WO2006034542A1

WO2006034542A1 - Alzheimer's and mild cognitive impairment diagnostics

Info

Publication number: WO2006034542A1
Application number: PCT/AU2005/001485
Authority: WO
Inventors: Zeinab Khalil
Original assignee: The National Ageing Research Institute Inc.
Priority date: 2004-09-27
Filing date: 2005-09-27
Publication date: 2006-04-06

Abstract

A method of Alzheimer's disease diagnosis and/or diagnosis of MCI in a patient comprising: (i) dermally administering to the patient an amount of an endothelial acting vasodilating agent effective to induce local microvascular dilation in a normal patient; and (ii) detecting local microvascular blood flow change following vasodilating agent administration; wherein Alzheimer's disease and/or MCI is indicated if vasodilating agent induced microvascular dilation is reduced in a statistically significant manner relative to dilation detected in a normal patient population. The invention also relates to a diagnostic kit for detecting Alzheimer's disease and/or MCI.

Description

ALZHEIMER'S AND MILD COGNITIVE IMPAIRMENT DIAGNOSTICS

FIELD OF THE INVENTION

The present invention relates to methods for diagnosis of Alzheimer's disease and/or MCI in patients and diagnostic kits for use in such methods.

BACKGROUND OF THE INVENTION

There are presently some 4.5 million Americans and around 160,000 Australians suffering from Alzheimer's disease. In the United States it is predicted that by 2050 this number will increase by almost 3 -fold to 13.2 million. This disease which results in progressive loss of memory and cognitive function is often associated with older age (although early onset forms of the disease do exist), is becoming a more and more prevalent disorder in ageing western societies. In the last year alone it was estimated that Alzheimer's disease resulted in costs of $US95 billion in lost productivity, personal and medical care in the United States. Apart from the economic cost, Alzheimer's disease is a disorder associated with enormous personal cost and trauma. For example, in later stages of the disease it is common for sufferers to experience memory loss to an extent that even immediate family and close friends can no longer be recognised. This is an incredibly traumatic scenario not only for the sufferer but for their carers, upon whom greater dependancy is placed as the disease progresses.

Mild Cognitive Impairment (MCI) is a risk state or indicator for progression to Alzheimer's disease. MCI is a transition state between the cognitive changes associated with the ageing process and those of a more serious nature that are associated with Alzheimer's. The disorder can be divided into two broad subtypes, amnestic MCI, which significantly affects memory, and nonamnestic MCI, which does not. Both subtypes are characterised by impaired language, attention and visuospatial function. Some reports have suggested that amnestic MCI converts to Alzheimer's at a rate of about 10 to 15 percent of amnestic MCI diagnosed patients per year. Other researchers have suggested that over time nearly all patients affected with MCI will progress eventually to Alzheimer's disease or another form of dementia.

Practice guidelines of 2001 from the American Academy of Neurology outline the following criteria for diagnosis of amnestic MCI - (a) a report from the individual, preferably confirmed by another person, of memory problems; (b) measurable, greater- than-normal, memory impairment detected with standard assessment tests; (c) normal overall thinking and reasoning skills; (d) ability to perform normal daily activities.

There have recently been some significant advances in Alzheimer's treatments that appear to offer hope for slowing progression of the disease. Other improved therapies are expected to become available over the next few years.

The effective use of therapeutic agents to halt or at least slow progression of the disease is of course contingent upon accurate disease diagnosis. Up until recently diagnosis of Alzheimer's disease has been fraught with difficulty as the diagnosis is based upon behavioural characteristics common to many other forms of dementia. While it is possible for various scans and tests to be conducted, an Alzheimer's disease diagnosis is currently only made after elimination of other forms of dementia and brain disorder. Conclusive Alzheimer's disease diagnosis is presently only possible following post-mortem autopsy. Similarly, even with the aid of the MCI diagnosis criteria mentioned above, accurate diagnosis of MCI is particularly difficult as there will always be difficulty in determining whether memory impairment is greater than normal or whether memory or cognitive changes are related to normal memory.

Accordingly, there is a pressing need for development of Alzheimer's and MCI diagnostic methods that will serve to provide an accurate diagnosis of the disorders at an early stage so that therapeutic regimes presently available, and those which will become available over the next years, can be adopted to halt or at least slow progression of the disease. It is with these factors in mind that the present invention has been conceived. It is well recognised that the pathology of Alzheimer's disease is associated with a reduction in brain cell number and the presence in the brain of neurofibrillary tangles and amyloid plaques, resulting in disorganisation of the neural network. The amyloid or senile plaques are formed by deposition in the brain of amyloid β protein (Aβ4). Without wishing to be bound by theory it seems that MCI is likely to be associated with the early stages of the same progression. Apart from taking a sample of brain tissue to test for presence of Aβ4 or it's toxic effects, which is obviously not a convenient or desirable proposition as a diagnostic tool, there is presently no known means of testing whether patients express abnormally high levels of Aβ4 protein that could lead to damaging effects.

