LU100877B1 - Method of performing differential diagnosis of neurogenerative diseases in a subject - Google Patents

Abstract

The invention is directed to an in vitro method for performing a differential diagnosis of neurodegenerative diseases in a subject, said subject being selected among subjects suffering from Alzheimer's disease, mental depression, dementia with Lewy Body, frontotemporal dementia, and/or vascular dementia. Said method comprises the steps of: (a) determining at least five criteria of said subject, (b) comparing said at least five criteria of said subject with reference values by calculating a global note in relation with each neurodegenerative disease, and (c) determining whether said subject suffers from Alzheimer's disease, mental depression, dementia with Lewy Body, frontotemporal dementia, vascular dementia or mixed dementia.

Description

METHOD OF PERFORMING DIFFERENTIAL DIAGNOSIS OF NEURODEGENERATIVE

DISEASES IN À SUBJECT Description Technical field

[0001] The invention is directed to a method for performing a differential diagnosis of neurodegenerative diseases in a subject.

Background art

[0002] The performance of a differential diagnosis allows a clinician to categorize the patient, who has then the opportunity to follow an adequate treatment in order to heal, or at least to improve his/her medical conditions.

[0003] The neurodegenerative diseases, which are characterized by the progressive loss of structure or function of neurons, are numerous, as shown by the following list: Alzheimer's disease, mental depression, amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson’s disease, progressive supranuclear palsy, Parkinson’s disease with dementia, dementia with Lewy Body, vascular dementia, etc.

[0004] With regard with Alzheimer’s disease, it has been noticed that an elevated concentration of noradrenaline can be an etiological for some patients (Fitzgerald P.J., Current Alzheimer Research, 2010, 7, 506-516).

[0005] The frontiers between those diseases are sometimes difficult to distinguish, especially in the case of Alzheimer’s disease so that the clinician in charge of the follow-up of the patients can hesitate and give an inappropriate treatment to one patient. The diagnosis can also be difficult to establish in the sense that it implies expensive and cumbersome experiments, such as medical imaging and/or lumbar punctures to collect a sample of cerebrospinal (CSF) fluid. It is also noted that the absence of syndrome does not “rule out” the presence of a disease. There are up to 30% misdiagnosis without biomarkers (Jack C. R., et al, Alzheimer’s & Dementia, 2018, 14, 532-562).

[0006] On the other hand, medical questionnaires are not sufficient for the clinician to be certain of the diagnosed disease.

[0007] A link between morphological and functional changes occurring in the monoaminergic ascending system and the physiopathology of AD has been suggested (Simic G. et al., Neuropathol. Appl. Neurobiol., 2009, 35, 532-554 and Trillo L. et al, Neurosci Biobehav. Rev., 2013, 37, 1363-1379). Noradrenergic neurons in the locus coeruleus (LC) are the main source of noradrenergic inputs to numerous regions throughout the brain. Due to its widespread efferent innervation, the LC projection system plays a pivotal regulatory role in processes such as stress and maintenance of cognitive performances (Sara S. J., Nat. Rev. Neurosci, 2009, 10, 211-223). This leads to the quest of catecholamines detection in order to develop new tools for neurodegenerative disease diagnosis. Catecholamines are monoamines derived from tyrosine and play the role of neuromodulators in the central nervous system and in the peripheral sympathetic nervous system, as well as hormones in the blood circulation.

[0008] Mixed dementia is also not very well treated since the patients are often diagnosed with just a single type of dementia. In this case, a physician will base the pharmaceutical decisions/prescriptions on the type of dementia that has beendiagnosed, which at the end, will not help the patient to improve his/her living conditions.

Summary of invention Technical Problem

[0009] The invention has for technical problem to alleviate at least one of the drawbacks present in the prior art. More particularly, the invention has for technical problem to avoid expensive and cumbersome experiments to diagnose a subject suffering from a neurodegenerative disease or from a mixed dementia. Said disease can be one of the following: Alzheimer’s disease, mental depression, dementia with Lewy Body, frontotemporal dementia, vascular dementia or mixed dementia.

Technical solution

[0010] The invention is directed to an in vitro method for performing a differential diagnosis of neurodegenerative diseases in a subject, said subject being selected among subjects suffering from Alzheimer’s disease, mental depression, dementia with Lewy Body, frontotemporal dementia, and/or vascular dementia. Said method comprises the steps of: (a) determining at least five criteria of said subject, (b) comparing said at least five criteria of said subject with reference values by calculating a global note in relation with each neurodegenerative disease, and (c) determining whether said subject suffers from Alzheimer’s disease, mental depression, dementia with Lewy Body, frontotemporal dementia, vascular dementia or mixed dementia.

[0011] According to a preferred embodiment, said subject suffering from Alzheimer's disease is selected among subjects suffering from Alzheimer's disease with a noradrenaline concentration [NA] comprised between 3000 pmol/l and 15000 pmol/l and among subjects suffering from Alzheimer's disease with a noradrenaline concentration [NA] comprised between 100 pmol/l and 2700 pmol/l.

[0012] According to a preferred embodiment, said step (a) is a step of determining at least eight criteria.

[0013] According to a preferred embodiment, said criteria are selected from a) an age of said subject, b) a score of said subject to a questionnaire adapted for screening cognitive function, preferentially a mini-mental state examination questionnaire, c) a dopamine concentration [D] in one blood sample of said subject, d) an adrenaline concentration [A] in one blood sample of said subject, e) a noradrenaline concentration [NA] in one blood sample of said subject, f) a dopamine expected concentration [D]®*** in one blood sample of said subject, g) an adrenaline expected concentration [A]°*Pécted in one blood sample of said subject, h) a noradrenaline expected concentration [NA]e*Pected in one blood sample of said subject.

