to block the functioning Kc_a channel, and may, for example, include apamin, charybdotoxin, or a combination thereof, depending upon the specific functioning calcium- dependent potassium channel involved.

In addition, in accordance with the present invention, a method is provided for diagnosing Alzheimer's disease which includes the step of detecting the presence or absence of one or more functioning small-conductance calcium-dependent potassium (SKc_a) channels in blood platelets of a human subject, the absence of a functioning SK__a channel in such platelets indicating a positive diagnosis for Alzheimer's disease.

Detection of the absence of the functioning SKc_a channel will be indicated by lack of inhibition by a SKc channel blocker which has the ability to block the functioning SKca channel, and may, for example, include apamin, charybdotoxin, or a combination thereof, depending upon the specific SKc_a channel involved.

In addition, in accordance with the present invention, a method is provided for diagnosing Alzheimer's disease which includes the step of detecting the presence or absence of a functioning charybdotoxin-sensitive potassium (Kc_h) channel in blood platelets of a human subject, the absence of a functioning Kc_h channel in such platelets indicating a positive diagnosis for Alzheimer's disease.

Detection of the absence of the functioning Kc_h channel will be indicated by lack of inhibition by charybdotoxin which has the ability to block the functioning K_Ch channel .

Detection of the presence or absence of the functional Kc_a channel, SKc_a channel and/or K_Ch channel may be determined by conventional techniques for measuring electrical currents in cells such as the patch clamp technique disclosed by Sakmann, B. et al²⁸. The presence or absence of the functional potassium channel may also be detected by (1) loading blood platelets with ⁸⁶Rb⁺, (2) stimulating ⁸⁶Rb⁺ efflux (from the platelets via K__a channels) with thrombin or ionomycin, (3) subjecting the thrombin- or ionomycin-stimulated ⁸⁶Rb⁺ efflux to the action of an appropriate potassium channel blocker, such as apamin, charybdotoxin or a combination thereof, depending upon the particular functional Kc_a channel involved, and (4) determining if the Kc_a channel blocker significantly inhibits the thrombin- or ionomycin-stimulated ⁸⁶Rb⁺ efflux to cause significant reductions in the ⁸⁶Rb⁺ efflux, a lack of significant inhibition and significant reduction in the ⁸⁶Rb⁺ efflux indicating a positive diagnosis for Alzheimer's disease.

Thus, where the Kc_a channel is a small-conductance calcium dependent potassium (SKc_a) channel, the potassium channel blocker employed will preferably be apamin or a combination of apamin and charybdotoxin (weight ratio apamin: charybdotoxin from about 4:1 to about 1:1, preferably from about 3:1 to about 1.5:1) .

Where the K_Ca channel is a charybdotoxin-sensitive potassium (Kc_h) channel, the potassium channel blocker employed will be charybdotoxin or a combination of charybdotoxin and apamin (weight ratio charybdotoxin: apamin from about 4:1 to about 1:1, preferably from about 3:1 to about 1.5:1).

Brief Description of the Figures

Figures la and lb are graphs showing the effects of apamin and charybdotoxin on thrombin-stimulated ⁸⁶Rb⁺ efflux in Control subjects (Figure la) and in patients with Alzheimer's disease (Figure lb) ; Figures 2a and 2b are graphs showing the effect of -dendrotoxin on thrombin-stimulated ⁸⁶Rb⁺ efflux in

Control subjects (Figure 2a) and in patients with Alzheimer's disease (Figure 2b); and

Figures 3a and 3b are graphs showing the effect of apamin and charybdotoxin on ionomycin-stimulated ⁸⁶Rb⁺ efflux in Control subjects (Figure 3a) and in patients with Alzheimer's disease (Figure 3b).

Example The following clinical experiments were carried out to determine if there is abnormal function of potassium channels in the platelets of patients with Alzheimer's disease.

Methods

Patient selection. Subjects with and without cognitive dysfunction were rigorously assessed annually, with a full range of blood tests to exclude metabolic or other causes of dementia and to assess cognitive function using the Cambridge Examination for Mental Disorders of the Elderly (CAMDEX)²⁵.

