# US 8,465,727 B2 · Biomarkers for the diagnosis of ALS

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- Number: US 8,465,727 B2
- Document ID: US8465727B2
- Status: Granted
- Kind: B2
- Application number: 12/899,235
- Filed: 2010-10-06
- Published: 2011-04-14
- Granted: 2013-06-18
- Earliest claimed: 2009-10-06
- Assignees: University of Pittsburgh—Of the Commonwealth System of Higher Education
- Applicants: Robert P. Bowser
- Inventors: Robert P. Bowser
- Web page: https://patentgrep.com/patent/US8465727B2

## CPC

- G01N 33/6893: {related to diseases not provided for elsewhere}
- A61P 25/02: for peripheral neuropathies
- A61P 25/18: Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- G01N 2800/285: Demyelinating diseases; Multipel sclerosis
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Methods for determining the onset of ALS in a subject are provided. One method includes analyzing a sample obtained from the subject for the presence or amount of one or more biomarkers indicative of ALS. In a preferred embodiment, the biomarkers are one or more of the following: C-reactive protein (CRP), cystatin c, plasminogen, complement C3, CysGly-transthyretin, and phosphorylated neurofilament heavy chain (pNFH). The sample is typically cerebral spinal fluid (CSF). The levels or concentrations of the biomarkers can be used to determine the onset of ALS, monitor the progression of ALS, or monitor the progression of a treatment for ALS.

## Claims

### Claim 1

A method for selecting a subject for treatment of amyotrophic lateral sclerosis (ALS) comprising
measuring the concentration of phosphorylated neurofilament heavy chain (pNFH) in a blood or cerebral spinal fluid sample obtained from the subject;
measuring the concentrations of complement C3 (C3) in the sample;
determining a ratio of pNFH to C3; and
selecting the subject for treatment if the ratio of pNFH/C3 is greater than 0.000125 and the concentration of pNFH is greater than 0.635 ng/ml,
wherein the concentrations of pNFH and C3 are measured by immunoassay or mass spectrometric measures.

### Claim 2 (depends on 1)

The method of claim 1 further comprising the step of administering a treatment to the patient.

### Claim 3 (depends on 1)

The method of claim 1 wherein the concentration of pNFH and C3 are measured by ELISA.

### Claim 4 (depends on 1)

The method of claim 1 wherein the concentration of pNFH and C3 are measured by mass spectrometric measures including Surface Enhanced Laser Dissociation/Ionization Time-of-Flight Mass Spectroscopy (SELDI-TOF-MS) or Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LS-MS/MS).

### Claim 5 (depends on 1)

The method of claim 1 wherein the specificity of the method for selecting the subject with ALS is at least 95%.

### Claim 6

A method for assisting in diagnosing amyotrophic lateral sclerosis (ALS) in a subject comprising:
measuring the concentration of phosphorylated neurofilament heavy chain (pNFH) in a blood or cerebral spinal fluid sample obtained from the subject;
measuring the concentration of complement C3 (C3) in the sample;
measuring the concentration of C Reactive Protein (CRP) in the sample;
determining a ratio of pNFH to C3;
determining a ratio of pNFH to CRP;
wherein a concentration of pNFH greater than 0.635 ng/ml and a ratio of pNFH/C3 greater than 0.000125 and a ratio of pNFH/CRP greater than 0.3755 is indicative of ALS,
wherein the concentrations of pNFH, C3, and CRP are measured by immunoassay or mass spectrometric measures.

### Claim 7 (depends on 6)

The method of claim 6 wherein the concentration of pNFH, C3, and CRP are measured by ELISA.

### Claim 8 (depends on 6)

The method of claim 6 wherein the concentration of pNFH, C3, and CRP are measured by mass spectrometric measures including Surface Enhanced Laser Dissociation/Ionization Time-of-Flight Mass Spectroscopy (SELDI-TOF-MS) or Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LS-MS/MS).

### Claim 9 (depends on 6)

The method of claim 6 wherein the specificity of the method for diagnosing the subject with ALS is about 100%.