It has become increasingly apparent over recent times that the microvasculature plays an important role in the pathology of Alzheimer's disease (1). In fact it has been suggested that the cerebral vasoactive properties of Aβ4 constitute a possible mechanism for vascular mediated injury associated with Alzheimer's disease. In vitro studies on isolated aorta (2) and middle cerebral arteries (3) have demonstrated a possible role for Aβ4 in cerebrovascular dysfunction. More recently studies in rat skin microvasculature using the amyloid β fragments Aβ4 32-35 (1), Aβ44 1-40 (4), Aβ4 25-35 (5) (6) (7) have shown that administration of such fragments can result in endothelial activated microvascular constriction, via a mechanism mediated by endothelin-1 (ET-I) and free radical action on endothelial cells.

Although it was previously thought unlikely that endogenous Aβ4 protein in Alzheimer's sufferers could exhibit a peripheral microvascular effect (Suo, Z et al., "Soluble Alzheimers - amyloid constricts the cerebral vasculature in vivo"; Neuroscience Letters, Volume 257 (1998); pp 77-80), the present inventors have now demonstrated this to be the case. Furthermore, the present inventors have devised a technique by which the peripheral endothelium driven microvascular constriction activity of Aβ4 can be utilised in the context of a diagnostic test. - A -

Accordingly, it is an object of the present invention to provide a diagnostic method and/or a diagnostic kit for diagnosis of Alzheimer's disease and/or Mild Cognitive Impairment (MCI). Other objects of the present invention will become apparent from the following detailed description thereof.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention there is provided a method of Alzheimer's disease diagnosis and/or diagnosis of MCI in a patient comprising: (i) dermally administering to the patient an amount of an endothelial acting vasodilating agent effective to induce local microvascular dilation in a normal patient; and

(ii) detecting local microvascular blood flow change following vasodilating agent administration; wherein Alzheimer's disease and/or MCI is indicated if vasodilating agent induced microvascular dilation is reduced statistically significantly relative to dilation detected in a normal patient population.

Preferably the endothelial activating vasodilating agent is selected from substance P, bradykinin and serotonin. A particularly preferred endothelial acting vasodilating agent is acetylcholine.

Administration of the endothelial acting vasodilating agent may be topical, by injection or by electrophoresis. Preferably administration is by electrophoresis.

Detection of local microvascular blood flow change may preferably be conducted by laser Doppler flowmetry and statistical significance of microvascular dilation reduction relative to dilation detected in a normal patient population may conveniently be established by conducting one-way multivariate analysis of variance (MANOVA). According to another embodiment of the present invention there is provided a method of Alzheimer's disease diagnosis and/or diagnosis of MCI in a patient comprising:

(i) dermally administering to a patient by electrophoresis a solution of between

5% v/v and 50% v/v acetylcholine in a physiologically suitable medium for a period of between 10 seconds and 40 seconds at a current of between 0.1mA and

0.4mA over a skin area of between 0.2cm and 3.0cm ; and

(ii) detecting local microvascular blood flow change following acetylcholine administration, by laser Doppler flowmetry; wherein Alzheimer's disease and/or MCI is indicated if acetylcholine induced microvascular dilation is reduced in a statistically significant manner relative to dilation detected in a normal patient population.

According to a still further embodiment of the present invention there is provided a method of Alzheimer's disease diagnosis and/or MCI diagnosis in a patient comprising: (i) dermally administering to a patient by electrophoresis a saline solution and then after a delay a solution of between 5% v/v and 50% v/v acetylcholine in the saline solution for a period of between 10 seconds and 40 seconds at a current of between 0.1mA and 0.4mA over a skin area of between 0.2cm² and 3.0cm²; and (ii) determining local microvascular blood flow following each of saline and acetylcholine administration, by laser Doppler flowmetry;

(iii) calculating a ratio of microvascular blood flow following acetylcholine administration to microvascular blood flow following saline administration (E:S ratio); wherein Alzheimer's and/or MCI is indicated if E:S ratio is below a threshold value determined by calculating E:S ratios under similar conditions for normal patient and Alzheimer's disease and/or MCI affected populations.

According to a still further embodiment of the present invention there is provided a diagnostic kit when used in diagnostic methods as outlined above. According to a still further embodiment of the present invention there is provided a diagnostic kit for detecting Alzheimer's disease and/or MCI comprising:

(i) a solution of acetylcholine in a physiologically acceptable medium and contained in an electrode chamber allowing administration of the solution to a patient by electrophoresis; and

(ii) an electrophoresis administration unit adapted to receive the acetylcholine solution containing electrode chamber.

In a preferred embodiment of the invention the electrode chamber is additionally adapted for storage of the acetylcholine solution.

FIGURES

The invention will be further described with reference to the figures, wherein:

Figure 1 shows the ratio of endothelial vascular blood flow response to administration of acetylcholine against water, in both control and AD patients.

Figure 2 shows the endothelial vascular response to ACh in control and transgenic mice overexpressing amyloid β protein.

Figure 3 shows the ratio of endothelial vascular blood flow response to administration of acetylcholine against saline, in both control and AD patients.

