WO2018178474A2 - Procédé de diagnostic de la démence frontotemporale et de la maladie d'alzheimer - Google Patents

Procédé de diagnostic de la démence frontotemporale et de la maladie d'alzheimer Download PDF

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WO2018178474A2
WO2018178474A2 PCT/ES2018/070261 ES2018070261W WO2018178474A2 WO 2018178474 A2 WO2018178474 A2 WO 2018178474A2 ES 2018070261 W ES2018070261 W ES 2018070261W WO 2018178474 A2 WO2018178474 A2 WO 2018178474A2
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value
intensity
spectral
raman
derivative
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WO2018178474A3 (fr
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Pedro CARMONA HERNÁNDEZ
Adolfo TOLEDANO GASCA
Eduardo LÓPEZ TOBAR
Marina Molina Santos
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad Complutense de Madrid
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Universidad Complutense de Madrid
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/3577—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing liquids, e.g. polluted water
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease

Definitions

  • the present invention relates to diagnostic methods of frontotemporal dementia and Alzheimer's disease.
  • Frontotemporal dementia is a clinical syndrome caused by the degeneration of the frontal lobe of the human brain, which can spread to the temporal lobe. It is one of three syndromes caused by frontotemporal lobular degeneration, and the second most common cause of early onset dementia after Alzheimer's disease.
  • the diagnosis of DFT is primarily of a clinical type (including behavioral changes and alterations in language) and neuropsychological tests (Liscic et al. Arch. Neurol. 64, 535-540 (2007)) with correct diagnostic results below 80% . Neuroimaging techniques are also used, which require very specialized personnel and are very expensive.
  • AD Alzheimer's disease
  • a neurodegenerative disease of insidious and age-related onset and its pathogenesis can affect the brains of patients long before symptoms are fully expressed, possibly years and even decades before diagnosis.
  • the clinical diagnosis of AD is mainly based on the exclusion of other diseases (Struyfs et al. Front. Neurol. 6, article 138 (2015)), and in relation to autopsy confirmation the clinical diagnostic criteria of AD reach on average 81% sensitivity and 70% specificity (Knopman et al. Neurology 56, 1 143-53 (2001)).
  • these figures mostly come from specialized clinical centers and diagnostics based on follow-up periods of several years. In the early stages of the disease and when diagnostic work is performed in non-specialized centers, a much lower diagnostic accuracy can be expected. Therefore the
  • REPLACEMENT SHEET (RULE 26) Definitive diagnosis of AD can only be made through a post-mortem pathological examination of the brain. In addition, a single type of pathology can produce different cognitive results, making diagnosis difficult. The challenge of finding effective treatments for AD and other dementias is parallel to exploring the detection of preclinical changes as early as possible and accurately identifying the pathology (or pathologies) responsible. The analysis of cerebrospinal fluid (CSF) levels of amyloid peptides of 42 amino acids ( ⁇ ⁇ ) and tau protein (T-tau) increases the diagnostic certainty for AD (Blennow et al. Nat. Rev. Neurol. 6,131-44 ( 2010)).
  • CSF cerebrospinal fluid
  • ⁇ ⁇ amyloid peptides of 42 amino acids
  • T-tau tau protein
  • vibrational spectroscopy provides specific information on the composition and molecular structure of biological tissue components, such as, for example, proteins, nucleic acids and lipids. This structural information is unique to each biological substance and therefore vibrational spectroscopy can provide "fingerprint" information about the overall biochemical state of a biological tissue such as blood, and therefore can provide useful clinical tests that are simple. , fast and minimally invasive.
  • Infrared spectroscopy has been used for diagnostic purposes of AD by lymphocyte analysis (Carmona et al. Anal. Bioanal. Chem. 402, 2015-2021 (2012)) and blood plasma (Peuchant et al. Translat. Res. 152 , 103-1 12 (2008); Carmona et al. J. Alzheimer's Dis. 34, 91 1-920 (2013)), but none of these studies addresses the differential diagnosis between DFT and AD.
  • REPLACEMENT SHEET (RULE 26) linked to frontotemporal dementia, in order to distinguish between healthy subjects, patients with this disease and patients with Alzheimer's disease.
  • the invention relates to a method for the diagnosis of frontotemporal dementia by Raman spectroscopy in a blood sample comprising:
  • the invention in a second aspect, relates to a method for the diagnosis of frontotemporal dementia by infrared spectroscopy in a blood sample comprising: a) recording an infrared spectrum of a previously obtained human blood sample;
  • REPLACEMENT SHEET (RULE 26) b) obtain an infrared spectral value of at least one of the following regions of the infrared spectrum:
  • the invention relates to a method for the diagnosis of frontotemporal dementia according to aspect 1 which further comprises the steps of a method according to aspect 2 applied on a sample of the same subject.
  • the invention relates to a method for the diagnosis of a neurodegenerative disease by Raman spectroscopy in a blood sample in which the neurodegenerative disease is frontotemporal dementia or Alzheimer's disease comprising:
  • REPLACEMENT SHEET (RULE 26) R3 region between 1000-390 cm “1 wherein said spectral value is selected from an intensity value, a value of a Raman intensity ratio between bands, an area value of said spectral region and / or a value of a frequency, Y
  • c) diagnose a neurodegenerative disease selected from frontotemporal dementia and Alzheimer's by comparison of the Raman spectral value obtained in stage b with a corresponding reference spectral value, or by comparison by multivariate analysis of the Raman spectral value obtained in stage b with a spectral value corresponding Raman reference, wherein said method comprises determining at least one Raman spectral value selected from the group formed by the average of the first derivative at intensity 1662 cm “1 and 1660 cm “ 1 (VR3); the average of the first derivative of the intensity at 745 cm “1 and 744 cm “ 1 (VR12); the value of the first derivative of the intensity at 760 cm “1 (VR15); the value of the first derivative of the intensity at 416 cm-1 (VR25) and the ratio of the area of the band to 1555 cm-1 with respect to the area of the band towards 1525 cm “1 (VR8).
  • the invention relates to a method for the diagnosis of a neurodegenerative disease, wherein the neurodegenerative disease is frontotemporal dementia or Alzheimer's disease by infrared spectroscopy in a blood sample comprising: a) recording an infrared spectrum of a previously obtained human blood sample;
  • REPLACEMENT SHEET (RULE 26) wherein said infrared spectral value is selected from absorbance, intensity, an area value of said spectral region and / or a percentage value of areas between bands of said spectral region; and c) diagnose a neurodegenerative disease selected from frontotemporal dementia and Alzheimer's by comparison of the infrared spectral value obtained in stage b with a corresponding infrared reference value, or by comparison by multivariate analysis of the infrared value obtained in stage b with an infrared spectral value corresponding reference, wherein said method comprises determining at least one infrared spectral value selected from the group formed by the ratio of the absorbance at 1 156 cm “1 to the absorbance of the maximum towards 1 171 cm “ 1 (VIR2) and the ratio of the first derivative of the intensity at 1 152 cm “1 to the first derivative at least towards the intensity 1 171 cm “ 1 (VIR3).
  • the invention relates to a method for the diagnosis of a neurodegenerative disease wherein the neurodegenerative disease is frontotemporal dementia or Alzheimer's disease according to the fourth aspect which further comprises the steps of a method according to the fifth aspect applied to a Sample of the same subject.
  • the invention relates to a method for the differential diagnosis of frontotemporal dementia and Alzheimer's by Raman spectroscopy in a blood sample of a patient who has previously been diagnosed with a neurodegenerative disease between frontotemporal dementia or Alzheimer's which comprises
  • REPLACEMENT SHEET (RULE 26) R3 region between 1000-390 cm “1 wherein said spectral value is selected from an intensity value, a value of a Raman intensity ratio between bands, an area value of said spectral region and / or a value of a frequency , and c) differentially diagnose frontotemporal dementia or Alzheimer's by comparison of the Raman spectral value obtained in stage b with a corresponding reference spectral value, or by comparison by multivariate analysis of the Raman spectral value obtained in stage b with corresponding reference Raman spectral values .
  • the invention relates to a method for differential diagnosis of frontotemporal dementia and Alzheimer's by infrared spectroscopy in a blood sample of a patient who has previously been diagnosed with a neurodegenerative disease comprising: a) recording a spectrum infrared of a previously obtained human blood sample;
  • the invention relates to a method for the differential diagnosis of frontotemporal dementia and Alzheimer's disease according to the seventh aspect that additionally comprises the steps of a method according to the eighth aspect applied on a sample of the same subject.
  • the tenth aspect the invention relates to a method for the diagnosis of Alzheimer's disease by Raman spectroscopy in a blood sample comprising:
  • said spectral value is selected from, an intensity value, a value of a Raman intensity ratio between bands, an area value of said spectral region and / or a value of one frequency, and c) diagnose Alzheimer's by comparison of the Raman spectral value obtained in stage b with a corresponding reference spectral value, or by comparison by multivariate analysis of the Raman spectral value obtained in stage b with a corresponding Raman reference spectral value, in wherein said method comprises determining at least one spectral value selected from the group formed by average of the first derivative at intensity 1662 cm “1 and 1660 cm “ 1 (VR3); average of the first derivative at intensity 745 cm “1 and the intensity 744 cm “1 (VR12), the value of the
  • REPLACEMENT SHEET (RULE 26) first derivative at intensity 760 cm “1 (VR15); the value of the first derivative at intensity 416 cm “ 1 (VR25); the height of the band towards 1555 cm “1 (VR7) and the ratio of the area of the band towards 1555 cm “ 1 to the area of the band towards 1525 cm “1 (VR8).
  • the invention relates to a method for the diagnosis of Alzheimer's disease by infrared spectroscopy in a blood sample comprising: a) recording an infrared spectrum of a previously obtained human blood sample;
  • the invention in a twelfth aspect, relates to a method for the diagnosis of Alzheimer's disease according to the tenth aspect that additionally comprises the steps of a method according to the eleventh aspect applied on a sample of the same subject.
  • the invention relates to an apparatus for plasma analysis comprising:
  • the invention relates to a computer program comprising a code suitable for carrying out the diagnostic methods according to the invention.
  • the invention relates to a computer support containing the computer program according to the invention.
  • the invention relates to a compound suitable for the treatment of frontotemporal dementia for use in the treatment of a subject who has been diagnosed with a frontotemporal dementia by a method according to the invention.
  • the invention relates to a compound suitable for the treatment of Alzheimer's for use in the treatment of a subject to whom Alzheimer has been diagnosed by a method according to the invention.
  • REPLACEMENT SHEET (RULE 26) Figure 1. Raman mean spectra, in the 3150-350 cm “1 region , of peripheral blood plasma obtained from healthy controls (-) and from patients with DFT () and mild AD
  • Figure 7 Infrared mean spectra, expressed in second derivative in the 1640-1620 cm “1 region , of peripheral blood plasma obtained from healthy controls (-) and from patients with DFT (-) and mild AD ( ⁇ ) ⁇
  • Figure 8 Infrared mean spectra in the 1 185-1 140 cm "1 region of peripheral blood plasma obtained from healthy controls (-) and patients with DFT () and mild AD ( ⁇ ) ⁇ Figure 9.
  • ROC curve of the canonical discriminant function of the combination of infrared and Raman spectra of peripheral blood plasma samples Classification of 151 healthy controls (class I) against 74 DFT patients and 70 EA patients (class ll + lll) .
  • REPLACEMENT SHEET (RULE 26) Figure 10.