### Claim 10

A method for selecting a subject for treatment of amyotrophic lateral sclerosis (ALS) comprising:
measuring the concentration of phosphorylated neurofilament heavy chain (pNFH) in a blood or cerebral spinal fluid sample obtained from the subject;
measuring the concentration of complement C3 (C3) in the sample
measuring the concentration of C Reactive Protein (CRP) in the sample,
determining a ratio of pNFH to C3;
determining a ratio of pNFH to CRP;
selecting the subject for treatment when the concentration of pNFH is greater than 0.635 ng/ml and the ratio pNFH/C3 is greater than 0.000125 and the ratio of pNFH/CRP is greater than 0.3755 is indicative of ALS,
wherein the concentrations of pNFH, C3, and CRP are measured by immunoassay or mass spectrometric measures.

### Claim 11 (depends on 10)

The method of claim 10 wherein the concentration of pNFH, C3, and CRP are measured by ELISA.

### Claim 12 (depends on 10)

The method of claim 10 wherein the concentration of pNFH, C3, and CRP are measured by mass spectrometric measures including Surface Enhanced Laser Dissociation/Ionization Time-of-Flight Mass Spectroscopy (SELDI-TOF-MS) or Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LS-MS/MS).

### Claim 13 (depends on 10)

The method of claim 10 wherein the specificity of the method for selecting a subject with ALS is about 100%.

### Claim 14

A method for assisting in diagnosing amyotrophic lateral sclerosis (ALS) in a subject comprising:
measuring the concentrations of phosphorylated neurofilament heavy chain (pNFH) in a blood or cerebral spinal fluid sample obtained from the subject;
measuring the concentration of complement C3 (C3) in the sample;
determining a ratio of pNFH to C3,
wherein a ratio greater than 0.000125 is indicative of ALS,
wherein the concentrations of pNFH and C3 are measured by immunoassay or mass spectrometric measures.

### Claim 15 (depends on 14)

The method of claim 14 wherein the concentration of pNFH and C3 are measured by ELISA.

### Claim 16 (depends on 14)

The method of claim 14 wherein the concentration of pNFH and C3 are measured by mass spectrometric measures including Surface Enhanced Laser Dissociation/Ionization Time-of-Flight Mass Spectroscopy (SELDI-TOF-MS) or Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LS-MS/MS).

### Claim 17 (depends on 14)

The method of claim 14 wherein the specificity of the method for diagnosing the subject with ALS is at least 95%.

### Claim 18

A method for assessing the efficacy of a treatment for amyotrophic lateral sclerosis (ALS) comprising
administering a drug to a subject having ALS,
measuring the concentrations of pNFH and complement C3 (C3) in a blood or cerebrospinal fluid sample obtained from the subject before and after administration of the drug;
wherein the treatment is effective if the ratio of pNFH to C3 decreases after administration of the drug relative to the ratio of pNFH to C3 prior to administration of the drug,
wherein the concentrations of pNFH and C3 are measured by immunoassay or mass spectrometric measures.

### Claim 19 (depends on 18)

The method of claim 18 wherein the concentration of pNFH and C3 are measured by ELISA.

### Claim 20 (depends on 18)

The method of claim 18 wherein the concentration of pNFH and C3 are measured by mass spectrometric measures including Surface Enhanced Laser Dissociation/Ionization Time-of-Flight Mass Spectroscopy (SELDI-TOF-MS).

### Claim 21

A method for assisting in diagnosing amyotrophic lateral sclerosis (ALS) in a subject comprising:
measuring the concentration of phosphorylated neurofilament heavy chain (pNFH) in a blood or cerebral spinal fluid sample obtained from the subject;
measuring the concentration of complement C3 (C3) in the sample;
measuring the concentration of C Reactive Protein (CRP) in the sample;
determining a ratio of pNFH to C3;
determining a ratio of pNFH to CRP;
wherein a concentration of pNFH greater than 0.635 ng/ml, and a ratio of pNFH/C3 greater than 0.000125, or a ratio of pNFH/CRP greater than 0.3755 is indicative of ALS, wherein the concentrations of pNFH, C3, and CRP are measured by immunoassay or mass spectrometric measures.