Figure 4 shows the ratio of endothelial vascular blood flow response to administration of acetylcholine against saline, in both control and AD patients, where patients under treatment for AD are excluded from the AD patient sample. Figure 5 shows the ration of endothelial vascular blood flow response to administration of acetylcholine against saline, in both control and AD patients, where patients under treatment for AD and who suffer from cardiovascular disease are excluded from the AD patient sample.

Figure 6 shows a bar graph of ratio of endothelial response to saline response for different clinically affected patient groups, demonstrating the effect of dementia on E/S ratio.

Figure 7 shows a bar graph of ratio of endothelial response to saline response for patient groups with different Mini Mental State Examination (MMSE) scores.

Figure 8 shows a bar graph of ratio of endothelial response to saline response for patient groups with different levels of cognitive impairment.

Figure 9 shows a plot of E:S ratios for normal (control) and MCI affected patients.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As indicated above the present invention relates to a method of diagnosis and diagnostic kits for diagnosis of Alzheimer's disease (AD) and/or Mild Cognitive Impairment (MCI).

Throughout this specification, for the sake of simplicity, reference to Alzheimer's disease is also intended to imply reference to MCI, where the context is appropriate. This procedure can be also used to monitor the effectiveness of treatment over time. Although the diagnostic may be performed upon non-symptomatic patients it is more likely that the diagnostic method will be adopted on patients demonstrating some clinical evidence of dementia, preferably at an early stage of dementia onset. It may also be appropriate for patients with a family history of AD to be subjected to the diagnostic method in order that they may have an opportunity to adopt lifestyle practices and whatever treatment options that are available at an early stage, in order to prevent progression of the disease that may be developing, although not yet behaviourally evident. It may also be appropriate for AD patients to be subjected to this testing in order to monitor effectiveness of treatment in improving endothelial microvascular responses in these patients.

The diagnostic methods according to the invention involve the dermal administration to the patient of an endothelial acting vasodilating agent. The term "dermal" is intended to encompass all modes of administration of endothelial acting vasodilating agents to the dermis or skin that will allow the agent or agents concerned to exhibit their pharmacological activity on dermal endothelial cells. The nature of the administration route adopted will of course depend upon the pharmacokinetics of the agent concerned. For example smaller chemical compounds or lipophilic compounds could be absorbed into endothelial cells by direct topical application, in the form of an ointment, oil, salve or the like. Larger compounds or lipophobic compounds may require administration by way of transdermal injection, by electrophoresis (in the case of charged compounds) or with the aid of a lipophilic carrier or liposomes etc. It is to be understood, however, that these modes of administration are mentioned by way of example only. Further details of administration modes and appropriate carriers, diluents, additives and the like are provided in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co, Easton Pennsylvania, USA, the disclosure of which is included herein in its entirety by way of reference.

The amount of endothelial acting vasodilating agent administered may depend upon the nature and activity of the compound concerned, the mode of administration and patient specific factors such as the age, weight, height, sex and general health and well-being of the patient. Other factors material to the amount of endothelial acting vasodilating agent administered to a patient would be apparent to a skilled physician. By way of example, amounts of from about 0.1ng/kg bodyweight to about l,000μg/kg bodyweight are contemplated to be useful in the diagnostic method. More preferably, amounts between about lng/kg bodyweight to about lOOμg/kg bodyweight and even more preferably amounts of about 10ng/kg bodyweight to about lOμg/kg bodyweight are envisaged. It is of course important for effecting the diagnostic method according to the invention that the amount of the endothelial acting vasodilating agent administered is that which would be effective to induce local microvascular dilation in a normal patient. By a "normal patient" it is intended to mean a patient not suffering from AD, and of generally good health. Ideally the normal patient against which the appropriate amount of compound administration should be judged for a patient upon which the diagnostic method is being conducted, would be a patient of approximately the same or similar age, weight, height, sex and general state of health as the patient upon which the diagnostic method is being conducted. It will of course not be possible to match all variables of normal patients with patients under diagnosis, but a skilled physician would readily understand the important criteria that would need to be matched in order to determine the appropriate amount of endothelial acting vasodilating agent that would be effective to induce local microvascular dilation in a normal patient.

By the term "endothelial acting vasodilating agent" it is meant to encompass all physiologically well tolerated agents that can be administered dermally and exhibit detectable endothelial-mediated microvascular dilation in the area local to administration in normal patients, at feasible administration amounts. Such compounds may be known or as yet unidentified, but as long as they meet the requirements outlined herein can be adopted for use in the diagnostic methods according to the invention. Preferred endothelial acting vasodilating agents include substance P, bradykinin and serotonin and a particularly preferred endothelial acting vasodilating agent is acetylcholine (ACh).

In the case of the use of ACh, administration will preferably be by way of electrophoresis in the form of an ACh solution in saline or preferably, distilled water. For example, solutions of between about 5% and about 50% ACh v/v (preferably between about 15% v/v and 40% v/v), in either distilled water or saline are envisaged. By the term "physiologically suitable medium" it is intended to mean a solvent suitable for administration via electrophoresis to a human patient in combination with ACh, and particularly including, but not necessarily limited to, distilled water and saline solutions. Other pharmaceutically and electrophoretically suitable solvents, carriers, diluents or additives, as referred to in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co, Easton Pennsylvania, USA, may be included within the physiologically suitable mediums according to the invention.