Diagnoses were made according to criteria of the National Institutes of Neurology and Communicative Disorders-Alzheimer ' s Disease and Related Disorders Association Work Group (NINCDS-ADRDA) ¹³ and the criteria described in the Diagnostic and Statistical Manual of Mental Disorders, Third Edition, Revised (DSM-IIIR)¹⁴ in addition to data from CT scans,¹⁵ _' ²⁶ and the SPET regional cerebral blood flow data¹⁵-¹⁶. Those with an OPTIMA (Oxford Project to Investigate Memory and Aging) diagnosis of 'probable AD' not only had no evidence of any other significant metabolic or psychiatric process that was thought to contribute to the dementia, but also had evidence of both significant medial temporal lobe atrophy on CT²⁶, without evidence of moderate or greater white matter change on axial images, and moderate or greater parietotemporal blood flow deficits on SPET¹⁵-¹⁶. If there was clinical evidence of another process in addition to or other than AD, which the clinician considered could have contributed to the clinical presentation or have given rise to it, then a diagnosis of 'possible AD' was made (four cases, one with ischaemia and three with possible frontal lobe dementia) . If, however, the clinical presentation could have been attributed to either AD or another dementia, but the imaging and longitudinal data were highly suggestive of AD on the basis of previously reported necropsy-confirmed cases¹⁶, and overall the clinician's impression was of AD, then a diagnosis of 'probable AD' was made (four cases, two of frontal lobe dementia, one of vascular disease, and one of hypoxia) . In six cases the diagnosis was clearly 'probable AD' . In a previous necropsy-confirmed cohort, the use of these criteria, using CT and SPET changes alone, without taking into account the clinical history or cognitive profile, had a sensitivity of over 85% and a specificity of over 95%¹⁶. In the cases with 'probable AD' studied in this series, all of whom remain alive, the likelihood that AD accounted for the dementia is thought to be extremely high. Controls were selected to be age-and sex-matched (Table 1), had no evidence of cognitive dysfunction, i.e. their CAMCOG scores were over 79/108²⁷, complained of no memory problems, and did not have the combination of a minimum medial temporal lobe width on CT of less than the fifth centile for controls of the same age with a moderate or greater parietotemporal perfusion deficit on SPET. Subjects recruited as part of OPTIMA were 14 patients with dementia of the Alzheimer type and 14 non-demented age- and sex-matched controls (details in Table 1) ; each experiment was performed on 11 or 12 of these individuals. Platelet perfusion-1⁷-. Blood was drawn by venepuncture and anticoagulated with acid-citratedextrose (ACD) . Platelets were prepared by centrifugation within an hour of venesection and incubated with ⁸⁶Rb⁺ for 2 h. The platelet suspension (2.5 x 10⁷ platelets per ml) was then injected into perfusion chambers, where the cells settled and became immobilized on inert filters (Millipore) . The platelets were then continuously perfused with Krebs solution, to allow them to stabilize for 20 min before the start of each experiment. The perfusion buffer was then changed for 5 min to Krebs solution containing thrombin (0.3 IU/ l) or ionomycin (1 μM) , after which the perfusion was switched to the original solution and continued for another 20 min. From 0 to 10 min after the addition of thrombin or ionomycin the perfusate was collected at 1-min intervals and thereafter at 2-min intervals. The solutions used for perfusion were bubbled with 95% O₂ and 5% CO₂ throughout the experiment (pH 7.4) . At the end of the experiment the polycarbonate filters were retrieved and placed in scintillation vials, to which 3.5 ml of Aquasafe 500 scintillation fluid was added. The radioactivity in the perfusates and filters was determined by liquid scintillation counting in a Beckmann LS 6000 SE counter and the loss of radioactivity from the cells was calculated. The amount of ⁸⁶Rb⁺ (pmol) in each fraction of perfusate collected was determined using the specific activity of the isotope. The radioactivity was measured in each fraction and the results were plotted as the cumulative efflux of ⁸⁶Rb⁺ against time. Buffers and drugs. ACD contained citric acid (15 g ) , trisodium citrate (25 g) , and dextrose (20 g) in 1 1 of distilled water. Krebs buffer contained (mmol/1) : NaCl (119); KC1 (4.6); CaCl₂ (1.5); NaH₂P0 (1.2); MgCl₂ (1.2); aHC0₃ (15); and glucose (11). Apamin, charybdotoxin, α- dendrotoxin, iberiotoxin, ionomycin, and human thrombin were purchased from Sigma Chemical Company, Poole, Dorset. ⁸⁶RbCl was purchased from Amersham International pic (Amersham, Bucks) .