[0014] According to a preferred embodiment said blood sample of said subject is one plasma sample of said subject.

[0015] According to a preferred embodiment, said dopamine, adrenaline and noradrenaline concentrations are determined with an HPLC equipped with an electrochemical detector.

[0016] According to a preferred embodiment, said dopamine expected concentration is obtained by applying the following equation

[D]expected = min(K,; [ED] T [DJaitzheumer |) wherein K4 is a constant comprised between 5 and 25, and wherein [D] aizheimeriS 454 pmol/l.

[0017] According to a preferred embodiment, said adrenaline expected concentration is obtained by applying the following equation [A]expected = min(K,; [4] T [A aizhermer |) wherein K4 is a constant comprised between 5 and 25, and wherein [A] aizheimer iS 483 pmol/l.

[0018] According to a preferred embodiment, said noradrenaline expected concentration is obtained by applying the following equation [NA]expected = min(K,; |[NA] T [NAlaizhermer |) wherein K4 is a constant comprised between 5 and 25, and wherein [NA] Aizheimer IS 4339 pmol/l.

[0019] According to a preferred embodiment, said reference values used in step (b) are as follows: a. the age of said subject is comprised within the range 40.2 years and 89.8 years, b. the score of said questionnaire adapted for screening cognitive function is comprised between 0 and 30, c. the dopamine concentration is comprised between 111 pmol/l and 1334 pmol/l, d. the adrenaline concentration is comprised between 66 pmol/l and 2366 pmol/l, e. the noradrenaline concentration is comprised between 874 pmol/l and 8681 pmol/l, f. the dopamine expected concentration is comprised between 2 pmol/l and 838 pmol/l, g. the adrenaline expected concentration is comprised between 8 pmol/l and 1788 pmol/l, h. the noradrenaline expected concentration is comprised between 2 pmol/l and 4727 pmol/l.

[0020] According to a preferred embodiment, said method further comprises the step of attributing a correction factor & to said reference values, said correction factor 5 being comprised between 0.50 and 1.50.

[0021] According to a preferred embodiment, said step (b) of calculating a global note in relation with each neurodegenerative disease comprises the following sub-steps: a) determining a virtual value VSriterion by neurodegenerative disease in function of each criterion, b) attributing a weighting factor to each virtual value V£iterion determined in the previous sub-step, c) determining a sum of each of said weighted virtual values so as to obtain one global note in relation with each neurodegenerative disease.

[0022] According to a preferred embodiment, said virtual value V£iiterion by neurodegenerative disease in function of each criterion is zero if one measured value VAL“terion of said subject for each criterion is outside the range of the reference values defined above, or said virtual value Vgiterion py neurodegenerative disease in function of each criterion is determined for each criterion by applying the following equation if the measured value VALSiterion of said subject for each criterion is within the range of the reference values defined above: criterion _ vaLcriterion_ My criterion Vaisease = 14 Keren gern wherein VALSiterion is the measured value of one subject, said measured value corresponding to each criterion, said criterion being defined above,

wherein MINIS!terion js the minimum reference value with respect to each neurodegenerative disease in function of each criterion, wherein MAXIST!erion is the maximum reference value with respect to each neurodegenerative disease in function of each criterion, and wherein K is a mathematical parameter, K being preferentially comprised between 0 and 0.1.

[0023] According to a preferred embodiment, the weighting factor (w+) in relation with the virtual value with respect to the age of said subject and to the score of said subject is comprised between 0.1 and 0.9, preferentially is equal to 0.5 and the weighting factor (wz) in relation with the virtual value with respect to the concentrations and the expected concentrations comprised between 1.1 and 3.0, preferentially is equal to 2.

[0024] According to a preferred embodiment, said step of determining whether said subject suffers from Alzheimer’s disease, mental depression, dementia with Lewy Body, frontotemporal dementia, vascular dementia or mixed dementia is performed by comparing the global notes obtained in relation with each of neurodegenerative diseases.

[0025] According to a preferred embodiment, said method further comprises, between said step (c) and said step (d), a step (c’) of determination whether said subject suffers from arterial hypertension.

[0026] In general, the particular embodiments of each object of the invention are also applicable to other objects of the invention. To the extent possible, each object of the invention is combinable with other objects.

Advantages of the invention

[0027] The invention is particularly interesting in that the differential diagnosis of a patient, when said diagnosis is precise, allows the patient to be adequately followed-up by the clinician. Once the patient has been diagnosed with the correct disease she/he suffers from, she/he can receive the best medical care that is going to help her/him to improve her/his medical conditions. While the differential diagnosis allows for detection of a mixed dementia, it is also an advantage to provide adequate “mixed treatment” for responding more efficacy to such health conditions, especially since it is currently difficult to detect such mixed dementia conditions and to know which disease is implicated when a mixed dementia condition is detected.

[0028] The use of blood test allows for the ease of repeatability of the diagnosis, allowing the patient to be followed up, to be diagnosed early (and it is known than an early diagnosis favours the treatment and greatly improves the condition of life of the patient and his/her family).

Brief description of the drawings

[0029] Figure 1: Principles of the method of the present invention.

[0030] Figure 2: Distribution of the subjects in the panel used in the determination of the method.

[0031] Figure 3: Cloud of points using the global note from the point of view from the group suffering from Alzheimer’s disease with a noradrenaline concentration [NA] comprised between 3000 pmol/l and 15000 pmol/l (AD1).

[0032] Figure 4: Cloud of points using the global note from the point of view from the group suffering from Alzheimer’s disease with a noradrenaline concentration [NA] comprised between 100 pmol/t and 2700 pmol/l (AD2).

Description of an embodiment

[0033] The present invention has allowed for the development of an efficient differential diagnosis method.

