Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   

 

  Pharmaceutical Patents  

 

Title:  Diagnosis of tauopathies
United States Patent: 
7,387,879
Issued: 
June 17, 2008

Inventors: 
Vanmechelen; Eugeen (Nazareth-Eke, BE), Vanderstichele; Hugo (Ghent, BE)
Assignee: 
Innogenetics N.V. (Ghent, BE)
Appl. No.: 
10/682,103
Filed: 
October 9, 2003


 

Pharm/Biotech Jobs


Abstract

The present invention provides a method for the diagnosis of tauopathies in an individual and/or for the differential diagnosis of a tauopathy versus a non-tauopathy based on the detection of the ratio of phospho-tau (181)/total tau in said individual. The present invention further provides a phospho-peptide for standardization in a method of the invention.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to a method for the diagnosis of a tauopathy in an individual, said method involving: determining the ratio of phospho-tau (181)/total tau in said individual; inferring that said individual is suffering a tauopathy by comparing the obtained ratio of phospho-tau (181)/total tau in said individual with the ratio of phospho-tau (181)/total tau in control individuals, whereby an altered ratio of phospho-tau (181)/total tau compared to said ratio in control individuals being an indication of tauopathy.

The present invention also relates to a method for the differential diagnosis of a tauopathy versus a non-tauopathy in an individual, said method involving: determining the ratio of phospho-tau (181)/total tau in said individual; inferring that said individual is suffering a tauopathy by comparing the obtained ratio of phospho-tau (181)/total tau in said individual with the ratio of phospho-tau (181)/total tau in individuals suffering a non-tauopathy or with the phospho-tau (181)/total tau ratio in control individuals, whereby an altered ratio of phospho-tau (181)/total tau compared to said ratio in individuals suffering a non-tauopathy or in control individuals being an indication of tauopathy.

It is an aim of the present invention to provide a method for the diagnosis of a tauopathy in an individual.

It is another aim of the present invention to provide a method for the diagnosis of Alzheimer's disease, Pick's disease, sporadic Frontotemporal dementia and/or Frontotemporal dementia with Parkinsonism linked to chromosome 17 in an individual.

It is another aim of the present invention to provide a method for the differential diagnosis of a tauopathy versus a non-tauopathy.

It is another aim of the present invention to provide a method for the differential diagnosis of a tauopathy versus a non-tauopathy neurodegeneration.

It is another aim of the present invention to provide a method for the differential diagnosis of a tauopathy versus vascular dementia, Creutzfeldt Jacob Disease, stroke and/or neurotoxicity in patients with leukemia.

It is another aim of the present invention to provide a method for the differential diagnosis of Alzheimer's disease, Pick's disease, sporadic Frontotemporal dementia and/or Frontotemporal dementia with Parkinsonism linked to chromosome 17 versus vascular dementia, Creutzfeldt Jacob Disease, stroke and/neurotoxicity in patients with leukemia.

It is another aim of the present invention to provide an in vitro method as described above.

It is another aim of the present invention to provide a phospho-peptide for use in standardization.

It is another aim of the present invention to provide a phospho-peptide for use in standardization in a method to detect phospho-tau (181).

It is another aim of the present invention to provide a phospho-peptide for use in standardization in a method as described above.

It is another aim of the present invention to provide a diagnostic kit for use in a method as described above.

It is another aim of the present invention to provide a peptide, a method and/or a diagnostic kit for the testing or screening of drugs, for therapeutic monitoring and/or for the determination of the effectiveness of a certain treatment for a tauopathy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for the diagnosis of a tauopathy in an individual, said method comprising the step of: determining the ratio of phospho-tau (181)/total tau in said individual; inference that said individual is suffering a tauopathy by comparing the obtained ratio of phospho-tau (181)/total tau in said individual with the ratio of phospho-tau (181)/total tau in control individuals, an altered ratio of phospho-tau (181)/total tau compared to said ratio in control individuals being an indication.