  • the inventors have developed diagnostic methods that allow plasma samples to be classified among DFT patients and AD patients or healthy subjects with a sensitivity and specificity greater than 80%.
  • the invention relates to a method for the diagnosis of frontotemporal dementia by Raman spectroscopy in a blood sample (first method of the invention) comprising: a) recording a Raman spectrum of a previously obtained human blood sample ; b) obtain a Raman spectral value of at least one of the following regions of the Raman spectrum:
  • REPLACEMENT SHEET (RULE 26) multivariate of the Raman spectral value obtained in step b with a corresponding Raman reference spectral value.
  • Diagnostic method or “Diagnose”, as used herein, refers to both the process of trying to determine and / or identify a possible disease in a subject, that is, the diagnostic procedure, as well as opinion which is reached through this process, that is, the diagnostic opinion. As such, it can also be considered as an attempt to classify the status of an individual into separate and distinct categories that allow medical decisions to be made about treatment and prognosis.
  • diagnosis of Alzheimer's disease although it is preferred that it be so, does not need to be correct for 100% of the subjects to be diagnosed or evaluated. However, the term requires that it can be identified that a statistically significant part of the subjects suffer from Alzheimer's disease.
  • Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80% , at least 90% or at least 95%. P values are preferably 0.05, 0.01, 0.005 or less.
  • Frontotemporal dementia defines as a neurodegenerative disease characterized by a progressive change in personality and behavior and / or an early and progressive alteration of language. Frontotemporal dementia is caused when nerve cells in the temporal lobes of the brain die and the pathways that connect them change; The loss of chemical messengers also occurs. Over time, the brain tissue of the frontal and temporal lobes shrinks. In the early stages they are usually non-specific and difficult to recognize as the first manifestations of dementia. Thus, other explanations are usually suggested, such as depressive disorders, manic episodes or psychosocial problems. The mood of euphoria, especially when associated with increased loquacity and agitation, can be confused at first with a state of mania or hypomania. In the initial phase of the disease, these alterations of behavior and
  • REPLACEMENT SHEET (RULE 26) Personality usually prevail over cognitive deficits, which makes it difficult to diagnose early.
  • the conventional neurological examination is normal and the complementary tests that provide information are magnetic resonance imaging and the neuropsychological examination "The term” subject "or” individual “or” patient "refers to a human being.
  • the diagnostic methods of the present invention are vibrational spectroscopic methods.
  • “Vibrational spectroscopic method” as used herein refers to the analysis of molecular properties based on vibrations at the molecular level.
  • Raman is an effective dispersion / dispersive technique in the range of 50-4000 cm 1 and involves a frequency shift of the incident light (Raman shift).
  • the electromagnetic spectrum can be analyzed in the infrared zone that ranges from 100 to 5000 cm "1 and where vibrational and rotational excitations occur.
  • the optical orientation of the light beam entering the monochromator (scattered ray) with respect to the beam of incident light in the sample can be 90 degrees or 180 degrees (retro-reflection)
  • the Raman scattered light is analyzed by a retro-reflection optics.
  • Step a) of the first method of the invention comprises recording a Raman spectrum in a previously obtained human blood sample.
  • the term "Raman spectrum” refers to the collection of wave numbers comprising the spectral region between 300 and 3100 cm "1 , although the spectral region may be wider provided that it comprises said anterior spectral region.
  • the authors of the present invention have found the presence of 3 spectral regions that correlate with the presence or not of frontotemporal dementia, which comprise the regions defined in R.1 to R.3.
  • the Raman spectrum is recorded using a 785 nm exciter laser line.
  • Step b) comprises obtaining a Raman spectral value of at least one of the following regions of the Raman spectrum:
  • spectral value refers to any measurable parameter that can be obtained from a spectrum and that can be used to compare various spectra.
  • the spectral value to be determined is selected from intensity value, value of a ratio of Raman intensities between bands, area value of a spectral region and a frequency value.
  • intensity value refers to the intensity of a peak at a given wave number.
  • the expression "band around a frequency value expressed in cm “ 1 " refers to the band in the Raman vibrational spectrum whose intensity is obtained in the frequency range determined by vi ⁇ X cm “1 , where X is a variable frequency value that is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 cm “1 or greater.
  • X has a value of 2.
  • a band around a frequency value of, for example, 2936 cm “ 1 refers to the maximum band that is obtained in the range from 2934 to 2938 cm “1.
  • said term may also be referred to as” band approaching a frequency value expressed in cm “1 ".
  • the increase of ⁇ X cm “1 in a band frequency is indicated to define a frequency range amplitude of the same band that can be measured in various spectrometers that differ in the frequency measurement accuracy. Therefore, it should be understood that the value of
  • REPLACEMENT SHEET (RULE 26) Frequencies vi that appear in this specification may vary by ⁇ X cm '1 with respect to said frequency value depending on the spectrometer used.
  • the term “Raman intensity obtained around a frequency value vi expressed in cm” 1 " is understood to refer to the intensity value of the Raman spectrum obtained in the maximum of the band that appears in the frequency range determined by vi ⁇ X cm "1 .
  • X has a value of 2
  • a Raman intensity obtained around a frequency value of 2936 cm “1 corresponds to the intensity value of the maximum obtained in the frequency range between 2934 cm “ 1 and 2938 cm “ 1.
  • the Raman intensity of a band in this invention is understood as the height of said band, since the intensity of a band is proportional to its height.
  • the term "area”, as used herein, refers to the use of the area defined by a peak of the spectrum that can be calculated by drawing a baseline through the peak and measuring the area enclosed in the peak. The baseline is normally drawn based on the points before and after the peak in the spectrum.
  • the area used for normalization is the area of the spectral region between 600 and 3100 cm “1 , or the area of the amide band I in the region between 1700 and 1600 cm “ 1 .
  • the term “frequency” used herein refers to the so-called “number of waves", which is a magnitude of frequency that indicates the number of times a wave vibrates in a unit of distance. The unit used in this invention is cycles per centimeter or reciprocal centimeters, cm "1 .
  • the first method of the invention comprises determining at least one spectral value selected from the group consisting of
  • REPLACEMENT SHEET (RULE 26) - VR3 average of the first derivative of the intensity at 1662 cm “1 and 1660 cm "
  • - VR5 the height of the band towards 1525 cm “1 ;
  • - VR6 the area of the band towards 1555 cm “1 ;
  • - VR12 average of the first derivative of the intensity at 745 cm “1 and 744 cm “1 ;
  • - VR13 the value of the first derivative of the intensity at 747 cm “1 ;
  • - VR17 the value of the first derivative of the intensity at 762 cm “ 1;
  • - VR18 the ratio of the intensity of the maximum towards 758 cm “ 1 to the intensity at 744 cm “1 ;
  • REPLACEMENT SHEET (RULE 26) - VR20: the ratio of the intensity to 494 cm “1 to the intensity of the maximum towards 512 cm "1 ;
  • the first method of the invention comprises determining at least two spectral values selected from the group consisting of VR1 to VR29 as defined above. In another embodiment, the first method of the invention comprises determining at least three, preferably at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28 or 29 spectral values selected from the group consisting of VR1 to VR29 as defined above. The invention contemplates any combination of VR1 to VR29.
  • the first method of the invention comprises determining at least one Raman spectral value selected from the group consisting of the ratio of the area of the Raman spectral profile between 910 and 980 cm “1 (VR10), the frequency of the band in the region of the spectrum between 740 and
  • REPLACEMENT SHEET 750 cm “1 (VR1 1), the ratio of Raman intensities obtained at the maximum of the band towards 758 cm-1 and 744 cm-1 (VR18), the height of the band towards 1555 cm “ 1 (VR7), the area of the band towards 1555 cm “1 (VR6), the value of the first derivative of the intensity towards 416 cm “ 1 (VR25) and the ratio of the Raman intensities obtained towards 409 cm “1 and 423 cm “ 1 ( VR29).
  • the first method of the invention comprises determining the ratio of the area of the Raman spectral profile between 910 and 980 cm “1 (VR10), the frequency of the band in the region of the spectrum between 740 and 750 cm “1 (VR11), the ratio of Raman intensities obtained at the maximum of the band towards 758 cm “ 1 and 744 cm “1 (VR18), the height of the band towards 1555 cm “ 1 (VR7), the value of the first derivative of the intensity towards 416 cm “1 (VR25) and the ratio of the Raman intensities obtained towards 409 cm “ 1 and 423 cm “1 (VR29).
  • the method of diagnosis of frontotemporal dementia in a blood sample is based on infrared spectroscopy (second method of the invention) and comprises
  • said infrared spectral value is selected from an absorbance or intensity value, an area value of said spectral region and / or a value of percentages of areas between bands of said spectral region; and c) diagnose frontotemporal dementia by comparison of the infrared spectral value obtained in stage b with a corresponding infrared reference value, or by comparison by analysis
  • REPLACEMENT SHEET (RULE 26) multivariate of the infrared value obtained in step b with a corresponding infrared spectral reference value.
  • IR spectrum refers to an IR spectrum that comprises the spectral regions between 1000 and 1200 cm “1 and between 1600-1700 cm “ 1 , which may be spectrum consist of a broader spectral region that comprises the previous spectral regions, those spectra comprising at least the spectral region between 910 and 1700 cm "1 being preferred.
  • Step a) of the second method of the invention comprises recording an infrared spectrum of a previously obtained human blood sample.
  • the "infrared spectral value” refers to a value obtained from the infrared spectral profile of one of the regions defined in IR.1 and IR. 2, such as, for example, are a percentage of area of a component band of the spectral profile of said region, an absorbance value or the normalized area of the spectral profile of said region.
  • the infrared spectral value is selected from an absorbance or intensity value, an area value of said spectral region and / or a percentage value of areas between bands of said spectral region.
  • band around a frequency expressed in cm- 1 The terms “band around a frequency expressed in cm- 1 ", "intensity value” and “area value” have been defined above and are equally applicable to infrared spectra.
  • Absorbance also called optical density
  • Absorbance is defined as the (logarithmic) relationship between the intensity of infrared light that hits a sample and the intensity of that same light that is transmitted through that sample.
  • absorbance When the infrared spectrum is expressed in absorbances, the absorbance of a band at a given frequency is directly proportional to the height of that band. Therefore, the area relationships between two bands is equivalent to the height relationships between those bands. Finally, the percentage of area between bands is a way of expressing the relationships of areas between those same bands.
  • the second method of the invention comprises determining at least one infrared spectral value selected from the group consisting of:
  • - VIR3 the ratio of the first derivative of the intensity to 1 152 cm “1 to the first derivative of the intensity at the minimum towards 1171 cm “1
  • -VIR4 the ratio of the first derivative of the intensity to 1 154 cm “1 to the first derivative in the minimum of the intensity towards 1 171 cm “1 ;
  • -VIR7 average of the first derivative of the intensity towards 1160 cm “1 and 1 162 cm “ 1 normalized with respect to the first derivative of the intensity at the minimum towards 1 171 cm “1 ;
  • -VI R8 ratio of the area between 1165 cm “1 and 1 145 cm “ 1 to the area between 1 180 cm “1 and 1 165 cm “ 1 expressed the spectrum in second derivative;
  • the second method of the invention comprises determining at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or 9 infrared spectral values selected from the group consisting in VIR1 to VIR9 as defined above.
  • the invention contemplates any combination of VIR1 to VIR9.
