TECHNICAL FIELD

The present application relates to diagnosis of pulmonary disorders, and more particularly, to improved diagnosis of asthma versus chronic obstructive pulmonary disease (COPD) using metabolomic analysis.

BACKGROUND

Diseases of the respiratory system are among the leading diseases in terms of their impact on society. According to the 1998/99 National Population Health Survey, there were around 2,474,400 Canadians diagnosed with asthma and 498,900 with COPD. These illnesses resulted in 454 and 9,398 deaths, respectively. COPD is the 5th leading cause of death in Canada, and the only one that is increasing in prevalence. The prevalence of asthma is increasing worldwide, and it is the most common chronic disease of childhood. These conditions place a significant burden on the healthcare system, accounting for over $4 billion annually in direct and indirect costs.

A feature common to most diseases of the respiratory system is some form of lung inflammation. Lung inflammation consists of specific inflammatory cells and by products generated by cellular activity. Thus, specific lung diseases are often diagnosed not only by their clinical presentation, but also by the type of inflammation measured. Inflammatory cells release enzymes and other proteins in the airway, which can be measured and are specific to the cell type (i.e. mast cell tryptase or eosinophil cationic protein). The pathology of asthma is different from that seen in COPD, and the degree of inflammation and cellular damage varies with disease severity. For example, patients with asthma often have sputum samples positive for cells called eosinophils, and those with COPD or pneumonia present with increased sputum neutrophils. Differentiating asthma from other causes of chronic airflow limitation such as Chronic Obstructive Pulmonary Disease (COPD) can be difficult in a typical outpatient setting.

In addition, the degree of inflammation and cellular damage varies with asthma or COPD severity.

The treatments for each disease are designed to address this inflammation (i.e. corticosteroids or biologics versus antibiotics).

Unfortunately for clinicians, detecting this inflammation in individual patients is often difficult. Instead, clinicians rely on physiological (i.e. spirometry, peak flow, airway hyperreactivity (AHR)) or functional measurements (i.e. symptoms, or quality of life) to assess response to therapy. These tests, while useful, appear to be somewhat insensitive to changes in inflammatory status that later become clinically relevant.

While accurate airway inflammation measurements from bronchoscopy are possible, it is invasive and unavailable in the daily clinical setting. Thus, research has focused on non-invasive measures of inflammation such as induced sputum, eNO, and various inflammatory markers in body fluids. While experience with sputum has shown valuable results, significant barriers remain to its use clinically, including limited availability in most centers and the inability of young children and even many adults to expectorate. Exhaled NO (eNO) overcomes some of these barriers; however, while eNO shows correlation with asthma inflammation and outcomes experimentally, it requires time, co-operation, age greater than 4 years and, unless very carefully performed, may measure sinus rather than airway values. Ultimately, eNO still lacks the sensitivity and specificity of induced sputum. While other tests for asthma inflammation in blood or urine have been studied, i.e. urine leukotrienes, or eosinophil protein X, they lack the sensitivity required for clinical practice. Overall, a simple, non-invasive, readily available and sensible test for patients with airway inflammation is currently not widely available.

Using objective measurements of airway inflammation to guide asthma therapy can produce superior therapeutic results compared with traditional measures alone (e.g., symptoms and lung function).

For clinicians in a typical outpatient setting, detecting airway inflammation is often difficult. As such, differentiating asthma from other respiratory diseases like Chronic Obstructive Pulmonary Disease (COPD) can be difficult as both have somewhat similar clinical presentations. Objective measurements for differentiation of asthma and COPD are not used in a typical doctor's office. Most clinicians continue to rely upon patient history and examination before making a diagnosis and administering trials of therapy. If the incorrect diagnosis is made, then the prescribed treatment will likely be ineffective, and exposing patients to un-necessary side-effects and the patients respiratory problem will likely persist costing the health care system money and causing longer suffering by the patient.