Thrombin and ionomycin. Solutions of thrombin were prepared freshly in distilled water for each experiment and further diluted to a concentration of 0.3 IU/ml in Krebs solution. Stock solutions of ionomycin (10 mM in dimethylsulphoxide) were prepared and stored in aliquots at 4°C. On the day of the experiment ionomycin was further diluted in Krebs solution to a concentration of 1 μM. Apamin, charybdotoxin, iberiotoxin, and - dendrotoxin. Apamin, charybdotoxin, iberiotoxin, and α- dendrotoxin were freshly prepared for each experiment . Apamin, (100 nM) , charybdotoxin (300 nM) , iberiotoxin (300 nM) , and α-dendrotoxin (200 nM) were reconstituted in distilled water and stored in aliquots at -20°C. On the day of the experiment the toxins were further diluted in Krebs solution. Apamin was pre-incubated with the platelets (added at time -20 min) and charybdotoxin, iberiotoxin, , and α-dendrotoxin were added with thrombin

(at 0 min) for a period of 5 min. Data presentation and analysis . The results in

Figures 1-3 are shown as cumulative effluxes of ⁸⁶Rb⁺ from 0 to 14 min. The data are shown as means ± SEMs (n = number of experiments with platelets obtained from different volunteers) . The efflux data were analyzed using analysis of variance with repeated measures.

K± channel fluxes . Platelets were prepared and their K⁺ channel fluxes studied as described by DeSilva, H.A. et al¹⁷, by loading fresh platelets with ⁸⁶Rb⁺ (used as a radioactive analogue of κ⁺)¹⁸ _' ¹⁹ and stimulating ⁸⁶Rb⁺ efflux with thrombin and ionomycin. It has already been shown that thrombin and ionomycin stimulate ⁸⁶Rb⁺ efflux from platelets via K⁺ channels, and that the efflux occurs via Kca channels, sensitive to the highly selective inhibitors apamin and charybdotoxin (i.e. small-conductance calcium-dependent, SK_Ca, channels and charybdotoxin- sensitive, Kc_h channels) , and via voltage-gated (K_v) channels, sensitive to α-dendrotoxin²⁰ _' ²¹.

Uptake of rubidium by platelets. The uptake of ⁸⁶Rb⁺ by the platelets was the same in both groups (not shown) . Since over 90% of this uptake in platelets is inhibitable by ouabain and attributable to the sodium/potassium pump (de Silva & Aronson, unpublished observations) , this result suggests that the sodium/potassium pump functions normally in AD.

Non-stimulated & ± efflux. Non-stimulated cumulative ⁸⁶Rb⁺ efflux was linear with time (open circles; Figures 1-3) and did not differ between AD patients and controls. This efflux is partly mediated by the Na⁺/K⁺/2C1^~ co-transport system¹⁷.

Thrombin-stimulated ^j>Rb-t efflux. Figures la and lb show the effects of apamin and charybdotoxin on thrombin- stimulated ⁸⁶Rb⁺ efflux. Figure la relates to Control subjects, and shows that thrombin 0.3 IU/ml (4-filled circles) increased ⁸⁶Rb⁺ efflux over the non-stimulated efflux (1-open circles) . Apamin 100 nM (3-open squares) and charybdotoxin 300 nM (2-open triangles) inhibited the stimulated ⁸⁶Rb⁺ efflux (n=ll; P <0.0001).