The detection of local microvascular blood flow change following vasodilating agent administration involves the monitoring of microvascular blood flow in the region of administration of the endothelial acting vasodilating agent, both prior to administration and following administration. By way of example it may be appropriate to monitor local microvascular blood flow for between 5 seconds and 10 minutes prior to administration of the endothelial acting vasodilating agent and then again for a similar period following administration. In one form of the invention, the microvascular blood flow may be monitored continuously both prior to, during and following administration of the vasodilating agent, or microvascular blood flow may be monitored to detect maximum height of the response flow, for the time period until 50% desensitization or until return to baseline level. In the case of administration by electrophoresis it may be appropriate to monitor microvascular blood flow following administration of the physiologically suitable medium (eg. saline) so as to take into account any effect on microvascular blood flow of the electrophoresis of the medium in determining the blood flow changes attributable to the vasodilating agent.

Historically a variety of different techniques have been used to monitor and quantify changes in blood flow. These techniques have been reviewed by a number of authors (Oberg PA, Laser-Doppler flowmetry. Biomed Eng 18:125-163 1990., Johnson JM, The cutaneous circulation. In: Shephard AP & Oberg PA (eds) Laser-Doppler blood flowmetry, pp:121-139, Kluwer Academic, Boston, 1990) and include various different methods of plethysmography-venous occlusion, mercury in rubber strain gauge, impedence, photo- electric-thermometry and thermography, Doppler ultrasound flowmetry, electromagnetic flow measurement, thermal and dye-dilution, thermal clearance, radioisotope infusion techniques and direct cell velocity observation using video-microscopic tissue monitoring. Most of these techniques are invasive in nature, technically sophisticated and both demanding and time consuming to perform. In a preferred embodiment of the invention the microvascular blood flow is monitored by utilising laser Doppler flowmetry. Details of laser Doppler flowmetry (LDF) techniques are provided in Oberg PA, Laser-Doppler flowmetry, Biomed Eng 18:125-163 1990, and Johnson JM, The cutaneous circulation. In: Shephard AP & Oberg PA (eds) Laser-Doppler blood flowmetry, pp:121-139, Kluwer Academic, Boston, 1990, the disclosures of which are included herein in their entirety by way of reference. In an example of the invention adopted by the present inventors a Primed Periflux (PF-2B) laser Doppler flowmeter (Stockholm, Sweden) was utilised. Another LDF model is the DRT4 (Moor Instruments, England), which measures total tissue perfusion. Other techniques for monitoring superficial capillary blood flow are also applicable.

In order to determine whether the patient expresses unusually high levels of circulating Aβ4 or compounds that may be derived from it, the presence of which would be detected by the diagnostic methods according to the invention and which are indicative of Alzheimer's disease, it is necessary to compare the level of dermal microvascular blood flow change following vasodilating agent administration in the patient under diagnosis, with results obtained from a normal patient population. The normal patient population will preferably include at least 5, particularly preferably at least 10, more particularly preferably at least 30 non-Alzheimer's disease sufferers for whom dermal microvascular blood flow change has been determined following administration of an endothelial acting vasodilating agent under the same conditions as those applied to the patient under diagnosis. By a statistical analysis of the blood flow change result obtained for the patient under diagnosis comparative to the results for the normal patient population it will be possible to determine whether the microvascular dilation induced by the vasodilating agent administration in the patient under diagnosis is reduced statistically significantly compared to the normal patient population. If there is statistically significant reduction in local microvascular dilation, Alzheimer's disease is indicated. Appropriate statistical methods to be adopted in such analysis would be apparent to persons skilled in the art, but by way of example it is mentioned that in one embodiment of the invention a one-way multivariate analysis of variance (MANOVA) analysis is adopted. Details of this statistical approach are provided in "Using multivariate statistics", by Barbra G Tabachnick and Linda S Fidell, Publisher, Harper and Row, New York, 1989, the disclosure of which is included herein in its entirety by way of reference.

For example, in the case of administration by electrophoresis the comparison of microvascular flow change with that obtained for a normal patient population may be conducted by calculating a ratio of skin endothelial microvascular blood flow following acetylcholine administration to microvascular blood flow following saline administration (E:S ratio), wherein Alzheimer's disease is indicated if E:S ratio is below a threshold value determined by calculating E:S ratios under similar conditions for normal patient (and Alzheimer's disease affected) populations.

Microvascular dilation is of course correlated with increased microvascular blood flow and microvascular constriction is correlated with decreased microvascular blood flow.