Metabolomics is the study of metabolic pathways and biochemical molecules created in a living system. By measuring changes in metabolites, biochemical effects induced by a disease or its therapeutic intervention can be determined. However, typically it is not just a change in one metabolite that allows for identification of a disease state or the effects of therapeutic intervention. Rather, a combination of metabolites must be identified and analysed in order for relevant information to be obtained.

Accordingly, there remains a need to provide a reliable and easy-to-use method of objective measurements for improved diagnosis of asthma versus chronic obstructive pulmonary disease (COPD) that can overcome the limitations of the prior art.

SUMMARY

A metabolomic approach for improved diagnosis of asthma versus chronic obstructive pulmonary disease (COPD) is provided. Multivariate statistical analysis of data from urine can identify distinct metabolomic profiles that correlate with the clinical phenotypes of asthma versus COPD.

Analysis of metabolites in urine, as provided herein, can have the ability differentiate patients with asthma versus COPD both in the Emergency Department (ED) at the time of exacerbation and in follow-up post treatment.

Broadly stated, in some embodiments, a method is provided for diagnosing an asthma disease state in a subject comprising: obtaining metabolomic data on an obtained biological sample from the subject to obtain a subject profile; b. performing a statistical analysis on the metabolomic data to compare the subject profile to a predetermined asthma disease state profile and to a predetermined Chronic Obstructive Pulmonary Disease (COPD) disease state profile, wherein the analysis does not comprise identification of components of the biological sample; and c. providing a diagnosis of the asthma disease state when the statistical analysis identifies the subject profile as more similar to the predetermined asthma disease state profile than to the predetermined COPD disease state profile.

Broadly stated, in some embodiments, a method is provided for diagnosing an asthma disease state in a subject comprising: measuring a concentration of at least 11 of the metabolites as disclosed herein in an obtained biological sample from the subject to determine a subject profile; comparing the subject profile, by a statistical analysis, to a predetermined asthma disease state profile and to a predetermined Chronic Obstructive Pulmonary Disease (COPD) disease state profile; and providing a diagnosis of the asthma disease state when the statistical analysis identifies the subject profile as more similar to the predetermined asthma disease state profile than to the predetermined COPD disease state profile.

Broadly stated, in some embodiments, a method is provided for diagnosing a Chronic Obstructive Pulmonary Disease (COPD) disease state in a subject comprising: a. obtaining metabolomic data on an obtained biological sample from the subject to obtain a subject profile; b. performing a statistical analysis on the metabolomic data to compare the subject profile to a predetermined asthma disease state profile and to a predetermined Chronic Obstructive Pulmonary Disease (COPD) disease state profile, wherein the analysis does not comprise identification of components of the biological sample; and c. providing a diagnosis of the COPD disease state when the statistical analysis identifies the subject profile as more similar to the predetermined COPD disease state profile than to the predetermined asthma disease state profile.

Broadly stated, in some embodiments, a method is provided for diagnosing a Chronic Obstructive Pulmonary Disease (COPD) disease state in a subject comprising: a. measuring a concentration of at least 11 of the metabolites as disclosed herein in an obtained biological sample from the subject to determine a subject profile; b. comparing the subject profile, by a statistical analysis, to a predetermined asthma disease state profile and to a predetermined Chronic Obstructive Pulmonary Disease (COPD) disease state profile; and c. providing a diagnosis of the COPD disease state when the statistical analysis identifies the subject profile as more similar to the predetermined COPD disease state profile than to the predetermined asthma disease state profile.

Broadly stated, in some embodiments, a method is provided for creating a predetermined profile for differentiating between an asthma disease state and a Chronic Obstructive Pulmonary Disease (COPD) disease state in a subject comprising: measuring the concentration of at least 11 of the metabolites as disclosed herein in each of a plurality of urine samples obtained from asthma patients and COPD patients; performing a statistical analysis on the concentration values of (a) to differentiate between the asthma disease state and the COPD disease state.