Figure lb relates to patients with Alzheimer ' s disease and shows that thrombin 0.3 IU/ml (4-filled circles) increased ⁸⁶Rb⁺ efflux over the non-stimulated efflux (1-open circles) to the same extent as in controls (P = 0.996). Apamin 100 nM (3-open squares) and charybdotoxin 300 nM (2-open triangles) had no significant effect on the stimulated ⁸⁶Rb⁺ efflux (n=12; P = 0.941). Figures 2a and 2b show the effect of α-dendrotoxin on thrombin-stimulated ⁸⁶Rb⁺ efflux. Figure 2a relates to Control subjects and shows that thrombin 0.3 IU/ml (filled circles) increased ⁸⁶Rb⁺ efflux over the non-stimulated efflux (open circles) . α-dendrotoxin 200 nM (open triangles) inhibited the thrombin-stimulated efflux (n=ll; P <0.0001) . Figure 2b relates to patients with Alzheimer' s disease and shows that thrombin 0.3 IU/ml (filled circles) increased ⁸⁶Rb⁺ efflux over the non-stimulated efflux (open circles) . α-dendrotoxin 200 nM (open triangles) inhibited the thrombin-stimulated efflux (n=12; P <0.0001).

Control subjects. In control subjects, thrombin stimulated an increase in ⁸⁶Rb⁺ efflux from platelets (Figures la and 2a) . In 8 of 11 control subjects, apamin and charybdotoxin inhibited the thrombin-stimulated ⁸⁶Rb⁺ efflux by at least 18% and 16% respectively, while in the other three subjects these toxins had minimal effects (less than 10% inhibition) . When the data from all the control subjects were pooled, both apamin and charybdotoxin caused significant reductions in ⁸⁶Rb⁺ efflux (Figures la; Table 2) . In addition, α-dendrotoxin inhibited thrombin- stimulated ⁸⁶Rb⁺ efflux from the platelets of all the controls (Figure 2a; Table 2). These results are similar to the effects of these toxins on thrombin-stimulated ⁸⁶Rb⁺ efflux in the platelets of young volunteers²⁰ _' ²¹, and they confirm that there are SKc_a, K__h/ and K_v channels in normal human platelets .

Alzheimer's disease. Thrombin also stimulated ⁸⁶Rb⁺ efflux from the platelets of 12 patients with AD (Figures lb and 2b), to the same extent as in controls (cf. Figures la and 2a with Figures lb and 2b) . In contrast to the results in controls, apamin and charybdotoxin had minimal effects on thrombin-stimulated ⁸⁶Rb⁺ efflux (less than 10% inhibition) in 9 of 12 patients with AD, while in the other three patients each toxin inhibited the thrombin-stimulated efflux by at least 16%. When the data from all the patients with AD were pooled, neither apamin nor charybdotoxin caused significant reductions in ⁸⁶Rb⁺ efflux (Figure lb; Table 2) . In contrast, α-dendrotoxin inhibited the thrombin-stimulated ⁸⁶Rb⁺ efflux from the platelets of all the patients with AD (Figure 2b; Table 2) . Ionomvcin-stimulated ^≤-Rb± efflux.

Figures 3a and 3b show effect of apamin and charybdotoxin on ionomycin-stimulated ⁸⁶Rb⁺ efflux. As seen in Figure 3a, Control sub iects : Ionomycin 1 μM (5-filled circles) increased ⁸⁶Rb⁺ efflux over the non- stimulated efflux (1-open circles) . Apamin 100 nM (4-open squares) and charybdotoxin 300 nM (3-open triangles) inhibited the stimulated ⁸⁶Rb⁺ efflux (P <0.0001). Apamin and charybdotoxin combined (2-filled squares) inhibited the stimulated efflux more than either toxin alone (n=ll; P <0.0001) .