In a preferred embodiment of the invention the electrophoretic administration of acetylcholine in a physiologically suitable medium is conducted for between about 5 seconds and about 2 minutes, preferably between about 10 seconds and about 40 seconds, particularly preferably for approximately 30 seconds. A current of between about 0.05mA and about 0.5mA, preferably between about 0.1mA and about 0.4mA and, particularly preferably, a current of approximately 0.2mA may be utilised for the electrophoresis. Preferably the skin area over which an electrophoretic electrode is applied, and across which the acetylcholine will be administered, is between about 0.25cm² and about 3.0cm², preferably between about 0.2cm² and about 3.0cm². It should be recognised, however, that these variables are mentioned by way of example only and that depending upon the acetylcholine (or other endothelial acting vasodilating agent) solution adopted, the patient concerned and the nature of the electrophoresis apparatus utilised, it may be appropriate to adopt electrophoresis conditions outside the ranges mentioned above. In another aspect of the invention there is provided an Alzheimer's disease diagnostic kit that may be utilised in relation to the diagnostic methods referred to herein. In a preferred form the diagnostic kit comprises a solution of acetylcholine in a physiologically acceptable medium that is contained within an electrode chamber. The kit may also comprise a physiologically suitable medium (eg. saline). The electrode chamber being compatible with an electrophoresis administration unit so that the acetylcholine containing chamber can easily be placed in position and connected to the electrophoresis administration unit for administration of medium and/or acetylcholine via electrophoresis to the skin of a patient under diagnosis. Preferably the medium and/or acetylcholine solution can be supplied and stored within an electrode chamber, such that the chambers are produced at relatively low cost and may be disposable after use. It may also be equipped with appropriate covering means to prevent leakage of the solution therewithin, to ensure sterility of the solution and to protect the solution from other environmental factors (such as light) if necessary.

The invention will now be described further with reference to the following non-limiting Examples:

Example 1 Endothelial vascular response to acetylcholine in human AD and control patients

Subjects:

This study was approved by the University of Melbourne, Human Ethics Committee. All volunteers were asked to refrain from smoking on the day of the study and not to consume caffeine-containing drinks for at least 2 h before the start of the measurements. All subjects gave fully informed and witnessed written consent. Subjects taking vasoactive medication or suffering from conditions involving endothelial disfunction (such as hypertension, hypercholesterolemia, diabetes, atherosclerosis, congestive heart failure) were excluded. Experimental protocol:

All studies were performed in a temperature-controlled room (22⁰C) with the subjects sitting in a comfortable chair with their right arm resting comfortably on a soft pillow. During a 30 min acclimatisation period, the volar surface of the forearm was gently cleaned with an alcohol wipe and then swabbed with deionized water. Following acclimatisation, the forearm skin red blood cell flux responses to electrophoresis of ACh was measured.

Electrophoresis protocol: Two Perspex electrode chambers were attached to the middle third of the forearm (2cm apart) by means of a double sided adhesive ring. Each chamber consists of an inner ring with a small hole drilled through the centre to accommodate the placement of a small optical fibre to measure blood flow (blood flux) in the area of the skin directly beneath the probe via a Perimed Periflux (PF-2B) laser Doppler flowmeter, (Stockholm, Sweden). One chamber was filled with 200ul of vehicle (distilled water) and the other was filled with 200ul of 20% ACh (dissolved in distilled water). An indifferent electrode was attached to the volar aspect of the subject's wrist. A battery-powered electrophoresis Unit (Phoresor II - PM700) was connected to the chamber by alligator clips attached to two parallel metal infusion ports. The Unit provides a direct current for drug electrophoresis. First forearm skin red blood cell flux was recorded immediately before the start of electrophoresis until a stable baseline was achieved. Drug was delivered using an anodal current (0.2 mA for 30 Sec). The blood flux was recorded continuously. The electrophoresis unit was connected to one of the chambers to deliver either vehicle or ACh in a random manner, with a waiting period of 20-30 min between the two stimuli. Blood flow measurements:

Basal blood flow as well as the vascular response to ACh was measured using laser Doppler flowmetry (LDF). LDF is a non-invasive method of measuring skin microvascular blood flow following physiological and pharmacological interventions. LDF allows the continuous measurement of changes in blood flow over time. The results can be recorded on a chart recorder and analysed manually (when the periflux machine is used) or recorded directly onto a computer and analysed off-line using the DRTSOFT software package (when the Moor Instrument is used). Basal blood flow was recorded for 10 to 20 minutes to obtain stable measurements. Responses to ACh were recorded for a maximum of 30 minutes by which time the response had declined to base line. Overall the procedure was completed within 1 hour.

Statistical Analysis:

Data was analysed using one-way MANOVA. Multivariate analysis of variance was used to investigate the differences among different types of dementia or different degrees of dementias of a particular type (Independent variables) in terms of their effect on endothelial vascular response (Dependent variable).

The validity of this test could be described in terms of its sensitivity and specificity. The % of persons with AD who test positive by the clinical criteria (reduced endothelial responses) - this represents the sensitivity of the test. The % of persons without AD who were correctly categorised as negative by the criteria - this represents the specificity of the test.

Understanding the validity will help in predicting the presence of the condition.

Positive Predictability (PV+) is the proportion of those with a positive test who have AD. Negative Predictability (PV-) is the proportion of those with a negative test who do not have AD.