The present invention also relates to a method for the differential diagnosis of a tauopathy versus a non-tauopathy in an individual, said method comprising the steps of: determining the ratio of phospho-tau (181)/total tau in said individual; inference that said individual is suffering a tauopathy by comparing the obtained ratio of phospho-tau (181)/total tau in said individual with the ratio of phospho-tau (181)/total tau in individuals suffering a non-tauopathy or with the phospho-tau (181)/total tau ratio in control individuals, an altered ratio of phospho-tau (181)/total tau compared to said ratio in individuals suffering a non-tauopathy or in control individuals being an indication.

The present invention is based on the finding that the ratio of phospho-tau (181)/total tau in CSF from patients suffering AD and in CSF from patients suffering certain forms of FTD is significantly altered compared to the phospho-tau (181)/total tau ratio in CSF from control individuals. The present invention is further based on the finding that the ratio of phospho-tau (181)/total tau in CSF from patients suffering AD is significantly altered compared to the phospho-tau (181)/total tau ratio in CSF from patients suffering stroke. The indication that the phospho-tau (181)/total tau ratio in patients with a tauopathy is altered, forms a basis for the development of a diagnostic test for the diagnosis of a tauopathy in an individual and/or for the differential diagnosis of individuals suffering a tauopathy versus individuals suffering a non-tauopathy.

`A tauopathy` is any form of dementia that is associated with a tau pathology.

Alzheimer's disease and certain forms of Frontotemporal dementia (Pick's disease, sporadic Frontotemporal dementia and Frontotemporal dementia with Parkinsonism linked to chromosome 17) are the most common forms of tauopathy. In accordance, the present invention relates to any method as described above, wherein the tauopathy is Alzheimer's, Pick's disease, sporadic Frontotemporal dementia and Frontotemporal dementia with Parkinsonism linked to chromosome 17. Other tauopathies include but are not limited to Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD) and Subacute sclerosing panencephalitis.

In a specific embodiment, the present invention relates to a method for the diagnosis of Azheimer's disease in an individual, said method comprising the steps of: determining the ratio of phospho-tau (181)/total tau in said individual; inference that said individual is suffering Alzheimer's disease by comparing the obtained ratio of phospho-tau (181)/total tau in said individual with the ratio of phospho-tau (181)/total tau in control individuals, an altered ratio of phospho-tau (181)/total tau compared to said ratio in control individuals being an indication.

A `non-tauopathy` is any status of the brain that is not associated with a tau pathology. In an embodiment of the invention, said non-tauopathy is a non-tauopathy neurodegeneration. A non-tauopathy neurodegeneration is any form of neurological disorder that is not associated with a tau pathology. Non-tauopathy neurodegenerations include but are not limited to vascular dementia, Creutzfeldt Jacob Disease, stroke and/or neurotoxicity in patients with leukemia.

Therefore, in a specific embodiment, the present invention relates to a method for the differential diagnosis in an individual of Alzheimer's disease versus stoke, said method comprising the steps of: determining the ratio of phospho-tau (181)/total tau in said individual; inference that said individual is suffering Alzheimer's disease and not stroke by comparing the obtained ratio of phospho-tau (181)/total tau in said individual with the ratio of phospho-tau (181)/total tau in individuals suffering a stroke, an altered ratio of phospho-tau (181)/total tau compared to said ratio in individuals suffering a stroke being an indication.

Phospho-tau (181) includes all tau molecules that carry a phosphate on the threonine at position 181. The numbering with respect to the amino acid sequence refers to the longest tau isoform hTau40 (Goedert et al., 1989).

Total tau refers to all forms of tau and includes tau in any state of phosphorylation.

The present invention thus relates to tau and phospho-tau (181) for use as neurological markers for the diagnosis of a tauopathy and/or for the differential diagnosis of a tauopathy versus a non-tauopathy.

Based on the level of phospho-tau (181) and total tau in an individual, the ratio of phospho-tau (181)/total tau in said individual can then be determined.

The ratio of phospho-tau (181)/total tau can be detected in vitro as well as in vivo.

The method for the in vitro detection of the ratio of phospho-tau (181)/total tau in an individual comprises the steps of: obtaining a sample from said individual; determining the ratio of phospho-tau (181)/total tau in said sample; inference that said individual is suffering a tauopathy by comparing the obtained ratio of phospho-tau (181)/total tau in said individual with the phospho-tau (181)/total tau ratio in a sample from individuals suffering a non-tauopathy or with the phospho-tau (181)/total tau ration in a sample from control individuals, an altered ratio of phospho-tau (181)/total tau compared to said ratio in individuals suffering a non-tauopathy or in control individuals being an indication.