  • REPLACEMENT SHEET (RULE 26) In a preferred embodiment of the second method of the invention, it comprises determining at least one infrared spectral value selected from the group consisting of the percentage of spectral profile area between 1639 and 1623 cm “1 with respect to the area between 1672-1623 cm “ 1 expressed in second derivatives (VIR1), the ratio of absorbance at 1 156 cm “1 with respect to the absorbance of the maximum towards 1171 cm “ 1 (VI R2) and the ratio of the first derivative at 1 152 cm “1 to the first derived at a minimum towards 1171 cm “1 (VIR3).
  • the second method of the invention comprises determining the percentage of spectral profile area between 1639 and 1623 cm “1 with respect to the area between 1672-1623 cm “ 1 expressed in second derivatives (VIR1) and the ratio of the absorbance at 1156 cm “1 with respect to the absorbance of the maximum towards 1,171 cm “ 1 (VI R2).
  • a third method of diagnosis of frontotemporal dementia comprises the stages of the first method of the invention and additionally the stages of the second method of the invention applied on a sample of the same subject.
  • the third method of the invention comprises determining at least one Raman spectral value selected from the group consisting of VR1 to VR29 as defined above and at least one infrared spectral value selected from the group consisting of VIR1 to VIR9.
  • the invention contemplates any combination of VR1 to VR29 together with VIR1 to VIR9.
  • the third method of the invention comprises preferably determining at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at minus 23, at least 24, at least 25, at least 26, at least 27, at least 28 or 29 spectral values selected from the group consisting of VR1 to VR29 as defined above and at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 spectral values selected from the group consisting of VIR1 to VIR9 as defined above.
  • REPLACEMENT SHEET (RULE 26) Any combination of VR1 to VR29 and VIR1 to VIR9 are contemplated in the present invention.
  • the third method of the invention comprises determining the Raman spectral values corresponding to the frequency value of the band in the region of the Raman spectrum between 740 and 750 cm “1 (VR1 1), the value of the first derived from the intensity towards 416 cm “1 (VR25), the area of the band towards 1555 cm “ 1 (VR6) and the infrared spectral values corresponding to the absorbance ratio at 1156 cm “1 at the absorbance of the maximum towards 1 171 cm “1 (VIR2), the ratio of the first derivative to 1 152 cm “ 1 to the first derivative in the minimum towards 1 171 cm “1 (VIR3) and the percentage of spectral profile area between 1639 and 1623 cm “ 1 with respect to the area between 1672-1623 cm “1 expressed in second derivatives (VIR1).
  • the methods of the present invention in order to obtain a spectral value, can contemplate the normalization and deconvolution of the spectra.
  • the invention preferably contemplates correcting the baseline of the Raman spectrum and / or infrared spectrum and normalizing said spectrum with respect to its total area or with respect to the area of the amide band I in order to determine the spectral value.
  • normalization refers to the elimination of systematic differences between measurements.
  • One of the most significant sources of systematic error in Raman and infrared spectral measurements arises from the variations in total intensity. Standardization can be useful to prevent differences in spectral value due to differences in the amount of sample tested or due to variability in the quality of the sample used as well as to eliminate unwanted sources of systematic variation in measurements of Raman, and the like.
  • Normalization can be carried out using any band that does not change between patients with frontotemporal dementia and patients who do not suffer from frontotemporal dementia. In a preferred embodiment, normalization can be carried out by dividing the value of the
  • REPLACEMENT SHEET (RULE 26) peak spectrum of interest between the value of the full spectrum.
  • the entire spectral region is between the wavelength of 600 and 3100 cm -1.
  • step a) of the method of the invention the baseline of the Raman and / or infrared spectrum is corrected and said spectrum is normalized with respect to the area of the amide band I.
  • Amide I refers to the region 1700-1600 cm -1.
  • any of the normalization values mentioned above are used.
  • the reference value is preferably normalized using the same normalization value.
  • the reference value is the intensity of the Raman band at a frequency of about 2936 cm "1 in a control patient (that is, a patient without frontotemporal dementia)
  • the intensity of the band of the Control spectrum has to be obtained by normalizing in the same way as the intensity of the band of the sample under study, in this case, the reference value to be used in step c) to determine if the intensity of the band increases is the normalized reference value.
  • the intensity value is determined after normalizing the value of the spectrum with respect to the total area of the Raman spectral profile.
  • deconvolution refers to a method by which the spectrum is resolved in its constituent elements, thereby allowing the identification of signals that would otherwise remain masked.
  • it is convenient to eliminate the fluorescence signals generated in the spectrum by biological substances contained in the blood samples that at least partially mask the Raman spectra of the samples in question .
  • REPLACEMENT SHEET (RULE 26) means of optimization of the microspectrometer to be used, the fluorescence suppression cannot be 100%. Therefore, it is convenient to obtain the spectrum by using laser lines with near-infrared excitation, such as a YAG neodymium laser line at 1064 nm for example. In a preferred embodiment, the Raman spectrum is recorded using an infrared laser excitation line near 750-1400 nm.
  • Step c) of the methods of the invention comprises comparing the Raman and / or infrared spectral value obtained in step b) with a corresponding reference spectral value or by comparison by multivariate analysis, and more preferably it is performed by linear combinations of the spectral values.
  • step c) comprises comparing the Raman and / or infrared spectral value obtained in step b) with a corresponding reference spectral value.
  • reference spectral value refers to the cut-off value (associated with the greater sensitivity and specificity) obtained by means of the ROC curve for the spectral variable of a region.
  • reference spectral refers to the corresponding discriminant cut-off point (C), that is, the mean value of the centroids of the two groups I and II (DI and Dll); each centroid being the discriminant score (discriminant function value) for the averages of each of the discriminant variables in each group.
  • the centroid of a group is a point whose coordinates are the averages, in the group, of each of the discriminant variables. If the discriminating score Di for sample i is such that the Di-DI difference in absolute value is less than the C-DI difference in absolute value, said sample is considered to belong to group I, and will belong to group II otherwise .
  • reference spectral value refers to a laboratory value used as a reference for the values / data obtained from samples.
  • the reference value (or reference level) can be an absolute value, at a relative value, a value that has an upper and / or lower limit, a series of
  • REPLACEMENT SHEET (RULE 26) values, an average value, a median, an average value, or a value expressed by reference to a reference or control value.
  • a reference value may be based on the value obtained from an individual sample, such as, for example, a value obtained from a study sample but obtained at a point in the previous time.
  • the reference value can be based on a high number of samples, such as the values obtained in a population of samples or based on a set of samples including or excluding the sample to be tested.
  • the samples are of the same type of sample as the sample obtained from the subject under study, ie if the Raman spectrum of the subject in study was obtained from plasma, then the reference value is obtained from a Raman spectrum obtained from a set of plasma samples.
  • the reference spectral value is the spectral value of a sample obtained from a control patient, more particularly from a subject not suffering from frototemporal dementia, and more preferably from a healthy subject.
  • the reference sample obtained from a control patient as defined above is the same type of sample as the sample obtained from the subject under study, ie if the Raman spectrum of the subject under study has been obtained. of plasma, then the reference value is obtained from a Raman spectrum obtained from a plasma sample in the control subject.
  • the reference spectral value is the spectral value of a sample obtained from the patient under study but obtained at a point in the previous time and, in particular, at a point in time at which the patient did not show any symptoms.
  • the reference sample obtained from a patient at a point in the previous time as defined above is the same type of sample as the sample obtained from the subject under study, ie: if the Raman spectrum of the subject under study was obtained from plasma, then the reference value is obtained from a Raman spectrum obtained from a plasma sample of the same subject at the point in the previous time. The equivalent applies to determine an infrared spectrum.
  • the reference values may be the cut-off points of the ROC curves, whether ROC curves constructed with a single spectral parameter or ROC curves constructed with linear combinations (discriminant functions) of several parameters
  • REPLACEMENT SHEET (RULE 26) Spectral
  • the cut points are those included in Tables 1-3.
  • the person skilled in the art knows that the cut-off points can be chosen based on the specificity or sensitivity that is desired and for this the area under the ROC curve (AUC) is important as a quality criterion of a diagnostic method in the classification of samples.
  • step c) of comparing the value obtained in stage b with a corresponding reference value is performed by comparison by multivariate analysis, and more preferably it is performed by linear combinations of the spectral values.
  • the Raman and / or infrared reference value has been obtained in a healthy subject.
  • an increase in the spectral value VR6, VIR1 and / or VIR2 is indicative that the subject suffers from frontotemporal dementia.
  • a Raman spectral value is considered “increased” or “increased", when the Raman value of a band around, by way of non-limiting illustrative example 2396 cm-1, compared to the corresponding reference spectral value is at least 1 , 5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35 %, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 10%, at least 120%, at least 130%, at minus 140%, at least 150% or more.
  • a decreased VR1 1, VR25, and / or VIR3 spectral value is indicative that the subject suffers from frontotemporal dementia.
  • a Raman spectral value is "diminished", when a specific Raman spectral value, compared to the corresponding reference spectral value, is reduced by at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
  • step c) of the methods of the invention comprises comparing by multivariate analysis the Raman and / or infrared spectral value obtained in step b) with a corresponding Raman and / or infrared reference spectral value.
  • Comparison by multivariate analysis refers to the comparison by one of these three alternative methods: discriminant analysis, cluster analysis and neural networks. Comparison by discriminant analysis is preferred as a model consisting of a discriminant function (for two groups in this invention) based on linear combinations of the predictor variables that provide the best possible discrimination between the groups, since said discriminant analysis provides better classification results. of samples than those obtained by the other alternative methods of comparison.
  • discriminant function for two groups in this invention
  • the determination of the methods of the invention are carried out in a blood sample.
  • the blood sample is a plasma sample.
  • plasma refers to the liquid element of the blood, which represents 60% of the total blood volume and lacks red and white blood cells.
  • the present invention can be carried out in plasma substantially free of platelets.
  • a platelet rich plasma sample is preferred.
  • platelet rich plasma refers to plasma that has been enriched with platelets and generally contains more than 300-350,000 platelets / ⁇ .
  • REPLACEMENT SHEET (RULE 26) platelet rich plasma.
  • the various methods consist of obtaining blood in tubes with anticoagulant and subjecting them to different centrifugation conditions according to the different protocols, so that the blood separates into its basic components depending on the density, selecting the fraction corresponding to platelet rich plasma.
  • the blood plasma fraction to be analyzed can be obtained previously from a sample of human blood (preferably human peripheral blood) already obtained by means of any of the conventional blood fractionation procedures, such as centrifugation fractionation -filtration for example.
  • the plasma sample is dehydrated plasma
  • “Dehydrated biological fluid”, as used herein refers to a biological fluid sample that shows a removal of water content. In a preferred embodiment, the removal of water is complete.
  • the plasma sample is processed before recording the Raman and / or infrared spectrum. In an even more preferred embodiment, the processing is carried out by dehydration. In a preferred embodiment, dehydration is carried out by evaporation. In another embodiment, dehydration is carried out to dryness. In another preferred embodiment, the dehydrated biological fluid is dehydrated plasma.
  • the sample is crystallized.
  • the sample of the blood plasma fraction is prepared to record its Raman and infrared spectrum following a procedure comprising: a) Take an adequate amount of plasma that is deposited well spread on a CaF 2 crystal;
  • the dehydrated plasma is prepared following a procedure comprising extending a volume of between 6 and 100 ⁇ of a fraction of blood plasma on a CaF 2 or ZnSe crystal or on a metal support and allowing it to evaporate completely until dryness at a temperature between 10 and 25 ° C, preferably between 15 and 25 ° C.