[0034] The diseases that are diagnosed thanks to the method of the present invention are the following neurodegenerative diseases: from Alzheimer’s disease with a noradrenaline concentration [NA] comprised between 3000 pmol/l and 15000 pmol/l (AD1), Alzheimer's disease with a noradrenaline concentration [NA] comprised between 100 pmol/l and 2700 pmol/l (AD2), mental depression (PSY), dementia with Lewy Body (DLB), frontotemporal dementia (FTD), and/or vascular dementia (VD). The diagnosis is able to discriminate which neurodegenerative disease a patient is suffering from. The diagnosis method of the present invention also evidently allows for discriminating patients suffering from mild cognitive impairement. Such patients typically present cognitive problems, anxiety, burn- out, sleep apnea, alcoholim. They are refers as control patient or neurocontrol patient (Control/CTRL/NeuroControl/NeuroCtrl).

[0035] The sensitivity, which is the percentage of patients suffering from the disease and tested as positive among a population of patients identified as suffering from said disease using a reference test), as well as the specificity, which is the percentage of patients who do not suffer from the disease and who were tested as negative among a population of patients identified as not suffering from said disease using a reference test, have been determined for each of the screened neurodegenerative diseases that are diagnosed thanks to the test designed in the present invention. A test with 100% sensitivity will recognize all patients with the disease by testing them positive. A test with 100% specificity will exclude the disease from all healthy patients.

[0036] With regard to the Alzheimer's disease with a noradrenaline concentration [NA] comprised between 3000 pmol/l and 15000 pmol/l (AD1), the maximum sensitivity is 94.74% and the maximum specificity is 95.77%.

[0037] With regard to Alzheimer's disease with a noradrenaline concentration [NA] comprised between 100 pmol/l and 2700 pmol/l (AD2), the maximum sensitivity is

95.65% and the maximum specificity is 88.89%.

[0038] With regard to mental depression (PSY), the maximum sensitivity is 83.33% and the maximum specificity is 100.00%.

[0039] With regard to dementia with Lewy body (DLB), the maximum sensitivity is

100.00% and the maximum specificity is 98.80%.

[0040] With regard to fronto-temporal dementia (FTD), the maximum sensitivity is 70.0% and the maximum specificity is 93.75%.

[0041] With regard to vascular dementia (VD), the maximum sensitivity is 42.86% and the maximum specificity is 97.70%.

[0042] Finally, with regard to the control patients (see above for the definition of the control patients), the maximum sensitivity is 57.89% and the maximum specificity is 100.00%.

[0043] In order to achieve these highly significant statistical results, a reliable method of differential diagnosis of neurodegenerative disease has been performed. Figure 1 conceptually shows the method of the present invention and figure 2 shows the panel of subjects from which the reference values have been determined. The abbreviation OD stands for “Other Diseases” and includes in fact PSY, DLB, FTD and VD. PSY (mental depression) also includes patients suffering from bipolar disorders.

[0044] The following criteria have been used in the present method:

[0045] The age of the patients that are to be diagnosed is collected, either by asking it clearly to the patients, or by asking it to one of his/her acquaintances, or by checking it in his/her identification papers.

[0046] Then, the patients are asked to respond to a medical questionnaire with regard to their cognitive performance. These tests consist of a set of 20 to 30 questions which assess basic cognitive functions. Most assessments require the individual to give basic personal information (i.e., name, address, etc.), to answer simple questions based on common knowledge (i.e., who is the president of the USA?), and to remember simple items of information such as a list of three words or name and address to recall later. According to the response given by the individual, a score is given that helps the clinician to categorize the patient. The questionnaires can be one of the abbreviated mental test (AMT), the mini-mental state examination (MMSE) and the six-item cognitive impairment test (6CIT). For the purpose of the present method of differential diagnosis, the MMSE has been chosen, but any other suitable medical questionnaire used to assess basic cognitive functions could be used.

[0047] Secondly, a blood sample of each individual is collected and is analysed to determine the level of three catecholamines (CA) into the plasma of the patients. A simultaneous quantitation of epinephrine (adrenaline), norepinephrine (noradrenaline) and dopamine using a standardized HPLC method is performed. The HPLC is coupled with an electrochemical detection. The advantage of this screening is that the collection of blood sample is easy to perform on the individual, especially on the elderly, in comparison to the standard method of diagnosis of neurodegenerative diseases usually requiring the collection of cerebrospinal fluid. It is indeed known that a lumbar puncture is more invasive than blood collection and that lumbar puncture is not always feasible on all kind of patients.

[0048] The level of CA into the plasma of the individuals, the results of the MMSE as well as the age of the individuals are taken into account and processed in the following mathematical model.

[0049] These first five criteria (age, score to a medical questionnaire, adrenaline concentration ([A]), noradrenaline concentration ([NA]) and dopamine concentration ([D])) are now processed in order to determine which neurodegenerative disease the subject is suffering from.

[0050] A parameter based on the catecholamine concentrations can thus be determined in order to refine the diagnosis. It is indeed to be noted that the first five criteria are sufficient to provide the right diagnosis. In the case where a refinement is necessary, the experimentally-determined catecholamine concentrations will be process to determine the expected concentration of those catecholamines.

[0051] The determination of the expected concentration in the catecholamines is based on the measurement of the disparity between the measured concentration and a concentration of reference. The concentration of reference has been chosen to be the concentration of the catecholamines in the blood sample, more particularly the plasma, in patients suffering from Alzheimer’s disease (AD). A mean value of such concentration has been determined among the initial panel of 100 subjects that has been selected in the training model step.