The term `sample` refers to any source of biological material, for instance body fluids, brain extract, peripheral blood or any other sample comprising phospho-tau (181) protein. In an embodiment of the invention, the ratio of phospho-tau (181)/total tau is determined in vitro by analysis of the ratio of phospho-tau (181)/total tau in a body fluid sample of the patient. The term `body fluid` refers to all fluids that are present in the human body including but not limited to blood, lymph, urine and cerebrospinal fluid (CSF) comprising phospo-tau (181) protein. In another embodiment of the present invention the ratio of phospho-tau (181)/total tau is determined in a cerebrospinal fluid (CSF) sample taken from the patient. In accordance, the present invention relates to a method as described above, comprising the steps of: obtaining a cerebrospinal fluid sample from the individual; determining the ratio of phospho-tau (181)/total tau in said cerebrospinal fluid sample; inference that said individual is suffering a tauopathy by comparing the obtained ratio of phospho-tau (181)/total tau in said individual with the ratio of phospho-tau (181)/total tau in the CSF from individuals suffering a non-tauopathy or with the phospho-tau (181)/total tau ratio in the CSF from control individuals, an altered ratio of phospho-tau (181)/total tau compared to said ratio in the CSF from individuals suffering a non-tauopathy or in the CSF from control individuals being an indication.

Total tau can be quantified by any method known, including but not limited to the use of antibodies or else by a functional assay (Bramblett et al., 1992). Any monoclonal or polyclonal antibody that specifically recognizes total tau may be used for the quantification of total tau. Antibodies recognizing normally and abnormally phosphorylated tau include Alz50 (Ghanbari et al., 1990), HT7 (Mercken et al., 1992) and AT120 (Vandermeeren et al., 1993). But also other antibodies known in the art which recognize total tau can be used. A very fast and user-friendly method for the quantification of total tau is the INNOTEST hTau-Ag (Innogenetics, Gent, Belgium). Phospho-tau (181) can be quantified by any method known in the art, including but not limited to the use of antibodies. In a preferred embodiment, phospho-tau (181) is quantified by an immunoassay comprising at least the following steps: obtaining a sample from the patient; bringing said sample into contact with a monoclonal antibody specifically recognizing phospho-tau (181), under conditions being suitable for producing an antigen-antibody complex; detecting the immunological binding of said antibody to said sample.

In an even more preferred embodiment, phospho-tau (181) can be quantified by a sandwich ELISA comprising the following steps: obtaining a sample from the patient; bringing said sample into contact with a monoclonal antibody (primary antibody or capturing antibody) recognizing phospho-tau (181), under conditions being suitable for producing an antigen-antibody complex; bringing said sample into contact with a monoclonal antibody (secondary antibody or detector antibody) specifically recognizing phospho-tau (181), under conditions being suitable for producing an antigen-antibody complex; bringing the antigen-antibody complex into contact with a marker either for specific tagging or coupling with said secondary antibody, with said marker being any possible marker known to the person skilled in the art; possibly also, for standardization purposes, bringing the antibodies in contact with a purified phospho-tau protein or phospho-peptide reactive with both antibodies.

Advantageously, the secondary antibody itself carries a marker or a group for direct or indirect coupling with a marker.

The expression `recognizing`, `reacting with`, `immunological binding` or `producing an antigen-antibody complex` as used in the present invention is to be interpreted that binding between the antigen and antibody occurs under all conditions that respect the immunological properties of the antibody and the antigen.

The expression `specifically recognizing` as used in the present invention is to be interpreted that said antibody is capable of forming an immunological complex with phospho-tau (181) but not with a tau molecule that lacks the phosphorylation at threonine 181.

Any monoclonal antibody that specifically recognizes phospho-tau (181) can be used in said method for the quantification of phospho-tau (181). A preferred monoclonal antibody for use in the quantification of phospho-tau (181) is AT270 (International application published under WO 95/17429). But also other antibodies known in the art that specifically recognize phospho-tau (181) can be used.