  • the sample volume will depend on the type of support, for example CaF 2 or ZnSe crystal or metal support.
  • the volume of blood plasma fraction is between 6 and 14 ⁇ or between 10-100 ⁇ .
  • the volume is between 10-20 ⁇ , 20-30 ⁇ , 30-40 ⁇ , 40-50 ⁇ , 50-60 ⁇ , 60-70 ⁇ , 70-80 ⁇ , 80-90 ⁇ or 90-100 ⁇ .
  • the volume of a fraction of blood plasma is 10 ⁇ .
  • the surface of the glass or metal support is between 0.5 and 2 cm 2 , preferably 1 cm 2 .
  • the CaF 2 crystal is used.
  • CaF 2 crystal refers to a cubic crystal with a single first-order Raman line of 322 cm-1 at 300 ° K, shows high transmittance in the far UV range at Medium IR, low refractive index, high mechanical resistance and high laser damage threshold.
  • ZnSe crystal refers to a chemically inert, non-hygroscopic and highly pure product that is very effective in many optical applications due to its extremely low volume losses, high thermal shock resistance and stability in virtually all environments, it is easily machined.
  • a metal support is used, preferably the metal Ag, Au and Cu.
  • the surface preparation can be through electrochemical corrugation, metal coating of a nanostructured substrate or deposition of metal nanoparticles.
  • Evaporation to the dryness of the volume of said fraction can be carried out at a temperature between 10 and 25 ° C without observing any alteration of the
  • REPLACEMENT SHEET (RULE 26) samples for the purpose of the present invention. Evaporation is preferably performed at between 15 and 25 ° C, both limits being included, for the purpose of faster evaporation of the sample.
  • the blood fraction sample (preferably a blood plasma sample) suitable for Raman spectroscopic analysis of the present invention can be prepared before acquiring the spectroscopic data according to a procedure comprising the following steps: a. obtaining said blood fraction (preferably the blood plasma fraction) from a previously obtained human peripheral blood sample by means of any of the commonly used centrifugation-filtration blood fractionation procedures used; b. spread the plasma sample on a metal support or CaF 2 or ZnSe crystal with a square surface of 0.5 to 2 cm 2 , preferably 1 cm 2 ; C.
  • Methods for the diagnosis of a neurodegenerative disease where the neurodegenerative disease is frontotemporal dementia or Alzheimer's.
  • the invention relates to a method for the diagnosis of a neurodegenerative disease by Raman spectroscopy (fourth method of the invention) in a blood sample wherein the neurodegenerative disease is frontotemporal dementia or Alzheimer's disease comprising: a) recording a Raman spectrum of a previously obtained human blood sample;
  • REPLACEMENT SHEET (RULE 26) b) obtain a Raman spectral value of at least one of the following regions of the Raman spectrum:
  • Neurodegenerative disease in the context of the present invention refers to frontotemporal dementia and Alzheimer's. On certain occasions such diseases are not easily differentiable, however the methods of the invention do allow it to be differentiated effectively.
  • NINCDS-ADRDA National Institute of Neurological and Communicative Disorders and the Alzheimer 's Disease and Related Disorders Association
  • CDR clinical dementia
  • MMSE state exam mini mental
  • medial temporal atrophy determined by magnetic resonance imaging, MRI
  • AD Alzheimer's criteria for diagnosis in 1984 (McKhann et al., 1984, Neurology 34: 939-44):
  • AD The patient meets the criteria of probable AD and has histopathological evidence of AD through autopsy or biopsy.
  • Probable or prodromal AD Dementia has been established by clinical and neuropsychological examination. Cognitive alterations also have to be progressive and be present in two or more areas of cognition. The appearance of the deficits has been between the ages of 40 and 90 years and finally there must be an absence of other diseases that can produce a dementia syndrome.
  • AD Possible or non-prodromal AD: There is a dementia syndrome with an atypical appearance, presentation or progression; and without a known etiology; but it is not believed that comorbid diseases that can cause dementia are at the origin of it.
  • the fourth method of the invention comprises determining in addition to at least one Raman spectral value selected from the group formed by the average of the first derivative at intensity 1662 cm “1 and 1660 cm “ 1 (VR3); the average of the first derivative of the intensity at 745 cm “1 and 744 cm “ 1 (VR12); the value of the first derivative of the intensity at 760 cm “1 (VR15); the value of the first derivative of the intensity at 416 cm “ 1 (VR25) and the ratio of the area of the band towards 1555 cm "1 with respect to the area of the band towards 1525 cm “ 1 (VR8), at least one other Raman spectral value selected from the group formed by
  • - VR1 the ratio of Raman intensities obtained towards 2936 cm “1 and towards 1658 cm “1
  • - VR2 the ratio of Raman intensities obtained towards 1671 cm “1 and towards 1658 cm “1 ;
  • - VR6 the area of the band towards 1555 cm “1 ;
  • - VR7 the height of the band towards 1555 cm “1 ;
  • REPLACEMENT SHEET (RULE 26) VR14: the value of the first derivative of the intensity at 757 cm '1 ;
  • VR16 the value of the first derivative of the intensity at 761 cm "1 ;
  • VR17 the value of the first derivative of the intensity at 762 cm "1 ;
  • VR18 the ratio of the intensity of the maximum towards 758 cm “1 to the intensity at 744 cm “1 ;
  • VR19 the ratio of the height of the band towards 644 cm “1 to the height of the band towards 622 cm “1 ;
  • VR20 the ratio of the intensity to 494 cm “1 to the intensity of the maximum towards 512 cm "1 ;
  • VR28 the value of the first derivative of the intensity at 496 cm “1 ; and VR29: the ratio of the intensity at 409 cm-1 to the intensity at 423
  • the fourth method of the invention comprises additionally determining at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at minus 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23 or at least 24 spectral values selected from the group consisting of VR1-VR2, VR4-VR7, VR9-VR14, VR16-24, VR 26-29 as defined above.
  • the invention contemplates any combination of VR1-VR2, VR4-VR7, VR9-VR14, VR16-24, VR 26-29 as defined above.
  • REPLACEMENT SHEET (RULE 26) combination of VR3, VR12, VR15, VR25 and VR8 together with VR1 -VR2, VR4-VR7, VR9- VR14, VR16-24, VR 26-29.
  • the method comprises further determining a spectral value of the region of the Raman spectrum selected from the group formed by the frequency of the Raman band between 740 and 750 cm “1 (VR1 1) and the ratio of the intensity of the maximum towards 758 cm “1 and the intensity at 744 cm “ 1 (VR18).
  • the fourth method of the invention comprises determining the Raman values corresponding to the average of the first derivative at intensity 1662 cm “1 and 1660 cm “ 1 (VR3); the average of the first derivative of the intensity at 745 cm “1 and 744 cm “ 1 (VR12); the value of the first derivative of the intensity at 760 cm “1 (VR15); the value of the first derivative of the intensity at 416 cm “ 1 (VR25) and the ratio of the area of the band to 1555 cm “1 with respect to area of the band towards 1525 cm “1 (VR8), the frequency of the Raman band between 740 and 750 cm “ 1 (VR1 1) and the ratio of the intensity of the maximum towards 758 cm “1 and the intensity at 744 cm “ 1 (VR18).
  • the invention in another aspect, relates to a method of diagnosing a neurodegenerative disease, wherein the neurodegenerative disease is frontotemporal dementia or Alzheimer's disease by infrared spectroscopy (fifth method of the invention) in a blood sample comprising: a) record an infrared spectrum of a previously obtained human blood sample; b) obtain an infrared spectral value of at least one of the following regions of the infrared spectrum: IR1 1700-1600 cm "1
  • REPLACEMENT SHEET (RULE 26) c) diagnose a neurodegenerative disease selected from frontotemporal dementia and Alzheimer's by comparison of the infrared spectral value obtained in stage b with a corresponding infrared reference value, or by comparison by multivariate analysis of the infrared value obtained in stage b with an infrared spectral value corresponding reference, wherein said method comprises determining at least one infrared spectral value selected from the group formed by the ratio of the absorbance at 1156 cm “1 to the absorbance of the maximum towards 1 171 cm “ 1 (VIR2) and the ratio of the first derivative of the intensity at 1 152 cm “1 to the first derivative at least towards the intensity 1171 cm " 1 (VIR3).
  • the fifth method of the invention comprises additionally determining at least one infrared spectral value selected from the group consisting of:
  • -VIR5 the ratio of the first derivative of the intensity to 1 156 cm “1 to the first derivative in the minimum of the intensity towards 1 171 cm “1 ;
  • -VIR6 average of the first derivative of the intensity towards 1158 cm “1 and 1 160 cm “ 1 normalized with respect to the first derivative of the intensity at the minimum towards 1 171 cm “1 ;
  • the fifth method of the invention comprises determining at least 2, at least 3, at least 4, at least 5, at least 6 and at least 7 infrared spectral values selected from the group consisting of VIR1, VIR4-VIR9 as defined above.
  • the invention comprises any combination of VIR2, VR3 with VIR1, WIR4-VR9.
  • the fifth method of the invention comprises additionally determining the spectral value of the region of the infrared spectrum corresponding to the percentage of spectral profile area between 1639 and 1623 cm “1 with respect to the area between 1672-1623 cm “ 1 expressed in second derivatives (VIR1).
  • the fifth method of the invention comprises determining the infrared spectral values corresponding to the ratio of the absorbance at 1156 cm -1 to the absorbance of the maximum towards 1,171 cm “1 (VIR2) and the percentage of area of the Spectral profile between 1639 and 1623 cm “1 with respect to the area between 1672-1623 cm “ 1 expressed in second derivatives (VIR1).
  • the invention relates to a method for the diagnosis of a neurodegenerative disease wherein the neurodegenerative disease is frontotemporal dementia or Alzheimer's disease (sixth diagnostic method) comprising the steps according to the fourth method of the invention and additionally the Stages of the fifth method of the invention applied on a sample of the same subject.
  • the sixth method of the invention comprises determining at least one Raman spectral value selected from the group formed by the average of the first derivative at intensity 1662 cm “1 and 1660 cm “ 1 (VR3); the average of the first derivative of the intensity at 745 cm “1 and 744 cm “ 1 (VR12); the value of the first derivative of the intensity at 760 cm “1 (VR15); the value of the first derivative of the intensity at 416 cm “ 1 (VR25) and the ratio of the area of the band to 1555 cm “1 with respect to area of the band towards 1525 cm “1 (VR8) and at least one infrared spectral value selected from the group formed by the ratio of absorbance to 1 156 cm “ 1 to the absorbance of the maximum towards 1 171 cm “1 (VIR2) and the relationship of the first
  • the sixth method of the invention comprises determining at least 2, at least 3, at least 4 or the 5 spectral values selected from the group consisting of VR3, VR12, VR15, VR25 and VR8 and at least 1 or 2 Spectral values selected from the group formed by VIR2 and VIR3.
  • the invention comprises any combination VR3, VR12, VR15, VR25 and VR8 together with VIR2, VIR3.
  • the sixth method of the invention comprises additionally determining at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at minus 22, at least 23, at least 24 or at least 25 spectral values selected from the group consisting of VR1-VR2, VR4-VR7, VR9-VR11, VR13-VR14, VR16-VR24, VR26-VR29 and additionally at least 1, at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7 infrared spectral values selected from the group consisting of VIR1, VIR4-VIR9 as defined above.