[0052] The adrenaline expected concentration corresponds to the following mathematical function: [A]expected = min(K,; [A] = [Alaizneumer |) (1a)

meaning that the adrenaline expected concentration corresponds to the minimum value between Ki; and the modulus of the difference between the measured adrenaline concentration in the blood sample ([A]) with the mean adrenaline concentration in the blood sample for patients suffering from AD ([A] Alzheimer): wherein Ky is a mathematical constant equal to 15. K; can be also comprised between 10 and 20 or between 5 and 25. In fact, Ks can be all the integers between 5 and 25, and [AlAizhermer is the average adrenaline concentration in the blood sample for patients suffering from AD. In fact, [A] Aizheimer @mounts to 483 pmol/l.

[0053] The noradrenaline expected concentration thus corresponds to the following mathematical function: [NAJe*Pected = min(K,; [NA] - [NA] Atzhermer |) (1b) meaning that the noradrenaline expected concentration corresponds to the minimum value between Ki; and the modulus of the difference between the measured noradrenaline concentration in the blood sample ([NA]) with the average noradrenaline concentration in the blood sample for patients suffering from AD ([NA] Aiznermer) wherein K, is a mathematical constant equal to 15. K4 can be also comprised between 10 and 20 or between 5 and 25. In fact, Kı can be all the integers between 5 and 25, and [NA] Aizhermer is the average noradrenaline concentration in the blood sample for patients suffering from AD. In fact, [NA] Aizhermer @mounts to 4339 pmol/l.

[0054] The dopamine expected concentration thus corresponds to the following mathematical function: [D]expected = min(K;; |[P] = [DJaizheumer |) (1c) meaning that the dopamine expected concentration corresponds to the minimum value between K; and the modulus of the difference between the measured dopamine concentration in the blood sample ([D]) with the average dopamine concentration in the blood sample for patients suffering from AD ([D]aizhermer). wherein K is a mathematical constant equal to 15. K; can be also comprised between 10 and 20 or between 5 and 25. In fact, Ks can be all the integers between 5 and 25, and [Platzhemer is the average dopamine concentration in the blood sample for patients suffering from AD. In fact, [D]aizheumer AMOUNts to 454 pmol/l.

[0055] Those values obtained for one subject, either measured (for the age, the score, [A], [NA] and [D]) or determined ([Ajexected, [NAJe<Pected, and [D]%Pected), are compared to a set of reference values. The eight criteria have been measured and/or determined and the following reference tables (Table | and Table Il) have been established.

[0056] Table | shows the minimum reference values that have been determined in respect to the five criteria (age, score to the medical questionnaire, [NA], [A] and [D]) in relation with the neurodegenerative disease. The minimum for the three calculated criteria used in case of the refinement of the diagnosis method is also indicated (IN AJe<pected [AJexrected and [D]expected),

[a pa 2063 66 | 111 | 20 | 9 | 1a _ 1023 | 143 | 153 | 2 | 9 | 8 | 1534 DB [617] 8 [1065[104 | 204 | 55 | 8 | 2 | FID [490] 0 [e874 | 123 | 246 60 | 43 | 7 | 1518 Table |: Reference table showing the lowest value of each criterion in function of the neurodegenerative disease.

[0057] Table II shows the maximum reference values that have been determined in respect to the five criteria (age, score to the medical questionnaire, [NA], [A] and [D]) in relation with the neurodegenerative disease. The maximum for the three calculated criteria used in case of the refinement of the diagnosis method is also indicated (IN A]expected [AJexpected and [DJexrected), | PR | eee LA | | PR (AE 8681 | 1721 4255 | 1228 2539 | 1372 3691 1257 7217 4727 5744 | 1271 |1003| 3225 | 669 | 577 | 3706 | 1526 1175 | 1032 6331 |2366 [1334] 3675 | 1788 Table Il: Reference table showing the highest value of each criterion in function of the neurodegenerative disease.

[0058] Therefore, for subject suffering from Alzheimer's disease with a noradrenaline concentration [NA] comprised between 3000 pmol/l and 15000 pmol/l (AD1), the reference values for the criteria are as follows: - the age is comprised between 57.8 years and 87.7 years; - the score at the MMSE is comprised between 20 and 29 ; - [NA] is comprised between 2963 pmol/l and 8681 pmol/l ; - [A] is comprised between 66 pmol/l and 1721 pmol/l ; - [D] is comprised between 111 pmol/l and 878 pmol/l ; - [NAJexpected is comprised between 29 pmol/l and 4255 pmol/l ; - [AJe<ected is comprised between 9 pmol/l and 1228 pmol/l ; - [Djé*Pected is comprised between 14 pmol/l and 415 pmol/l.

Said reference values can be in fact written as being comprised between MINIS" and MAXI,

[0059] For subject suffering from Alzheimer’s disease with a noradrenaline concentration [NA] comprised between 100 pmol/l and 2700 pmol/l (AD2), the reference values for the criteria are as follows: - the age is comprised between 51.9 years and 89.8 years; - the score at the MMSE is comprised between 4 and 27 ; - [NA] is comprised between 1023 pmol/l and 2539 pmol/l ;

7 io comprised between 143 pmol/l and 1372 pmol/l ; LU100877 7 Ae oon between 153 pmol/l and 656 pmol/l ; terres 8 comprised between 2 pmol/l and 804 pmol/l ; A comprised between 9 pmol/l and 932 pmol; | Said refer 5 comprised between 8 pmol/l and 279 pmol/l efe ues can be i i ;

[0060] MINISD# "0" and MAXIE fact writen as being comprised between or subject suffering from ment i criteria are as follows: al depression (PSY), the reference values for the the SI comprised between 40.2 years and 74.5 years; - [NA] is © m e MMSE is comprised between 22 and 30.0 ; on prise between 1534 pmol/l and 3691 pmol/l ; 1 comprise between 191 pmol/l and 407 pmol/l ; - ay a between 276 pmol/l and 519 pmol/l et 5 comprised between 19 pmol/l and 1257 pmolll ; “pipes je comprised between 13 pmol and 127 pmol. | etween 13 pmol/l and 127 Said reference values c i pmol/ Cie an be i i