Broadly stated, in some embodiments, a diagnostic model is provided for differentiating between an asthma disease state and a Chronic Obstructive Pulmonary Disease (COPD) disease state in a subject, the diagnostic model comprising at least 11 of the metabolites as disclosed herein.

Broadly stated, in some embodiments, a use of a diagnostic model as described herein is provided for diagnosing an asthma disease state and/or a COPD disease state in a subject in a subject.

In some embodiments, a computer readable medium is provided, the computer readable medium comprising instructions for carrying out the methods and/or the uses according to the methods and/or the uses as described herein.

In some embodiments the biological sample is urine.

In some embodiments the statistical analysis id a partial least squares discriminant analysis.

In some embodiments the concentration of at least one metabolite is determined using one or more or a combination of spectrometric and spectroscopic techniques selected from the group consisting of liquid chromatography, gas chromatography, high performance liquid chromatography, capillary electrophoresis, mass spectrometry, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, high performance liquid chromatography-mass spectrometry, capillary electrophoresis-mass spectrometry, raman spectroscopy, near infrared spectroscopy, and nuclear magnetic resonance spectroscopy.

In some embodiments a method of quantifying at least one metabolite with a phenol or amine functional group is provided comprising preparing a urine sample from a patient comprising, diluting the sample with ACN, buffer and12C-DNS-Cl; heating the sample; adding NaOH to the sample; further heating the sample; acidifying the sample; increasing the volume of the sample; spiking the sample with13C-IS; and increasing the volume of the sample with 50% ACN; running the prepared urine sample through the mass spectrometer to obtain a measurement of the at least one metabolite with a phenol or amine functional group; determining the concentration of creatinine in the sample; and normalizing the measurement of the at least one metabolite with a phenol or amine functional group using the concentration of creatinine in the sample.

In some embodiments a method of quantifying at least one metabolite with a carboxylic acid functional group is provided, the method comprising preparing a urine sample from a patient comprising, diluting the sample with ACN; combining the sample with TEA and12C-DmPA; heating the sample; cooling the sample to room temperature; and diluting the sample with formic acid, ACN and13C-DmPA standard solution; running the prepared urine sample through the mass spectrometer to obtain a measurement of the at least one metabolite with a carboxylic acid functional group; determining the concentration of creatinine in the sample; and normalizing the measurement of the at least one metabolite with a carboxylic acid functional group using the concentration of creatinine in the sample.

In some embodiments a method of diagnosing asthma versus COPD is provided comprising obtaining a biological sample from a patient; identifying the concentration of metabolites present in the biological sample using the method of either one or both of the quantification methods described above to create a subject profile; comparing the subject profile, by a statistical analysis, to a predetermined asthma disease state profile and to a predetermined Chronic Obstructive Pulmonary Disease (COPD) disease state profile; and providing a diagnosis of asthma or COPD when the statistical analysis identifies the subject profile as more similar to one of the predetermined disease state profiles than to the other predetermined disease state profile.

In some embodiments the metabolomic data includes information on at least 11 metabolites.

In some embodiments the at least 11 metabolites include 3-Hydroxyisovalerate, Glutamine, Arginine, Lactic acid, Glycolate, Tyrosine, 2-oxoglutarate, Glycine, Histidine, 1-methylhistamine and Taurine.

In some embodiments the at least 11 metabolites further include any number of the VIP metabolites.

In some embodiments the method may further include administering a treatment for asthma or COPD to the subject, as appropriate based on the diagnosis. In some embodiments the treatment for asthma comprises one or more of high dose corticosteroids and biological therapy targeting asthmatic inflammation. In some embodiments the treatment for COPD comprises one or more of anti-muscarinic inhaler, antibiotics, lung surgery and lung transplantation.

In some embodiments the step of obtaining metabolomic data is performed using the method of either one or both of the quantification methods described above.

In some embodiments the step of measuring the concentration of at least 11 metabolites is performed using the method of either one or both of the quantification methods described above.