  • the invention contemplates any combination of
  • the sixth method of the invention comprises determining the Raman spectral values corresponding to the value of the first derivative of the intensity at 760 cm-1 (VR15); the ratio of the area of the band towards 1555 cm “1 to the area of the band towards 1525 cm “ 1 (VR8) and the infrared spectral values corresponding to the absorbance at 1 156 cm “1 to the absorbance of the maximum towards 1 171 cm “ 1 (VIR2); the ratio of the first derivative to 1 152 cm “1 to the first derivative at least towards 1 171 cm “ 1 (VIR3); and percentage of the area between 1639 cm “1 and 1623 cm “ 1 with respect to the area between 1672 cm “1 and 1623 cm “ 1 in the spectrum expressed in second derivative (VIR1).
  • a decreased VR15 and / or VIR3 spectral value is indicative that the subject suffers from a neurodegenerative disease where the neurodegenerative disease is frontotemporal dementia or disease
  • an increased VR8, VIR1 and / or VIR2 spectral value is indicative that said subject suffers from a neurodegenerative disease where the neurodegenerative disease is frontotemporal dementia or Alzheimer's disease.
  • the terms "infrared spectroscopy”, “infrared spectral value”, “absorbance value”, “intensity value”, “area value of said spectral region”, “percentage value of area between bands” and their particularities have been described previously and are equally applicable to these methods of the invention.
  • the Raman and / or Infrared reference value has been obtained in a healthy subject.
  • step c) is performed by comparison by multivariate analysis, and more preferably is performed by linear combinations of the spectral values.
  • the invention relates to a method for the differential diagnosis of frontotemporal dementia and Alzheimer's by Raman spectroscopy (seventh method of the invention) in a blood sample of a patient who has previously been diagnosed with a neurodegenerative disease between frontotemporal dementia or Alzheimer's disease that includes
  • REPLACEMENT SHEET wherein said spectral value is selected from an intensity value, a value of a ratio of Raman intensities between bands, an area value of said spectral region and / or a value of a frequency, and c) differentially diagnose frontotemporal dementia or Alzheimer's by comparison of the Raman spectral value obtained in stage b with a corresponding reference spectral value, or by comparison by multivariate analysis of the Raman spectral value obtained in stage b with corresponding reference Raman spectral values.
  • differentiated diagnosis refers to the procedure by which a certain disease, nosological entity, syndrome, or any state of health or disease is identified by excluding other possible causes that present a clinical picture similar to the one the patient suffers from.
  • frontotemporal dementia Alzheimer, “subject”, “blood sample” “vibrational spectroscopic method”, “Raman spectrum”, “spectral value”, “intensity value”, “band around a frequency value expressed in cm-1, Raman intensity obtained around a frequency value vi expressed in cm -1, “Relationship of intensities between bands”, “area”, “frequency” and their particularities have been previously defined, and are equally applicable to the diagnostic methods of a neurodegenerative disease that is frontotemporal dementia or Alzheimer's disease.
  • the seventh method of the invention comprises determining at least one spectral value selected from the group consisting of
  • REPLACEMENT SHEET (RULE 26) - VR3 average of the first derivative of the intensity at 1662 cm “1 and 1660 cm "
  • - VR5 the height of the band towards 1525 cm “1 ;
  • - VR6 the area of the band towards 1555 cm “1 ;
  • - VR12 average of the first derivative of the intensity at 745 cm “1 and 744 cm “1 ;
  • - VR13 the value of the first derivative of the intensity at 747 cm “1 ;
  • - VR17 the value of the first derivative of the intensity at 762 cm “1 ;
  • - VR18 the ratio of the intensity of the maximum towards 758 cm “ 1 to the intensity at 744 cm “1 ;
  • REPLACEMENT SHEET (RULE 26) - VR20: the ratio of the intensity to 494 cm “1 to the intensity of the maximum towards 512 cm "1 ;
  • the seventh method of the invention comprises determining at least two spectral values selected from the group consisting of VR1 to VR29 as defined above.
  • the seventh method of the invention comprises determining at least 3, preferably at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28 or 29 spectral values selected from the group consisting of VR1 to VR29 as defined above.
  • the invention contemplates any combination of VR1 to VR29.
  • the seventh method of the invention comprises determining at least one Raman spectral value selected from the group formed by the ratio of intensities obtained towards 2936 cm “1 and towards 1658 cm “ 1 (VR1), the value
  • REPLACEMENT SHEET (RULE 26) of the first derivative of the intensity at 760 cm “1 (VR15) and the ratio of the intensity to 494 cm “ 1 to the intensity of the maximum towards 512 cm “1 (VR20).
  • the seventh method of the invention comprises determining the Raman spectral value corresponding to the ratio of intensities obtained towards 2936 cm “1 and towards 1658 cm “ 1 (VR1).
  • the invention relates to a method for the differential diagnosis of frontotemporal dementia and Alzheimer's by infrared spectroscopy (eighth method of the invention) in a blood sample from a patient who has previously been diagnosed with a neurodegenerative disease comprising : a) record an infrared spectrum of a previously obtained human blood sample;
  • said infrared spectral value is selected from an intensity value, an absorbance value, an area value of said spectral region and / or a percentage value of areas between bands of said spectral region; and c) differentially diagnose frontotemporal dementia or Alzheimer's, by comparison of the infrared spectral value obtained in stage b with an infrared reference value that allows the distinction between frontotemporal dementia and Alzheimer's, or by comparison by multivariate analysis of the infrared value obtained in stage b with infrared reference spectral values of frontotemporal dementia and Alzheimer's
  • the eighth method of the invention comprises determining at least one infrared spectral value selected from the group consisting of:
  • VIR1 percentage of the area between 1639 cm “1 and 1623 cm '1 with respect to the area between 1672 cm ' 1 and 1623 cm “ 1 in the spectrum expressed in second derivative.
  • - VIR2 the ratio of the absorbance at 1 156 cm “1 to the absorbance of the maximum towards 1 171 cm "1 ;
  • the eighth method of the invention comprises determining at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or 9 infrared spectral values selected from the group consisting in VIR1 to VIR9 as defined above.
  • the invention contemplates any combination of VIR1 to VIR9.
  • the eighth method of the invention comprises determining at least one infrared spectral value selected from the group formed by the ratio of the area between 1 165 cm '1 and 1 145 cm “1 to the area between 1 180 cm ' 1 and 1 165 cm “1 expressed the spectrum in second derivative (VIR8), the ratio of the area between 1 166 cm “ 1 and 1 145 cm “1 to the area between 1 185 cm “ 1 and 1 145 cm “1 (VI R9) and the ratio of the first derivative of the intensity to 1 154 cm “1 to the first derivative in the minimum intensity towards 1 171 cm “ 1 (VIR4).
  • the eighth method of the invention comprises determining the infrared spectral values corresponding to the ratio of the area between 1 165 cm “1 and 1 145 cm “ 1 to the area between 1 180 cm “1 and 1 165 cm “ 1 expressed the spectrum in second derivative (VIR8) and the ratio of the area between 1 166 cm “1 and 145 cm “ 1 to the area between 1 185 cm “1 and 1 145 cm “ 1 (VI R9).
  • the invention in another aspect, relates to a method for differential diagnosis of frontotemporal dementia and Alzheimer's disease (ninth method of the invention) comprises the steps of the seventh method of the invention and additionally the steps of the eighth method of the invention applied to a sample of the same subject.
  • the ninth method of the invention comprises determining at least one spectral value selected from the group consisting of VR1 to VR29 as defined above and at least one spectral value selected from the group consisting of VIR1 to VIR9.
  • the ninth method of the invention comprises preferably determining at least two, at least three, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at minus 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28 or 29 spectral values selected from the group consisting of VR1 to VR29 as defined above and at least 2, at least 3, at minus 4, at least 5, at least 6, at least 7, at least 8 or at least 9 spectral values selected from the group consisting of VIR1 to VIR9 as
  • REPLACEMENT SHEET (RULE 26) They are defined above.
  • the invention contemplates any combination of VR1 to VR29 together with VIR1 to VIR9.
  • the ninth method comprises determining the Raman spectral values corresponding to the ratio of Raman intensities obtained towards 2936 cm “1 and towards 1658 cm “ 1 (VR1); the value of the first derivative of the intensity at 760 cm “1 (VR15); the ratio of the intensity at 494 cm “ 1 to the intensity of the maximum towards 512 cm “1 (VR20); and the infrared spectral values corresponding to the ratio of the first derivative to the intensity 1 154 cm “1 to the first derivative at least towards 1 171 cm “ 1 (VIR4); the ratio of the area between 1 165 cm “1 and 1 145 cm “ 1 to the area between 1 180 cm “1 and 1 165 cm “ 1 expressed the spectrum in second derivative (VIR8) and the ratio of the area between 166 cm “1 and 1 145 cm “ 1 to the area between 1 185 cm “1 and 1 145 cm “ 1 ( VI R9)
  • a decreased VR20 and / or VIR8 spectral value corresponding to the ratio of Raman intensities obtained towards 2936 cm “1 and
  • the Raman reference value and / or infrared reference value has been obtained from a subject that has previously been diagnosed with frontotemporal dementia or a subject that has previously been diagnosed with Alzheimer's
  • step c) is performed by comparison by multivariate analysis, and more preferably is performed by linear combinations of the spectral values.
  • the invention relates to a method for the diagnosis of Alzheimer's disease by Raman spectroscopy (tenth method of the invention) in a blood sample comprising: a) recording a Raman spectrum of a previously obtained human blood sample ; b) obtain a Raman spectral value of at least one of the following regions of the Raman spectrum:
  • REPLACEMENT SHEET intensity 416 cm “1 (VR25); the height of the band towards 1555 cm “ 1 (VR7) and the ratio of the area of the band towards 1555 cm “1 to the area of the band towards 1525 cm “ 1 (VR8).
  • the tenth method of the invention comprises determining in addition to at least one value selected from the group formed by average of the first derivative at intensity 1662 cm “1 and 1660 cm “ 1 (VR3); average of the first derivative at intensity 745 cm “1 and intensity 744 cm “ 1 (VR12), the value of the first derivative at intensity 760 cm “1 (VR15); the value of the first derivative at intensity 416 cm “1 (VR25); the height of the band towards 1555 cm “1 (VR7) and the ratio of the area of the band towards 1555 cm “ 1 to the area of the band towards 1525 cm “1 (VR8), additionally determine at least one spectral value selected from the group formed by
  • - VR5 the height of the band towards 1525 cm “1 ;
  • - VR6 the area of the band towards 1555 cm “1 ;
  • REPLACEMENT SHEET (RULE 26)
  • VR1 1 the frequency of the band in the region between 740 and 750 cm
  • VR14 the value of the first derivative of the intensity at 757 cm "1 ;
  • VR16 the value of the first derivative of the intensity at 761 cm "1 ;
  • VR17 the value of the first derivative of the intensity at 762 cm "1 ;
  • VR18 the ratio of the intensity of the maximum towards 758 cm “1 to the intensity at 744 cm “1 ;
  • VR19 the ratio of the intensity of the band towards 644 cm to the intensity of the band towards 622 cm “1 ;
  • VR20 the ratio of the intensity to 494 cm “ to the intensity of the maximum towards 512 cm “1 ;
  • VR22 the value of the first derivative of the intensity at 413 cm - VR23: the value of the first derivative of the intensity at 414 cm
  • VR28 the value of the first derivative of the intensity at 496 cm
  • VR29 the ratio of intensity at 409 cm-1 to intensity at 423
  • the tenth method of the invention comprises additionally determining at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at minus 1 1, at least 12, at least 13, at
  • REPLACEMENT SHEET (RULE 26) minus 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 or at least 23 spectral values selected from the group consisting of VR1-VR2, VR4-VR6, VR9-VR11, VR13-14, VR 16-24, VR26-VR29 as defined above.