[0061] MINI and MAXIS{{rrion, in fact written as being comprised between or subject suffering from dementia wi ; a for the criteria are as follows: la with Lewy body (DLB), the reference values 7 the 20e 5 Comprised between 61.7 years and 81.6 years; _ [NA] is com Te MSE is comprised between 8 and 24 ; . [Al is com prise between 1065 pmol/l and 7217 pmol/l ; re comp ised between 104 pmol/l and 418 pmol/l ; -[N de a oo between 204 pmol/l and 375 pmol/l [Ales is © peste between 55 pmol/l and 4727 pmol/l ; “rte | ween 8 pmol/l and 158 1 t Bree comes between oi rast cle values can be i i .

[0062] MINIDLE and MAXIgjigrion, in fact wrilten as being comprises beiween or subject suffering from fronto-te i for the criteria are as follows: mporal dementia (FTD), the reference values the score at the ised between 40.0 yours and 76.5 years; is comprised between 0 " -IN and 27.0 ; J ee comprised between 874 pmol/l and 5744 pmolll ; 58 comprise between 123 pmol/l and 1271 pmol/l ; - RAT car between 246 pmol/l and 1003 pmol/l niet ig a comprised between 60 pmol/l and 3225 pmol/l ; [Dl is comprised between 7 pmol and 577 pmol. | . etween 7 pmol/l and 577 Said reference values ca i pmol/l cle n be i ;

[0063] MINIF7" and MAXIgrion. in fact writer as being comprised Letween or subject suffering from va oriteria are as follows: scular dementia (VD), the reference values for the - e i : "be 506 compres poweer 5.8 years and 67 ma omprised between 21 ; [NA] is comprised between 1 and 29; ; - 518 pmol/l and 370 - [A 6 pmol/l : a 8 comprised between 240 pmol/l and 1526 omoll - - [DLs comprised between 199 pmol and 533 pmol | et comprised between 226 pmol and 1175 pmol/ ; - [D]expected ; ween 145 pmol/l and 10 Cl [D]exrected is comprised between 43 pmol/l and 230 pr

Said reference values can be in fact written as being comprised between MINIÇfiterion and MAXI{fiterion,

[0064] For a healthy control, the reference values for the criteria are as follows: - the age is comprised between 40.2 years and 84.7 years; - the score at the MMSE is comprised between 22 and 30 ; - [NA] is comprised between 925 pmol/l and 6331 pmol/l ; - [A] is comprised between 164 pmol/l and 2366 pmol/l ; - [D] is comprised between 208 pmol/l and 1334 pmol/l ; - [NAJe*ected is comprised between 27 pmol/l and 3675 pmol/l ; - [AJexPected is comprised between 71 pmol/l and 1788 pmol/l ; - [Djé*Pected is comprised between 22 pmol/l and 838 pmol/l. Said reference values can be in fact written as being comprised between MINIEEReon trol and MAXIfealthy control :

[0065] A correction factor 8 can be attributed to these lowest and highest reference values. Said correction factor 5 can be comprised between 0.50 and 1.50. Preferentially, said correction factor & can be comprised between 0.75 and 1.25. If the correction factor 6 is equal to 1, then the reference values that are used in the diagnostic method of the present invention are in fact the reference values as shown in Table | and in Table Il.

[0066] The correction factor 5 is used to take into consideration any possible deviation that can occur during the measurements of the catecholamine concentrations or the possible errors made by inadvertence at the determination of the MMSE score. In fact, for a correction factor & different from 1, the values of Table | and Table Il slightly differ. iteration of the method of the present invention on one subject with different reference tables (obtained via different correction factor 5) can provide a better robustness of the results. For instance, if a patient is diagnosed with fronto-temporal dementia with 3 different reference tables, then the certainty of the diagnosis is increased in comparison if said patient would have been diagnosed as suffering from this specific disease by reference to only 1 reference table.

[0067] The reference values, or the corrected reference values, are used to compare the at least five criteria (or even the eight criteria). The comparison step is in fact determinant to express a global note (or a global score) for each neurodegenerative disease. To obtain the global note, which will be used to diagnose the subject, a virtual value is firstly determined for each criterion.

[0068] If the subject presents a value for a criterion which is outside the range of the reference values (the lower limit being expressed in Tabie |, or in a corrected Table | if a correction factor 6 has been applied; the upper limit being expressed in Table il, or in a corrected Table Il in case where a correction factor § is applied), then the virtual value is set to zero. In other words, the selected criterion does not count.

[0069] If the subject presents a value for a criterion which is inside the range of the reference values (as indicated above, in accordance with the reference values of (corrected) Table | and (corrected) Table 11), then, the following mathematical equation is applied: i i criterion _ criterion Vilease " =1+K TET aS (2) wherein VAL®"ren is the measured value corresponding to a criterion. For instance, if the virtual value for the criterion of age of the subject is to be determined, VAL represents the actual age of said subject. However, if the virtual value for the level of adrenaline in the blood sample of the subject is to be determined, then VAL is the actual measured level of adrenaline in the blood sample (or in the plasma) of the subject.

MINISriterionand MAXISiterion represent respectively the lowest and the highest reference values obtained, possibly adjusted with the correction factor 5. In fact, the lowest reference values (without applying the correction factor 8) are those listed in Table | and the highest reference values are those listed in Table Il (see also paragraphs below said tables).

K is a mathematical parameter, varying between 0 and 0.1. Preferentially, K is comprised between 0 and 0.05.