For standardization purposes, a tau protein or peptide phosphorylated at threonine 181 can be used. This can be obtained by any method such as extraction from brain or in vitro phosphorylation of normal tau. Since it is difficult to determine accurately the degree of phosphorylation of specific phospho-sites concentrated in the proline region, in an embodiment of the invention, a synthetic phospho-peptide is used for standardization. Said synthetic phospho-peptide should be able to form an immunological complex with the antibodies used in the immunoassay.

The present invention thus also relates to a phospho-peptide comprising at least two epitopes that are recognized by a monoclonal antibody, said phospho-peptide being liable to form an immunological complex with said monoclonal antibodies in a sandwich ELISA. Previous work has shown that, although a peptide contains an epitope for a certain monoclonal antibody, said monoclonal antibody does not always recognize said peptide (DeLeys et al., 1996). The present inventors were able to define a phospho-peptide with two epitopes such, that indeed said phospho-peptide is able to form an immunological complex with the monoclonal antibodies recognizing said epitopes. In addition, the present inventors were able to define both epitopes such that the phospho-peptide is able to form an immunological complex with the monoclonal antibodies recognizing said epitopes in a sandwich ELISA.

The term `peptide` refers to a polymer of amino acids (aa) and does not refer to a specific length of the product. In an embodiment of the invention, the length for the phospho-peptide is between 15 and 100 amino acid. In a preferred embodiment of the invention, the phospho-peptide contains 20 to 50 amino acids. In another preferred embodiment of the invention, the phospho-peptide contains 30 to 40 amino acids.

The peptide of the invention can be produced by any method known in the art such as classical chemical synthesis as described by Houbenweyl (1974) and Atherton and Shepard (1989), by any commercially available method such as described in the examples section, or by means of recombinant DNA techniques as described by Sambrook et al. (1989).

A phospho-peptide is a peptide that carries a phosphate on at least one amino acid. The use of the phospho-peptide of the invention allowed the present inventors to determine the relation of phospho-peptide to specific phospho isoforms and to assess the degree of phosphorylation of specific phospho-sites (see example 1, 1.5). The use of the phospho-peptide of the invention will allow the quantification of particular molecular forms of tau in a standardized way.

Phosphorylated peptides can be made by any method known. They can be made post-assembly, by reaction for example with di-t-butyl diisopropyl diisopropylphosphoaramidite and oxidation with t-butyl hydroperoxide of unprotected serine and threonine residues. They can also be made by incorporation of phosphorylated amino acids during peptide synthesis. Recently, new phosphorylated serine derivatives (N-.alpha.-Fmoc-O-benzyl-L-phosphoSer) are commercially available (Calbiochem-Novabiochem AG, San Diego, Calif. 92121) to synthesize directly phosphopeptides without post-assembly phosphorylation.

In an embodiment, the present invention relates to a phospho-peptide liable to form an immunological complex with monoclonal antibody HT7 and monoclonal antibody AT270, comprising at least: the minimal epitope of HT 7: ProProGlyGlnLys (SEQ ID NO 1); and the minimal epitope of AT270: ProProAlaProLysThr(p)Pro (SEQ ID NO 2).

In an even more preferred embodiment, the present invention relates to a phospho-peptide as described above, comprising the following sequence:

The present invention further relates to the use of said phospho-peptide in a method for measuring the level of phospho-tau (181).

The present invention further relates to the use of said phospho-peptide in a method for the diagnosis of a tauopathy and/or for the differential diagnosis of a tauopathy versus a non-tauopathy.

The present invention further relates to the use of said phospho-peptide in a method for the diagnosis of Alzheimer's disease.

The present invention further relates to the use of said phospho-peptide in a method for the differential diagnosis of Alzheimer's disease versus stroke.

The method for the in vitro detection of the ratio of phospho-tau (181)/total tau in an individual can also be used for testing or screening of drugs, for therapeutic monitoring and/or to evaluate the effect of a certain treatment on the tauopathy in said individual.