  • the invention contemplates any combination of VR3, VR12, VR15, VR25, VR7 and VR8 together with VR1-VR2, VR4-VR6, VR9-VR11, VR13-14, VR 16-24, VR26-VR29.
  • the tenth method of the invention comprises obtaining the Raman spectral values corresponding to the average of the first derivative at intensity 1662 cm “1 and 1660 cm “ 1 (VR3); average of the first derivative at intensity 745 cm “1 and intensity 744 cm “ 1 (VR12), the value of the first derivative at intensity 760 cm “1 (VR15); the value of the first derivative at intensity 416 cm “ 1 (VR25); the height of the band towards 1555 cm “1 (VR7) and the ratio of the area of the band towards 1555 cm “ 1 to the area of the band towards 1525 cm “1 (VR8).
  • the invention relates to a method for the diagnosis of Alzheimer's disease (eleventh method of the invention) by infrared spectroscopy in a blood sample comprising: a) recording an infrared spectrum of a previously obtained human blood sample;
  • said infrared spectral value is selected from an absorbance value, an intensity value, an area value of said spectral region and / or a percentage value of areas between bands of said spectral region; and c) diagnose Alzheimer's
  • REPLACEMENT SHEET (RULE 26) by comparison of the infrared spectral value obtained in stage b with a corresponding infrared reference value, or by comparison by multivariate analysis of the infrared value obtained in stage b with a corresponding infrared reference spectral value, wherein said method comprises determining at least an infrared spectral value selected from the group formed by ratio of the area between 1 165 cm “1 and 1145 cm '1 to the area between 1 180 cm “ 1 and 1 165 cm “1 expressed the second derivative spectrum (VIR8), the ratio of the first derivative at 1 154 cm “1 to the first derivative in the minimum towards 1 171 cm “ 1 (VIR4); average of the first derivative towards 1 158 cm “1 and 1 160 cm “ 1 normalized with respect to the first derivative in the minimum towards 1,171 cm “1 (VIR6).
  • the eleventh method of the invention in addition to the determination of at least 1, at least 2 or the 3 infrared spectral values selected from the group formed by ratio of the area between 1 165 cm “1 and 1 145 cm “ 1 to the area between 1 180 cm “1 and 1165 cm “ 1 expressed the spectrum in the second derivative (VIR8), the ratio of the first derivative to 1 154 cm “1 to the first derivative in the minimum towards 1,171 cm “ 1 (VIR4 ); average of the first derivative towards 1 158 cm “1 and 1 160 cm “ 1 normalized with respect to the first derivative in the minimum towards 1 171 cm “1 (VIR6), comprises additionally determining at least one value selected from the group consisting of
  • - VIR2 the ratio of the absorbance at 1 156 cm “1 to the absorbance of the maximum towards 1 171 cm “1
  • - VIR3 the ratio of the first derivative of the intensity to 1 152 cm “1 to the first derivative of the intensity at the minimum towards 1171 cm “1 ;
  • the eleventh method of the invention in addition to the determination of at least 1, at least 2 or the 3 infrared spectral values selected from the group formed by ratio of the area between 1 165 cm “1 and 1 145 cm “ 1 to area between 1 180 cm “1 and 1 165 cm-1 expressed the spectrum in second derivative (VIR8), the ratio of the first derivative to 1 154 cm “ 1 to the first derivative in the minimum towards 1 171 cm “1 (VIR4 ); average of the first derivative towards 1 158 cm-1 and 1 160 cm “1 normalized with respect to the first derivative at least towards 1 171 cm-1 (VIR6) comprises determining at least 2, at least 3, at least 4 , at least 5 or at least 6 infrared spectral values selected from the group consisting of VIR1-VIR3, VIR5, VIR7 and VIR9.
  • the eleventh method of the invention comprises the infrared spectral values corresponding to the ratio of the area between 1 165 cm “1 and 1 145 cm “ 1 to the area between 1180 cm “1 and 1 165 cm “ 1 expressed the spectrum in second derivative (VI R8) and the percentage of the area between 1639 cm “1 and 1623 cm “ 1 with respect to the area between 1672 cm “1 and 1623 cm “ 1 in the spectrum expressed in second derivative (VIR1).
  • the invention relates to a method of diagnosis of Alzheimer's disease (twelfth method of the invention) comprising the steps of the tenth method of the invention and additionally the steps of the eleventh method of the invention applied on a sample thereof. subject.
  • the twelfth method of the invention comprises determining at least one spectral value selected from the group consisting of (VR3); average of the first derivative at intensity 745 cm “1 and intensity 744 cm “ 1 (VR12), the value of the first derivative at intensity 760 cm “1 (VR15); the value of the first derivative at intensity 416 cm “1 (VR25); the height of the band towards 1555 cm “1 (VR7) and the ratio of the area of the band towards 1555 cm “ 1 to the area of the band towards 1525 cm “1 (VR8) and at least one spectral value selected from the group formed by ratio of the area between 1165 cm "1 and 1 145 cm “ 1 to the area between 1 180 cm “1 and 1 165 cm “ 1 expressed the spectrum in second derivative (V
  • REPLACEMENT SHEET (RULE 26) at 1 154 cm “1 to the first derivative in the minimum towards 1,171 cm “ 1 (VIR4); average of the first derivative towards 1158 cm “1 and 1 160 cm “ 1 normalized with respect to the first derivative at least towards 1171 cm “1 (VIR6).
  • the twelfth method of the invention comprises additionally determining at least one spectral value selected from the group consisting of VR1-VR2, VR4-VR6, VR9-VR1 1, VR13-14, VR 16-24, VR26-VR29 and additionally at least one infrared spectral value selected from the group consisting of VIR1 -VIR3, VIR5, VIR7 and VIR9.
  • the twelfth method of the invention comprises obtaining the Raman spectral values corresponding to the value of the first derivative at intensity 760 cm “1 (VR15) and the ratio of the area of the band towards 1555 cm-1 to the area of the band towards 1525 cm “1 (VR8) and the infrared spectral values corresponding to the ratio of the first derivative at intensity 1 154 cm “ 1 to the first derivative at least towards intensity 1 171 cm “1 (VIR4) average of the first derivative towards intensity 1 158 cm “1 and intensity 1 160 cm “ 1 normalized with respect to the first derivative at least towards intensity 1 171 cm “1 (VIR6) and percentage of the area between 1639 cm “ 1 and 1623 cm “1 with respect to the area between 1672 cm “ 1 and 1623 cm “1 in the spectrum expressed in second derivative (VIR1).
  • a decreased VR15 and / or VIR4 spectral value is indicative that the subject suffers from Alzheimer's.
  • an increased VR8, VIR1 and / or VIR6 spectral value is indicative of the subject suffering from Alzheimer's.
  • step c) is performed by comparison by multivariate analysis, and more preferably is performed by linear combinations of the spectral values.
  • the invention in another aspect, relates to an apparatus for plasma analysis comprising:
  • the devices that form the Raman spectrometer are known in the prior art, basically a laser and a CCD (charge-coupled devices) photomultiplier.
  • the equipment can be formed by two interchangeable monochromatic light sources (for example, He-Ne laser, Ar laser and laser diode) connected to an excitation optical fiber that guides the light to the optical head and focuses it on the samples.
  • the light scattered throughout the sample is collected through the same optical head and through the collection optical fiber is guided to the monochromator that separates it spatially and spectrally.
  • the CCD detects the signal diffracted by the monochromator and transforms the photons of the scattered light into a digital electrical signal and the spectrum is sent to a computer.
  • the purpose of the computer system of the apparatus of the invention is, among others, to control the acquisition of the dispersed Raman signal and its subsequent analysis, including mathematical processing.
  • the computer system also controls the parameters of the CCD, the laser beam focus system and its power, among others.
  • the computer system may additionally allow a certain degree of manipulation of the spectra, such as for example baseline correction, comparison between several spectra, area calculations and Fourier transforms, comparison of the spectral value of the sample to be analyzed with the reference spectral value or comparison by multivariate analysis with values that allow differentiating between a blood sample associated with Alzheimer's disease or frontotemporal dementia and a blood sample associated with Alzheimer's disease, among others.
  • the apparatus of the invention comprises a Raman spectrometer and an IR spectrometer.
  • the IR spectrometer can be of dispersive or Fourier transformation type.
  • said IR spectrometer is a Fourier transform spectrometer.
  • the apparatus of the invention further comprises a microscope.
  • the apparatus of the invention further comprises a videomicroscope having a beam splitter that allows the light to be directed both to a webcam and to a Raman head. In order to focus the laser and collimate Raman's light, a microscope objective can be used.
  • the invention relates to a computer program comprising a code suitable for performing any of the diagnostic methods of the invention.
  • the invention relates to a computer support containing the computer program of the invention.
  • the invention relates to a computer system provided with means for implementing the diagnostic method according to the invention.
  • the computer system may include:
  • REPLACEMENT SHEET (RULE 26) (b) at least one processor to run the computer program.
  • the computer system may include one or more general processors or processors that have particular purposes and associated memory, including volatile and non-volatile memory devices.
  • Machine-readable physical storage media useful in various embodiments of the invention may include any machine-readable physical storage media, for example solid state memory (such as flash memory), machine-readable magnetic and optical storage devices and media. , and memory using other persistent storage technologies.
  • a machine-readable medium can be any tangible medium that allows the computer to access computer programs and data.
  • Machine-readable media may include erasable or non-erasable tangible volatile and non-volatile media implemented in any method or technology that can store information, such as machine-readable instructions, program modules, programs, data, data structures and database information. of data.
  • the machine-readable medium includes but is not limited to RAM (random access memory), ROM (read-only memory), EPROM (erasable programmable read-only memory), EEPROM (read-only memory electrically erasable programmable), flash memory or other memory technology, CD-ROM (compact disc read-only memory), DVD (digital versatile discs) or other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and nonvolatile memory, and any other tangible media that can be used to store information and can be read by machine, including any suitable combination of the above.
  • RAM random access memory
  • ROM read-only memory
  • EPROM erasable programmable read-only memory
  • EEPROM read-only memory electrically erasable programmable
  • flash memory or other memory technology
  • CD-ROM compact disc read-only memory
  • DVD digital versatile discs
  • the present invention can be implemented in a stand-alone computer or as part of a networked computer system.
  • the Raman and / or Infrared spectra used as reference can be used to record, record and retrieve electronically or digitally.
  • the system may compare the data in a "comparison module" that can use a variety of programs and software formats available for operational comparison to compare spectra determined in the Determination module with reference data.
  • the comparison module is configured to use pattern recognition techniques to compare the sequence information of one or more inputs with one or more reference data patterns.
  • the comparison module can be configured to use existing disposable or commercially available software to compare patterns and can be optimized for particular data comparisons that are made.
  • the comparison module provides a machine-readable comparison result that can be processed in the machine-readable form by means of predefined criteria, or user-defined criteria, to provide a report comprising content based in part on the comparison result that can be stored and accessed as required by a user using a visual presentation module.