Results are shown in figure 1. Example 2

Endothelial vascular response to acetylcholine in transgenic mice

Methodology: Transgenic mice expressing the cDNA construct encoding the Aβ sequence and the cytoplasmic domain of human amyloid precursor protein under the control of human β- actin promoter were created (Li et al 1999) (Li Q et al., "Intracellular accumulation of detergent soluble amyloidgenic A fragment of Alzheimer's disease precursor protein in the hippocampus of aged transgenic mice", J. Neurochem. Volume 72 (1999); pp 2479-2487.)

One line expresses the wild type (C 100. WT) sequence the other expresses the familial AD associated mutation at position 717 of APP 770 (C100.V717F). Transgenic (Tg) mice with an average weight of 30g were initially anaesthetised with pentobarbitone sodium (Nembutal 20mg/kg i.p.). Additional doses of anaesthetic (5mg/kg) were administered to ensure that the mice remained under a constant state of surgical anaesthesia. This method of anaesthesia has previously been shown not to alter the basal vasodilation response. At the end of the experiment, the animals were sacrificed by barbiturate overdose.

The blister model and ACh perfusion: In order to gain access to the microvasculature, a blister was induced on the mice hind footpads using a metal suction cap heated to 4O⁰C and vacuum pressure of approximately 4OkP. Blister induction requires that the animal's feet are secured to the vacuum chamber for 20 min which produces a blister without damage to the microvessels. Once the blister is formed the epidermis is separated from the dermis. The removal of the blister epithelium allows access by superfusion to the extracellular space surrounding nerve terminals. Each mouse foot is secured in a perspex chamber with inlet and outlet ports. Perfusion of Ringer's solution and peptides over the blister is maintained at 4ml/hr by a peristaltic pump (Microperpex S, LKB, Sweden). Both perfusate and body temperature were kept at 37⁰C. An initial equilibration with Ringer's solution for 20 min was allowed before each experiment, during which time a stable baseline was established. Vasodilation and vasoconstriction response to the perfused substance were measured as percentage change in relative blood flux over time, using Laser Doppler flowmetry (Moor Instruments, England) via a probe placed in a central port immediately over the blister base. Blood flux was recorded immediately into a computer containing the DRT4 software. Sodium Nitroprusside (SNP; 100 μM) was perfused at the beginning of each experiment to control for individual variability in smooth muscle reactivity between mice. The vascular response to ACh was examined in Tg mutant (CT100.V717.F) mice while non-Tg mice were used as controls. Following stabilisation of the baseline, endothelial cell function was examined using ACh (30 min) while smooth muscle cell function was examined using SNP (10 min). Data (mean + S.E.M.) are presented as percentage change in baseline flux over a 30 min period. Statistical analysis was performed using One-way ANOVA. The data for smooth muscle reactivity as determined by the initial perfusion of SNP was included as a covariate in the statistical analysis.

Results are shown in Figure 2.

Example 3

A double blind study to determine the diagnostic utility of a non-invasive skin test for early diagnosis of AD

Patients with mild cognitive symptoms were recruited from an outpatient memory clinic operating at Melbourne Extended Care & Rehabilitation Service and assessed using gold standard clinical tools for AD diagnosis. An age-matched control sample of patients was derived from a pre-existing database of 700 healthy older adults. A non-invasive skin test to determine the ratio of a vascular response to a provocative agent compared to saline (ratio E/S) as adopted in example 1, with the exception that the vehicle used was saline solution rather than water, was examined for diagnostic utility.

Ratio E/S was significantly lower for AD patients (1.9+0.4), (n=58) compared to controls

(7.6+0.4), (n=68) (see Fig 3). When the patient sample was filtered to exclude AD patients receiving treatment for the disease matched by control on age and gender (n=39 in each group), the corresponding ratios remained significant (1.8+0.4 and 8.1+1.9, respectively, see Fig 4). Further filtering to exclude cardiovascular disease increased the significance level in ratio E/S to pOOOl (1.2±0.2, n=17 and 10.1±2.2, n=32 for AD and controls respectively, see Fig 5). The interrater and retest reliability was 0.89. Using a cutoff ratio of 2 for the ratio E/S, an 82% diagnostic sensitivity and 97% diagnostic specificity was observed. We conclude that the test has clinical utility as an early diagnostic marker of AD.

Example 4

A study to validate the use of the skin test for early diagnosis of patients with mild cognitive impairment and those with clinical evidence of Alzheimer's disease

Peripheral markers of vascular nature may be a useful diagnostic adjunct to the clinical detection of those with Alzheimer's disease. In this study, we compared the peripheral endothelial responses of people with early AD and normal control subjects. The results support our hypothesis that endothelial alterations can be detected early in the course of the disease. To validate the test, we compared the vascular reactivity of patients with clinically confirmed AD to that of people who have normal cognition and those with other forms of dementia. In addition, we studied a small sample of normal elderly people at risk of developing AD, to establish the predictive value of the test.