[0070] In order to determine the global note in relation with each neurodegenerative disease, the virtual value in relation with all of the five criteria (age, score, [A], [NA], [D]) and/or with the three supplementary criteria (([A]®Pected, [NAJexPected, and [DJjé<rected) is to be determined in accordance with the previous mathematical equation. Five or eight virtual values are thus calculated.

[0071] A weighting factor is then attributed to each of the virtual values. The virtual values in relation with the age and with the score are less relevant than the virtual values in relation with the concentration or the expected concentration. It is indeed more objective to determine scientifically a concentration of a catecholamine in the blood than asking questions to a person. The age is also a more subjective parameter since its relation with respect to the disease is rather variable and depends on the individuals rather than a determined law. For those reasons, the weighting factor for the virtual values in respect of the age and the score of the medical questionnaire has been set for instance to 0.5, while the weighting factor for the virtual values in respect with the concentrations or the expected concentration has been set for instance to 2.

[0072] The global note in relation with each neurodegenerative disease is merely obtained by adding all the weighted virtual values together. The mathematical writing is thus as follows: GLOBAL NOTE = w,Vage + WiVscore + Wa (Via) + Vina + Vin) (3) +w, (Vyajexvected + Vin ajexpected + Vipjexvected) wherein wı is a number comprised between 0.1 and 0.9, preferentially between

0.3 and 0.7. For instance, w is equal to 0.5.

wherein wz is a number comprised between 1.1 and 3.0, preferentially between

1.5 and 2.5. For instance, wz is equal to 2.

[0073] The global note in relation with each neurodegenerative disease obtained for a sample of 13 subjects is indicated in Table lil. To make the diagnosis, the clinician compares the values of the obtained global notes for each of the six neurodegenerative diseases and concludes that the patient suffers from a specific disease if the global note for this specific disease is the highest among the six global notes. The last three columns of Table Ill show the conclusion of the diagnosis, the 1° diagnosis being the one which is the most probable for the patient. When the interval between the global notes is too weak, typically when the interval is inferior to 2.00 (preferentially inferior to 1.00, more preferentially inferior to 0.50), then a second diagnosis is made. Such patients, where two diagnosis can be made, or even when a third diagnosis can be made (also shown in Table Ill), generally suffers from mixed dementia. It has also been determined that patient suffering from mental depression (PSY) also shown signs of Alzheimer's disease (AD), mental depression being precursory signs of Alzheimer's disease (AD).

AD | Ab2 [sv [ous | ro | Vo [Control [ 3% 29 | 57

35.50 | 35.73 | 30.56 | 30.71 | 35.48 | 40.64 | 40.48 | VD [Control] |

30.46 | 35.76 | 41.45 | 20.52 | 40.65 | 30.76 | 40.44

25.32 | 35.64 | 40.61 | 25.36 | 35.40 | 20.40 | 40.23 | PSY |Control| | | 4 |20.20 | 30.57 | 40.44 | 30.78 | 30.38 | 25.27 | 25.18 |PsY| | | | 5 | 40.53 | 30.58 | 41.35 | 20.35 | 40.73 | 25.75 | 35.55 | 6 | 30.32 | 35.66 | 41.08 | 30.55 | 35.44 | 25.47 | 35.53 |PsY| | |

30.46 | 35.57 | 40.79 | 35.62 | 30.32 | 35.57 | 35.44 |PSY| | | | 8 | 30.36 | 30.55 | 30.76 | 40.86 | 30.62 | 35.46 | 3536 |DLB| | | | 9 | 30.36 | 30.50 | 10.05 | 40.91 | 30.35 | 15.28 | 25.36 [DLB| | |

30.32 | 40.77 | 20.48 | 41.10 | 30.63 | 35.34 | 35.32 |DLB | AD2 | |

25.20 | 40.56 | 35.73 | 40.72 | 40.39 | 25.27 | 30.26

25.21 | 40.70 | 30.64 | 25.47 | 40.52 | 25.32 | 30.27 |AD2| FD | |

25.46 | 20.45 | 20.43 | 20.44 | 40.66 | 25.66 | 35.40 [FTD| | | Table Ill: Global note in relation with each neurodegenerative disease calculated for 13 subjects and conclusion of the differential diagnosis method of the present invention.

[0074] In fact, for each patient, the distance between the score of the patient and the cloud of patients in each category is measured. For instance, when in formula (2) the reference values for AD1 are taken into account (namely the value for MINI disease and the value for MAXISI SE 2" are those corresponding to MINISTER and to MAXISRE™", Le, for example, in case where the criterion of [NA] is concerned, 2963 and 8681, respectively), one representation as the one depicted in figure 3 can be drawn. In this case, all the patients suffering from AD1 have high and concentrated score. In the other three categories (AD2, OD and Control), there are some subjects who also show scores similar to the scores of the patients in the AD category. But, this does not mean that they would eventually be diagnosed as suffering from AD, because they may have obtained a better score in another category.

[0075] That is the reason why the global note must be calculated by taking into account all the neurodegenerative diseases under study. For instance, when the reference values in formula (2) are used for the control group, a representation as the one shown in figure 4 is drawn for the very same group of patient.

[0076] By applying this algorithm to each patient, the final suggested diagnosis is the diagnosis of the cloud of points (amongst the 8 possible original clouds) to which the patient is the closest according to the distance measured. If a second-best diagnosis (as shown in Table Ill) (and possibly a third-best diagnosis) has a close enough score, this second-best, and potentially the third-best, may also be indicated to the clinician.

[0077] It is therefore possible for an individual to be diagnosed from a neurodegenerative disease or from mixed dementia. The attribution of a global note for each neurodegenerative disease and for each patient allows the clinician not only to categorize the patient in one class of disease (for instance, by assessing that said patient suffers from AD) but also to give an insight to what other disease this same patient could suffer from (based on the second-best and potentially the third-best diagnosis).