The method for the early in vivo detection of the ratio of phospho-tau (181)/total tau in an individual comprises the steps of determining the ratio of phospho-tau (181)/total tau in said individual and comparing it to the ratio of phospho-tau (181)/total tau in control healthy individuals. In an embodiment, phospho-tau (181) and total tau can be quantified by in vivo imaging. Phospho-tau (181) and total tau can be quantified in situ by non-invasive methods including but not limited to brain imaging methods described by Arbit et al. (1995), Tamada et al. (1995), Wakabayashi et al. (1995), Huang et al. (1996), Sandrock et al. (1996), Mariani et al. (1997). These in vivo imaging methods may allow the localization and quantification of phospho-tau (181) and total tau, for example, by use of labeled antibodies respectively specifically recognizing phospho-tau (181) or recognizing total tau.

Phospho-tau (181) and total tau can also be used as markers for in vivo imaging for testing or screening of drugs, for therapeutic monitoring and/or to evaluate the effect of a certain treatment on the tauopathy in said individual.

The present invention further relates to a diagnostic kit for the diagnosis of a tauopathy in an individual and/or for the differential diagnosis of a tauopathy versus a non-tauopathy comprising at least an antibody specifically recognizing phospho-tau (181).

In another embodiment, the present invention relates to a diagnostic kit as described above comprising at least: an antibody specifically recognizing phospho-tau (181); an antibody recognizing tau.

In another embodiment, the present invention relates to a diagnostic kit as described above comprising at least a phospho-peptide according to the invention.

In another embodiment, the present invention relates to a diagnostic kit as described above comprising at least: an antibody specifically recognizing phospho-tau (181); a phospho-peptide according to the invention.

In another embodiment, the present invention relates to a diagnostic kit as described above comprising at least: an antibody specifically recognizing phospho-tau (181); an antibody recognizing tau; a phospho-peptide according to the invention.

A preferred kit for the diagnosis of a tauopathy in an individual is based on an immunoassay and comprises: a monoclonal antibody (primary antibody) which forms an immunological complex with an epitope of phospho-tau (181); a monoclonal antibody (secondary antibody) which specifically recognizes phospho-tau (181); a marker either for specific tagging or coupling with said secondary antibody; appropriate buffer solutions for carrying out the immunological reaction between the primary antibody and the test sample, between the secondary antibody and the test sample and/or between the bound secondary antibody and the marker; a phospho-peptide according to the invention.

The present invention further relates to the use of a diagnostic kit as described above for the diagnosis of a tauopathy in an individual and/or for the differential diagnosis of a tauopathy versus a non-tauopathy.

The present invention further relates to the use of a diagnostic kit as described above for the diagnosis of Alzheimer's disease, Pick's disease, sporadic Frontotemporal dementia and/or Frontotemporal dementia with Parkinsonism linked to chromosome 17.

The present invention further relates to the use of a diagnostic kit as described above for the differential diagnosis of Alzheimer's disease, Pick's disease, sporadic Frontotemporal dementia and/or Frontotemporal dementia with Parkinsonism linked to chromosome 17 versus vascular dementia, Creutzfeldt Jacob Disease, stroke and/or neurotoxicity in patients with leukemia.

The present invention also relates to the use of total tau and phospho-tau (181) as neurological markers for the manufacture of a diagnostic kit for the diagnosis of a tauopathy and/or for the differential diagnosis of a tauopathy versus a non-tauopathy.

The present invention also relates to the use of a phospho-peptide, a method and/or a diagnostic kit of the invention for the testing or screening of drugs, for therapeutic monitoring and/or for the determination of the effectiveness of a certain treatment for a tauopathy.

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

The reference to any prior art in this specification is not, and should not be taken as an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.
 

Claim 1 of 13 Claims

1. A diagnostic kit comprising a phospho-peptide wherein the phospho-peptide consists of 15 to 50 amino acids in length, wherein the phospho-peptide is liable to form an immunological complex with monoclonal antibody HT7 and monoclonal antibody AT270, wherein the phospho-peptide comprises: (a) the minimal epitope of HT7: ProProGlyGlnLys (SEQ ID NO: 1); and (b) the minimal epitope of AT270: ProProAlaProLysThr(p)Pro (SEQ ID NO:2); and wherein the phospho-peptide is in a container.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]