As seen in Figure 3b, Alzheimer's disease; Ionomycin 1 μM (5-filled circles) stimulated ⁸⁶Rb⁺ efflux over the non-stimulated efflux (1-open circles) to the same extent as in controls (P = 0.960). Apamin 100 nM (4-open squares) and charybdotoxin 300 nM (3-open triangles) , either alone or in combination (2-filled squares) , had no significant effect on the stimulated ⁸⁶Rb⁺ efflux (n=ll; P = 0.883). Control subjects. In 9 of 11 control subjects, apamin and charybdotoxin inhibited the ionomycin-stimulated ⁸⁶Rb⁺ efflux by at least 18% and 22% respectively, while in two subjects these toxins had minimal effects (less than 12% inhibition) . When the data from all the controls were pooled, both apamin and charybdotoxin caused significant reductions in ⁸⁶Rb⁺ efflux (Figure 3a; Table 2) . The two toxins combined had a greater effect than either toxin alone (Figure 3a; Table 2) . These results are similar to those seen in young volunteers²⁰ _' ²¹, and they confirm the presence of SKc_a and Kc_h channels in human platelets .

Alzheimer's disease. Ionomycin also stimulated ⁸⁶Rb⁺ efflux from the platelets of 11 patients with AD (Figure 3b), to the same extent as in controls (cf. Figures 3a and 3b) . However, apamin, charybdotoxin, and their combination had minimal effects on the ionomycin-stimulated ⁸⁶Rb⁺ efflux in 8 of 11 patients with AD (less than 12% inhibition) . When the data from all the patients with AD were pooled, apamin and charybdotoxin, either alone or in combination, had minimal effects on ⁸⁶Rb⁺ efflux (Figure 3b; Table 2) .

Although abnormalities of K⁺ channels have been reported in AD in neural cells (reduced post-mortem binding of ¹²⁵I-apamin to hippocampal neurones²²) and in fibroblasts (absence of a 113 pS channel sensitive to tetraethylammonium⁹) , this study is the first to show functional abnormalities of K⁺ channels in the platelets of patients with AD. The ⁸⁶Rb⁺ effluxes in platelets in response to both thrombin and ionomycin were quantitatively normal in AD, and the thrombin-stimulated efflux showed normal sensitivity to inhibition with α-dendrotoxin, suggesting that the K_v channels are normal in platelets in AD. However, the lack of inhibition of both thrombin- stimulated and ionomycin-stimulated effluxes by apamin and charybdotoxin suggests either that the SK_Ca and K channels are not present in platelets in AD or, if they are present, that they are not sensitive to inhibition by these toxins .

If SKc_a and Kc_h channels are present in platelets in AD, but unresponsive to inhibition, that would be consistent with the observation that the binding of ¹²⁵ι- apamin is reduced in post-mortem hippocampal neurones in AD²². This might be due to a change in the structure of the binding sites of the inhibitors. Alternatively, it might be due to an abnormality of the specific interaction of calcium with the channels, since the efflux stimulated by ionomycin, which increases the intracellular concentration of calcium, was not inhibitable.

Furthermore, the K_v channels were not affected, suggesting that Kc_a channels are selectively impaired in AD.

Alternatively, SK_Ca and Kc_h channels may not be present at all in AD. However, if that is so, then ⁸⁶Rb⁺ efflux must be occurring through other K⁺ channels, since the thrombin-stimulated and ionomycin-stimulated effluxes were quantitatively normal. However, normal human platelets do not contain large-conductance calcium- dependent (BKc_a) channels²¹, and iberiotoxin, a selective inhibitor of BKc channels, had no effect on ionomycin- stimulated effluxes in platelets from any individual (Table 2) , while the only other type of K⁺ channel found in platelets, K_v channels²⁰ _' ²¹, responded normally to α- dendrotoxin. These observations argue against upregulation of other normal channels in AD. On the other hand, metabolites of the beta-amyloid precursor protein (β-APP) are capable of de novo formation of K⁺ channels²³ that are insensitive to some inhibitors¹, and the ⁸⁶Rb⁺ efflux detected in AD might be via such channels .

Absence or non-functionality of the K__a channels might also explain the observation²⁴ that platelets from patients with AD had a higher thrombin-stimulated rise in intracellular Ca²⁺, since that might represent an exaggerated attempt to switch on non-existent or non- functiona1 channe1s .