Summary of the results

We investigated the utility of a simple skin test for early diagnosis of AD in a sample of 169 patients with cognitive impairment and dementia and in 168 cognitively intact healthy volunteers. A non-invasive skin test to determine the ratio of endothelial microvascular response to acetylcholine compared to saline (ratio E/S) was examined for diagnostic utility. Mean ± SEM of ratios E/S were 8.8+0.9 for controls (n=168), 1.4+0.1 for AD patients (n=76) and 2.9+0.5 for other dementia (n=94). Using the optimal cut-off point of E/S ratio of 1.9 determined by Receiver Operating Characteristic Curves, an 80% diagnostic sensitivity and specificity for AD have been observed. The interrater and retest reliability was 0.89. Furthermore, 15 subjects were randomly drawn from a longitudinal healthy ageing study initially recruited in 1996. Five of those subjects met the criteria for MCI after eight years of follow up using a battery of cognitive tests. When tested for their E/S ratio in a blind fashion, the skin test successfully identified those subjects. We suggest that this simple skin test can be applied as diagnostic adjunct in patients with mild cognitive symptoms or those with clinical evidence of AD.

Subj ects/recruitment

Two samples were used in the current study. The validation sample consisted of 168 cognitively intact healthy volunteers derived from a pre-existing database at the National Ageing Research Institute of 500 healthy older adults. All subjects had skin temperature, medication history, BP, pulse and MMSE performed at time of assessment. A sample of 169 patients with cognitive impairment and early dementia were recruited from an outpatient memory clinic operating at Melbourne Extended Care and Rehabilitation Service. Complete diagnostic information on the type and severity of cognitive impairment was available for this sample. All subjects had a full clinical assessment (Physical exam, blood tests, CT scan, informant history +/- neuropsychological exam) performed by geriatrician or psychogeriatrician. The Mini Mental State Examination (MMSE) score was available on all patients who were classified according to ICDlO and NINCDS-ADRDA criteria, representing the current research gold standard for dementia diagnosis. A second sample of 15 healthy older people was also studied to determine the predictive value of the test. This sample was drawn randomly from an ongoing study of cognitive ageing. All of the individuals in this sample were free of cardiovascular, endocrine and other systemic disorders. In this sample, 5 individuals met the clinical criteria for mild cognitive impairment and 10 were classified as having normal cognitive performance. These classifications were based on the assessment of cognitive status over the eight years prior to the current study, Investigators who conducted the skin test were blinded to each individual's cognitive status.

Statistical Analysis

Vascular data was calculated as the ratio of endothelial response to saline response for each subject. This ensured the saline response (dependent on individual vascular reactivity to electrophoresis) was incorporated as an internal control in each individual response. MANOVA was used to investigate the differences between different diagnoses and or differing degrees of cognitive impairment (Independent variables) in terms of their effect on endothelial vascular response (Dependent variable).

Detailed Results

Main outcome measure: Ratio of skin endothelial vascular response to acetylcholine compared to saline response (ratio E/S). Some subjects from each group were re-tested 1-2 weeks later to determine reliability.

Validation Results: Mean ± SEM of ratios E/S were 8.8+0.9 for controls (n=168), 1.4+0.1 for AD patients (n=76) and 2,9+0.5 for other dementia (n=94). Most subjects in the latter group were diagnosed with mixed dementia with only 6 subjects identified as pure vascular dementia (E/S ratio of 2.4+0.8), 9 subjects identified to have both vascular and AD dementia (E/S ratio of 1.7+0.4), 7 subjects identified to be FTD (E/S ratio of 2.1+1.2), and 6 subjects identified to be DLBD (E/S ratio of 1.3+0.3) (see Fig 6).

Using the optimal cut-off point of E/S ratio of 1.9 determined by Receiver Operating Characteristic Curves, an 80% diagnostic sensitivity and specificity for AD have been observed. The interrater and retest reliability was 0.89.

The MCI group was explored and the controls with MMSE less than 30 were separated into groups according to their MMSE score. From the control group, 19 subjects had a MMSE <30. Of those, the ratio E/S for subjects with MMSE 27, 26 & 25 were 5.4+2.3, 3.0+1.9 and 1.5+0.3 with n=12, 5 and 2 respectively (see Fig 7).

From the clinic group, 13 patients were diagnosed with vascular cognitive impairment (VCI) (E/S ratio of 4.9+1.5) and 18 patients were diagnosed with MCI (E/S ratio of 2.7±1.0). Of the latter group, 8 subjects had MMSE=>27(E/S ratio of 4.2+1.9) and 10 subjects had MMSE=<26 (E/S ratio of 1.5+0.8) (see Fig 8). Predictive validity for MCI

The distribution of E/S ratios in individuals classified as normal or MCI from the second sample is shown in Figure 9. None of the healthy individuals showed ES ratios of less than 3.0. The median E/S ratio in the MCI group was (1.61+0.4) and for controls was (8.3+0.8) and the group difference was significant (p=0.000).