  • a visual presentation module makes it possible to visually present the content based in part on the comparison result for the user, in which the content is an indicative report of the results of the comparison of the spectrum obtained from the sample of the patient of interest with the spectrum of a healthy subject.
  • the visual presentation module allows to visually present a report or content based in part on the comparison result for the end user, in which the content is an indicative report of the results of the comparison of the spectrum of the patient with the reference spectrum
  • the comparison module may include an operating system (eg, UNIX, Windows) in which a management system is executed of relational databases, an Internet application and an Internet server application.
  • the Internet application may include the executable code needed to
  • REPLACEMENT SHEET (RULE 26) generate database language instructions [for example, conventional query language (SQL) instructions].
  • the computer instructions can be implemented in software, firmware or hardware and include any type of programmed stage undertaken by modules of the information processing system.
  • the computer system may be connected to a local area network (LAN) or a wide area network (WAN).
  • LAN local area network
  • WAN wide area network
  • a comparison module provides machine-readable data that can be processed in a machine-readable manner by means of predefined criteria, or user-defined criteria, to provide content. Recovered that can be stored and accessed as required using a visual presentation module.
  • the computerized system may include or may be operatively connected to a visual presentation module, such as a computer monitor, a touch screen or a video visual presentation system.
  • the visual presentation module allows the user to present instructions to the system user, allowing the user to observe the system inputs and allowing the system to display the results to the user as part of a user interface.
  • the computerized system may optionally include or may be operatively connected to a printing device to make printed copies of the information provided by the system.
  • an Internet browser may be used to provide a user interface to allow the user to interact with the system to enter information, to make requests and to display the recovered content.
  • the various functional modules of the system may be adapted to use a web browser to provide a user interface.
  • a user can make requests to retrieve data from data sources, such as databases, and interact with the comparison module to make comparisons and look for pattern matches.
  • the user can indicate and click on user interface elements such as buttons, drop-down menus, scroll lines, etc., conventionally used in graphical user interfaces to interact with the user interface.
  • REPLACEMENT SHEET (RULE 26) system and make the system carry out the methods of the invention.
  • the requests made with the user's web browser can be transmitted in a network to a web application that can process or format the request to make a query in one or more databases that can be used to provide the relevant information regarding the spectra generated, the content recovered, process this information and generate the results.
  • the invention relates to the use of a compound suitable for the treatment of frontotemporal dementia for the manufacture of a medicament for the treatment of a subject who has been diagnosed with a frontotemporal dementia by any method according to the invention.
  • the invention relates to a compound suitable for the treatment of frontotemporal dementia for use in the treatment of a subject who has been diagnosed with frontotemporal dementia by any method according to the invention.
  • the invention relates to a method of treating frontotemporal dementia in a subject comprising administering a compound suitable for treating frontotemporal dementia to the subject in need, in which said subject has been diagnosed as frontotemporal dementia according to a method of the invention. .
  • the invention relates to the use of a compound suitable for the treatment of Alzheimer's for the manufacture of a medicament for the treatment of a subject to whom Alzheimer has been diagnosed by any method according to the invention.
  • the invention relates to a compound suitable for the treatment of Alzheimer's for use in the treatment of a subject who has been diagnosed with Alzheimer's by any method according to the invention.
  • the invention relates to a method of treating Alzheimer's in a subject comprising administering a compound suitable for treating Alzheimer's to the subject in need, in which said subject has been diagnosed with Alzheimer's according to a method of the invention.
  • a method of treating Alzheimer's in a subject comprising administering a compound suitable for treating Alzheimer's to the subject in need, in which said subject has been diagnosed with Alzheimer's according to a method of the invention.
  • frontotemporal dementia and "Alzheimer” have been previously defined and are equally applicable to these aspects of the invention.
  • Compound suitable for treating frontotemporal dementia refers to any drug or compound that allows treating frontotemporal dementia, that is to reduce, improve, slow progress or eliminate symptoms of frontotemporal dementia.
  • the compound suitable for treating frontotemporal dementia is a compound suitable for treating Alzheimer's.
  • the compound suitable for treating frontotemporal dementia is a compound suitable for treating Alzheimer's is not a cholinesterase inhibitor such as as donepezil (Aricept®), rivastigmine (Exelon®) and galantamine (Razadyne®) that are acetylcholine inhibitors esterase, or benzodiazepines such as stazolam (ProSom®), diazepam (Valium®), flurazepam (Dalmane®), midazolam (Dormicum®, Versed®), temazepam (Restoril®, Normison®), triazolam (Halcion®), alprazolam ( Xanax®), chlordiazepoxide (Librium®), clonazepam (Rivotril®, Klonopin®), clorazepate (Tranxene®), lorazepam (Ativan®, Temesta®, Tavor®), oxa
  • SSRIs serotonin reuptake inhibitors
  • fluoxetine Prozac®
  • sertraline Zoloft®
  • paroxetine Paxil®
  • fluvoxamine Luvox®
  • citalopram Celexa®
  • escitalopram Lexapro®
  • Other non-SSRI antidepressants may also be used such as trazodone (Desyrel®), venlafaxine (Effexor®), duloxetine (Cymbalta®), bupropion (Wellbutrin®) or mirtazapine (Remeron®).
  • antisychotics that block the effect of dopamine, such as olanzapine (Zyprexa®), quetiapine (Seroquel® or Ketipinor®), risperidone (Risperdal®), ziprasidone (Geodon®), aripiprazole (Abilify®), paliperidone (INVEGA®).
  • REPLACEMENT SHEET (RULE 26) "Compound suitable for treating Alzheimer's” comprises any drug that allows treating Alzheimer's, that is, a treatment to reduce, improve, slow progress or eliminate symptoms of Alzheimer's disease.
  • suitable therapy to treat Alzheimer's disease is selected from the group consisting of a cholinesterase inhibitor, an N-methyl-D-aspartate (NMDA) receptor antagonist, anti-p-amyloid immunotherapy, a gamma-secretase inhibitor and an anti-tau therapy.
  • NMDA N-methyl-D-aspartate
  • Cholinesterase inhibitor refers to a chemical compound that inhibits cholinesterase or anticholinesterase, which prevents the destruction of the released acetylcholine, thereby causing an increase in the concentration and duration of neurotransmitter effects.
  • the two types of cholinesterase are acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE).
  • ACHE acetylcholinesterase
  • BCHE butyrylcholinesterase
  • the complete cholinesterase sequence in humans has the registration number P06276 in the Uniprot database (January 20, 2016).
  • the compound used according to the invention is an acetylcholinesterase inhibitor.
  • AChE inhibition affects basic processes that have been implicated in the pathogenesis of AD.
  • AChE inhibition affects the processing of amyloid precursor protein (APP) and attenuates the toxicity induced by ⁇ through mechanisms that include disruption of ⁇ production, disruption of ⁇ 1 levels -40 and ⁇ 1-42 and the formation of the soluble form of the amyloid precursor protein.
  • APP amyloid precursor protein
  • suitable therapies for treating mild to moderate AD include cholinesterase inhibitors such as Cognex® (tacrine), Aricept (donepezil), Exelon® (rivastigmine) or Razadine® (galantamine), between others.
  • cholinesterase inhibitors such as Cognex® (tacrine), Aricept (donepezil), Exelon® (rivastigmine) or Razadine® (galantamine), between others.
  • Moderate to severe AD can be treated with Namenda® (memantine), which acts as NMDA receptor antagonists. Since NMDA antagonists work differently from cholinesterase inhibitors, both types of medications can
  • REPLACEMENT SHEET (RULE 26) Recipe in combination. These medications can help delay or prevent AD symptoms from getting worse.
  • the therapies according to the present invention additionally include music therapy, physical therapy, psychomotor education, occupational therapy or animal therapy, among others.
  • Three cholinesterase inhibitors are currently marketed worldwide to treat Alzheimer's disease, specifically donepezil, galantamine and rivastigmine.
  • Donepezil (1-benzyl-4 - [(5,6-dimethoxy-1-indanon) -2-yl] -methyl-piperidine) is a reversible acetylcholinesterase (AChE) inhibitor.
  • Galantamine [4aS- (4a ⁇ , 6 ⁇ , 8aR * )] - 4a, 5,9, 10,1 1, 12-hexahydro-3-methoxy-1 1 -methyl-6H-benzofuro [3a, 3,2 - ef] benzacepin-6-ol
  • Galantamine is an isolated alkaloid of the snowdrop, Galanthus nivalis. It is a highly selective, reversible and competitive acetylcholinesterase inhibitor.
  • Rivastigmine ((S) -N-ethyl-3- [1- (dimethylamino) ethyl] -N-methyl-phenyl-carbamate) is a reversible, non-competitive acetylcholinesterase and butyrylcholinesterase inhibitor.
  • Additional compounds have been described to inhibit cholinesterase, for example edrophonium, demecarium, ambenonium, neostigmine bromide, dehydroevodiamine chloride, serolin, imperatorin, scopoletin (SCT), huperizine A (Hup A), heptilstigmine tartrate (MF-201), suronacrine maleate (HP-128), UCB-1 1056, berberine iodide, norpyridostigmine, chylostigmine (HP-290, NXX-066), THB-013,
  • REPLACEMENT SHEET (RULE 26) PD-142676, terestigmine tartrate (CHF-2060), thiacimserine, MF-8615, MF-268 bitartrate, anseculin hydrochloride (KA-672.HCI), ensaculia hydrochloride, icopezil maleate (CP-1 18954), serine salicylate, physostigmine salicylate, JWS-USC-75IX, P1 1467, P-10358, bis (7) -tacrine, HMR-2420, CP-126998, TV-3279, MSF, THA-C8, subergorgic acid, suberogorgin , SPH-1286, huperzine B (Hup B), pyridostigmine bromide (Ro-1 -5130), huprine Y, coronaridine, RS-1233, kobophenol A, bis (12) - huperine, RS-1259, ITH-4012
  • NMDA receptor antagonist refers to a chemical compound that inhibits the reaction generated by the polysynaptic discharge of nociceptive primary afferent fibers.
  • Memantine (3,5-dimethyltricyclo [3.3.1 .13.7] decan-1-amino or 3,5-dimethylamantane-1-amino) stands out among the antagonists.
  • NMDA antagonists are nimodipine (3- (2-methoxyethyl) 5-propan-2-yl 2,6-dimethyl-4- (3-nitrophenyl) -1, 4-dihydropyridine-3,5-dicarboxylate), dizocilpine, AP-5 (2- amino-5-phosphonopentanoic acid), AP-7 (2-amino-7-phosphonoheptanoic acid), CHF 3381 (n- (2-indanyl) -glycineamide hydrochloride) and ifenprodyl (4- [2 - (4-benzylpiperidin-1-yl) -1-hydroxypropyl] phenol) and could be used to treat AD according to the invention.
  • anti-amyloid immunotherapy refers to a therapy that uses the immune system to slow down or stop the damage caused by the accumulation of ⁇ -amyloid.
  • Said immunotherapy comprises the administration of anti-beta-amyloid antibodies.
  • Anti-beta-amyloid antibodies useful for the treatment according to the present invention include those that directly destroy the amyloid plaque (the microglia is stimulated by immunization and devours the previously formed amyloid plaques), those that capture the amyloid
  • REPLACEMENT SHEET (RULE 26) (The formation of the antigen-antibody complex in peripheral blood sequesters the amyloid outside the brain and prevents deposition) or those that inhibit aggregation (the formation of the antigen-antibody complex prevents the amyloid from aggregating in senile plaques).