The frequencies of alleles ε2, ε3 , and ε4 of the apolipoprotein E gene in the patients and controls are shown in Table 3. As expected, significantly more patients with AD had one or two ε4 alleles . Nine of the 13 individuals (patients and controls) who had one or two ε4 alleles had abnormalities of inhibition of Kc_a channels, compared with only five of the 15 who had no ε4 alleles

(P=0.06), suggesting a link between the K⁺ channel abnormalities and the presence of the ε4 allele. No such relationship was found between the K⁺ channel abnormalities and either the ε2 or the ε3 allele. In conclusion, there were abnormalities of inhibition of SKc_a and Kc_h channels in the platelets of patients with AD compared with matched controls. Thus, K__a channel abnormalities provide a marker for patients with AD. Table 1. Patient data. The data are given as mean (sd) . There were no significant differences between the groups .

Alzheimer ' s disease Controls

Measure (n = 14) (n = 14)

Age (y) 67.1 (7.5) 67.9 (7.7) Sex (M/F) 9/5 9/5

Systolic blood pressure (mmHg) 154 (16) 140 (15)

Diastolic blood pressure (mmHg) 90 (14) 81 (14)

Serum Sodium (mM) 139 (3) 139 (2)

Serum potassium (mM) 3.9 (0.4) 4.0 (0.3)

Serum Calcium (mM) 2.43 (0.10) 2.32 (0.13)

Serum urea (M) 5.7 (1.3) 6.3 (2.7)

Serum creatinine (μM) 104 (12) 103 (24)

Blood glucose (mM) 5.9 (2.1) 5.6 (1.1)

Platelet count (xl0⁹/l) 233 (58) 233 (62)

Haemoglobin (g/dl) 13.9 (0.8) 14.2 (1.1)

Mean cell volume (fl) 89 (4) 90 (5)

White cell count (xl0⁹/l) 6.9 (1.5) 6.5 (1.5)

Total serum cobala ins (ng/1) 270 (90) 315 (125)

Table 2. Inhibition of thrombin-stimulated and ionomycin-stimulated &Rb± effluxes by apamin. charybdotoxin, α-dendrotoxin, and iberiotoxin in platelets of patients with AD and age- and sex- matched controls . Data are given as median (interquartile range) percentages. Statistical comparisons by rank sum tests .

Thrombin-stimulated ⁸⁶Rb⁺ efflux

Inhibition in Inhibition in Alzheimer ' s

Toxin controls (%) disease (%) P value

Apamin 26 (20-27) 0 (0-0) <0.01

Charybdotoxin 22 (18-29) 0 (0-4) <0.01 α-dendrotoxin 26 (20-29) 19 (18-27) NS

Ionomycin-stimulated ⁸°Rb⁺ efflux

Inhibition in Inhibition in Alzheimer's

Toxin controls (%) disease (%) P value

Apamin 30 (20-31) 1 (0-20) <0.01

Charybdotoxin 28 (20-34) 4 (1-26) <0.01

Apamin +

Charybdotoxin 51 (34-52) 2 (0-40) <0.01

Iberiotoxin 2 (0-3) 0 (0-4) NS

Table 3. Numbers of normal and abnormal results in patients and controls. An abnormal result was defined as less than 15% inhibition by apamin and charybdotoxin. Statistical comparisons were by chi-square test. For comparison the distributions of apolipoprotein E alleles in the two groups are also shown.

Alzheimer's P

Agonist disease Controls value

Thrombin No. with an abnormal result

<0.02*

No. with a normal result

Ionomycin No. with an abnormal result

=0.01*

No. with a normal result

Apolipoprotein E allele frequencies

£2 /2 0 2

E2 /3 1 0 ε3 /3 4 8 <0 . 03 t ε3 /4 5 4 ε4/4 4 0

*chi-squared test tchi- squared test for trend References

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Claims

What is claimed is:

1. A method for diagnosing Alzheimer's disease, which comprises obtaining a sample of platelets from a human subject, and detecting the presence or absence of functioning calcium-dependent potassium (Kc_a) channels of specified slope conductance in such platelets, the absence of said functioning Kc_a channel indicating a positive diagnosis for Alzheimer's disease.