Discussion/Conclusion

Patients with AD and DLBD appear to have a prominent peripheral endothelial cell dysfunction with an inability to respond to an endothelial provocative factor, such as acetylcholine. Patients with pure vascular dementia have an E/S ratio that is significantly higher than the optimal cut-off point of E/S ratio of 1.9 determined by Receiver Operating Characteristic Curves. E/S ratios for MCI fell between AD and controls. This is consistent with the pathogenesis of AD. Within individuals with MCI there was an association between E/S ratio and clinical status (as assessed by MMSE). This supports further the idea that blood vessel pathology in the periphery changes as CNS AD load also changes before this load is sufficient to give rise to the clinical syndrome of dementia. The E/S ratio was abnormal in individuals even with very mild MCI (e.g. identified from a subtle decline memory function over the eight years prior to assessment). This suggests that this measure is also sensitive to differentiating normal from abnormal aging.

We contend that this novel skin test can be applied as a diagnostic adjunct in patients with mild cognitive symptoms suggestive of a possible dementia disorder or as a diagnostic adjunct for demented patients in whom the history, physical examination, and laboratory studies are in agreement with a diagnosis of probable AD. Our results also show that the E/S ratio is a useful preclinical biomarker of AD.

Overall, these results support the hypothesis that endothelial alterations can be detected early in the course of the disease. It is to be recognised that the present invention has been described by way of example only and that modifications or alterations to the diagnostic methods and kits described herein, that would be obvious to persons skilled in the art based upon the present disclosure, are considered to fall within the scope and spirit of the invention.

REFERENCES

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4. Poliviou, H and Khalil, Z;"β-amyloid protein modulates the vascular activity of acetylcholine at the level of the microvasculature"; Alzheimer's Reports; Volume 1, Number 4 (1998); pp 233-239

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Claims

CLAIMS:

1. A method of Alzheimer's disease diagnosis and/or diagnosis of MCI in a patient comprising:

(i) dermally administering to the patient an amount of an endothelial acting vasodilating agent effective to induce local microvascular dilation in a normal patient; and

(ii) detecting local microvascular blood flow change following vasodilating agent administration; wherein Alzheimer's disease and/or MCI is indicated if vasodilating agent induced microvascular dilation is reduced in a statistically significant manner relative to dilation detected in a normal patient population.

2. The method according to claim 1 wherein the endothelial acting vasodilating agent is selected from substance P, bradykinin and serotonin.

3. The method according to claim 1 wherein the endothelial acting vasodilating agent is acetylcholine.

4. The method according to any one of claims 1 to 3 wherein administration is topical or by injection.

5. The method according to claim 3 wherein administration is by electrophoresis.

6. The method according to claim 5 wherein a solution of between about 5% v/v and about 50% v/v acetylcholine in a physiologically suitable medium is administered for a period of between about 5 seconds and about 2 minutes at a current of between about 0.05mA and about 0.5mA over a skin area of between about 2.0cm2 and about 3.0cm2.

7. The method according to any one of claims 1 to 6 wherein the detection of local microvascular blood flow change is by laser Doppler fiowmetry.

8. The method according to any one of claims 1 to 7 wherein statistical significance of microvascular dilation reduction relative to dilation detected in a normal patient population is established by conducting one-way multivariate analysis of variance (MANOVA).

9. The method according to any one of claims 1 to 8 for diagnosis of MCI.

10. The method according to any one of claims 1 to 8 for diagnosis of Alzheimer's disease.

11. A method of Alzheimer's disease diagnosis and/or diagnosis of MCI in a patient comprising:

(i) dermally administering to a patient by electrophoresis a solution of between 5% v/v and 50% v/v acetylcholine in a physiologically suitable medium for a period of between 10 seconds and 40 seconds at a current of between 0.1mA and 0.4mA over a skin area of between 2.0cm2 and 3.0cm2; and

(ii) detecting local microvascular blood flow change following acetylcholine administration, by laser Doppler fiowmetry; wherein Alzheimer's disease and/or MCI is indicated if acetylcholine induced microvascular dilation is reduced statistically significantly relative to dilation detected in a normal patient population.

12. The method according to claim 11 for diagnosis of MCI.

13. The method according to claim 11 for diagnosis of Alzheimer's disease.

14. A method of Alzheimer's disease diagnosis and/or MCI diagnosis in a patient comprising: (i) dermally administering to a patient by electrophoresis a saline solution and then after a delay a solution of between 5% v/v and 50% v/v acetylcholine in the saline solution for a period of between 10 seconds and 40 seconds at a current of between 0.1mA and 0.4mA over a skin area of between 0.2cm² and 3.0cm²; and

(ii) determining local microvascular blood flow following each of saline and acetylcholine administration, by laser Doppler flowmetry;

15. The method according to claim 14 for diagnosis of MCI.

16. The method according to claim 14 for diagnosis of Alzheimer's disease.

17. A diagnostic kit when used in a method according to any one of claims 1 to 16.

18. An diagnostic kit for detecting Alzheimer's disease and/or MCI comprising: (i) a solution of acetylcholine in a physiologically acceptable medium and contained in an electrode chamber allowing administration of the solution to a patient by electrophoresis; and

19. The kit according to claim 18 wherein the electrode chamber is additionally adapted for storage of the acetylcholine solution. The kit according to either claim 18 or claim 19 wherein the solution comprises between about 5% v/v and about 50% v/v acetylcholine.