[0078] An additional criterion that is used is the determination whether the subject in question suffers from arterial hypertension. It has been indeed noticed that the prevalence of arterial hypertension is four times less in patients suffering from

AD. This could be linked to the formation of aggregate in the brain and the low clearance of those aggregates (due to (extremely) low blood pressure in these patients). Therefore, the determination of the blood pressure in the subject can be a further parameter in order to determine whether the subject suffers from AD.

[0079] The accurate detection and identification of catecholamines simultaneously in the blood lays a solid foundation for the study of the potential links between AD and catecholamines. In this exploratory study, with a cohort of patients (n = 100) suffering from dementia and other neurodegenerative diseases, we demonstrate the interest of the plasma catecholamine assay for the development of less invasive and accessible solution for the diagnosis of Alzheimer's disease (AD1+AD2), other neurodegenerative diseases, such as PSY, DLB, FTD, VD and also mixed dementia. The statistical exploitation of the signature of catecholamines, coupled with simple data such as age, the mini-mental state examination (MMSE) and the determination of blood pressure, makes it possible to reach for the diagnosis of subject suffering from Alzheimer's disease with a noradrenaline concentration [NA] comprised between 3000 pmol/l and 15000 pmol/l (AD1) with high sensitivity and specificity performances, namely with

94.74% and 95.77% respectively and also for the diagnosis of subject suffering from Alzheimer's disease with a noradrenaline concentration [NA] comprised between 100 pmol/l and 2700 pmol/l (AD2) (95.65% and 88.89% of sensitivity and specificity respectively).

[0080] The diagnosis method of the present invention is very useful not only to diagnose a subject, but it is also a tool for providing the follow-up of those subjects. In particular, it is highlighted that the preparation of samples is performed easily, since it is based on a simple blood test. For the medical research point of view, this diagnosis method is also very practical, since, due to the simple detection method used, new treatment and new medicines can be easily assessed.

[0081] Analytical method

[0082] Blood collected in lithium heparin tubes was centrifuged at 3500 rpm for 10 min at +4°C. 1 mL plasma and 100 pL of 2,3-dihydroxybenzoic acid (DHBA) internal standard (from 100 nM DHBA stock solution) was added to extraction tubes with 100 mg of aluminum oxide previously activated with a mixture of 500 uL TRIS 3M and 100 uL EDTA 10 % (5:1) according to a known method (Anton A. H., et al., J. Pharmacol. Exp. Ther., 1962, 138(3), 360-375). Samples were stirred for 10 min and centrifuged at 2000 rpm at 4°C. The aluminum oxide with bound catecholamines was then washed 3 times with 5 mL of distilled water followed by centrifugation. The elution of catecholamines was achieved by the addition of 500 HL of HCIO4 0.2 N and subsequent centrifugation at 2000 rpm for 10 min at 4°C. Aliquots of 100 pL were injected into the HPLC system (Waters 515 HPLC Pump, Waters Model 717 autosampler injector), which was equipped with a Purospher® STAR RP-C18 endcapped (5 um). The mobile phase consisted of 50 mM sodium acetate buffer containing 0.9 mM sodium lauryl! sulfate, 0.3 mM EDTA, 17.5 mM acetic acid and 12 % methanol (vol/vol) at pH 3.6. Electrochemical detection (Coulochem Il detector) was performed using an ESA 5010 cell with a glassy carbon working electrode set at a potential of +360 mV vs. Ag/AgCl.

[0083] Description of sample preparation

[0084] The catecholamines (adrenaline, noradrenaline and dopamine) are extracted from the plasma matrix by adsorption on alumina before the HPLC analysis. Sample preparation is simple, because pH-adjustment of the plasma samples is not necessary. Moreover, samples preparation requires only washing steps with Wash Buffer. A selected HPLC column in combination with mobile phase, optimised for this particular separation, allows for sure and reliablechromatographic quantification. With a certified HPLC kit, one person can analyse up to 100 plasma samples per day.

[0085] Extraction: Label a sample clean up cartridge appropriately for each sample. Add

0.5 mi extraction buffer to each cartridge and shake briefly. Then add 1 mi plasma (total capacity of the cartridge is 1.5 ml) and 50 pi Internal Standard (= 600 pg DHBA). Close the cartridge with the top plug and mix for 10 min. Then remove the bottom plug from the cartridge and remove the plasma supernatant by using a vacuum equipment or centrifugation (place the cartridge in a disposable centrifugation tube and centrifuge 1 min at 2000 rpm).

[0086] Wash steps: Re-mount the bottom plug and remove the top plug. Add 1 ml Wash Buffer. Close the cartridge again and mix for 30 s (vortex). Then remove the bottom plug from the cartridge and remove the plasma supernatant by using a vacuum equipment or centrifugation (place the cartridge in a disposable centrifugation tube and centrifuge 1 min at 2000 rpm). Repeat this step 2 more times. After the last (third) wash step dry the cartridges well by centrifugation (2 min at 4000 rpm). To ensure that the Wash Buffer is removed completely, tap at the cartridge; the alumina should loosen from the frit. Discard bottom plug.

[0087] Elution: Prior to elution attach the plastic tube supplied with the kit to the outlet of the sample clean up column. Add 120 pl Elution Buffer, shake briefly, and wait for min. Then mix for 30 s (vortex). Place the sample clean up column (with the plastic tube attached) in a fresh vial and centrifuge 1 min at 2000 rpm. The eluted sample is collected in the attached tube (This tube can be directly placed into a Waters WISP autosampler).