2. The method as defined in Claim 1 wherein the absence of said functioning Kc_a channel is indicated by lack of inhibition by a potassium channel blocker which has the ability to block the functioning Kc_a channel.

3. The method as defined in Claim 2 wherein the potassium channel blocker is apamin or charybdotoxin or a combination thereof.

4. The method as defined in Claim 1 wherein the presence or absence of the calcium-dependent potassium channel is determined by (1) loading blood platelets with ⁸⁶Rb⁺, (2) stimulating ⁸⁶Rb⁺ efflux (from the platelets via Kc_a channels) with thrombin or ionomycin, (3) subjecting the thrombin- or ionomycin-stimulated ⁸⁶Rb⁺ efflux to the action of a potassium channel blocker, and (4) determining if the potassium channel blocker significantly inhibits the thrombin- or ionomycin-stimulated ⁸⁶Rb⁺ efflux to cause significant reductions in the ⁸⁶Rb⁺ efflux, a lack of significant inhibition and significant reduction in the ⁸⁶Rb⁺ efflux indicating a positive diagnosis for Alzheimer's disease.

5. A method for diagnosing Alzheimer's disease, which comprises detecting the presence or absence of one or more functioning small-conductance calcium-dependent potassium (SK_ ) channels in blood platelets of a human subject, the absence of a functioning SKc_a channel in such platelets indicating a positive diagnosis for Alzheimer's disease.

6. The method as defined in Claim 5 wherein the absence of a functioning SK_Ca channel is indicated by lack of significant inhibition by a SKc_a channel blocker which has the ability to block the functioning SKc_a channel.

7. The method as defined in Claim 6 wherein the potassium channel blocker is apamin.

8. The method as defined in Claim 6 wherein the potassium channel blocker is a combination of apamin and charybdotoxi .

9. The method as defined in Claim 5 wherein the presence or absence of the SK__a channel is determined by (1) loading blood platelets with ⁸⁶Rb⁺, (2) stimulating ⁸⁶Rb⁺ efflux (from the platelets via K_a channels) with thrombin or ionomycin, (3) subjecting the thrombin- or ionomycin-stimulated ⁸⁶Rb⁺ efflux to the action of a SKc_a channel blocker, and (4) determining if the SK_Ca channel blocker significantly inhibits the thrombin- or ionomycin- stimulated ⁸⁶Rb⁺ efflux to cause significant reductions in the ⁸⁶Rb⁺ efflux, a lack of significant inhibition and significant reduction in the ⁸⁶Rb⁺ efflux indicating a positive diagnosis for Alzheimer's disease.

10. A method for diagnosing Alzheimer's disease, which comprises detecting the presence or absence of a functioning charybdotoxin-sensitive potassium (Kc_h) channel in blood platelets of a human subject, the absence of a functioning Kc_h channel in such platelets indicating a positive diagnosis for Alzheimer's disease.

11. The method as defined in Claim 10 wherein the absence of a functioning K_h channel is indicated by lack of significant inhibition by charybdotoxin which has the ability to block the functioning K__h channel .

12. The method as defined in Claim 10 wherein the absence of a functioning Kc_h channel is indicated by lack of significant inhibition by a combination of charybdotoxin and apamin.

13. The method as defined in Claim 10 wherein the presence or absence of the charybdotoxin-sensitive channel is determined by (1) loading blood platelets with ⁸⁶Rb⁺, (2) stimulating ⁸⁶Rb⁺ efflux (from the platelets via Kc_a channels) with thrombin or ionomycin, (3) subjecting the thrombin- or ionomycin-stimulated ⁸⁶Rb⁺ efflux to the action of a K_h channel blocker, and (4) determining if the Kc_h channel blocker significantly inhibits the thrombin- or ionomycin-stimulated ⁸⁶Rb⁺ efflux to cause significant reductions in the ⁸⁶Rb⁺ efflux, a lack of significant inhibition and significant reduction in the ⁸⁶Rb⁺ efflux indicating a positive diagnosis for Alzheimer's disease.