  • anti-familial immunotherapy comprises the use of immunogenic compositions comprising one or more peptides of ⁇ -amyloid (for example ⁇ (1-5), ⁇ (1-6), ⁇ (1-12), ⁇ ( 13-28), ⁇ (25-35), ⁇ (35-42), ⁇ (33-42) and ⁇ (33-40))
  • ⁇ ( ⁇ - ⁇ ) refers to a peptide derived from the peptide beta-amyloid consisting of amino acids in position X to amino acid in position Y).
  • Beta-amyloid peptides can be administered either in the form of conjugates (for example, conjugates to KLH or an albumin) or in the form of a composition with an adjuvant (for example, Freund's complete adjuvant, Freund's incomplete adjuvant, QS21 , aluminum hydroxide gel, MF59, calcium phosphate, liposine, saponin, squalene, L121, monophosphoryl lipid A (MPL) in Emulsigen, polysorbate 80, cholera toxin (CT), LTK and LTK63).
  • conjugates for example, conjugates to KLH or an albumin
  • an adjuvant for example, Freund's complete adjuvant, Freund's incomplete adjuvant, QS21 , aluminum hydroxide gel, MF59, calcium phosphate, liposine, saponin, squalene, L121, monophosphoryl lipid A (MPL) in Emulsigen, polysorbate 80, cholera toxin (CT), LTK
  • a vaccine useful in the present invention would be the AN-1972 vaccine, which consists of A42 which is an artificial copy of the human beta-amyloid protein, which when injected stimulates the immune system to clean abnormal deposits of the brain of sick rodents. This stimulates the formation of antibodies that adhere to the neuritic plaques to be subsequently digested by the cellular elements of the immune system (microglia, astrocytes).
  • gamma secretase inhibitors with gamma secretase being an enzyme involved in the synthesis of ⁇ amyloid.
  • Gamma secretase inhibitors refers to a compound that inhibits gamma secretase which is an enzyme involved in the synthesis of ⁇ amyloid.
  • Gamma secretase inhibitors useful in the present invention are, among others, LY-450139 (CAS No. 425386-60-3), also called Semagacestat, DAPT (CAS No. 208255-80-5), CTS-21166 or MK 8931.
  • an "anti-tau therapy” would be analogous to an anti-amyloid-beta immunotherapy against extracellular plaques.
  • Such therapy includes tau kinase inhibitors, potentiation of phosphatase activity to enhance microtubule stability, alteration or blockage of tau hyperphosphorylation, decrease or inhibition of tau aggregates and filament formation.
  • the compound is an antibody.
  • REPLACEMENT SHEET (RULE 26) anti-tau, as disclosed in Yanamandra K. et al., Annals of clinical and translational Neurology, volume 2, topic 3, pages 278-288, March 2015.
  • Example 1 Procedure for obtaining peripheral blood plasma fraction.
  • Example 2 Measurement of Raman and Infrared spectra of the blood plasma fraction.
  • REPLACEMENT SHEET (RULE 26)
  • the Raman spectra have been measured in the region of 3150-350 cm “1 by a dispersive spectrometer of the Renishaw signature, InVia Raman microscope model, using the 785 nm laser line of maximum 500 mw power and an effective power in the sample of the 10%
  • the microscopic objective used was Leica 100X / 0.85 N Plan Epi (short distance).
  • the spectra analyzed are the average of six accumulations with a resolution of 2 cm "1 and 21 s of exposure time.
  • Figure 1 shows plasma Raman spectra of healthy controls and patients with mild and moderate DFT and AD. Showing the entire spectral scale, these spectra do not show large differences between them, and therefore it is necessary to consider small spectral intervals to observe significant differences.
  • Figure 2 shows that Raman intensity towards 1671 cm “1 increases slightly when passing from healthy controls to patients with DFT or AD, due to the formation of polypeptide structure in ⁇ -sheet. A small decrease in maximum intensity is also noted. around 1658 cm "1 when passing from healthy controls to sick subjects, due to a relative decrease in a-helical protein structure.
  • Figure 3 shows that the band located 744 cm “1 in the spectrum of healthy controls suffers a slight shift towards higher frequencies when passing to the spectra of patients with DFT or AD. This spectral change is also accompanied by an increase of the intensity of the maximum towards 758 cm “1 .
  • Figure 4 shows a lower intensity of the band towards 1525 cm "1 , which is assigned to carotenoids, compared to the spectra corresponding to DFT and EA.
  • This decrease in intensity in the spectra of these degenerative diseases is it can be attributed to the oxidative stress present in these diseases, since the free radicals generated in the oxidative stress decrease the electronic conjugation of the carotenoids and therefore the intensity of the mentioned band.
  • This figure also shows a change of intensity of the band towards 1555 cm "1 which may be due to an alteration in the concentration of tryptophan-containing proteins or a change in the tertiary structure of these proteins with tryptophan amino acid residues.
  • FIG. 5 shows a clear difference in the intensity of the tension band CH towards 2936 cm “1 between the spectrum of the DFT and that of the EA, which means that this band has classifying power to distinguish DFT patients from patients with AD, and therefore it is useful for the differential diagnosis of this invention.
  • This variation of the voltage intensity CH can also be attributed to variations in the tertiary protein structure.
  • the intensity that also changes significantly when passing from DFT to the EA is 494 cm “1 as shown in Figure 6, and therefore this signal also contributes to the differential diagnosis between DFT and EA.
  • the infrared spectra were measured on a Perkin-Elmer Fourier transform spectrometer, model 1725X.
  • the registration conditions were as follows: registration range: 4000-400 cm “1 ; accumulation of 32 spectra; spectral resolution of 2 cm “ 1 .
  • the possible contribution of ambient water vapor bands was corrected by subtracting the water vapor spectrum with an appropriate factor until the absence of steam bands in the region between 2000-1800 cm "1 .
  • Figure 7 shows the mean plasma spectra of healthy controls and patients with DFT and mild AD, where it is seen that the intensity of the profile between 1640 and 1623 cm “1 of the spectrum of healthy controls is lower than that of the spectrum of DFT and EA
  • Figure 8 shows a clear difference between DFT and EA in the spectral profile between 1 166 and 1 145 cm “1 , and therefore this region contributes to the differential diagnosis between these two neurodegenerative diseases.
  • Table 1 includes the areas under the ROC curves resulting from comparisons of two groups of subjects, using only Raman spectroscopy. Each ROC curve corresponds to the canonical discriminant function obtained from the discriminant analysis by comparing two groups of subjects, with a 95% confidence interval. This discriminant analysis was carried out using the stepwise inclusion modality and using the Wilks lambda method, and based on all the spectral values or variables defined above in the regions R.1, R.2 and R.3. Class I, II or III, or class (II + III), related to cognitive status, and as speci fi c variables (spectral values) mentioned above were considered as dependent or grouping variables (Var. Group).
  • the cut-off point of the ROC curve comparing healthy controls versus the group consisting of patients with DFT and patients with AD indicates that those blood plasma samples with equal or superior discriminant function at -0.4450 they would be positive, corresponding to patients with DFT or AD, and samples with discriminant function below this value would correspond to healthy controls.
  • the classification according to this method would be 80% sensitivity and 68% specificity (Table 1).
  • REPLACEMENT SHEET (RULE 26) Table 1. Areas under the ROC curves resulting from the comparison of different groups of subjects by Raman spectroscopy. The VR values correspond to the Raman values defined above,
  • Example 4 ROC curves of the canonical discriminant function of infrared spectra of peripheral blood plasma samples. Table 2 includes the areas under the ROC curves resulting from comparisons of two groups of subjects, using only infrared spectroscopy. Each ROC curve corresponds to the function
  • REPLACEMENT SHEET (RULE 26) Canonical discriminant obtained from the discriminant analysis comparing two groups of subjects. This discriminant analysis was performed using the stepwise inclusion mode and using Wilks' lambda method, and based on all the spectral values or variables defined above in the IR.1 and IR.2 regions. Class I, II, or III or class (II + III), relative to cognitive status, were considered dependent or grouping variables (Var. Group), and the spectroscopic parameters (spectral values) mentioned above as independent variables.
  • REPLACEMENT SHEET (RULE 26) Example 5.
  • Table 3 includes the areas under the ROC curves resulting from comparisons of two groups of subjects, using the combination of infrared spectroscopy with Raman spectroscopy.
  • Each ROC curve corresponds to the canonical discriminant function obtained from the discriminant analysis by comparing two groups of subjects.
  • This discriminant analysis was performed using the stepwise inclusion mode and using the Wilks lambda method, and based on all the spectral values or variables defined above in the Raman regions (R.1, R.2 and R.3) and infrared (IR.1 and IR.2).
  • Table 3 Areas under the ROC curves resulting from the comparison of different groups of subjects through the combination of infrared spectroscopy and Raman spectroscopy.
  • the VR values correspond to the Raman values defined above and the VIR values correspond to the infrared values defined above.
  • Table 3 shows that the combination of both spectroscopic techniques leads to a marked improvement in the correct classification of the samples when it comes to comparing not only healthy controls with patients of class II, III, or class (ll + lll), but also DFT patients versus patients with AD, which is of special interest from the point of view of a reliable DFT-EA differential diagnosis.
  • the differentiation between patients with DFT and patients with EA combining Raman and infrared spectroscopy can be done in two ways: a) using the ROC curve that compares to healthy controls versus patients with DFT, and the ROC curve comparing healthy controls versus patients with AD; and b) the ROC curve comparing healthy controls versus the class integrated by DFT and EA, and the ROC curve comparing patients with EA versus patients with DFT.
  • Table 4 Changes in the infrared and Raman classifying spectral parameters, in the comparison of groups of plasma samples belonging to healthy subjects, patients with DFT and patients with AD, and areas under the respective ROC curves usable for diagnostic classifications.
  • the VR values correspond to the Raman values defined above and the VIR values correspond to the infrared values defined above. ⁇ indicates increase, i indicates decrease.
  • the group of healthy subjects is distinguished from the group consisting of patients with DFT and AD (DFT + EA) by several parameters, including VR8 that may be associated with oxidative stress because the band located towards 1525 cm “1 (Fig. 4 ) is attributable to tension vibrations of carbon-carbon double bonds with electronic conjugation, as is the case in carotenoids.
  • VR8 that may be associated with oxidative stress because the band located towards 1525 cm “1 (Fig. 4 ) is attributable to tension vibrations of carbon-carbon double bonds with electronic conjugation, as is the case in carotenoids.
  • the decrease in the intensity of this band when passing from healthy controls to the integrated group of patients (DFT + EA) can be explained by the action of oxidative stress that is present in

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Abstract

La présente invention concerne un procédé pour le diagnostic de la démence frontotemporale, un procédé de diagnostic d'une maladie neurodégénérative, la maladie neurodégénérative étant une démence frontotemporale ou la maladie d'Alzheimer et un procédé pour le diagnostic différentiel de la démence frontotemporale et d'Alzheimer et un procédé pour le diagnostic d'Alzheimer, ces procédés étant basés sur la spectroscopie Raman et/ou infrarouge qui consistent à enregistrer un spectre Raman et/ou infrarouge d'un échantillon de sang.
PCT/ES2018/070261 2017-03-31 2018-03-27 Procédé de diagnostic de la démence frontotemporale et de la maladie d'alzheimer Ceased WO2018178474A2 (fr)

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