Claims (16)

Claims

1. An in vitro method for performing a differential diagnosis of neurodegenerative diseases in one subject, said subject being selected among subjects suffering from Alzheimer's disease, mental depression, dementia with Lewy Body, frontotemporal dementia, and/or vascular dementia, said method comprising the steps of: a) determining at least five criteria of said subject, b) comparing said at least five criteria of said subject with reference values by calculating a global note in relation with each neurodegenerative disease, and c) determining whether said subject suffers from Alzheimer's disease, mental depression, dementia with Lewy Body, frontotemporal dementia, vascular dementia or mixed dementia.

2. The method according to claim 1, wherein said subject suffering from Alzheimer’s disease is selected among subjects suffering from Alzheimer's disease with a noradrenaline concentration [NA] comprised between 3000 pmol/l and 15000 pmol/l and among subjects suffering from Alzheimer's disease with a noradrenaline concentration [NA] comprised between 100 pmol/l and 2700 pmol/l.

3. The method according to any one of claims 1-2, wherein said step (a) is a step of determining at least eight criteria.

4. The method according to any one of claims 1-3, wherein said criteria are selected from a) an age of said subject, b) a score of said subject to a questionnaire adapted for screening cognitive function, preferentially a mini-mental state examination questionnaire, c) a dopamine concentration [D] in one blood sample of said subject, d) an adrenaline concentration [A] in one blood sample of said subject, e) a noradrenaline concentration [NA] in one blood sample of said subject, f) a dopamine expected concentration {Djé*Pected in one blood sample of said subject, g) an adrenaline expected concentration [A]e*Pected in one blood sample of said subject, h) a noradrenaline expected concentration [NAJé*ected in one blood sample of said subject.

5. The method according to claim 4, wherein said blood sample of said subject is one plasma sample of said subject.

|

6. The method according to any one of claims 4-5, wherein said dopamine, adrenaline | and noradrenaline concentrations are determined with an HPLC equipped with an electrochemical detector.

7. The method according to any one of claims 4-6, wherein said dopamine expected concentration is obtained by applying the following equation [D]expected = min(K;; |[D] - [DJatzneimer |) wherein Ki is a constant comprised between 5 and 25, and wherein [D] aizheimeris 454 pmol/l. "=

8. The method according to any one of claims 4-7, wherein said adrenaline expected concentration is obtained by applying the following equation [A]expected = min(K,; |L4] - [AJaizhermer |) wherein K4 is a constant comprised between 5 and 25, and wherein [Al aizneimer IS 483 pmol/l.

9. The method according to any one of claims 4-8, wherein said noradrenaline expected concentration is obtained by applying the following equation [NA]expected = min(K,; |[NA] T [NA] aizhevmer |) wherein K; is a constant comprised between 5 and 25, and wherein [NA] aizheimer IS 4339 pmol/l.

10. The method according to any one of claims 4-9, wherein said reference values used in step (b) are as followed: a) the age of said subject is comprised within the range 40.2 years and 89.8 years, b) the score of said questionnaire adapted for screening cognitive function is comprised between 0 and 30, c) the dopamine concentration is comprised between 111 pmol/l and 1334 pmol/l, d) the adrenaline concentration is comprised between 66 pmol/l and 2366 pmol/l, e) the noradrenaline concentration is comprised between 874 pmol/l and 8681 pmol/l, f) the dopamine expected concentration is comprised between 2 pmol/l and 838 pmol/l, g) the adrenaline expected concentration is comprised between 8 pmol/l and 1788 pmol/l, h) the noradrenaline expected concentration is comprised between 2 pmol/l and 4727 pmol/l.

11. The method according to any one of claims 1-10, further comprising the step of attributing a correction factor & to said reference values, said correction factor & being comprised between 0.50 and 1.50.

12. The method according to any one of claims 1-11, wherein, said step (b) of calculating a global note in relation with each neurodegenerative disease comprises the following sub-steps: oo a) determining a virtual value Vise" by neurodegenerative disease in function of each criterion, oo b) attributing a weighting factor to each virtual value Veste" determined in the previous sub-step, c) determining a sum of each of said weighted virtual values so as to obtain one global note in relation with each neurodegenerative disease.

13. The method according to claim 12, wherein 1 said virtual value VSriterion by neurodegenerative disease in function of each criterion is zero if one measured value VALS" of said subject for each criterion is outside the range of the reference values in accordance with claim 9, or wherein said virtual value VSTiterion by neurodegenerative disease in function of each criterion is determined for each criterion by applying the following equation if the measured value VAL£iterion of said subject for each criterion is within the range of the reference values in accordance with claim 9 : —————"—"""—""———

; : criterion_ criterion wherein VALEiteron is the measured value of one subject, said measured value corresponding to each criterion, said criterion being defined in claim 3, wherein MINI®ron is the minimum reference value with respect to each neurodegenerative disease in function of each criterion, wherein MAXISterion is the maximum reference value with respect to each neurodegenerative disease in function of each criterion, and wherein K is a mathematical parameter, K being preferentially comprised between 0 and 0.1.

14. The method according to any one of claims 12-13, wherein the weighting factor (wa) in relation with the virtual value with respect to the age of said subject and to the score of said subject is comprised between 0.1 and 0.9, preferentially is equal to 0.5 and wherein the weighting factor (wz) in relation with the virtual value with respect to the concentrations and the expected concentrations comprised between 1.1 and 3.0, preferentially is equal to 2.

15. The method according to any one of claims 1-14, wherein said step of determining whether said subject suffers from Alzheimer's disease, mental depression, dementia with Lewy Body, frontotemporal dementia, vascular dementia or mixed dementia is performed by comparing the global notes obtained in relation with each of neurodegenerative diseases.

16. The method according to any one of claims 1-15, wherein said method further comprises, between said step (c) and said step (d), a step (c’) of determination whether said subject suffers from arterial hypertension.